Salinity Stress and Calcium in Pomegranate: Impacts on Growth, Ion Homeostasis, and Photosynthesis

Abstract

Salinity has significant impacts on crops, a problem that is exacerbated under climate change conditions. For this reason, research is focused on possible ways to mitigate the impacts by adapting cultivation methods such as administering appropriate materials or formulations to plants. Therefore, this study investigated the effects of calcium (Ca²⁺) supplementation on the growth, physiology, and chemical composition of pomegranate plants (Punica granatum L. cv. ‘Wonderful’) grown under salinity stress. Young self-rooted plants were cultivated in pots containing a sand/perlite (1:1) mixture and irrigated with Hoagland’s nutrient solution amended with NaCl (0, 60, or 120 mM) and CaCl₂·2H₂O (0 or 10 mM). Salinity significantly reduced the fresh and dry weight of aboveground tissues; photosynthetic performance; chlorophyll content; and potassium (K), calcium (Ca), and magnesium (Mg) concentrations, particularly under high NaCl levels. Sodium (Na) accumulation increased in all plant parts, while nitrogen (N), manganese (Mn), and zinc (Zn) concentrations were elevated in basal leaves. Calcium supplementation mitigated several of these adverse effects, especially under moderate salinity. It helped maintain leaf biomass, supported K⁺ retention in roots, partially improved chlorophyll concentration, and limited Na⁺ accumulation in certain tissues. However, Ca²⁺ application did not consistently reverse the negative impacts of severe salinity (120 mM NaCl), and in some cases, interactions between Ca²⁺ and other nutrients such as Mg²⁺ were antagonistic. These findings confirm the inherent salt tolerance of pomegranate and demonstrate that calcium plays a partially protective role under salinity, particularly at moderate stress levels. Further research is needed to optimize Ca²⁺ use in saline agriculture and enhance sustainable cultivation of pomegranate in salt-affected soils.

1. Introduction

A major problem in many regions is the accumulation of salts in cultivated soils [1]. Saline soils contain water-soluble salts that hinder plant growth, primarily chlorides and sulfates of Na, Ca, and Mg [2]. Among these, Na-based salts are the most detrimental, as Na negatively affects soil structure, reducing aeration and limiting plant development [3].

Salinity is an escalating threat to agriculture [4], being one of the most critical environmental factors that limit crop growth and productivity [5]. Approximately 6% of the earth’s total surface is covered by saline soil, and this percentage is becoming increasingly larger as arable land expands worldwide [6].

Salinity impairs plant growth through several physiological mechanisms. These include: (a) osmotic stress—salinity lowers soil water potential, reducing water availability to roots and leading to water deficit if water absorption and/or transpiration are not sufficiently regulated; (b) ion toxicity—Na⁺ and Cl⁻ accumulate under saline conditions, causing toxicity symptoms in salt-sensitive plants; and (c) ion imbalance—high salt concentrations interfere with the uptake, transport and assimilation of essential nutrients such as K⁺ and Ca²⁺, leading to nutritional deficiencies and impaired physiological functions [7].

The adverse effects of salinity manifest throughout the plant, either as visible damage or reduced productivity. Some plants have evolved mechanisms to exclude or tolerate salt at the cellular level [8]. Generally, salinity delays germination, stunts growth, and leads to smaller plants [9]. Common symptoms include chlorosis, leaf desiccation, defoliation, and necrosis of young shoots [7]. Salinity also reduces photosynthetic efficiency and protein synthesis, while disturbing energy and lipid metabolism [8]. These effects are often linked to impaired stomatal conductance [10]. Furthermore, salinity shifts biomass allocation towards roots, increasing the root-to-shoot ratio [11]. As plants mature, their tolerance may increase due to enhanced osmotic adjustment capabilities [12,13,14]. The severity of salinity effects depends on factors such as species, genotype, and plant age. The harmful effects of salinity on pomegranate trees have also been well documented. Plants exposed to increasing salinity show reduced height, fewer leaves, and smaller stem diameters [15,16,17]. Döring and Lüdders observed that as NaCl concentration in nutrient solutions increased, plant growth declined, with Na and Cl accumulating predominantly in roots and leaves, respectively. Salinity also affected carbohydrate concentrations in the roots and leaves of pomegranate plants [18,19].

To mitigate salinity stress, calcium (Ca) plays a vital role in plant physiology. Ca is abundant in most soils, and deficiencies are rare under natural conditions. In mature leaves, Ca can constitute over 10% of dry weight [7]. It is essential for maintaining membrane integrity, forming cell walls, synthesizing proteins, and supporting cell division and meristem activity. Additionally, Ca²⁺ regulates pH and ion exchange, and it is crucial for pollen germination and pollen tube growth, as well as fruit respiration [7,20,21]. Moreover, Ca²⁺ functions as a secondary messenger, transmitting signals triggered by environmental stimuli, pathogens, and mechanical injuries through transient increases in cytoplasmic Ca²⁺ levels [7]. Under saline conditions, the importance of calcium is further amplified. Ca levels often rise with increasing soil salinity [22], and its application can alleviate salinity stress [23,24]. This effect is attributed to Ca²⁺’s regulatory role in metabolism [25] and its ability to compete with Na for membrane binding sites, thereby stabilizing membranes and maintaining K⁺ and Na⁺ balance [26]. Salinity also disrupts nutrient uptake in glycophytes by directly affecting transport mechanisms or through ion toxicity in the growth medium [27]. Competition among ions and changes in membrane selectivity impair nutrient absorption. Therefore, understanding the interplay between salinity and calcium in plant nutrition is essential for successfully cultivating pomegranate under saline conditions.

Pomegranate (Punica granatum L.) adapts very well to the Mediterranean climate and to the climate of semi-arid regions, a pattern observed since ancient times. The total area of pomegranate production worldwide exceeds 300,000 ha, with more than 76% found in five countries (India, Iran, China, Turkey, and the USA) [28]. In recent decades, there has been a great tendency for this crop to acquire an important position in many Mediterranean countries, including Greece [29]. Pomegranate plants generally have no special soil requirements. They can thrive even in saline soils or when irrigated with brackish water. However, to achieve optimal yield and fruit quality, they require deep, well-drained, medium-textured soils, such as clay, sandy loam, and light clay loam, with a pH of 5.5–7.0 and adequate organic matter content [30]. Despite this adaptability, pomegranate cultivation remains vulnerable to salinity, which poses a growing challenge to agriculture worldwide, particularly in regions where soil salinization is prevalent.

