Impact of Exogenous Applications of Salicylic Acid on the Tolerance to Drought Stress in Pepper (Capsicum Annuum L.) Plants

Background: Drought is also one of the most widespread abiotic stresses that adversely effects the growth and development of plants. To investigate the effect of salicylic acid and drought stress on several physiological and chemical reactions in sweet pepper plants, the experiment was achieved as a factorial based on a completely randomized design in greenhouse. Drought stress levels were non-stress conditions (irrigation with eld capacity), moderate stress (30% eld capacity irrigation) and intense water stress (60% eld capacity irrigation) and three concentrations of salicylic acid included 0 (as control), 0.5 and 1 mM were sprayed on the plant in three to four leaf stages. Results: The results showed that drought decreased fresh and dry weight of shoots and roots, leaf relative water content (RWC), fruit diameter and length, the index including chlorophyll and leaf area and increased electrical conductivity (EC), antioxidant activity, total phenolic content, ascorbate, polyphenol oxidase (PPO) and ascorbate peroxidase (APX) activity. After application of foliar salicylic acid, all of the above parameters, except the electrical conductivity content, increased. Conclusions: From the results of this experiment it is concluded that salicylic acid provides a better tolerance for drought stress in pepper plant through its inuence on vegetative, biochemical and physiological characteristics. correlation root fruit fruit signicant leaf area correlation leaf negative with fruit diameter fruit fruit diameter with fruit length and leaf area but signicant observed ascorbic correlation observed for fruit with leaf area and correlation observed with polyphenol Meantime, AsA. phenolic

The relative humidity (R.H. %) and mean temperature during the growth seasons were 60-70% and 18/ 25 °C (day/ night) respectively. SA was used at three levels, namely 0 (sprayed with distilled water as acontrol), 0.5 and 1mM. The SA was sprayed upper and lower leaves of fourth leaf stage. Tween-20 was used as a surfactant. The rst and second spray application of SA was at three days before and two weeks after the drought stress began. In the early stages of plant growth, all pots were watered until eld capacity. Accordingly, it lasted approximately four months three days after the foliar spray until sampling. The experiment consisted of three levels of drought stress: full irrigation (control group), 60% and 30% of eld capacity, moderate and severe stress respectively. We have not used any fertigation in this experiment. All pots were weighed on a daily basis. These conditions were contended until the end of the investigation.
For the determination of physic-chemical parameters, 27 plants were harvested in the green stage (80% maturity). Leaf samples were harvested by three replicates and each replicate was obtained from three pots, and therefore nine plants existed in each treatment group. The samples were quickly frozen in liquid nitrogen. They were stored at -80 °C.

Growth Parameters
The leaf area (LA) of leaves was measured using an area meter (AM 300 Bioscienti c Ltd.UK) fully grown leaves that expanded from the main stem (Phimchan et al. 2012). The leaf chlorophyll index (CHLI) was assessed according to the method described in the study of Wang et al. (2017) from the last fully expanded leaf of sweet pepper by SPAD502. The shoots fresh weight of pepper plants were assessed by a digital analytical balance. The dry weight of shoots were determined after being dried in an oven at 60°C (Ahmed et al. 2014). The roots of the sweet pepper plants were carefully separated and then washed thoroughly with distilled water several times and then measured with a digital analytical balance. Then, they were placed in an oven at 60°C to assess the dry weight of roots (Ahmed et al. 2014). A method by Barranco et al. (2005) was applied for determination of leaf relative water content (RWC). According that, the fresh leaves were cut into 0.1 g disks. The contaminants and residues of samples were removed thoroughly with distilled water. Then, the leaf samples were placed to the test tubes with 10 ml distilled water. The tubes were placed for 30 min at 30°C, subsequently the initial electric conductivity of the solution (Ec 1 ) was measured. For calculating secondary electric conductivity (Ec 2 ), the tubes heated for 15 min at 100°C in bath of water. nely; the total electrical conductivity was calculated using the formula: EL (%) = (EC 1 /EC 2 ) × 100.

