Positive effects of plant growth regulators on physiology responses of Fragaria × ananassa cv. ‘Camarosa’ under salt stress

ABSTRACT

This experiment was conducted to investigate the impact of foliar spraying salicylic acid (SA) and methyl Jasmonate (MJ) on the physiology responses of strawberry (Fragaria × ananassa cv. ‘Camarosa’) grown under salinity stress. According to results, SA and MJ significantly reduced the injuries caused by salt stress, possibly through promoting K⁺ accumulation as well as decreasing the electrolyte leakage and Na⁺ contents in the leaves. The best protective effects resulted from 0.75 mM SA and 0.25 mM MJ treatments. These results indicate that SA and MJ can effectively improve the defense system and antioxidant capacity of strawberry in the salt affected environments.

KEYWORDS:

• Strawberry
• salt stress
• salicylic acid
• methyl jasmonat

Introduction

Strawberry is an important commercial fruit crop, widely grown in all temperate regions of the world including Iran. Strawberry production in Iran is more than 32,000 ton, and the cultivation area is approximately 2400 ha (FAO, 2012). The cultivation area of this berry has been continuously increasing in Iran for the last decades. At present, strawberries are grown in two main provinces of Iran (Kurdistan and Golestan). However, owing to its economic importance and the demand for locally grown berries, growers in other provinces are starting to produce more strawberries. One of the key production constraints for strawberry is high levels of salinity. In fact, this fruit species is considered to be one of the most salt sensitive of all crops (Sun et al., 2015). High levels of salinity are often found in the soils of arid and semi-arid regions and irrigation water. Most of the cultivated land in Iran is placed in such regions and hence is faced with different levels of salinity stress. This situation is becoming more problematic with the effects of global warming on salinity in such regions (Pankova and Konyushkova, 2013).

Owing to increasing use of poor quality water for irrigation and soil salinization, salinity is becoming a major abiotic stress, limiting growth and productivity of plants in many areas of the world. Depending on the severity and duration, salinity stress changes various physiological and metabolic processes that ultimately inhibit crop production (Gupta and Huang, 2014). More than 6% of the world’s total land area and 23% of the cultivated land worldwide are affected by salt stress, and the amount is increasing day by day (FAO, 2005). Therefore, the progressive accumulation of salt in the cultivated soil as a result of irrigation and climate changing increases the importance of this stressful factor (Pankova and Konyushkova, 2013).

High levels of NaCl have negative effects on enzymes and cell membranes. In response to salinity stress, the production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radical, is enhanced (Faghih et al., 2017). Salinity-induced ROS formation disturbs the cellular redox system in favor of oxidized forms, and can lead to oxidative damages in various cellular components such as proteins, lipids, and DNA, interrupting vital cellular functions of plants. Excessive amounts of ROS can enhance membrane lipid peroxidation and electrolyte leakage (Gunes et al., 2007; Rahat et al., 2011).

Initially, soil salinity is known to repress plant growth in the form of osmotic stress, which is then followed by ion toxicity. One of the most detrimental effects of salinity stress is the accumulation of Na⁺ and Cl⁻ ions in tissues of plants exposed to soils with high NaCl concentrations. Owing to the limiting assimilation, transport, and distribution of mineral nutrients within the plants, excessive uptake of Na⁺ and Cl⁻ ions might cause significant physiological disorders (Gupta and Huang, 2014).

Salicylic acid (SA) is considered as a hormone-like substance, which plays an important role in the regulation of plant growth and development, seed germination, fruit yield, glycolysis, flowering, and heat production in thermogenic plants (Eraslan et al., 2007). Ion uptake and transport (Figen et al., 2007), as well as photosynthetic rate, stomatal aperture, and transpiration (Khan et al., 2003) could also be affected by SA application. Alteration in the activity of antioxidant enzymes in vivo is one of the direct physiological effects of SA. Exogenous application of SA can regulate the activities of antioxidant enzymes and mitigates salinity stress by improving antioxidant defense system (Idrees et al., 2011).

