Effect of Salinity on Physiological and Biochemical Parameters of Leaves in Three Pomegranate (Punica Granatum L.) Cultivars

Abstract

Salinity is one of the most important abiotic stresses affecting crop yield. It is important to exploit pomegranates’ potential against salts because they are considered beneficial plants for human health due to their antioxidants and they are often exposed to severe salinity stress in the field. Three pomegranate cvs. were chosen as model plants for assessing the impact of different salt stress in the cultivation. The aim of this study was to evaluate the physiological and biochemical response of three pomegranate varieties (Punica granatum L.) (Wonderful, Ermioni, and Grenada) under different saline conditions. The plants were grown in a sand/perlite substrate in a 1:1 ratio and, throughout the experiment, were irrigated with a Hoagland nutrient solution, modified to contain four concentrations (0, 25, 50, and 75 mM) of NaCl, KCl, and K₂SO₄. At the end of the experiment, we measured the (a) concentrations of carotenoids and porphyrin of leaves; (b) phenols and flavonoids contents, and antioxidant capacity of leaves; (c) lipid peroxidation level; (d) leaf water potential; and (e) proline concentration. Ermioni contained the maximum concentration of proline phenols and flavonoids and antioxidant capacity in all salts. Furthermore, reductions in chlorophyll and carotenoid concentration were recorded in all cultivars. Grenada possessed the lowest porphyrin concentration. In conclusion, our results showed that Grenada was the most salt-susceptible cultivar. Salinity treatment triggered the enhancement in lipid peroxidation in the sensitive cultivar, while no change in lipid peroxidation level was observed in the tolerant cultivars. These data provide further support to the hypothesis that a mechanism exists that excludes salinity from the roots of tolerant cultivars, as well as an internal mechanism of tolerance that minimizes the accumulation of lipid peroxides through a higher proline content related to osmoregulation and membrane stabilization.

1. Introduction

Salinity is one of the major environmental problems worldwide. The effects of salinity depend on genetic and physiological factors, such as plant species, cultivars, growth stage, salt deposition, and excess of minerals in the soil solution, which can be a substantial constraint on crop growth and yield. The devastating effects of salinity on plant growth include toxic accumulation of salts, nutrient imbalance, low osmotic potential of soil solution, and/or their combination [1,2]. Salt concentration is adversely increasing the salinization on a global scale, with severe effects on plants that suffer significant damage when cultivated in salt-affected soils. [3]. Furthermore, the main plant-growth processes, such as photosynthesis, protein synthesis, ion balance, and lipid metabolism, are affected by salinity [4,5,6]. It is difficult to determine plant tolerance to salinity, because it is directly dependent on different physiological interactions within the plant [7].

Pomegranate (Punica granatum L.) belongs to the Lythraceae family and has important economic, nutritional, and medicinal properties [8]. It is adapted to arid and semiarid regions and is widely cultivated in the Mediterranean area. Furthermore, pomegranate can be cultivated under adverse climatic and soil conditions [9], as it has been classified as medium susceptible to salinity. Pomegranate fruits are a rich source of minerals and phenolics [10] distributed in exocarp, seeds, and arils [11], and thus it is considered a ‘super food’. Pomegranate fruits have numerous medicinal properties due to their high phenolic content [12]. The antioxidants consist of flavonoids, anthocyanins, and tannins, which are all phenolic compounds [13]. Furthermore, pomegranate is a rich source of phytochemical compounds, such as ellagitannins, punicalagin, anthocyanins, punicic acid, and tannins [14], which have antioxidant capacity [15]. Estimations of the total phenolic and flavonoid contents and total antioxidant capacity (FRAP) under saline conditions in pomegranate cvs. are scarce. Analysis of the phenolic compounds and evaluation of the antioxidant capacity in leaves of the three pomegranate cvs. have not been conducted.