Fruit physical properties
The morphological characteristics of pepper fruits such as length and diameter of fruit were measured. The measurements of diameter and length of the fruits were taken in maximum width and length. All measurements were by using a Caliber (Thuy and Kenji 2015).

Determine relative electrical conductivity
This test was performed by referring to the method developed by Deshmukh et al. (1991). The fresh leaves Cut (0.1 g) by disk and remove contaminants with distilled water. Then the leaf samples were placed to the test tubes containing 10 ml of distilled water and incubated for 30 min at 30°C. Then the initial electric conductivity of the solution (Ec 1 ) was measured. Subsequently the tubes heated for 15 min at 100°C in a bath of water and the secondary electric conductivity (Ec 2 ) was calculated. Eventually the total electrical conductivity was determined by the following formula: EL (%) = (EC 1 /EC 2 ) × 100 Measurement of Non-enzymatic antioxidants Measurement of AsA content A modi ed procedure by Luwe et al. (1993) was estimated for determination AsA content of pepper leaves. Initially, the samples pepper leaves (0.5 g) were poured by mortar and pestle in liquid nitrogen ant then homogenized in cold trichloroacetic acid (TCA, 1% w/v) and centrifuged at 12000×g for 20 min at 4°C.
Finely, the absorbance was read at 265 nm.

Assessment of Total phenolic content
The total phenol content was estimated per the method of Kahkonen et al. (1999). The leaves of fresh pepper (0.4 g) were homogenized in 4 ml of methanol and 0.1 Mol l -1 HCl mixtures. The mixture was centrifuged at 15000×g for 20 min at 4°C. The supernatant was mixed with distilled water (1:10) and by adding 2 ml diluted solution with 400 μl supernatant. The mixture was then added 1.6 ml of sodium carbonate (Na 2 CO 3 7.5%) to the above solution. The solution was stored in the dark for 30 min and centrifuged at 5000×g for 5 min. Eventually, the absorbance of the mixture was measured at 765 nm. The total phenolic content was estimated by Gallic acid mg ml -1 fresh material.

Antioxidant property
The DPPH was used to determine antioxidant activity of pepper leaves as described by Ab et al. (1998). The fresh leaves (0.2 g) were ground homogeneously with a mortar and pestle with 2 ml of ethanol at 4°C. About half of the solution was blended with a solution consist of 0.5 ml of 100 mm acetate buffer (pH 5.5) and 0.25 ml of 0.5 mM DPPH. The absorbance was recorded at 517 nm after 30 min. The antioxidant properties were calculated using the formula: Assaying the activity of antioxidant enzymes Preparation of extract: The pepper plant leaf was crushed in a mortar containing 50 ml of sodium phosphate buffer and then, centrifuged at 16,000 g at 10°C. The supernatant was to determine antioxidant enzyme activities APX and PPO.

Measurement of polyphenol oxidase activity
Reaction mixture containing 900 μl of catechol and 40 μl of 0.01 M sodium phosphate buffer with pH 6.8 was prepared and then 100 μl of enzyme extract was added. Then, the absorbance was recorded at 400 nm in 25°C for 3 min (Jiang et al. 2002).
Assaying of ascorbate peroxidase enzyme For this assay, 0.1 ml of enzyme extract was added to a mixture containing 0.1 ml of 0.5 mM ascorbate, 0.2 ml of H 2 O 2 1%, 2.5 ml of 50 mM potassium phosphate buffer and 0.1 mM EDTA and then the absorption was recorded at 290 nm at 25°C (Asada 1992).

Statistical analysis
All statistical analyses were performed by analysis of variance (ANOVA) using SPSS statistical 10 software. Differences between treatments were separated by Duncan's multiple range tests. The analyses were carried out to determine signi cant variation between the means at a signi cance level of P <0.05.