JA and its methyl ester (JA-Me) are endogenous growth regulators identified in many plant species, and induce a wide variety of physiological and developmental responses (Engelberth et al., 2001). Jasmonates activates plant defense mechanisms in response to insect-driven wounding, various pathogens, and environmental stresses, such as drought, low temperature, and salinity (Ghorbani et al., 2011). It is reported that exogenous application of JA antagonistically has regulated the expression of salt stress inducible proteins (Sheteawa, 2007) and has dramatically reduced sodium concentration in rice seedling (Kang et al., 2005).

The present work was carried out to study the effects of foliar spraying of SA and MJ on oxidative stress induced by NaCl in strawberry cv. ‘Camarosa’ that is one of the most important cultivars in Iran.

Materials and methods

Plant materials and treatments

In a pot experiment, strawberry (Fragaria × ananassa cv. ‘Camarosa’) plants were grown in plastic pots (two plants per pot) containing 2.5 kg of soil, peat, and perlite (1:1:1, v/v/v) mixture. Texture, pH, and electrical conductivity of soil mixture were silt-loam, 7.3, and 2.28 dS m⁻¹, respectively. The pots were kept in the greenhouse condition, and plants were irrigated every three days alternately with salty water containing 0, 30, and 60 mM NaCl. In order to avoid salt accumulation in the media, each irrigation step was continued to pass 1/3 of the water through the pots. In addition, after every two irrigation steps and before the third step, the media was rinsed with distilled water. Plant injury symptoms appeared 14 days after salinity stress. Two weeks after exerting salinity stress, plants were treated with foliar spraying of SA (0, 0.10, 0.50, and 0.75 mM) and MJ (0.25, 0.50, and 0.75 mM), respectively. SA and MJ treatments were repeated after seven days from the first treatment. With appearance symptoms of salt injury in plants, physiological and biochemical factors were investigated.

The layout was a factorial experiment in a complete randomized design with three replications and two plants per replication (a plastic pot).

Electrolyte leakage

To assess the membrane permeability, electrolyte leakage was determined based on the method described by Lutts et al. (1996). Six leaf discs were taken from the newly fully expanded leaves on one randomly chosen plant per replicate sample (pot). For removal of surface contaminations, samples were washed three times with distilled water, and then leaf discs were placed in a test tube containing 10 ml of distilled water. These samples were incubated at room temperature in a shaker for 24 h. After incubation, electrical conductivity of the solution (EC₁) was read. The same samples were then autoclaved at 120°C for 20 min, and a second EC was recorded (EC₂). Electrolyte leakage was measured using an electrical conductivity meter (CC-501, Elmetron, Zabrze, Poland) and calculated as EC₁/EC₂ and expressed as a percentage.

Proline determination

Proline was determined according to the method described by Bates et al. (1973). Briefly, 0.1 g fresh leaves were homogenized in 10 ml 3% (v/v) aqueous sulphosalicylic acid and filtered through a Whatman No. 2 filter paper. Two ml of the filtrate was then mixed with 2 ml acid-ninhydrin reagent and 2 ml glacial acetic acid in a test tube and the mixture was placed in a water bath for 1 h at 100°C. The reaction mixture was extracted with 4 ml toluene, and the chromophore-containing toluene fraction was aspirated, cooled to room temperature, and its absorbance was measured at 520 nm using a Shimadzu UV 160A spectrophotometer (Shimadzu Corp., Kyoto, Japan). Appropriate proline standards (Sigma Chemical Co., St. Louis, MO, USA) were included in order to calculate the concentration of proline in each leaf sample.

Analysis of ion concentration

At the end of the experiment period, roots and leaves of plants were dried at 80°C for 48 h, weighed, and ground to pass a 40-mesh sieve. The grounded materials (0.5 mg) were placed in a muffle furnace at 550°C for 5 h, and the ash was then dissolved in 10 ml 2 mol/L HCl and the volume was adjusted to 100 ml with distilled water. Na⁺ and K⁺ were determined using a flame photometer (PEP7, Jenway, Dunmow, UK).

Statistical analysis

Data were analyzed for significant differences using a factorial analysis of variance, with salicylic acid and methyl jasmonate and NaCl levels as the main factors. Statistical analysis was compared using the least significant differences (LSD) test at P ≤ 0.05. All the analyses were conducted using SAS software version 9.1.3 (SAS Institute Inc., Cary, NC, USA).