In this context, the present study aimed to evaluate the potential of exogenous calcium (Ca) application to mitigate salinity-induced stress in pomegranate (Punica granatum L. cv. ‘Wonderful’) by assessing its effects on plant growth, ion homeostasis, and photosynthetic performance. The findings are intended to contribute to a better understanding of the physiological and nutritional mechanisms involved and to support the development of improved cultivation strategies for pomegranate under saline conditions.

2. Materials and Methods

Six-month-old pomegranate (cv. Wonderful) plants, propagated by cuttings, uniform in height and girth, were transplanted to 3 L plastic pots containing a 1:1 sand/perlite mixture. The experimental plants were maintained outdoors in the Experimental Farm of the Department of Agricultural Development (Orestiada City, Greece), under a 3 m high lath house covered with plastic on the top to avoid rain (providing shading 20%). The experiment was initiated on April 29th, and the mean maximum and minimum temperatures for the months of May, June, and July were 28, 31.9, and 34 °C and 12.4, 15.5, and 19 °C, respectively. The maximum and minimum temperatures for these months were 33, 37, and 40 °C and 8, 9, and 16 °C, respectively. Plants were initially irrigated with good quality tap water (0.62 dS m⁻¹) for 21 days. Afterwards, the plants were irrigated every two or three days with 250 mL of modified half-strength Hoagland nutrient solution, containing NaCl alone or in combination with CaCl₂·2H₂O. The nutrient solution contained (in ppm): N 112, K⁺ 117.5, Ca²⁺ 80, P 31, S 16, Mg²⁺ 12, Fe²⁺/Fe³⁺ 1.0, Cl^- 0.885, B 0.135, Mn²⁺ 0.055, Zn²⁺ 0.065, Cu²⁺ 0.015, and Mo 0.025. In detail, the six treatments—each including six replicates (two plants per replicate)—were as follows (

Table 1. Composition of nutrient solutions (NaCl and CaCl₂; 2H₂O) applied per treatment to pomegranate plants.

	Treatments	NaCl (mM)	CaCl2·2H2O (mM)
1	C	0	0
2	Ca10	0	10
3	S60	60	0
4	S120	120	0
5	S60/Ca10	60	10
6	S120/Ca10	120	10

):

This experimental design was chosen because NaCl is the predominant salt in reclaimed water, and CaCl₂ mitigates potential calcium deficiencies [31]. Regarding the electrical conductivity (EC) of the solutions, values were 1.39, 2.85, 6.16, 11.33, 8.80, and 13.99 dS m⁻¹, respectively, while the corresponding pH values were 5.53, 4.32, 6.63, 7.54, 6.33, and 6.27. Every 10 days, 500 mL of water was supplied to each plant to leach out any accumulated salts. After 60 days from the beginning of the experimental treatments, the experiment was terminated once typical visual symptoms of salt toxicity (e.g., leaf necrosis, chlorosis, and stunted growth) became evident (Figure 1). At this time-point, comprehensive physiological assessments were performed as described in subsequent subsections.

2.1. Plant Growth Measurements

Baseline measurements (recorded prior to treatment initiation) included plant height, trunk girth (measured 3–4 cm above soil), and counts of leaves and nodes. At harvest, these parameters were reassessed, along with lateral stem number and length. One leaf from the top four nodes of each plant was photographed for digital morphometric analysis (Image Pro Plus 4.5, Media Cybernetics, Rockville, MD, USA) to determine area, length, width, and perimeter. Additional leaf samples were collected for subsequent analyses of pigment content and membrane leakage (see Section 2.2 and Section 2.3). Plants were then uprooted and separated into leaves (basal and upper halves), stems, and roots. After fresh weight (FW) determination, tissues were washed with deionized water, oven-dried at 75 °C for 48 h, and reweighed to determine dry weight (DW).

2.2. Chlorophyll Content and Photosynthesis Parameters

Chlorophyll extraction was performed on leaf discs (3 cm²) collected from mature top leaves at noon under full sunlight. Samples were immersed in 15 mL of 96% ethanol (Merck KGaA, Darmstadt, Germany) at 78 °C until complete discoloration. Chlorophyll (a + b) concentration was determined spectrophotometrically at 649 nm and 665 nm using the Wintermans and de Mots [32] formula as follows:

Chlorophyll a + b (in mg/g f.w.) = 6.10 × (A₆₆₅) + 20.04 × (A₆₄₉) × 0.015/f.w.

Chlorophyll a + b (in mg/cm²) = 6.10 × (A₆₆₅) + 20.04 × (A₆₄₉) × 0.015/leaf area

(A₆₆₅) and (A₆₄₉) represent absorbance values read at 665 and 649 nm wavelengths, respectively.

Additionally, both old and new leaves were used for CCI (Chlorophyll Content Index) measurements with a portable CCM-200 device (Opti-sciences Inc., Hudson NH, USA, https://www.adc.co.uk/wp-content/uploads/2013/09/CCM-200-plus-manual-1.pdf, (accessed on 25 June 2025).

Photosynthetic parameters, including net photosynthetic rate (P_n), transpiration rate (E), and stomatal conductance (g_s), were measured in mature top leaves using a portable gas analyzer (ADC BioScientific LC pro, Hoddesdon, Herts EN11 ONT, UK). Water use efficiency (WUE) was calculated as the P_n/g_s ratio, expressed in μmol CO₂ per mmol H₂O.

2.3. Electrolyte Leakage of Cell Membranes

To determine cell membrane permeability, 0.02 g and 5 mm long samples of fresh top leaves cut into narrow strips, were used. These samples were placed into plastic tubes with a lid and filled with 10 mL deionized water. The tubes were stirred for 24 h, and then the electrical conductivity and K concentration of the solutions were determined (C1 and K1 values, respectively). Afterwards, the samples were transferred to glass tubes with 10 mL of deionized water and placed into an autoclave furnace at 120 °C under a pressure of 103.1 kPa for 20 min. Then, the samples were cooled to 25 °C, and a second measurement of EC and K concentration took place (C2 and K2, respectively). Thus, the membrane permeability was calculated by the mathematical formulas C1/C2, C1/C1+C2, K1/K2, and K1/K1+K2.

