Effects of Ascorbic Acid and Reduced Glutathione on the Alleviation of Salinity Stress in Olive Plants

ABSTRACT

The aim of this study was to evaluate the effects of low molecular mass antioxidants and NaCl salinity on growth, ionic balance, proline, and water contents of ‘Zard’ olive trees under controlled greenhouse conditions. The experiment was carried out by spraying 2 mM of ascorbic acid (Asc) and 3 mM of reduced glutathione (GSH) on the plants that were treated with two salinity levels (0 and 100 mM NaCl) on their root medium. Plant growth parameters (leaf fresh weight, leaf dry weight, leaf number, total fresh weight, and total dry weight) were significantly improved by Asc compared with growth parameters in GSH and control plants. Higher concentrations of Na⁺ and Cl^– were observed in salt-stressed plants, while Na⁺ and Cl^– concentrations were decreased in the olive leaves that were sprayed with Asc. Salinity in the root zone caused a considerable decline in both K⁺ concentration and K/Na ratio. K⁺ concentration and K/Na ratio were significantly increased by application of Asc on plant leaves. Salinity caused an increase in electrolyte leakage (EL) compared with the control plants. Lowest EL and tissue water content (TWC) was obtained in Asc-sprayed plants, whereas TWC was increased in salt-stressed plants. Plants were subjected to salt stress and showed a higher relative water content (RWC) than the control plants. Salt stress induced proline accumulation in olive leaves. In conclusion, exogenous application of Asc is recommended to improve tolerance of olive plants under saline conditions.

KEYWORDS:

• Ascorbic acid
• ion homeostasis
• NaCl salinity
• olive
• reduced glutathione
• water content

Introduction

Salinity stress drastically affects plant growth and nutrient accumulation (dos Santos and Caldeira, 1999). The deleterious effects of salinity stress can be because of its osmotic effect, which can be considered as a decrease in water activity, specific ions toxicity, and inhibition of essential ions uptake (Marschner, 2012). These primary stresses are able to stimulate the reactive oxygen species (ROS) generation (Meloni et al., 2003), change hormonal homeostasis (Munns, 2002), alter metabolism of carbohydrates (Gao et al., 1998), decrease the activity of specific enzymes (Tabatabaei, 2006), and impair photosynthesis (Chartzoulakis et al., 2002). Due to these metabolic modifications, decline in both cell division and elongation can occur. In worse situations, cell division and elongation can be fully impaired, which eventually result in cell death of plant tissues (Munns, 2002).

The olive tree (Olea europaea L.) is a sclerophyllous species belonging to the Mediterranean region (Gullo and Salleo, 1988). In most Mediterranean coastal areas with high olive tree plantations, the increased need for good quality water for urban use restricted the use of fresh water for irrigation. On the other hand, in those areas, large quantities of low quality water (mostly saline) are available, which are usually used for olive tree irrigation. Since olive is categorized as a moderately tolerant plant to salinity stress (Maas and Hoffman, 1977; Rugini and Fedeli, 1990), there is a tendency to use that kind of water for irrigation of olive trees (Chartzoulakis et al., 2002). Therefore, improvement of salt tolerance in olive trees using breeding programs or using proper horticultural practice is claimed for extending its cultivation area.

Salt stress like other abiotic stresses causes oxidative stress through an induction in the ROS production (Hernandez et al., 1995). To control the level of ROS and to maintain cells under stress conditions, plant tissues contain a network of low molecular mass antioxidants, such as ascorbic acid (Asc) and glutathione (GSH) (Blokhina et al., 2003).

Asc is a small, water-soluble antioxidant molecule, which has a role as a primary substrate in the cyclic pathway for enzymatic detoxification of hydrogen peroxide (Noctor and Foyer, 1998). It acts as a cofactor of violaxantin de-epoxidase, therefore sustaining dissipation of excess excited energy (Smirnoff, 2000). Asc can act as a reductant for hydroxylation of proline residues during extension biosynthesis (Noctor and Foyer, 1998). It regenerates tocopherol from the tocopheroxyl radical, and helps membrane protection (Thomas et al., 1992). Moreover, Asc has some non-antioxidant functions in the cell. It mediates in control of cell division, progression of cell cycle from G1 to S phase (Liso et al., 1988; Smirnoff, 1996), and elongation of the cells (De Tullio et al., 1999).