Results and discussion

Effects of SA and MJ on electrolyte leakage

Cellular membrane dysfunction, resulting from salt stress, is well expressed by higher permeability of ions and electrolytes, which can be readily measured by the efflux of electrolytes (Lutts et al., 1996). Membrane permeability was determined by measuring electrolyte leakage. According to our results, the effects of salinity and hormonal treatments were statistically significant at electrolyte leakage (Table 1). Salinity significantly increased leaf electrolyte leakage and, as expected, the highest amount of leaf electrolyte leakage was observed in the plants treated by 60 mM of NaCl (average of all treatments 90.12%) (Table 2). Similar results were obtained by Kaya et al. (2002) and Lutts et al. (1996), who reported that high salt concentration had increased the membrane permeability in sensitive rice varieties. Application of SA and MJ significantly decreased leaf electrolyte leakage. However, the lowest leaf electrolyte leakage was obtained from plants treated with 0.75 mM SA (average for three levels of salinity 63.99%) followed by 0.25 mM MJ (average for three levels of salinity 61.04%), which had 19.25% and 22.98% lower leaf electrolyte than control plants, respectively (Table 2). There was a significant interaction between salinity and hormonal treatments (Table 1). The lowest leaf electrolyte leakage value was obtained from plants treated with 0.75 mM and 0.10 mM SA in 30 and 60 mM NaCl (73.02% and 88.70%, respectively), and 0.25 mM and 0.75 mM MJ in 30 and 60 mM NaCl (55.82% and 81.62%, respectively) (Table 2). Positive effects of SA on the stability of membrane and maintenance membrane functions (El-Tayeb, 2005; Stevens et al., 2006; Baninasab and Baghbanha, 2013), decreasing leaf electrolyte leakage (Stevens et al., 2006) and reducing membrane permeability and lipid peroxidation (Horvath et al., 2007) have been reported in several plant species under salt stress condition. Maintaining integrity of the cellular membranes under salt stress is considered as an integral part of the salinity tolerance mechanism (Stevens et al., 2006). Protective effect of SA and MJ on membrane permeability may be somewhat resulted from the role of these two compounds in activating the antioxidant enzymes, which in turn protect plants against the generation of ROS and membrane injury or may result in the synthesis of other substances that have protective effects on plants growing under stress.

Table 1. Analysis of variance (ANOVA) of NaCl salinity (N), hormonal treatments (T) and their interaction (N×  T) for the leaf electrolyte leakage (EC₁/EC₂), leaf proline, Na content of leaves (NaL), K content of leaves (KL), Na content of roots (NaR), K content of roots (KR), of strawberry under salt stress.

		F values
Source of variance	df	EC1/EC2	LP	NaL	KL	NaR	KR
N	2	244.65**	133.95**	997.37**	6.28**	85.90**	16.16**
T	6	6.84**	63.97**	5.29**	20.80**	1.68 ns	4.31**
N × T	12	6.63**	22.85**	7.87**	7.14**	2.34*	1.89 ns

ns: non significance.

*Significance at 0.05 probability level.

**Significance at 0.01 probability level.

Table 2. Effect of hormonal treatments (HT) on electrolyte leakage (EC₁/EC₂) and leaf proline (LP) of strawberry under salt stress.

NaCl salinity (mM)	HT (mM)	EC1/EC2 (%)	LP (µmol/g FW)
	Control	70.10 ± 6.58d	0.152 ± 0.016gh
	0.10 SA	22.17 ± 1.14g	0.074 ± 0.007 ij
	0.50 SA	24.62 ± 5.51fg	0.113 ± 0.010h–j
0	0.75 SA	27.27 ± 7.56fg	0.106 ± 0.003h–j
	0.25 MJ	35.72 ± 6.96 f	0.062 ± 0.006j
	0.50 MJ	51.12 ± 4.61e	0.060 ± 0.012 j
	0.75 MJ	29.30 ± 1.19fg	0.063 ± 0.001 j
	Control	80.25 ± 0.96cd	0.167 ± 0.019f–h
	0.10 SA	86.70 ± 7.39a–c	0.139 ± 0.002g–i
	0.50 SA	79.30 ± 6.55cd	0.148 ± 0.005gh
30	0.75 SA	73.02 ± 6.86d	0.392 ± 0.030b
	0.25 MJ	55.82 ± 5.86e	0.187 ± 0.030d–g
	0.50 MJ	86.30 ± 5.04a–c	0.102 ± 0.011h–j
	0.75 MJ	82.72 ± 5.31a–d	0.248 ± 0.009cd
	Control	87.40 ± 1.07a–c	0.218 ± 0.014d–f
	0.10 SA	88.70 ± 3.15a–c	0.198 ± 0.015d–g
	0.50 SA	94.80 ± 1.23ab	0.180 ± 0.029e–g
60	0.75 SA	91.65 ± 1.83a–c	0.773 ± 0.001a
	0.25 MJ	91.57 ± 2.08a–c	0.285 ± 0.050c
	0.50 MJ	95.07 ± 1.26a	0.140 ± 0.006gh
	0.75 MJ	81.62 ± 1.41b–d	0.242 ± 0.066c–e