Moreover, the amino acid proline is known to widely occur in higher plants and normally accumulates in large quantities in response to environmental stresses [16]. Proline contributes to stabilizing subcellular structures (e.g., membranes and proteins), scavenging free radicals, and buffering cellular redox potential under salinity conditions [17].

The aim of this work was (a) to investigate whether salinity stress can induce differential proline accumulation and evaluate the level of lipid peroxidation in three pomegranate cvs. that have differing responses to salinity stress; and (b) to reveal any existing tolerance mechanism, in addition to the antioxidant capacity, that is upregulated and functions to protect the salinity-tolerant cultivar.

Hence, the present investigation contributes to the literature by looking for differences among the cvs. Wonderful, Ermioni, and Grenada, which are commercial pomegranate cvs. cultivated in Greece.

2. Materials and Methods

A greenhouse experiment was conducted at the farm of Aristotle University of Thessaloniki, Greece (40°32′04.16″ N, 22°59′41.33″ E), during the period of March to September 2014. The experiment included three cvs. (Wonderful, Ermioni and Grenada). We applied three salts (NaCl, KCl, and K₂SO₄), four salt concentrations (0, 25, 50, and 75 mM) and eight replications (plants) per treatment. The plants were planted in a sand/perlite (1:1) medium. The experiment was conducted in a heated greenhouse, and the plants (2 years old), after severe pruning to one stem of 30 cm in length, were transplanted in plastic pots (25 cm upper diameter, 17 cm lower diameter, and 25 cm in height), filled with 1:1 sand/perlite mixture and placed on benches in the experimental greenhouse. The experimental conditions in the greenhouse were RH 60–70%, a temperature of 20–25 °C, and PPFD 900 μmol m⁻² s⁻¹ measured at the top of the plants with a quantum sensor. The experimental plants were irrigated with 25% Hoagland nutrient solution [18] modified to include 0, 25, 50, and 75 mM of NaCl, KCl, and K₂SO₄ salts. Every 2–3 days, each plant was irrigated with 400 mL of nutrient solution and every 15 d with 400 mL deionized water to leach out any accumulated salts. The position of the experimental plastic pots (8 replications) on the bench were changed to avoid position effects on light intensity. At the termination of the experiment (150 d), the biochemical parameters, such as total porphyrins, total phenols, flavonoids, FRAP values, and carbohydrates in leaves and water potential, were measured in 8 plants per treatment.

2.1. Total Carbohydrate Concentration

Total carbohydrates were measured according to [19]. For their extraction, 0.5 g of frozen leaf blade material was placed in a 25 mL glass test tube, and in each tube, 15 mL of 80% ethanol was added. All tubes were incubated in a 60 °C water bath for 30 min. The extract was filtered through Whatman No. 1 filter paper, and total carbohydrates were measured using anthrone reagent (Plummer 1987).

2.2. Porphyrin Concentration

Total porphyrins were determined by measuring optical density (OD) at 575, 590, and 628 nm, which are the absorption peaks of protoporphyrin, magnesium-protoporphyrin, and proto-chlorophyllide, respectively. The sum of the above determines the total porphyrin concentration as follows:

(A)

Protoporphyrin = [(12.25 × A₆₆₅ − 2.55 × A₆₄₉) × volume of supernatant (mL) × dilution factor/sample weight (g)]/892 × 1000

(Β)

Mg-Protoporphyrin = [(20.31 × A₆₄₉ − 4.91 × A₆₆₅) × volume of supernatant (mL) × dilution factor/sample weight (g)]/906 × 1000

(Γ)

Protochlorophyllide = [(196.25 × A₅₇₅ − 46.6 × A₅₉₀ − 58.68 × A₆₂₈) + (61.81 × A₅₉₀ − 23.77 × A₅₇₅ − 3.55 × A₆₂₈) + (42.59 × A₆₂₈ − 34.32 × A₅₇₅ − 7.25 × A₅₉₀)] × dilution factor/sample weight (g)]

Total porphyrins = (A) + (Β) + (Γ)

2.3. Total Phenols

Phenols were extracted from 0.3 g of fresh tissue in 80% methanol, assayed using Folin-Ciocalteu reagent, following a standard method, and are expressed as milligrams of gallic acid equivalent (GAE g⁻¹ FW), which was used for the standard curve with a range of 0–125 μM [20].