Glutathione is a non-protein sulphur-containing tripeptide, which acts as storage and is the transporting form of reduced sulphur (Tausz et al., 2004). GSH function as an antioxidant received much more attention by the researchers. Central nucleophilic cysteine residue is responsible for high reductive potential of GSH. It scavenges cytotoxic H₂O₂ and reacts non-enzymatically with other ROS, including singlet oxygen, superoxide, and hydroxyl radicals (Larson, 1988).

Since the two mentioned antioxidants (Asc and GSH) have several positive impacts on the physiological parameters of plants, therefore, the hypothesis of the current study was to improve tolerance of olive plants by use of ascorbic acid and reduced glutathione. There are no quantitative investigations on the effects of an exogenous applied antioxidant on tree resistance to salt stress. The purposes of this study were to determine: (i) the influence of salinity on growth, ionic balance, and proline content of olive trees; (ii) the effects of exogenous antioxidant applications on vegetative and physiological parameters of olive trees; and finally (iii) the improving effects of exogenous antioxidant applications on salinity tolerance of olive trees.

Materials and methods

Plant material, growth conditions, and treatments

‘Zard’ olives were transplanted from 1-year-old own-rooted plants into pots (12 l) containing perlite:sand:vermiculite (50:25:25, v:v) for hydroponic culture. During the experiment, the pots were kept in the glasshouse with a temperature of 30 ± 3 °C during the day and 20 ± 3 °C at night. The experiment was conducted by spraying 2 mM of ascorbic acid (Asc) (Borsani et al., 2001; Guo et al., 2006), 3 mM of reduced glutathione (GSH) (Borsani et al., 2001), and distilled water (control) on the plants that were treated with two salinity levels (0 and 100 mM NaCl) on their root medium. Salt concentrations were added to half strength of Hoagland solution (Hoagland and Arnon, 1950). Each treatment was replicated four times. The antioxidants and distilled water were finely sprayed on the leaves of olive trees in the controlled greenhouse condition. The plants were irrigated daily for 1 month with a half-strength of Hoagland solution. They were pruned to a single shoot per plant. Irrigation was done with 1 l of the half strength of Hoagland solution with 100 mM NaCl for salinity treatment and without NaCl for control plants. The electrical conductivity (EC) of the nutrient solution without NaCl was within the range of 2.7–2.8 ds m^–1; in the nutrient solution with 100 mM NaCl, it increased to 13.2 ds m^–1. The solution pH was adjusted to 6.5 by adding H₂SO₄. Since the new growth almost started 1 week after pruning, to be ensured that the new growth of the olive plants has occurred under salinity stress, salt treatments were continuously imposed 1 week after pruning. Antioxidants were applied to plant leaves 1.5 and 2 months (two times) after pruning. The glasshouse experiment lasted for 6 months. At the end of the 6th month, plants were removed from the substrate after removing leaves from the plants; leaf number, leaf weight, and total plant weight were measured. After weighing, they were dried at 80 °C in an air-forced oven for 48 h. During plant growth the visual symptoms of salt toxicity in the leaves (such as either necrosis or chlorosis) were visually assessed.

Tissue ion content

At the end of the experiment, leaves were harvested and analyzed for Cl^–, Na⁺, and K⁺. Tissue samples were extracted with dilute nitric acid. In the extract the Na⁺ and K⁺ concentrations were determined using a flame photometer and Cl^– concentration was obtained using a chloride meter (JENWAY, PCLM-3, Keison Products, UK).

Proline extraction and assay

Six months after the start of the experiment, determination of free proline content was done according to Bates et al. (1973). Fresh leaf samples (0.5 g) were homogenized in 3% (w/v) sulphosalicylic acid. Then the homogenate was filtered through filter paper. After the addition of acid-ninhydrin and glacial acetic acid, the resulting mixture was heated at 100 °C for 1 h in a water bath. Reaction was stopped using an ice bath. The mixture was extracted with toluene, and the absorbance of fraction with toluene aspired from the liquid phase was read at 520 nm. Proline concentration was determined using a calibration curve and expressed as µmol proline g^–1 Fwt.