Values followed by the same lowercase letters within a column are not significantly different using LSD at P ≤ 0.05.

Effects of SA and MJ on proline content

Under salt stress, plants accumulate several compatible solutes in the cytosol. Proline accumulation is an important mechanism for osmotic regulation under salt stress and has been correlated with stress tolerance (Huang et al., 2013). In our study, the proline content of leaves was significantly affected by salinity, SA, and MJ treatments (Table 2). Proline content increased in the NaCl-treated plants in parallel with the salt concentration, and the highest level being attained at 60 mM NaCl treatment in the control plants.

The highest proline content was obtained from leaves of plants treated by 0.75 mM SA and MJ (0.424 and 0.184, respectively) (Table 2). There was significant interaction between salinity and hormonal treatments (Table 1). In the 30 mM NaCl concentration, the highest proline content was recorded in the plants treated by 0.75 mM SA and MJ (0.392 and 0.248, respectively), while plants treated by 0.75 mM SA and 0.25 MJ had the highest amount of proline in 60 mM salinity (0.773 and 0.285, respectively) (Table 2). According to Sheteawa (2007), JA treatment improved salt tolerance in soybean through inducing the accumulation of nontoxic metabolites, sugar, free proline, and proteins. Manan et al. (2016) reported that MJ significantly increased proline content and ameliorated the deleterious effects of salinity on tomato plants by inducing the physiological and biochemical resistance. However, higher concentration of MJ in 60 mM NaCl salinity adversely affected the proline content of treated plants. In agreement with our finding, Salimi et al. (2016) reported that under stress condition, low concentrations of MJ are more efficient compared with the higher doses. In fact, MJ, at low concentrations, is more capable of enhancing physiological processes and improving plant tolerance by biochemical and molecular mechanisms; whereas, at higher levels, MJ may act as a stressful agent and intensify the salt stress.

Under salt stress, SA-treated strawberry plants cv. ‘Selva’ had both higher proline and glycine betaine contents and lower protein degradation (Jamali et al., 2016). In addition, proline concentration increased in SA-treated wheat seedlings under normal conditions (Farida et al., 2003). It has been reported that SA ameliorates the stress generated by NaCl through proline accumulation, which was involved in the synthesis of protective proteins that are necessary for the stress response (Li et al., 2014). A possible reason for proline accumulation during salinity stress could be an alteration in the activities of the enzymes involved in the biosynthesis and degradation of proline (Neelam and Saxena, 2009). In addition, it is reported that proline might act as a store of energy that can be rapidly broken down and used when the plant is relieved from stress or acts as an osmolyte and reduces the osmotic potential of the cell, so reducing toxic effects of ion uptake (Hare et al., 1998). In this case, the latter is more likely, because not only did the hormonal treatments enhance the proline content, but they also reduced the electric leakage of stressed plants. Therefore, proline can be considered as an important component of plant produced compounds from SA and MJ treatments in response to the salinity stress. In fact, proline reduces the detrimental effects of salinity and accelerates the recovery processes after stress.

Effects of SA and MJ on na⁺ and K⁺ contents

Salinity, SA, and MJ treatments significantly affected Na⁺ and K⁺ contents in both leaves and roots (Table 1). The NaCl treatment induced Na⁺ accumulation in leaves and roots of the strawberry, and the highest Na⁺ accumulation was consistently displayed in the plants subjected to the highest salinity (average of all treatments 12.34 and 13.05 mg g⁻¹ for leaves and root, respectively) (Table 3). All concentrations of SA and MJ decreased Na⁺ accumulation in the leaves of treated plants compared with the controls. However, 0.50 mM SA and MJ were the most effective treatments (average of all treatments 7.92 and 7.27 mg g⁻¹, respectively) (Table 3).