Correlation coe cient
The correlation coe cient was used for determining the natural and extensive relationships between the studied traits in this research. According to the results, some of the studied traits were signi cant ( Table 2). A positive signi cant correlation was reported between fresh weight of shoot with dry weight of shoot, fresh weight of root, dry weight of root, RWC, fruit diameter, fruit length and leaf area index. Also, fresh weight of shoot had a negative signi cant correlation with chlorophyll index. Meanwhile, the dry weight of shoots had a positive signi cant correlation with the fresh weight of roots, dry weight of roots, RWC, fruit diameter, fruit length, but there was a signi cant negative with chlorophyll index. In present study, fresh weight of roots correlated positively with dry weight of roots, relative water content, fruit diameter and fruit length. Meanwhile, a high signi cant correlation was found between root dry weight with relative water content, fruit diameter, fruit length, but had a negative signi cant correlation with leaf area index. In addition, we reported that a signi cant positive correlation was found between RWC with fruit diameter, fruit length, leaf area index, but had correlated negatively with chlorophyll index. Also, positive and signi cant correlation occurred for chlorophyll index with EC and ascorbic acid, but had signi cant negative correlation with fruit diameter and fruit length. A positive and signi cant correlation was observed for fruit diameter with fruit length and leaf area index, but a negative signi cant correlation was observed for ascorbic acid. A positive and signi cant correlation was observed for fruit length with leaf area index and negative signi cant correlation was observed with polyphenol oxidase, ascorbic acid and EC. Meantime, we reported that a signi cant positive correlation was observed between polyphenol oxidase with ascorbate peroxidase, ascorbic acid, EC, total phenolic content and antioxidant capacity. Ascorbate peroxidase shows a signi cant positive correlation with ascorbic acid, EC, total phenol and antioxidant activity. Also, positive and signi cant correlation occurred for Ascorbic acid with EC, total phenol and antioxidant activity. We reported that a signi cant positive correlation was observed between EC with total phenol and antioxidant activity. Ultimately, correlation analysis showed that total phenol has a positive and signi cant correlation with AsA. In a study on apple, Pearson correlation analysis in apples exhibited that the measured antioxidant activity can be attributed to the total phenolic measurement duo to relationship between each antioxidant and total phenolic content was very signi cant (Candrawinata et al. 2014).

Growth parameters
According to the data analysis of the present study (Table 3 and 4) drought stress and salicylic acid had a signi cant effect on fresh and dry weight of the shoots and roots, fruit length and diameter, leaf area index, chlorophyll index. Statistical results showed that with increasing drought stress, fresh and dry weight of the shoots and roots, fruit length and diameter, leaf area index, chlorophyll index decreased. As shown in Tables 3 and 4, foliar application of SA markedly increased the parameters mentioned above at all drought levels. Furthermore, the interaction effects of drought and salicylic acid treatments on these traits were signi cant except dry weight of shoot, fresh weight of root, fruit diameter, length fruit, chlorophyll index, leaf area index (Fig 1a and 1b and  table 4).

Relative water contents (RWC)
In the present study, RWC content in pepper plants under drought stress was signi cantly decreased. As the stress increased, this trait decreased (Table 3). Foliar application of SA signi cantly decreased the unfavorable effects of drought stress and thereby increased this trait (Table 3). In this feature, there was a signi cant interaction between drought stress and salicylic acid (Fig.1d).

Electrical conductivity
Results showed that drought stress and SA had a signi cant effect on electrolyte leakage. Drought stress increased electrolyte leakage into the intercellular space.
The highest enhance was observed at the severe drought stress as compared with controls. The foliar application of SA markedly reduced the electrical conductivity in stressed plants (Table 4). Also, the interactive effect of salicylic acid and drought stress was signi cant so that with increasing drought stress, electric conductivity content increased and after the use of SA has decreased (Fig. 1c).
Ascorbic acid Different concentrations of salicylic acid signi cantly increased the amount of ascorbic acid in pepper plants under drought and control conditions (Table 5) and this increase was greater in plants treated with salicylic acid under drought stress than non-stress conditions. Interaction of drought and salicylic acid treatments had signi cant effect on ascorbic acid content and showed that ascorbic acid content in pepper plants increased with enhancing salicylic and acid drought stress (Fig. 1e).