Electrolyte leakage (EL)

Electrolyte leakage was determined as described by Tuna et al. (2007). One gram of fresh leaf samples was washed three times with deionized water to remove surface-adhered electrolytes then placed in closed vials containing 10 ml of deionized water and incubated at 25 °C on a rotary shaker for 24 h; subsequently, electrical conductivity of the solution (L_t) was determined. Then samples were autoclaved at 120 °C for 20 min and the resulting electrical conductivities (L₀) were recorded after equilibration at 25 °C. The electrolyte leakage was calculated according to Eq. (1):

(1)

Water content measurement

Relative water content (RWC) was determined in the leaves, whereas tissue water content (TWC) was determined in whole of the plant. After 4 h floating of 0.5 g fresh leaf samples in deionized water, the turgid weights of samples were recorded. Then samples were dried in a hot air oven till constant weight is achieved (Schonfeld et al., 1988). RWC and TWC were respectively calculated according to Eqs. (2) and (3):

(2)

(3)

where LFW is leaf fresh weight; LDW is leaf dry weight; LTW is leaf turgid weight; FW is fresh weight; and DW is dry weight.

Statistical analysis

The data analysis was made using analysis of variance (ANOVA) in SAS 8.2 software (SAS Institute Inc, USA) and means of different treatments were compared using the least significant difference (LSD) test at P < 0.05.

Results

The results of analysis of variance for antioxidants, NaCl, and their interactions (antioxidants × NaCl) for the assessed parameters are given at the bottom of Tables 1–3.

Table 1. Effects of ascorbic acid (Asc), reduced glutathione (GSH), and distilled water (C) in interaction with salinity stress (0 and 100 Mm NaCl) on vegetative growth of olives grown in a mixture of perlite, sand, and vermiculite and irrigated with half strength of Hoagland solution.^z

Antioxidant × salinity	Leaf Fwt(g plant^–1)^y	Leaf Dwt(g plant^–1)	Leafnumber	Total Fwt(g plant^–1)	Total Dwt(g plant^–1)
Asc × NaCl 0	17 ± 3/7a	6 ± 1/38a	82 ± 17a	55 ± 6/8a	26 ± 2/2a
GSH × NaCl 0	13 ± 1/2ab	4 ± 0/46ab	53 ± 4/9ab	42 ± 1/7b	20 ± 0/8ab
C × NaCl 0	14 ± 1/4ab	4 ± 0/67ab	59 ± 6/4ab	41 ± 2/6b	20 ± 1/1ab
Asc × NaCl 100	7 ± 0/36bc	2/4 ± 0/17bc	36 ± 2/1bc	31 ± 1/1bc	16 ± 0/6bc
GSH × NaCl 100	4 ± 0/5c	1/2 ± 0/18c	19 ± 1/8c	25 ± 1/2c	12 ± 0/9c
C × NaCl 100 F probability	4 ± 0/36c	0/94 ± 0/05c	16 ± 1c	21 ± 2/3c	10 ± 0/9c
Antioxidant (Ant)	*	*	*	**	***
Salinity (S)	***	***	***	***	***
Ant × S	NS^x	NS	NS	NS	NS

^zThe data are means ± SEM.

^yMeans within each column not followed by the same letter present statistical difference as determined by least significant difference (LSD).

^xNS, not significant; *,**,***Significant at the 0.05, 0.01, and 0.001 probability level, respectively.

Table 2. Effects of ascorbic acid (Asc), reduced glutathione (GSH), and distilled water (C) in interaction with salinity stress (0 and 100 Mm NaCl) on ion concentrations and K/Na ratio in the olives grown in mixture of perlite, sand, and vermiculite and irrigated with half strength of Hoagland solution.^z