Table 3. Effect of hormonal treatments (HT) on Na⁺ and K⁺ contents of leaves and root of strawberry under salt stress.

		Leaf		Root
NaCl salinity (mM)	HT (mM)	Na^+ (mg g^−1)	K^+ (mg g^−1)	Na^+ (mg g^−1)	K^+ (mg g^−1)
	Control	2.36 ± 0.13kl	10.24 ± 0.81 f	6.01 ± 0.11hi	4.20 ± 0.47a–c
	0.10 SA	2.23 ± 0.32kl	23.91 ± 2.27 a	4.46 ± 0.24i	3.66 ± 0.08a–e
	0.50 SA	2.96 ± 0.32 k	25.90 ± 0.25 a	6.04 ± 0.28hi	4.65 ± 0.65a
0	0.75 SA	4.46 ± 0.26j	24.97 ± 2.38a	6.28 ± 0.44hi	3.14 ± 0.26c–g
	0.25 MJ	1.50 ± 0.24i	15.69 ± 0.60b–e	5.99 ± 0.43hi	4.09 ± 0.53a–d
	0.50 MJ	1.73 ± 0.17i	16.36 ± 0.60b–e	6.22 ± 0.23hi	4.50 ± 0.38ab
	0.75 MJ	3.01 ± 0.18k	13.89 ± 1.17e	6.96 ± 0.74gi	4.46 ± 0.49ab
	Control	11.38 ± 0.12cd	16.85 ± 0.22b–e	11.07 ± 0.60d–f	3.01 ± 0.30c–g
	0.10 SA	8.53 ± 0.25g–i	17.47 ± 0.27b–d	12.02 ± 0.27c–e	4.43 ± 0.25ab
	0.50 SA	7.75 ± 0.47hi	16.93 ± 0.41b–e	11.89 ± 1.56c–e	3.99 ± 0.37a–d
30	0.75 SA	9.99 ± 0.24ef	23.77 ± 2.14a	10.99 ± 0.38d–f	2.90 ± 0.08d–g
	0.25 MJ	9.26 ± 0.75fg	15.91 ± 0.33b–e	13.33 ± 1.07a–d	3.82 ± 0.41a–d
	0.50 MJ	7.53 ± 0.86i	14.31 ± 1.12c–e	11.40 ± 0.64c–f	2.92 ± 0.38d–g
	0.75 MJ	8.87 ± 0.61f–h	16.27 ± 1.57b–e	9.65 ± 2.24e–g	2.10 ± 0.06g
	Control	12.34 ± 0.25bc	15.08 ± 0.71c–e	8.81 ± 2.77f–h	1.96 ± 0.43g
	0.10 SA	13.67 ± 0.63a	17.56 ± 0.40bc	15.61 ± 0.10a	4.07 ± 0.71a–d
	0.50 SA	13.04 ± 0.20ab	18.80 ± 2.38b	14.32 ± 1.54a–c	3.42 ± 0.83b–f
60	0.75 SA	11.15 ± 0.64de	17.16 ± 0.41b–e	12.43 ± 1.11b–e	2.42 ± 0.39fg
	0.25 MJ	11.16 ± 0.52de	16.08 ± 0.63b–e	12.05 ± 1.00c–e	3.09 ± 0.44c–g
	0.50 MJ	12.55 ± 0.07ab	14.13 ± 0.53de	15.07 ± 0.67ab	2.54 ± 0.12e–g
	0.75 MJ	12.50 ± 0.04a–c	16.54 ± 0.15b–e	13.05 ± 0.16a–d	2.12 ± 0.41g

Values followed by the same lowercase letters within a column are not significantly different using LSD at P ≤ 0.05.