2.4. Inorganic Elements Analysis

For quantification of K, Ca, Mg, Na, Fe, Zn, and Mn, 0.5 g of milled, dried plant material was dry-ashed at 550 °C for 6 h. The ash was dissolved in 3 mL of 6 N HCl (Merck KGaA, Darmstadt, Germany) and diluted to 50 mL with deionized water. Elemental concentrations were determined using atomic absorption spectroscopy (Perkin-Elmer 2380; Perkin-Elmer, Salem, MA, USA) using standard methods.

Total nitrogen (N) content was analyzed using the Kjeldahl method [33]. One gram of milled, dried sample was digested with 20 mL concentrated H₂SO₄ (Merck KGaA, Darmstadt, Germany) in the presence of a catalyst at 390 °C for 130 min, followed by standard distillation and titration procedures.

Chloride content was determined by titrating aqueous extracts. Briefly, 0.2 g of milled tissue was extracted in 25 mL deionized water for 30 min. After filtration, 20 mL aliquots were titrated with 0.0141 N AgNO₃ (Merck KGaA, Darmstadt, Germany) solution using K₂CrO₄ (Merck KGaA, Darmstadt, Germany) as an indicator [34].

2.5. Statistical Data Analysis

The factorial experiment evaluated two salt concentrations (60 and 120 mM NaCl) combined with calcium supplementation (0 or 10 mM CaCl₂·2H₂O), plus a control, totaling eight experimental treatments. Each treatment consisted of six biological replicates, with two plants per replicate (n = 12 plants per treatment). All data were analyzed using one-way ANOVA followed by Duncan’s multiple range test (p < 0.05) for post hoc comparisons among the eight treatments. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Plant Growth Parameters

The evaluation of plant growth responses under salinity (S60, S120) and calcium (Ca10, S60/Ca10, S120/Ca10) treatments provides important insights into the physiological adjustments of pomegranate plants. A range of morphological traits, including biomass accumulation and allocation, was assessed to determine the impact of individual and combined stress factors. The results are presented below, categorized by fresh and dry weight parameters, as well as plant height and leaf number per plant.

Salinity and calcium treatments markedly influenced the fresh weight accumulation in different plant organs of pomegranate (

Table 2. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on growth parameters (fresh weight basis) of pomegranate (Punica granatum L. cv. Wonderful), including leaf fresh weight, stem fresh weight, root fresh weight, total plant fresh weight, and shoot-to-root ratio. Data represent the mean of six replicates. Different letters within each column indicate statistically significant differences at the 0.05 level.

Treatments	Leaf Fresh Weight (g)	Stem Fresh Weight (g)	Root Fresh Weight (g)	Plant Fresh Weight (g)	Shoot/Root
C	17.76 a	9.99 a	15.32 a	43.07 a	2.23 a
Ca10	11.80 b	6.71 b	11.07 a	29.58 b	1.69 a
S60	12.10 b	6.22 b	13.78 a	32.10 b	1.35 a
S60/Ca10	9.38 bc	4.46 b	10.25 a	24.09 b	1.46 a
S120	9.79 bc	5.19 b	10.65 a	25.64 b	1.41 a
S120/Ca10	7.95 c	4.38 b	9.06 a	21.39 b	1.34 a

). Leaf, stem, and total plant fresh weights were significantly reduced in all treated groups compared to the control, which consistently exhibited the highest values across these parameters. Moderate salinity (S60) and calcium application alone (Ca10) led to intermediate reductions in leaf fresh weight, whereas their combinations—particularly under high salinity (S120/Ca10)—resulted in the most pronounced decreases. Furthermore, all treated plants exhibited similarly reduced stem fresh weights compared to the control group, indicating that calcium alone (Ca10) or in combination with NaCl-induced salinity treatments (S60/Ca10, S120/Ca10) was not able to mitigate the adverse impact of salinity on stem biomass. Moreover, the shoot-to-root ratio on a fresh weight basis remained statistically unchanged, highlighting that although overall shoot growth declined under stress, it was proportionally balanced with root biomass across treatments (Table 2).

On a dry weight basis (

Table 3. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on total plant height, leaf number per plant and other growth parameters (dry weight basis) of pomegranate (Punica granatum L. cv. Wonderful), including leaf dry weight, stem dry weight, root dry weight, total plant dry weight, and shoot-to-root ratio. Data represent the mean of six replicates. Different letters within each column indicate statistically significant differences at the 0.05 level.

Treatments	Plant Height (cm)	Leaf Number per Plant	Leaf Dry Weight (g)	Stem Dry Weight (g)	Root Dry Weight (g)	Plant Dry Weight (g)	Shoot/Root
C	50.42 a	88.67 a	6.45 a	4.14 a	2.77 a	13.36 a	3.97 b
Ca10	46.82 a	61.92 bc	4.50 bc	2.86 b	1.95 b	9.31 b	3.77 bc
S60	42.13 a	73.17 ab	4.17 bc	2.52 b	1.88 b	8.57 b	3.59 bc
S60/Ca10	40.38 a	52.83 bc	6.75 a	1.80 b	1.71 b	10.26 ab	5.30 a
S120	42.83 a	60.42 bc	3.28 c	2.24 b	1.84 b	7.37 b	2.97 c
S120/Ca10	39.38 a	40.58 c	5.74 ab	1.90 b	1.70 b	9.34 b	4.54 ab

), treatment effects were similarly evident, particularly in aboveground biomass components. The control group showed the highest dry weights for leaves, stems, and total plant biomass. Notably, the S60/Ca10 treatment produced a leaf dry weight comparable to the control, without statistically significant differences, indicating that calcium may help maintain leaf biomass under moderate salinity rather than increasing it. On the other hand, high salinity (S120), either alone or in combination with calcium, consistently reduced leaf and stem dry weights, with the most severe reductions in leaves observed in the absence of calcium supplementation. Additionally, root dry weight followed the same pattern of reduction across all treatments relative to the control, though differences among the treated groups were not statistically significant. Furthermore, total plant dry weight mirrored the trends of its components, with S60/Ca10 maintaining a relatively higher biomass that did not significantly differ from the control, whereas all other treatments exhibited marked reductions. Importantly, the shoot-to-root dry weight ratio varied significantly, with S60/Ca10 displaying the highest value, indicating a preferential allocation of biomass to the aerial parts under these conditions. Conversely, the lowest ratio was observed in the high salinity treatment without calcium, reflecting a shift towards root investment or inhibited shoot growth under severe stress. These findings underscore the differential effects of salinity and calcium on biomass distribution, particularly under conditions of combined stress.