Antioxidant × salinity	K^+ (mg g^–1 Dwt)^y	Na^+ (mg g^–1 Dwt)	Cl^– (mg kg^–1 Dwt)	K/Na ratio
Asc × NaCl 0	35.62 ± 2.73a	15.28 ± 1.58a	1.6a	2.35 ± 0.35a
GSH × NaCl 0	20.61 ± 6.67c	20.52 ± 3.76a	2.01ab	1.08 ± 0.58b
C × NaCl 0	28.15 ± 7.65b	19.38 ± 6.86a	1.94ab	1.66 ± 0.93ab
Asc × NaCl 100	11.42 ± 0.76d	39.98 ± 5.42b	3.59b	0.30 ± 0.04c
GSH × NaCl 100	12.01 ± 1.36d	51.49 ± 23.77b	5.9c	0.26 ± 0.08c
C × NaCl 100 F probability	10.55 ± 0.65d	99.28 ± 7.50c	19.8d	0.08 ± 0.01c
Antioxidant (Ant)	*	***	**	*
Salinity (S)	***	***	***	***
Ant × S	**	***	*	*

^zThe data are means ± SEM.

^yMeans within each column not followed by the same letter present statistical difference as determined by least significant difference (LSD).

*,**,***Significant at the 0.05, 0.01, and 0.001 probability level, respectively.

Table 3. Effects of ascorbic acid (Asc), reduced glutathione (GSH), and distilled water (C) in interaction with salinity stress (0 and 100 Mm NaCl) on proline, electrolyte leakage (EL), relative water content (RWC), and tissue water content (TWC) in the olives grown in mixture of perlite, sand, and vermiculite and irrigated with half strength of Hoagland solution.^z

Antioxidant × salinity	Proline (µ mol g^–1 Fwt)^y	EL (%)	RWC (%)	TWC (%)
Asc × NaCl 0	8/9 ± 1/05a	7/3 ± 0/59a	91 ± 1/06a	51 ± 2/07ab
GSH × NaCl 0	8/9 ± 0/53a	7/6 ± 1/2a	94 ± 0/07ab	51 ± 0/48ab
C × NaCl 0	8/1 ± 0/93a	9/6 ± 1/14a	92 ± 1/51ab	52 ± 1/05a
Asc × NaCl 100	11 ± 0/7ab	10 ± 1/6a	93 ± 1/30ab	46 ± 0/36b
GSH × NaCl 100	15 ± 4/7b	13 ± 2/5ab	94 ± 0/24ab	50 ± 1/16ab
C × NaCl 100 F probability	9 ± 1/7a	20 ± 2/8b	96 ± 0/55b	52 ± 0/81a
Antioxidant (Ant)	NS^x	*	NS	*
Salinity (S)	*	***	*	*
Ant × S	NS	NS	NS	NS

^zThe data are means ± SEM.

^yMeans within each column not followed by the same letter present statistical difference as determined by least significant difference (LSD).

^xNS, not significant; *,***Significant at the 0.05 and 0.001 probability level, respectively.

Plant growth

Vegetative growth was considerably improved by application of Asc. The highest leaf fresh weight and leaf dry weight were observed in Asc-sprayed plants (Table 1). Leaf number considerably increased by application of Asc in comparison with leaf number in all treatments of salt-exposed plants (Table 1). For leaf vegetative parameters and leaf number, no significant differences were found between control plants in non-saline condition and Asc-sprayed plants in saline condition (Table 1). The highest total fresh and dry weights were recorded for Asc-sprayed plants in both saline and non-saline conditions. (Table 1). There were no significant differences between control plants in non-saline condition and Asc-sprayed plants in saline condition. These data show considerable effects of Asc on vegetative growth of olive trees in both saline and non-saline conditions. In salinity condition, no significant differences were found between GSH and control plants (Table 1).

Leaf ion content

The ion content and K/Na ratio were affected by antioxidants and salinity interactions. The concentration of K⁺ was increased by Asc application on olive leaves (Table 2). Under non-saline conditions, the highest and lowest K⁺ concentrations were observed in Asc- and GSH-sprayed plants, respectively. The concentration of K⁺ significantly decreased by NaCl application in root medium; however, no significant difference was observed between antioxidants under salinity condition (Table 2).