2.4. Total Flavonoids

Total flavonoids were extracted from 3 g of fresh tissue in 80% methanol and colorimetrically determined [21]. Rutin was used as the standard compound for quantification of total flavonoids. All values are expressed as milligrams of rutin per gram of fresh weight (FW). The data reported are the means of 8 replicates.

2.5. Antioxidant Capacity

To estimate antioxidant capacity, the ferric -reducing antioxidant potential (FRAP) assay was used [22]. The FRAP assay determines the capacity of antioxidants, as reductants, in a redox-linked colorimetric reaction of the reduction of the yellow Fe³⁺ 2,4,6-tripyridyl-s-triazine to a blue-colored Fe²⁺ complex at low pH, which is spectrophotometrically measured at 593 nm. The extracts were incubated at room temperature with the FRAP reagent, and their absorbance was recorded after 1 h. The reducing power is expressed as micromoles of ferrous sulfate (FeSO₄) per gram.

2.6. Leaf Water Potential

The leaf water potential was measured in the first two completely matured leaves, using a pressure chamber [23]. The measurements were performed at 09:00–10:00 a.m.

2.7. Determination of Lipid Peroxidation

The level of lipid peroxidation in leaves was measured as malondialdehyde (MDA) content, determined by reaction with 2-thiobarbituric acid (TBA)-reactive substances according to [16]. Tissue was homogenized in 0.3% TBA in 10% trichloroacetic acid (TCA) at 4 ± 1 °C. The concentration of MDA was calculated from the difference in the absorbance at 532 and 600 nm, using the extinction coefficient of 155 mmol⁻¹ cm⁻¹, which is expressed as nanomoles of MDA per gram of fresh weight.

2.8. Proline Estimation

Leaves were cut into small pieces, weighed, separately placed in glass vials containing 10 mL of 80% (v/v) ethanol, and heated at 60 ± 1 °C for 30 min. The extract was then filtered and diluted with 80% (v/v) ethanol up to 20 mL [17]. The leaf concentrations of free proline were determined in this extract following the acid-ninhydrin reagent method [19]. We transferred 2 mL of the aqueous alcohol extract into test tubes, and added 2 mL of acid-ninhydrin. With glass marbles on top to minimize evaporation, test tubes were maintained at 95 ± 1 °C for 60 min in a water bath and then allowed to cool at room temperature. Four milliliters of toluene was added to each replicate, and each was thoroughly mixed. After separation of solution layers, the toluene layer was carefully removed and placed in glass cuvettes, and absorption was determined at 518 nm. Ethanol extracts, as used for the Pro assay, were diluted 10 times with 80% (v/v) ethanol. The diluted extract was added drop-by-drop to 2 mL anthrone reagent in test tubes in an ice bath and left to mix the content. Fully mixed samples were incubated in a water bath at 90 ± 1 °C for 15 min and cooled, then absorbance was read in the spectrophotometer at 625 nm.

2.9. Statistical Data Analysis

The experimental layout included three cvs., three salts, four salt concentrations, and eight replicates (plants) per treatment. Data were subjected to analysis of variance (ANOVA). For comparison of means, the Duncan multiple range test was used (p ≤ 0.05) using the SPSS 24.0 statistical package (SPSS, Inc., Chicago, IL, USA).

3. Results

3.1. Carbohydrates and Porphyrins

Grenada displayed the greatest carbohydrate concentration in the 50 and 75 mM K₂SO₄ treatments and significantly differed from the other two cvs. (p ≤ 0.05) (Figure 1c). On the contrary, Ermioni had the highest carbohydrate concentration in leaves at 75 mM KCl (p ≤ 0.05) (Figure 1b).