Plant height (Table 3) was not significantly affected by any of the salinity or calcium treatments. The measured values ranged between 39.38 and 50.42 cm across treatments, with the highest observed in the control group. However, no statistically significant differences were detected between control and all the other treatments. In contrast, the number of leaves per plant (Table 3) responded sensitively to salinity and calcium treatments. The control treatment produced the highest leaf number per plant, while the moderate salinity treatment (S60) showed a comparable value. Treatments involving calcium alone or in combination with salinity (Ca10, S60/Ca10, and S120) resulted in reduced leaf numbers, all statistically lower than the control. The greatest reduction was observed under the combined high salinity and calcium treatment (S120/Ca10), which led to the lowest total leaf number per plant, significantly different from most other treatments.

3.2. Permeability of Leaf Cell Membranes

The addition of salt and calcium (either separately or in combination) to the nutritional solution elevated the leaf membrane permeability at the end of the experiment (

Table 4. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on permeability of leaf cell membranes in leaves of pomegranate (Punica granatum L. cv. Wonderful). Data represent the mean of four replicates. Different letters within the same column indicate statistically significant differences at the 0.05 level. C1 and C2 represent electrical conductivity, while K1 and K2 represent potassium concentrations, before (C1, K1) and after (C2. K2) heat and pressure treatment of the solutions, respectively.

	C1/C2	C1/C1+C2	K1/K2	K1/K1+K2
C	0.57 b	0.36 b	0.94 a	0.48 a
Ca10	0.66 ab	0.39 ab	0.98 a	0.49 a
S60	0.65 ab	0.39 ab	0.92 a	0.48 a
S60/Ca10	0.67 ab	0.40 ab	1.01 a	0.50 a
S120	1.01 a	0.49 a	0.95 a	0.49 a
S120/Ca10	1.57 a	0.52 a	0.99 a	0.50 a

). Nevertheless, this rise was statistically significant only in the S120-treated plants.

3.3. Leaf Chlorophyll

The treatment with the highest salinity with calcium (S120/Ca10) showed a substantial decline in the concentration of total chlorophyll per fresh weight or area unit in leaves (

Table 5. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on leaf chlorophyll parameters of pomegranate cv. Wonderful. Data represent the mean of six replicates. Different letters within each column indicate statistically significant differences at the 0.05 level.

Treatments	Chlorophyll a + b (mg/g Fresh Weight)	Chlorophyll a + b (mg/cm2)	CCI Units
C	3.84 a	0.040 a	40.21 a
Ca10	3.80 a	0.041 a	32.50 a
S60	4.19 a	0.037 a	30.26 a
S60/Ca10	2.56 ab	0.031 ab	30.34 a
S120	3.17 ab	0.034 ab	37.36 a
S120/Ca10	1.79 b	0.025 b	41.75 a

). What is more, the addition of calcium resulted in a little drop in the content of chlorophyll in each salinity treatment.

Finally, most salt-treated plants showed a minor but not significant decrease in chlorophyll units (CCI) in their leaves when compared to the control. Although plants treated with moderate salinity (S60 and S60/Ca10) exhibited numerically lower values, these were not statistically different from the control. Interestingly, the S120/Ca10 treatment showed the highest CCI value, despite its significant reduction in total chlorophyll content (Table 5).

3.4. Photosynthesis

The addition of salts to the nutritional solution considerably altered photosynthesis parameters in leaves (photosynthetic rate, transpiration rate, stomatal conductance, and water usage efficiency) (Figure 2). Specifically, all of the above parameters were found to be significantly and negatively influenced in the highest salinity treatments (S120/Ca10 and S120). Furthermore, medium salinity treatments (S60 and S60/Ca10) were found to have a deleterious effect on photosynthetic rates.

3.5. Chemical Composition of Plants

Salinity and calcium treatments markedly influenced the distribution and concentration of mineral nutrients in various parts of pomegranate plants. These effects varied across different plant parts, with notable shifts in the levels of macronutrients (K, Ca, Mg, Na) and micronutrients (Fe, Mn, Zn) in leaves, stems, and roots (

Table 6. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on nutrient element concentrations in basal and top leaves of pomegranate (Punica granatum L. cv. Wonderful). Data represent the mean of six replicates. Different letters within the same column and plant organ indicate statistically significant differences at the 0.05 level.

Treatments	N	K	Ca	Mg	Na	Fe	Mn	Zn
		(% Dry Weight)				(mg kg−1 Dry Weight)
		Basal leaves
C	1.91 c	1.31 ab	1.88 a	0.19 a	0.19 c	91.50 a	19.33 c	12.17 c
Ca10	1.82 c	1.38 a	1.92 a	0.20 a	0.14 c	118.17 a	26.50 bc	15.17 bc
S60	3.14 bc	1.22 bc	1.29 c	0.16 b	0.94 b	145.17 a	26.17 bc	13.67 c
S60/Ca10	5.21 a	1.16 c	1.52 b	0.16 b	0.94 b	90.00 a	25.00 bc	19.67 ab
S120	2.79 bc	1.09 cd	1.17 c	0.15 b	1.22 ab	129.83 a	30.83 ab	14.83 bc
S120/Ca10	4.85 a	0.97 d	1.22 c	0.15 b	1.40 a	106.77 a	34.51 a	22.87 a
		Top leaves
C	2.26 ab	1.31 a	1.20 a	0.16 a	0.16 c	50.50 a	13.33 c	11.17 b
Ca10	2.40 a	1.29 ab	1.08 a	0.13 b	0.09 c	51.00 a	17.83 ab	15.33 a
S60	2.42 a	1.21 b	0.59 c	0.13 b	0.78 b	46.50 a	17.83 ab	10.83 b
S60/Ca10	1.99 b	1.06 c	0.83 b	0.10 c	0.78 b	53.33 a	19.50 a	13.33 ab
S120	2.21 ab	1.02 c	0.48 c	0.09 cd	1.14 a	50.00 a	16.33 abc	10.33 b
S120/Ca10	2.19 ab	1.00 c	0.50 c	0.08 d	1.09 a	44.67 a	15.67 bc	11.83 b

,

Table 7. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on nutrient element concentrations in stems of pomegranate (Punica granatum L. cv. Wonderful). Data represent the mean of six replicates. Different letters within the same column indicate statistically significant differences at the 0.05 level.