The Na⁺ concentration was lower in unstressed plants when compared to salt-stressed plants. However, under salinity condition, application of antioxidant decreased the foliar Na⁺ concentration in comparison with control plants (Table 2). Concentration of Cl^– was reduced by application of Asc in both stressed and unstressed plants. In the non-saline condition, the highest and lowest Cl^– concentrations were respectively obtained in GSH (2.01)- and Asc (1.6)-treated plants. In the saline condition, the lowest and highest Cl^– concentrations were respectively obtained in Asc (3.59) and control (7.96) plants (Table 2). In the non-saline condition, the highest and lowest K/Na ratios were respectively observed in Asc and GSH-sprayed plants. In this case, Asc caused a 54% and 29% increase in the K/Na ratios in comparison with its ratio in GSH and control plants. The reduction in foliar K/Na ratio was more pronounced in salt-stressed plants. Under the salinity condition, the value of K/Na ratio was approximately three times higher than its value in control plants. However, for K/Na ratio, no significant difference was found between antioxidants under salinity conditions (Table 2).

Proline and water contents

Salt stress significantly affected free proline content in the leaves of olive trees. Apart from Asc-sprayed plants under salinity condition, higher proline content was found in the salinized GSH-sprayed plant in comparison with proline contents of all other treatments in both saline and non-saline conditions (Table 3).

The percentage of RWC in salinized control plants (96%) was significantly higher than its percentage in Asc-sprayed plants in non-saline conditions (91%) (Table 3).

The percentage of tissue water content (TWC) in Asc-sprayed plants, which was exposed to salinity stress, was lower than TWC in control plants in both saline and non-saline conditions (Table 3).

Electrolyte leakage (EL)

In non-saline conditions, there were no significant differences among treatments, while EL was significantly affected by application of treatments in saline conditions (Table 3). In salt-exposed plants, application of Asc resulted in 50% decrease in the percentage of EL compared to the control plants (Table 3).

Discussion

The vegetative growth of olive trees was improved by the spraying of Asc on leaves (Table 1). In the current study, salinity in root medium first resulted in chlorosis followed by necrosis and finally abscission of the leaves while Asc application partly prevented these symptoms in olive trees (data not shown). Asc is mediated in the process of cell division in plants. Asc content depends mainly on physiological status of plant as well as on environmental factors; moreover, its content is highly tissue dependent (Smirnoff, 1996). Improving effects of Asc applications on the vegetative growth of plant subjected to salinity (Shalata and Neumann, 2001) and drought (Dolatabadian et al., 2009) stresses have been previously reported. The treatment with exogenous Asc in tomato seedling led to formation of new leaves and roots after recovery from 9 h of salt treatment (Shalata and Neumann, 2001). Asc can directly remove superoxide, hydroxyl radicals, and singlet oxygen and reduce H₂O₂ to water via ascorbate peroxidase reaction (Noctor and Foyer, 1998). Dolatabadian et al. (2009) reported that foliar application of Asc could reduce stress-induced damage in corn leaf. Chlorophyll content usually decreases by stress conditions. It has been shown that nitrogen and chlorophyll contents in the leaf can be influenced by Asc (Zheng et al., 2006). Aliniaeifard and Tabatabaei (2010) found a positive correlation between foliar nitrogen concentration and chlorophyll content as well as between foliar nitrogen concentration and leaf number. Therefore, higher vegetative growth by Asc in current study can be because of the promoting effects of Asc on photosynthesis apparatus and as a result higher biomass production. Dehydroascorbate reductase (DHAR) catalyzes the reduction of dehydroascorbate (oxidized ascorbate) to Asc and thus mediates in the regulation of the Asc redox state (Smirnoff, 1996). It has been shown that increasing the expression of DHAR would result in generation of plants with increased Asc level and increased tolerance to salinity stress (Ushimaru et al., 2006). Exogenous application of Asc on tomato seedlings led to an increase in the number of tomato seedlings that are able to survive after a 9 h exposure to 300 mM NaCl (Shalata and Neumann, 2001). It has been suggested that decreased Asc level due to stress conditions would result in more sensitivity of photosynthesis apparatus to unfavorable conditions (Huang et al., 2005). In the current study, contrary to Asc, exogenous application of reduced glutathione did not improve the physiological parameters in olive trees. Ascorbate-deficient Arabidopsis mutant, vtc-1, has a lower level of Asc but higher level of glutathione after exposure to salt stress conditions. The lower Asc level in vtc-1 decreases its ability to cope with the adverse effect of salt stress on photosynthesis apparatus (Huang et al., 2005). Decreased Asc level in olive trees that were subjected to salinity stress has been reported by Valderrama et al. (2006). Therefore, it seems that exogenous application of Asc can compensate the decreased Asc level due to exposure to salinity stress, which resulted in better tolerance of olive trees under salinity condition.