NaCl did not significantly affect porphyrin concentration in any cv. (Figure 1d), while under KCl and K₂SO₄ treatments, the porphyrin concentration was reduced in all cvs. However, at the highest concentrations of KCl and K₂SO₄ (50 and 75 mM), Ermioni had the highest porphyrin concentration, which was significantly greater than those of the other two cvs. (p ≤ 0.05) (Figure 1e,f).

3.2. Total Phenols, Flavonoids, and FRAP Values

The concentration of total phenols of Ermioni was higher than that of the other two cvs. and differed significantly in all salt treatments, except only at 50 mM K₂SO₄ (p ≤ 0.05) (Figure 2a–c). Wonderful contained the lowest values of total soluble phenols. The same pattern was followed for total flavonoids, where Ermioni had a higher concentration than the other two cvs. (p ≤ 0.05) (Figure 2d–f).

The highest FRAP values were recorded in Ermioni, which contained significantly greater values than the other two cvs. (p ≤ 0.05). Wonderful possessed the minimum antioxidant capacity, which significantly differed from Ermioni and Grenada (p ≤ 0.05) (Figure 3a–c).

3.3. Water Potential

The highest positive values of water potential (Ψ) were found in the control treatment in all cvs. and treatments (Figure 4a–c). The highest values were recorded in Wonderful, which had significantly greater positive values than the other two cvs. (p ≤ 0.05). However, the minimum values were found in Ermioni and Grenada at the highest concentrations (50 and 75 mM) of KCl and K₂SO₄ (p ≤ 0.05), which were significantly greater than those of the Wonderful cv. (p ≤ 0.05) (Figure 4b,c).

3.4. Proline Concentration

The highest proline contents after salts treatment were found in the Ermioni and Wonderful cvs. (

Table 1. Effect of NaCl, KCl, and K₂SO₄ salts on proline content (mg g⁻¹ FW) in leaves of pomegranate cvs. Wonderful, Ermioni, and Grenada. Each value is the mean of 8 replications ± standard error for p ≤ 0.05.

Treatment	Proline Content (mg g−1 FW)
	Wonderful	Ermioni	Grenada
Control (0 mM NaCl)	0.15 a	0.13 a	0.16 a
25 mM NaCl	0.25 ab	0.29 ab	0.18 a
50 mM NaCl	0.42 b	0.49 b	0.24 ab
75 mM NaCl	0.65 c	0.65 c	0.31 ab
25 mM KCl	0.26 ab	0.32 ab	0.16 a
50 mM KCl	0.45 b	0.50 b	0.22 ab
75 mM KCl	0.68 c	0.67 c	0.32 ab
25 mM K2SO4	0.30 ab	0.35 ab	0.18 a
50 Mm K2SO4	0.48 b	0.56 b	0.23 ab
75 mM K2SO4	0.70 c	0.77 c	0.34 ab

Proline content (mg g^–1 FW) in leaves of pomegranate cvs. Wonderful, Ermioni, and under the effect salts, (a) NaCl (0, 25, 50, 75 mM), (b) KCl (0, 25, 50, 75 mM), (c) K₂SO₄ (0, 25, 50, 75 mM). Each value is the mean of 8 replications ± standard error for p ≤ 0.05.

). Salts did not significantly affect proline concentration in the Grenada cv. (NaCl, KCl, and K₂SO₄ treatments) (Table 1). At the highest concentrations (50 and 75 mM) of NaCl, KCl, and K₂SO₄ Ermioni and Wonderful cvs. contained the highest proline concentrations, which were significantly greater than that of the Ermioni cv. (p ≤ 0.05).

3.5. Lipid Peroxidation Estimation

The highest lipid peroxidation contents after salts treatment were found in the Grenada cv. (

Table 2. Effect of NaCl, KCl, and K₂SO₄ salts on lipid peroxidation (MDA) content (μmol g⁻¹ FW) in leaves of pomegranate cvs. Wonderful, Ermioni, and Grenada. Each value is the mean of 8 replications ± standard error for p ≤ 0.05.