Treatments	K	Ca	Mg	Na	Fe	Mn	Zn
	(% Dry Weight)				(mg kg−1 Dry Weight)
C	0.97 ab	1.08 a	0.097 bc	0.28 c	67.67 a	11.33 b	31.83 a
Ca10	1.09 a	1.27 a	0.085 d	0.22 c	59.17 a	11.17 b	31.83 a
S60	0.85 bc	1.22 a	0.111 a	0.78 ab	44.83 a	17.67 b	32.67 a
S60/Ca10	0.79 c	1.32 a	0.084 d	0.70 b	89.29 a	17.35 b	57.93 a
S120	0.74 c	1.11 a	0.108 ab	0.90 a	220.67 a	24.17 a	97.33 a
S120/Ca10	0.76 c	1.36 a	0.093 cd	0.87 a	43.35 a	17.61 b	39.33 a

and

Table 8. Effects of salinity (S60, S120) and calcium (Ca10), applied through the nutrient solution, on nutrient element concentrations in roots of pomegranate (Punica granatum L. cv. Wonderful). Data represent the mean of six replicates. Different letters within the same column indicate statistically significant differences at the 0.05 level.

Treatments	K	Ca	Mg	Na	Fe	Mn	Zn
	(% Dry Weight)				(mg kg−1 Dry Weight)
C	0.96 a	1.52 b	0.35 a	0.51 c	326.17 a	40.17 c	79.83 c
Ca10	1.22 a	1.75 a	0.37 a	0.39 c	274.00 a	38.17 c	95.83 bc
S60	0.93 a	1.33 cd	0.38 a	1.41 a	343.17 a	61.17 a	86.83 c
S60/Ca10	1.14 a	1.45 bc	0.32 a	1.02 b	307.50 a	45.17 bc	113.50 ab
S120	0.94 a	1.13 e	0.31 a	1.38 a	306.33 a	53.50 ab	82.17 c
S120/Ca10	1.11 a	1.24 de	0.34 a	1.56 a	329.67 a	57.83 a	119.50 a

).

In basal leaves, salinity significantly increased sodium (Na) concentration, particularly under the S120/Ca10 treatment, which recorded the highest Na levels (Table 6). This was accompanied by a decline in K, Ca, and Mg concentrations under salinity, with the most pronounced reductions observed in the S120 and S120/Ca10 treatments. The presence of supplemental calcium, especially in the S60/Ca10 treatment, partially mitigated these reductions for Ca but not for K and Mg. Moreover, while Fe concentration remained statistically unchanged across treatments, micronutrients such as Mn and Zn exhibited clear treatment-related trends. Notably, Mn and Zn concentrations increased under combined high salinity and calcium application, with S120/Ca10 showing the highest values for both elements in basal leaves.

A similar pattern was observed in top leaves (Table 6), where Na concentration increased significantly in response to salinity. More specifically, S120 and S120/Ca10 exhibited the highest Na accumulation, whereas Ca addition alone (Ca10) led to a reduction in Na concentration. Interestingly, K and Ca levels in top leaves declined progressively with increasing salinity, and this effect was not fully reversed by Ca supplementation. Mg concentration also declined under saline conditions, with the lowest values in the S120/Ca10 treatment. In terms of micronutrients, Fe content remained relatively stable, whereas Mn and Zn exhibited moderate increases under calcium-enriched conditions, particularly in the S60/Ca10 treatment.

In stem tissues (Table 7), Na accumulation followed the same increasing trend under salinity, reaching its peak in S120. Concurrently, K concentration decreased with increasing salinity, while Ca levels remained statistically stable across treatments. Magnesium (Mg) levels remained consistent among treatments, and Mn increased under high salinity, while Fe displayed variable responses without clear treatment effects.

In the roots (Table 8), Na concentration increased substantially under all salinity treatments, reaching its highest value in the S120/Ca10 treatment. However, unlike the shoot tissues, root K and Mg concentrations remained unaffected by salinity and/or calcium supplementation. Calcium (Ca) concentrations in the roots declined under salinity (S60 and S120) but remained somewhat higher when Ca was supplied (S60/Ca10 and S120/Ca10). Moreover, micronutrient accumulation, particularly Mn and Zn, was strongly enhanced under salinity, with the highest Zn concentration recorded in S120/Ca10. The elevation of Mn levels was also significant and prominent in the S60, S120, and S120/Ca10 treatments compared to the control, pointing to increased uptake or possibly reduced translocation from roots to shoots under salt stress.

The concentration of Cl in the stems increased in response to the addition of CaCl₂·2H₂O (Ca10) and NaCl (S60, S120) to the nutrient solution, with more pronounced accumulation observed in the S60 and S120 treatments compared to their respective combinations with calcium (S60/Ca10, S120/Ca10). In general, as salinity increased from 60 to 120 mM NaCl, stem Cl concentration rose significantly relative to the control, although no significant differences were detected between S60 and S120. A similar trend was observed in the roots, where all treatments resulted in a statistically significant increase in Cl concentration compared to the control. However, no significant differences were found between the S60 and S120, S60/Ca10, and S120/Ca10 treatments. Regarding nitrogen (N), salinity also had a notable effect on its concentration in plant tissues (Table 6). In basal leaves, salinity treatments combined with calcium (S60/Ca10 and S120/Ca10) led to significantly higher N concentrations compared to the other treatments (C, Ca10, S60, S120). In contrast, N concentration in the top leaves was not significantly affected by any of the treatments.