As expected, the Na⁺ content in the leaves and roots increased in parallel with salinity levels. Simultaneously, the accumulation of K⁺ in leaves and roots decreased gradually with the increase of salinity (Table 3). Accumulation of Na⁺ ions in leaves will be resulted in the reduction of osmotic potential, so contributes to the maintenance of the water potential differences between plant’s leaves and its media that is required to uptake water from the saline solution (Amini and Ehsanpour, 2005). A secondary effect of high concentrations of Na⁺ as well as Cl⁻ ions in the root medium is the suppression of uptake of essential nutrients such as K⁺ that results in lower productivity and may even lead to death (James et al., 2011). In fact, results of many studies indicate that plants that are exposed to NaCl, inevitably absorb a large amount of Na⁺, which subsequently causes a reduction in the contents of K⁺ (Asgari et al., 2012; Keutgen and Pawelzik, 2009). Na⁺ ions compete with K⁺ for uptake through common transport systems and this competition is usually in favor of Na⁺, as this element is usually considerably greater than K⁺ in the saline environments (Baninasab and Baghbanha, 2013). Exclusion or maintenance of low concentrations of toxic Na⁺ ions in different organs is considered as an essential phyto-physiological mechanism for salinity tolerance (Houmani and Corpas, 2016). In contrast to our results, it is reported that K⁺ contents increased in two strawberry cvs. ‘Korona’ and ‘Elsanta’ under elevated NaCl concentrations (Keutgen and Pawelzik, 2009). These authors suggested the existence of a more efficient K⁺ uptake in strawberry compared to other plants. In addition, they reported that ‘Korona’ had a significant increase of K⁺ content in leaves and crowns; whereas, ‘Elsanta’ showed an increase of K⁺ in fruits and petioles. Therefore, it is probable that strawberry cultivars vary in their responses to K⁺ uptake under salt stress.

However, in our study, the SA- and MJ-treated plants K⁺ concentrations increased in these two organs compared with the control plants. The highest amount of K⁺ in the leaves was observed in the plants treated by 0.75 mM SA (21.97 mg g⁻¹) and 0.10 mM MJ (15.90 mg g⁻¹); whereas, 0.10 mM SA (4.05 mg g⁻¹) and 0.25 MJ (3.67 mg g⁻¹) treatments resulted in the highest concentration of K⁺ in the roots (Table 3). An optimal K⁺/Na⁺ ratio is necessary to activate the enzymatic reactions in the cytoplasm for maintenance of plant growth and yield development. According to our results, 0.75 mM SA treatment showed the lowest value of Na⁺/K⁺ in the leaves of plants subjected to the 30 and 60 mM NaCl concentrations. On one hand, the lowest concentration of MJ (0.25 mM) was the most effective treatment of this compound in 0 and 60 mM NaCl levels. Our results are in agreement with several previous investigations’ findings, which indicated that SA treatment inhibited Na⁺ accumulation and stimulated K⁺ uptake in the shoots and roots of different plants such as wheat (Al-Hakimi and Hamada, 2001), barley (EI-Tayeb, 2005), maize (Gunes et al., 2007), and cucumber (Baninasab and Baghbanha, 2013) under salt stress. In addition, positive effects of JA on the accumulation of K⁺ and P ions were previously reported by Abu-Ghalia and El-Khalal (2001). Accumulation of these elements may contribute to the mitigative effects of JA under salt stress. Kang et al. (2005) reported that sodium concentration dramatically decreased by exogenous application of JA in rice plants. Negative correlation between Na⁺ and K⁺ as a result of hormonal treatment indicates that hormonal treatment could play significant role in modifying K⁺/Na⁺ selectivity under salt stress, which finally can reduce membrane damage and maintain water content.

Conclusion

The response of strawberry plants to SA and MJ treatment outlined in this study suggests that the application of SA and MJ induced changes in the antioxidant activity, Na⁺/K⁺ ratio, and proline content and these changes were associated with salt tolerance in the strawberry cv. ‘Camarosa’. SA applied at 0.75 mM and MJ at 0.25 mM were the most effective treatments participated in the salt tolerance, especially at higher concentrations of NaCl, and would protect strawberry plants partially against salt stress. Results of the present work suggest that SA and MJ, two readily available substances, could be used as plant protective agents in the salt affected environments, and these findings may have significant practical applications.

Acknowledgments

The authors thank Isfahan University of Technology for their support of this research. The authors acknowledge Mr. M. Ahmadi for their valuable help with this experiment.

Additional information

Funding

This work was supported by the Isfahan University of Technology.