In our study, EL was reduced in Asc-sprayed plants (Table 3). In agreement with our results, exogenous application of Asc caused a decrease in stress-induced increase in the EL following peroxidative damage to plasma membranes (Lechno et al., 1997; McKersie et al., 1990). Inhibitory effects of exogenous Asc on lipid peroxidation in sunflower seedling exposed to water stress have also been reported (Zhang and Kirkham, 1996).

In the present study, application of Asc improved ion homeostasis under normal and salinity conditions. Environmental conditions [e.g., light, vapor pressure deficit (VPD), water availability] can influence stomatal responses (Aliniaeifard et al., 2014; Aliniaeifard and van Meeteren, 2013, 2014; Merilo et al., 2014) and as a result they can have direct or indirect effects on photosynthesis. Under stress conditions, ions (e.g., K⁺ and Cl^–) and water effluxes would result in loss of guard cell turgor and consequently stomatal closure, while in the absent of adverse environmental conditions, as a result of K⁺ and Cl^– accumulation in guard cells, water would enter into the guard cells to induce a stomatal opening, which would be favorable for gas exchange, photosynthesis, and growth (Aliniaeifard et al., 2014; Aliniaeifard and van Meeteren, 2013, 2014; Armstrong and Blatt, 1995; Blatt, 2000; Ishikawa et al., 1983; MacRobbie, 2000; Schroeder et al., 2001a, 2001b). Water and salt stress-altered stomatal responses have been widely documented (Aliniaeifard et al., 2014; Aliniaeifard and van Meeteren, 2013, 2014; Merilo et al., 2014; Tabatabaei, 2006). Abscisic acid (ABA) usually acts as the main phytohormone for induction of stomatal closure (Aliniaeifard et al., 2014; Aliniaeifard and van Meeteren, 2013, 2014; Davies et al., 2005; Luan, 2002; Sauter et al., 2001). Guard cell ABA signal transduction for stomatal closure has also been extensively documented (Antoni et al., 2011; Fan et al., 2004; Hubbard et al., 2010; Joshi-Saha et al., 2011; Li et al., 2006; Luan, 2002; Pei and Kuchitsu, 2005). Application of ABA when the only source of CO₂ for photosynthesis is stomata would result in a decrease in photosynthesis (Aliniaeifard et al., 2014; Aliniaeifard and van Meeteren, 2013, 2014). It has been shown that ABA-induced stomatal closure involves production of hydrogen peroxide as an intermediate (Zhang et al., 2001). Chen and Gallie (2004) reported that stomatal closure could be reversed by Asc. A specific role for cell-wall-localized Asc for the perception of environmental stimuli has been suggested, which support regulation of stomatal dynamics by dehydroascorbate action in the leaves (Fotopoulos et al., 2006, 2008). Therefore, Asc via its scavenging effects on hydrogen peroxide can prevent ABA-induced stomatal closure. Chen and Gallie (2004) showed that diurnal level of Asc redox state correlates with the diurnal pattern of stomatal opening and closure. More open stomata were found in DHAR-overexpressing plants compared with stomata in wild type (Chen and Gallie, 2004). Since an increase in guard cells K⁺ concentration is necessary for induction of stomatal opening, it can be deduced that an increase in K⁺ concentration is a consequence of Asc effect on stomatal opening and ion homeostasis in the leaves.

Exogenous Asc gave only a non-significant reduction in Na⁺ accumulation in salinized tomato seedlings (Shalata and Neumann, 2001); however, in this study, exogenous Asc led to a considerable decrease in Na⁺ and Cl^– concentrations. It seems that Asc helped a plant to adjust its ion concentrations in order to cope with adverse effects of ion toxicity in the olive leaves.