Treatment	Lipid Peroxidation (MDA) Content (μmol g−1 FW)
	Wonderful	Ermioni	Grenada
Control (0 mM NaCl)	1.8 a	1.4 a	1.5 a
25 mM NaCl	2.4 a	2.1 a	2.7 ab
50 mM NaCl	3.2 ab	3.1 ab	4.5 b
75 mM NaCl	3.5 ab	3.3 ab	5.8 c
25 mM KCl	2.6 a	2.3 a	2.6 ab
50 mM KCl	3.6 ab	3.2 ab	4.8 b
75 mM KCl	3.4 ab	3.5 ab	6 c
25 mM K2SO4	2.7 a	2.2 a	2.8 ab
50 Mm K2SO4	3.1 ab	3.4 ab	5.0 b
75 mM K2SO4	3.7 ab	3.6 ab	6.1 c

Lipid peroxidation (MDA) content (μmol g⁻¹ FW in leaves of pomegranate cvs. Wonderful, Ermioni, and under the effect salts, (a) NaCl (0, 25, 50, 75 mM), (b) KCl (0, 25, 50, 75 mM), (c) K₂SO₄ (0, 25, 50, 75 mM). Each value is the mean of 8 replications ± standard error for p ≤ 0.05.

). Salts did not significantly affect lipid peroxidation concentration in Ermioni or Wonderful cvs. (NaCl, KCl, and K₂SO₄ treatments) (Table 1). At the highest concentrations (50 and 75 mM) of NaCl, KCl, and K₂SO₄, cv. Grenada had the highest lipid peroxidation content, which was significantly greater than that of the Ermioni and Wonderful cvs (p ≤ 0.05).

4. Discussion

The three pomegranate cvs. that we used in the present work differ in saline tolerance and provided a support for the study of the physiology and biochemistry of salt tolerance in pomegranates, avoiding the problem of comparisons between genetically unrelated cultivars. Salt stress usually affects the plant growth, development, and antioxindant capacity as well. This decrease in plant growth may occur due to excess accumulation of salts around the root zone, which affects the water uptake. Water plays an important role in the physiology of plants, and maintaining higher leaf water content is a physiological mechanism of enhancing salt stress tolerance. Water potential is a basic parameter that is commonly used to determine the tolerance for salinity stress. Our results showed that tolerant cvs. (Ermioni and Wonderful) had a lower reduction in Ψ compared with the sensitive cv. (Grenada) under salt stress. A similar decrease in Ψ under stress conditions was reported by many earlier researchers in other plants [24].

Salinity significantly reduced the carbohydrate concentration in all cvs. for all salts. The concentration of carbohydrates in the leaves was affected by salt treatments, suggesting that the salt affected some sugar-related metabolism in the leaves. Previous reports showed that exposure to abiotic stresses such as heavy metals [15] influenced the sugar metabolism. Our results agree with the findings of [3,25], who observed a similar decrease in the carbohydrate concentration due to salinity in pomegranate plants. Furthermore, many authors [26] described related results in pomegranate plants.

The porphyrin concentration was reduced at the highest KCl and K₂SO₄ concentrations, and Grenada was the most susceptible among the three cvs. The decrease in porphyrin concentration could be ascribed to the decrease in Fe absorption, which is a basic component of the porphyrin molecule due to its competition with the K ion of salts (KC and, K₂SO₄) and salt toxicity. Our findings are not in agreement with those of [27], that the Profeta Partanna pomegranate cv. did not show any differences in phenols and ellagic acid concentrations due to salinity. In addition, [28] reported similar results observing the salinity-activated biosynthesis of phenols in leaves. However, the close relationship between antioxidants and resistance to salinity in various plant species has been previously reported for peas [29], cotton [30], and rice [31].