4. Discussion

Pomegranate is generally a crop that can respond very well to soils with increased salinity [17,31,35]. In the present work, the plants showed no visible symptoms of leaf toxicity, chlorosis, or necrosis. However, the FW of leaves and stems was significantly reduced, by the addition of NaCl and CaCl₂·2H₂O salts to the nutrient solution. Root FW was not significantly affected in any of the salinity treatments, indicating a degree of stress tolerance or compensatory growth in the root system. The combination of NaCl and CaCl₂·2H₂O did not significantly alleviate the reductions in FW of the different vegetative parts, especially under high salinity (S120/Ca10), where pronounced declines were observed. The DW of leaves was significantly reduced by the inclusion of NaCl and CaCl₂·2H₂O salts to the nutrient solution, while the stems and root DW was significantly reduced only by the addition of NaCl. Notably, the S60/Ca10 treatment maintained leaf DW at levels comparable to the control, suggesting a potential protective role of calcium under moderate salinity, rather than an actual increase. Generally, salinity has negative effects on both the fresh [36] and dry weight of various plant parts [37]. Sun et al. [31] studying 22 pomegranate cultivars found that salt treatment decreased the leaf, stem, and shoot DW on average for all cultivars by 32%, 32%, and 32%, respectively. Similar results were found for explants of orange [38] and bitter almond [39]. Shiyab et al. [38] observed that increasing salinity negatively affected the number and length of lateral shoots in explants of oleander. In pomegranate, salinity has been reported to lead to a reduction in plant height, the length and number of internodes, shoot thickness, and leaf area [40,41]. Moreover, salt stress impacted negatively on the leaf number of pomegranate plants [42] and leaf area [40,41,43].

Adding Ca to the growth medium generally helped to improve the growth of pomegranate plants, an effect that has been also reported in other studies, e.g., [44]. Under saline conditions, Ca addition improves plant growth [45], root elongation [46], and shoot growth [47]. According to Islam et al. [45], application of Ca reverses the salt-induced changes in tomato plants through increasing osmoprotectants and the activation of antioxidant enzymes, as well as by optimizing mineral nutrient status.

With respect to leaf membrane permeability, our results indicated an increase in response to the addition of NaCl and CaCl₂·2H₂O salts, with a statistically significant effect only observed in the S120 treatment. This suggests that Ca²⁺ may provide partial protection at moderate levels but is not sufficient to fully prevent membrane destabilization under severe salt stress. This phenomenon is explained by the displacement of Ca²⁺ from membranes by Na⁺, leading to increased permeability [48]. Sharma et al. [49] consider that, by making bonds with the phospholipid bilayer, Ca²⁺ regulates the structure and function of membranes and, hence, stabilizes and promotes their structural integrity in plants subjected to stress conditions.

Saline conditions caused a decrease in chlorophyll a, chlorophyll b, and total chlorophyll contents in pomegranate [17,35,50]. Garcia-Sanchez et al. [51] pointed out that the reduction of leaf chlorophyll concentration is a result of the relationship of Cl and Na with citrus leaf stress. Moreover, it has been reported that salinity affects normal plant growth due to the lack of Mg since some enzymes need Mg²⁺ for chlorophyll composition, catalysis, and enzymes’ action [52]. Our results showed that Mg concentration in top leaves was reduced by up to 50% under 120 mM NaCl in nutrient solution. The addition of Ca mitigated the negative effects of NaCl in reducing chlorophyll concentration, which was also demonstrated in tomato plants [53].

The mechanisms underlying this synergy could be the following: (1) Maintenance of chlorophyll content (a + b) with calcium supplementation via stabilizing chloroplast membranes and thus preserving photosynthetic pigments [54]; reducing Na⁺ toxicity, which otherwise disrupts chlorophyll synthesis; and enhancing antioxidant enzyme activities, protecting chlorophyll from ROS-induced degradation [55]. (2) Improvement in plant dry weight, since calcium supports biomass via maintaining cell wall structure and turgor pressure, improving water uptake and transport by preserving root membrane integrity [56], and regulating enzyme activities involved in carbohydrate metabolism and growth. (3) Ionic balance and membrane function. Calcium promotes selective ion uptake, maintaining a favorable K⁺/Na⁺ ratio critical for photosynthesis and growth [56].

Some studies may report neutral or even negative effects of Ca on salinity stress mitigation concerning chlorophyll. Discrepancies can be attributed to 1. Genotype-specific responses; 2. Environmental conditions; 3. Growth stage; 4. Soil vs. hydroponics; and 5. Form (e.g., CaCl₂ vs. Ca(NO₃)₂), timing of Ca application, and frequency of application [57]. Interestingly, the mitigation effect of calcium is highly concentration-dependent: (a) Low to moderate Ca²⁺ level improves ion balance and membrane stability and alleviates NaCl toxicity, (b) excess Ca²⁺ may cause nutrient antagonism (e.g., with Mg²⁺ or K⁺) or can lead to osmotic imbalance or precipitation of phosphates in the nutrient solution [7,56].

Furthermore, while chlorophyll content index (CCI) values were numerically lower in the S60 and S60/Ca10 treatments, these differences were not statistically significant. Interestingly, S120/Ca10 exhibited the highest mean CCI value, despite recording the lowest total chlorophyll content per unit area or weight. This suggests a potential alteration in chlorophyll distribution or leaf structure under combined high salinity and calcium application. A possible explanation for that difference could be that CCI can be influenced by factors beyond just the presence of chlorophyll, including leaf thickness, etc.