Salinity stress in the present experiment caused deleterious effects on vegetative growth of olive trees. Negative effects of salt stress on fresh and dry weights of leaves, stems, and root have been previously reported in many plant species (Chaparzadeh et al., 2004; Chartzoulakis et al., 2002; Munns and Tester, 2008; Sucre and Suárez, 2011; Tabatabaei, 2006; Turhan and Eriş, 2007). The reduction in leaf growth is an early response of the plants to salinity (Munns and Termaat, 1986). Regarding the olive trees, considerable reduction in growth parameters has been reported for some olive cultivars (Ben Ahmed et al., 2010; Chartzoulakis et al., 2002; Tabatabaei, 2006; Tattini et al., 1992).

Under saline conditions, K⁺ concentration decreases in many glycophytes (Greenway and Munns, 1980). The accumulation of salt in the root zone induces the osmotic stress and impairs cell ion homeostasis by inhibition of nutrient uptake. In such situations, Na⁺ and Cl^– accumulate to some toxic levels within the leaves (Ben Ahmed et al., 2010; Greenway and Munns, 1980; Marschner and Marschner, 2012). Moreover, salinity can directly affect nutrient uptake, for example Na⁺ and Cl^– accumulation in the root zone can cause reduction in K⁺ and nitrate uptake, respectively (Marschner and Marschner, 2012). Salinity not only causes induction of ROS production (Chaparzadeh et al., 2004) but also causes reduction of non-enzymatic antioxidants, such as Asc and GSH (Valderrama et al., 2006).

In the current study, accumulation of proline was observed due to saline condition in the root zone. Proline accumulation in response to abiotic stresses is widely reported, and it may play a role in stress adaptation within the cell (Ashraf and Foolad, 2007; Ben Ahmed et al., 2010; Gilbert et al., 1998). In response to salinity osmotic adjustment involves the net accumulation of some solute (e.g., proline, glycine betaine, and some ions) in the cells (Ashraf and Foolad, 2007). Uptake of water into the cells because of the presence of these solutes enable them to maintain the turgor which needed for their physiological functions; this mechanism is an important adaptation for minimizing the detrimental effect of water deficit stresses (Ashraf and Foolad, 2007; Ben Ahmed et al., 2009, 2010).

In the present study Asc application led to a reduction in water content of the olive plants. By rapid water deprivation, the leaves that have more closed stomata can keep higher RWC compared to the leaves with more open stomata (Rezaei Nejad and van Meeteren, 2005). Valderrama et al. (2006) reported a considerable increase in H₂O₂ content in olive plants subjected to salinity stress induces closing of the stomata. Therefore, under salinity conditions we can expect closure of the stomata and increase in RWC. In agreement with our results, RWC in the leaf of Atriplex halimus was considerably higher in the presence of NaCl (Martínez et al., 2005). Moreover, Sucre and Suárez (2011) showed higher RWC (no significant differences) and leaf succulence but lower osmotic potential under high salinity condition. They suggest that the dehydration may not be the most important factor contributing to the decrease of osmotic potential. However, contrary to the result of the present study, there are some reports showing reduction of RWC under salinity conditions (Ben Ahmed et al., 2010; Kaya et al., 2007). In the present study, Asc changes water content of olive trees compared to control plants. In other words, lower tissue water content was observed by application of Asc. In accordance with our results, transgenic plants with increased DHAR expression exhibited stomatal opening and these plants are not capable of regulating water loss, whereas antisense plants with low DHAR expression have partially closed stomata and better control over transpiration (Chen and Gallie, 2004).

In conclusion, the results obtained in the present study indicated that salinity in the root medium caused a reduction in growth and ionic imbalance in olive trees, while exogenous application of Asc can improve growth via ionic balance and reduced electrolyte leakage of olive trees in saline conditions. Therefore, exogenous application of Asc is recommended to improve growth of olive plants under saline conditions.

Funding

We are very grateful to the research deputy of the University of Tabriz for financial support.

Acknowledgments

We are very grateful to the general director of Fadak olive company, A. Boland Nazar, for providing the olive trees, and to A. Baybordi and M. Yousefi for their technical assistance.

Additional information

Funding

We are very grateful to the research deputy of the University of Tabriz for financial support.