The ability of plants to inactivate ROS mainly depends on the antioxidant defense system, including nonenzymatic components [32]. The nonenzymatic components of the antioxidant defense system consist of various secondary metabolites, such as hydrophilic phenols and flavonoids, organic acids, lipophilic carotenoids, and water-soluble ascorbate. The existence of phenol metabolism is considered as one of the responses to abiotic stress [16,33].

Plant antioxidants are a source of bioactive compounds. They play an important role in plant functioning and mitigate the adverse effects of abiotic stresses, but they are also beneficial for human health [34]. Phenols are considered one of the most well-known antioxidants, and their increase in plant tissues under salinity probably explains their involvement in plant protection against oxidative damage due to salt stress [35,36]. Many authors [37] have reported that salinity causes disorders to plant metabolism, leading to the increased biosynthesis of phenols. The phenol structure is aromatic rings and free hydroxyls that can detoxify free radicals (ROS) [38,39]. In our experiment, the increase in phenol concentration was ascribed to antioxidant mechanisms in response to oxidative stress caused by salinity. However, the ROS produced by salinity were inactivated by various antioxidants from the photosynthetic electron transport chain within chloroplasts [40] and helped to detoxify ROS (S) [41,42].

Under saline conditions, plants produce excess ROS because of oxidative stress, and if the plants do not develop their defense mechanisms on time, ROS can damage DNA, membrane proteins, and lipids, resulting in cell death. The degree of oxidative cell damage is controlled by the ability to protect against oxidizing agents [43].

In some species, including in many crops such as barley and maize, there are varieties that are resistant to salinity, and they were found to have high concentrations of water-soluble phenols, flavonoids, anthocyanins, and various polyphenols in saline conditions, while the reverse effect occurs in species susceptible to salts [44]. The increase in proline content also seems to play an important role in hydroxyl radical scavenging, thus defending plants against oxidative damage, which is a common consequence of many abiotic stresses. Previous authors [1,45] reported findings similar to ours and observed a similar increase in the proline concentration due to salinity in plants.

Moreover, salinity is able to enhance oxidative damage such as lipid peroxidation in sensitive cultivars. Under salinity conditions, significant increases in lipid peroxidation were observed in the Grenada cultivar. Similar results were reported by [46], who found that salt treatment caused an increase in lipid peroxidation, as revealed by high TBAs contents in sensitive barley genotypes, but no significant effect was shown in the most salt-tolerant genotypes.

Thus, the production of and increase in phenol concentration may be a particularly good indicator of plant resistance to salinity. Ermioni appeared to be the most resistant to 25 and 50 mM salt concentrations compared with the other two cvs. for the NaCl and KCl salts. In the K₂SO₄ treatment, Wonderful was the most resistant to a concentration of 50 mM, while for 75 mM K₂SO₄, Ermioni was the most resistant.

5. Conclusions

The results of this study indicated that there is a wide variability among pomegranate cvs. regarding their tolerance to salinity.

The maximum concentrations of total phenolics, flavonoids, and FRAP were found in Ermioni at all salt concentrations. Grenada had the lowest porphyrin concentration for the KCl and K₂SO₄ treatments. The proline concentration positively correlated with the tolerance to salinity, while lipid peroxidation negatively correlated with the salinity tolerance. These results indicated that oxidative stress occurs due to elevated levels of ROS that cause damage to lipids, one of the possible mechanisms by which salinity toxicity in the pomegranate cells is expressed. That is, enhancement in lipid peroxidation in Grenada (the sensitive pomegranate cv.) was provoked by salinity, while lipid peroxidation during salinity stress in Ermioni and Wonderful (the tolerant cvs.) was lower. Based on these observations, salinity tolerance of the pomegranate cvs. may be associated with a mechanism that minimizes the accumulation of lipid peroxides. Moreover, the ability to increase the antioxidant capacity in order to limit cellular damage might be an important strategy to achieve salinity tolerance in pomegranate cvs. Therefore, further investigation is required to certify adaptive mechanisms of pomegranates for enhancing salt tolerance and productivity.