In pomegranate plants, the physiological parameters of photosynthesis [photosynthetic rate (P_n), transpiration rate (Ε), stomatal conductance (g_s), water use efficiency (WUE)] were adversely affected by salinity treatments, particularly under high NaCl concentrations (S120 and S120/Ca10). More specifically, stomatal conductance and water use efficiency were significantly reduced only under high salinity (S120 and S120/Ca10), whereas moderate salinity (S60 and S60/Ca10) caused a significant decline in P_n but did not significantly affect g_s or WUE. Tattini et al. [37] recorded similar results in Phyllirea plants. The reduction in the rate of photosynthesis under salinity conditions may be due to stomatal or non-stomatal factors [58]. Stomatal closure is the first reaction to inhibit photosynthesis in salinity conditions [59], and increased Cl⁻ ion concentration in leaves is the main cause of reduction in photosynthetic rate, which is a non-stomatal factor [60]. Pomegranate salt tolerance is mainly related to stomatal regulation, Na⁺/Cl⁻ uptake, and compartmentalization and osmolyte accumulation [61]. Thus, the observed reduction in photosynthesis was partly associated with reduced stomatal conductance under high salinity, although non-stomatal factors such as ion toxicity and pigment degradation may also have contributed under moderate stress. Another factor that contributed to this result may be the reduction in K⁺ concentration observed under salinity, since K plays a crucial role in enhancing photosynthetic capacity by elevating the concentration of photosynthetic pigments and promoting photosynthate production through stomatal opening. While the addition of calcium (Ca10) showed some capacity to mitigate declines in photosynthesis-related traits under moderate salinity, it was not sufficient to prevent significant impairments at higher NaCl concentrations.

Generally, salinity affects the ion balance in pomegranate tissues through impeding the ion uptake, accumulation, and transport [41]. Hence, the plants treated with NaCl and CaCl₂·2H₂O showed a significant increase in Cl concentration in stems and roots, especially under the S60 and S120 treatments. Similar results were observed in other plants [52,62]. However, when Ca was applied in combination with NaCl (S60/Ca10, S120/Ca10), Cl⁻ concentrations were generally lower than those recorded in NaCl-only treatments, suggesting a partial mitigating effect of Ca²⁺ on Cl accumulation. This is consistent with previous findings showing that Ca in the nutrient solution can modulate Cl⁻ toxicity by restricting its upward transport [63]. While some literature reports that calcium alone can reduce Na⁺ and Cl⁻ concentrations in pomegranate leaves [48], our data showed that Na accumulation in all plant parts increased with salinity and was highest in the S120/Ca10 treatment. This indicates that calcium was not able to significantly reduce Na⁺ levels under severe salinity in this study. Moreover, in orange plants grown under salinity conditions, Ca²⁺ was found to be effective in reducing the transport of Cl from roots to leaves, resulting in foliar protection under saline conditions [64,65], a phenomenon partially reflected in our findings for stems and roots but not clearly evident in leaves.

The concentration of N increased significantly in the basal leaves, while it was unaffected in the top leaves. Although several experiments performed under laboratory and greenhouse conditions have shown that salinity reduces N concentration in pomegranates [17,66], our findings suggest a differential response depending on leaf age or position. Nitrogen is the element that mostly determines plant growth. Thus, in the present work, the increase in basal leaf N concentration may have been due to the general decrease in vegetative growth observed in pomegranate plants, leading to a concentration effect, while its lack of change in top leaves may reflect limited competition between Cl⁻ and NO₃⁻ for uptake or translocation under the present salinity levels [67].

The concentration of K in the basal leaves, top leaves, and stems showed a significant decrease with the addition of NaCl alone or in combination with NaCl and CaCl₂·2H₂O. For plants growing at high concentrations of Na⁺ in the nutrient medium, a significant decrease in K⁺ concentration and K⁺:Na⁺ ratio has been observed in the plant tissue [41,68,69]. Due to their similar physicochemical properties, Na⁺ competes with K⁺ in plant uptake specifically through high-affinity potassium transporters (HKTs) and nonselective cation channels (NSCCs). Membrane depolarization caused by Na⁺ makes it difficult for K⁺ to be taken up by K⁺ inward-rectifying channels (KIRs) and increases K⁺ leakage from the cell by activating potassium outward-rectifying channels (KORs) [70]. The addition of CaCl₂·2H₂O alone to the nutrient solution enhanced the concentration of K in the basal leaves and the stems, but these increases were not statistically significant. In contrast, in the roots, treatment with 10 mM CaCl₂·2H₂O (Ca10) resulted in a statistically significant increase in K⁺ concentration compared to the control. According to several reports, supplemental Ca promotes K uptake and transport in plants [71,72]. Moreover, our results confirm that the beneficial effect of Ca²⁺ under salinity stress is more evident in the root tissues than in the shoot. While K⁺ concentrations in leaves and stems remained low under saline conditions, regardless of Ca addition, root K⁺ content was preserved or even improved with Ca application, supporting the view that Ca²⁺ plays a protective role in root ion homeostasis under stress [73,74].

Calcium increases the plant tissue resistance under various stress conditions including both biotic and abiotic stresses. Calcium uptake under salinity conditions is reduced due to ionic interactions, precipitation, and increased ionic strength [27]. Also, root growth and function can be negatively affected due to a high Na⁺/Ca⁺ ratio [25]. Humphery and Rodriguez [75] observed that the mechanism by which Ca²⁺ moves into roots is impaired under salinity conditions, and consequently, the Ca concentration is also reduced. In the present study, Ca concentration was significantly reduced in basal leaves, top leaves, and roots of pomegranate plants treated with NaCl, particularly under the highest salinity level (S120). In contrast, the inclusion of 10 mM CaCl₂·2H₂O to the nutrient solution led to an increase in Ca concentration in both basal leaves and roots either when applied alone (Ca10) or in combination with NaCl (S60/Ca10, S120/Ca10), though the effect was more pronounced in the latter. Calcium is important for cell biology under saline conditions because it maintains the integrity of cell membranes, regulates osmotic pressure, and maintains K⁺/Na⁺ selectivity [76]. Our findings align with these roles, as supplemental Ca appeared to mitigate ionic imbalances in tissues, particularly by supporting Ca retention in roots and lower leaves under salt stress. An increase in Ca concentration in plants treated with CaCl₂·2H₂O was also observed in other studies [77,78].