Total phenolics
According to the data analysis, drought stress had a signi cant effect on the total phenolic content of pepper plants (Table 5). After applying different concentrations of salicylic acid, total phenolic content in pepper plants was signi cantly under stress and control conditions ( Table 5). The interaction between drought stress and salicylic acid on total phenolic content was signi cant and showed that total phenol content increased with increasing drought stress and salicylic acid concentration in pepper plants (Fig. 2c).

Antioxidant capacity
The endogenous content of antioxidant activity remarkable increased in pepper plants with enhancing levels of drought compared to control conditions (Table   5). It markedly enhanced content of antioxidant activity in pepper plants when foliar SA was applied in pepper plants ( Table 5). The interactive impact of drought stress and salicylic acid on antioxidant activity was signi cant and showed that antioxidant activity in pepper plants increased with increasing drought stress and salicylic acid (Fig 2d).

Antioxidant Enzyme Activities
To inhibit reactive oxygen species in pepper plants under drought stress, their antioxidant system was activated and enzymes such as PPO and APX were signi cantly increased under both drought and SA treatments ( Table 5). As shown in Table 5, SA treatment stimulated both antioxidant enzymes more than control plants and the activity of these enzymes increased with increasing SA concentration. The interaction between drought stress and salicylic acid on antioxidant enzymes was signi cant as the amount of these enzymes increases with increasing drought and salicylic acid ( Fig. 2b and a).

Discussion
Drought is one of the main agents restricting the growth rate and physiological processes of plants (Loutfy et al. 2012). Agents such as plant species and genotypes, period of time and severity of drought stress, its age and developmental stage play a role in drought stress response. Drought stress can be evaluated by its effects on fruit morphological properties (El-Mageed et al. 2016). The growth of plant is caused by cell division, growth and differentiation. Drought stress reduces cellular turgor and disrupts cell growth processes and ultimately leads to poor plant growth. Plant growth regulators can be used to maintain proper water balance in the plant under drought conditions (Fahad et al. 2017).
In this study, results showed that drought stress decrease growth parameters such as fruit length and diameter of pepper plants, while SA increases growth parameters under drought stress. A study by Fariduddin et al. (2003) showed that the external spray of Salicylic acid increased growth rate in Brassica juncea plants and another study showed that salicylic acid was involved in processes such as enhancement of nutrient uptake In the present study, drought stress generally reduced LAI signi cantly (Table 4). This decrease in leaf area most likely resulted in lower light interception Yordanov et al. (2003) and a consequent decrease in photosynthesis. Although SA increased LAI signi cantly (Table 4), it could be via lowering developmental rate or delaying plant maturity Tasgin et al. (2003) and suggesting that foliar spray of SA at least partially compensated for harmful effects of drought stress. Thus, Senaratna et al. (2000) suggested that application of salicylic acid in bean and tomato plants could induce stress tolerance which is consistent with our ndings on pepper plants.
In the present study, shoot and root growth of pepper plants decreased signi cantly under drought stress (Table 3), the growth decrease is a suitable plant response to moisture depletion Efeoglu et al. (2009) and severe water scarcity affects cell elongation (Nonami 1998). Therefore, the growth decrease of pepper plants is a common symptom under drought stress. SA modulates the oxidative effects of stress that cause cell death and acts as a growth signal in cell resistance ). In a recent study, SA foliar spray reduced the adverse effects of stress and aided the natural growth of pepper plants ( Table 1). The role of SA in plant growth can be attributed to its effect on increasing cell division Sakhabutdinova et al. (2003) and we can say that, increasing the dry weight of the plant due to SA application indicates its overcoming of adverse effects of drought and improvement of plant growth Senaratna et al. (2000), which is consistent with our results in this study.
The results of our study showed that chlorophyll content decreased signi cantly on pepper plants under drought stress (Table 4). One study Panda et al.  Electrolyte leakage indicates damage to the cell membrane (Guo et al. 2006). The changes in cell lysis lead to impaired function as well as that indicate effect of environmental stress on plants. The disruption of cell membrane under environmental stress which can be explained by increase permeability and ion leakage from the membrane, that measured by the ow of electricity (Gupta et al. 2000). In this study, the amount of electrolyte leakage was increased by drought treatment (Table 4). A study on tomatoes under drought stresses showed that electrolyte leakage is due to impaired membrane integrity (Still et al., 2004). In this study, SA reduced the electrolyte leakage of pepper plants ( Table 4 Drought stress not only slows the growth of plants, but also changes the course of some metabolic processes. During long-term drought stress, the transfer of substances due to the decrease in available water results in changes in the concentration of some metabolites. As a result, the amount of water-soluble substances such as ascorbic acid increases. Ascorbic acid can prevent the plants from being oxidized to environmental stress due to the removal of free radicals from stress and its role in cellular stimulation and expansion and the absorption of substances into the cell that is effective in improving stress tolerance (Gallie 2013; Shan et al. 2011). In our study indicated that AsA content signi cantly increased under drought stress (Table 5) and exogenous SA played signi cant role in enhancing non-enzymatic components such as AsA (Table 5) . The results of this study showed that this metabolite increased due to application of both drought and SA treatments (Tables 5 and 6). Khalil et al. (2018) showed that total polyphenol content in Thymus vulgaris L. plants increased compared to control under salicylic acid as well as drought stress and our results are consistent with this nding.
In a recent study, our observations showed that the amount of antioxidant activity increased with increasing SA concentration and drought levels in pepper plants ( Table 5). Application of appropriate concentrations of SA reduces the harmful effects of oxidative stress by improving antioxidant capacity as well as the synthesis of protective compounds in plants (Hayat and Ahmed 2007). One study showed that plants exposed to drought stress exhibited greater total antioxidant capacity at higher PEG concentrations. Salicylic acid increased total antioxidant capacity in control and drought tolerant plants (Abbaspour and Ehsanpour 2016), the results are consistent with the results of this study.
Antioxidant enzymes are important components in ROS elimination (Navrot et al. 2007). Plants have an antioxidant system to reduce the damage caused by active oxygen (Qados 2015). Drought stress and salicylic acid treatments increased the amount of both enzymes studied in this study (Table 5). Foliar spray of maize plants with salicylic acid increased the activity of APX antioxidant enzyme under cadmium Krantev et al. (2008). In a study by Kang et al., 2003b, the increased activity of APX antioxidant enzyme in cold conditions was due to the increase in H2O2 and decreased by the enzyme and increased plant tolerance to stress.
Siddika et al. (2015) showed that the activity of PPO enzyme in wheat increased under drought conditions and in another study it was suggested that PPO enzyme is involved in reducing oxidative damage in plant. It can therefore be an index of adaptation to adverse environmental conditions Siddika et al. (2015), which is consistent with our results in this study.

Conclusion
The results of the current study on pepper showed that SA treatment plays an essential role in plant drought tolerance. As such, the application of the leaf SA by inducing Declarations we gratefully thank the University of Ilam for laboratory equipment.
Authors'contributions Zahra Khazaei and Asghar Estaji contributed to the conception and the design of the study. Zahra Khazaei performed all experiments and wrote the manuscript. Asghar Estaji supplemented and improved the manuscript as well as statistical analysis. All authors contributed to manuscript revision and read and approved the submitted version.

Funding
All experiments were performed in the Horticultural laboratory of University of Ilam, without external nancial support.

Availability of data and materials
All relevant data is contained within the manuscript. In addition, raw data from processed data will be made available by the authors, without undue reservation, to any quali ed researcher on request.

Consent for publication
All participants consented the con dential publication of their contributions in this study.

Competing interests
Tables    The same letters in each column indicate no signi cant difference at the 5% probability level in the Duncan test. LAI, CHLI, RWC, EC were the abbreviations of Leaf area index, Chlorophyll index, Relative water content, Electrical conductivity Table 5 Mean comparison of salicylic acid and drought stress effects on morphological parameters of pepper plants