The addition of NaCl and CaCl₂·2H₂O salts to the nutrient solution reduced the Mg concentration in the basal and top leaves of pomegranate plants. This reduction was most pronounced in the 120 mM NaCl (S120) and 10 mM CaCl₂·2H₂O (Ca10) treatments and remained low in the combined treatments (S60/Ca10 and S120/Ca10), indicating that neither Ca supplementation nor moderate salinity prevented this decline. A decline in leaf Mg concentration under salinity conditions has also been reported in pomegranate [35]. In stems, the Mg concentration was decreased by the addition of 10 mM CaCl₂·2H₂O, while the addition of NaCl alone (S60, S120) led to a moderate increase in the Mg concentration. However, when CaCl₂·2H₂O was combined with NaCl (S60/Ca10 and S120/Ca10), Mg levels declined again, indicating a possible antagonistic interaction between Ca and Mg uptake under both normal and saline conditions. The concentration of Mg in the roots of pomegranate plants was not statistically significantly affected by the addition of salts to the nutrient solution, which was also observed in orange plants [63]. The absorption rate of Mg is significantly reduced by K and Ca cations [7]. Thus, high concentrations of Ca²⁺ in the nutrient solution result in a reduction of Mg concentration in the leaves [79].

As NaCl and CaCl₂·2H₂O increased in the nutrient solution, Na concentration in all parts increased, too. Similar results were observed in many works conducted on pomegranate plants, showing an increase in Na in plant tissue with increasing NaCl concentration in irrigation water [31,43,50,80,81,82,83,84]. In the present work, Na concentration was higher in roots than in basal leaves, and higher in basal than in top leaves, a result that was also observed by Naeini et al. [80]. This suggests a regulated exclusion or compartmentalization mechanism, whereby roots act as a buffer to limit Na⁺ translocation to aboveground tissues. Sodium accumulation in roots is part of a protective mechanism of plants against Na toxicity, and only when salinity exceeds a certain threshold does Na accumulation increase in leaves [80]. These results agree with other studies on pomegranate [41,85,86] suggesting the ability of this species to accumulate Na⁺ in roots as a strategy to alleviate the detrimental effect of salt stress. Contrary to some earlier studies, in our work, Na⁺ concentration did not consistently decrease in all plant tissues upon Ca addition. However, the combination treatment under moderate salinity (S60/Ca10) resulted in lower Na⁺ levels in both roots and basal leaves compared to the respective NaCl-only treatment (S60), suggesting a partial mitigating effect of calcium under moderate salt stress. Similar findings have been reported in citrus species [87]. With the presence of Ca in the nutrient solution, plants obtain the ability to cope with the adverse effects of Na concentration [87]. Moreover, the presence of Ca is essential in maintaining K/Na selectivity, as well as in preventing the deleterious replacement of Ca²⁺ by Na⁺ in cell membranes [88].

Saline nutrient solutions increased the Fe concentration in basal leaves in all salt treatments. In top leaves, stems, and roots, the addition of salts caused fluctuations in Fe concentration, but these changes were not statistically significant, confirming that Fe responses to salinity may be tissue-specific and variable. Trace element solubility usually is diminished under salinity conditions, but aboveground trace element concentration may increase, decrease, or remain stable depending on plant species, tissue, salinity, trace element concentration, and environmental conditions [57]. There is evidence that salinity can cause Fe deficiency in plants [27], though this was not observed in the basal leaves of pomegranate in our study.

The addition of NaCl and CaCl₂·2H₂O salts to the nutrient solution led to a significant increase in Mn concentration in the basal leaves of pomegranate plants. Similarly, an increase was observed in the top leaves, which was statistically significant only in the treatments with a combination of NaCl and CaCl₂·2H₂O salts (S60/Ca10 and S120/Ca10), suggesting a synergistic effect. Regarding stems, Mn concentration presented a significant enhancement only in plants irrigated with high amounts of NaCl (S120), while CaCl₂·2H₂O alone did not significantly affect Mn levels in this tissue. In roots, the addition of salts led to an increase in Mn concentration, but this was significantly reduced when 10 mM CaCl₂·2H₂O was applied alone (Ca10), indicating a possible antagonism between Ca and Mn uptake. Some studies in tomato plants have shown that salinity has no effect [89] or increases [90] the concentration of Mn in the leaves and stems of the plants, depending on cultivar and environmental conditions.

Leaf Zn concentration led to an increase by the inclusion of NaCl and CaCl₂·2H₂O salts into the nutrient solution, but in top leaves, the increase was statistically significant only in the 10 mM CaCl₂·2H₂O treatment. Regarding the stems and roots, Zn concentration showed an increase that was statistically significant only in the 10 mM CaCl₂·2H₂O treatment. According to other studies, in citrus species, Zn concentration increased under salinity conditions [91], and our findings suggest that calcium supplementation may play a more decisive role than NaCl alone in modifying Zn uptake and distribution in pomegranate.

The above effects of saline treatments on the content of micronutrients in pomegranate plants can be attributed to the negative effects of salinity on ionic balance and osmotic potential [7,27], as well as potential competitive or synergistic interactions between macronutrients and trace elements in uptake and transport pathways.

5. Conclusions

The above results and the fact that visible symptoms appeared in pomegranate plants only under the treatments with high NaCl concentrations, and only at the final stage of the experiment, confirm the considerable tolerance of this species to salinity and its potential for cultivation in soils with relatively high salt content. However, salinity induced by NaCl clearly affected plant growth, physiological function, and chemical composition, indicating that even salt-tolerant species such as pomegranate may experience substantial sublethal stress. The role of calcium under saline conditions was shown to be beneficial in specific aspects, particularly under moderate salinity. Calcium supplementation improved ion homeostasis, partially alleviated Na⁺ accumulation, supported K⁺ retention in roots, and contributed to the maintenance of photosynthetic activity and chlorophyll content. However, its effect was not uniformly positive across all parameters or tissue types, and under high salinity (S120/Ca10), the addition of calcium was not sufficient to reverse growth inhibition or membrane destabilization. Therefore, calcium can be considered a potentially useful amendment under moderate salinity, but its mitigating capacity under severe salt stress appears limited. This underscores the need to better understand the interaction between salinity levels, calcium dosage, and plant developmental stages. Further research should be intensified to generate practical knowledge for the sustainable cultivation of pomegranate in saline environments and to optimize strategies—such as calcium supplementation—that may enhance plant tolerance and productivity under such stress conditions.