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. 2019 Aug 7;25(5):1149–1161. doi: 10.1007/s12298-019-00692-2

Nitric oxide mitigates salt stress effects of pepper seedlings by altering nutrient uptake, enzyme activity and osmolyte accumulation

Mostafakamal Shams 1, Melek Ekinci 1, Selda Ors 2, Metin Turan 3, Guleray Agar 4, Raziye Kul 1, Ertan Yildirim 1,✉
PMCID: PMC6745581  PMID: 31564778

Abstract

This study was planned to evaluate the role of exogenous application of sodium nitroprusside (SNP), a NO donor, on the deleterious effect of salinity in Capsicum annum L. seedlings. Different NO doses (0, 50, 100 and 150 µM SNP) were foliarly applied to pepper seedlings grown under the non-saline and saline conditions (50, 100 and 150 mM of NaCl). The photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), mineral element (Zn, Fe, B, K, Ca and Mg) uptake, plant growth and leaf relative water content (LRWC) were decreased by NaCl treatment, but NO treatments generally improved the observed parameters. 150 mM NaCl treatment caused overaccumulation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) by 87 and 100% respectively as compared to control. However, NO application (150 µM SNP) at 150 mM of NaCl significantly decreased H2O2 and MDA to 34 and 54%, respectively. The present study clarified that the exogenous NO treatment supported pepper seedlings against salinity stress by regulating the mineral nutrient uptake, antioxidant enzyme activity, osmolyte accumulation, and improving the LRWC and photosynthetic activity.

Keywords: Growth parameters, Mineral uptake, Nitric oxide, Pepper, Photosynthetic rate, Salinity

Introduction

Salinity is one of the main abiotic stress factors which affect crop physiological processes and enzymatic activities thereby limiting agricultural production worldwide. The adverse effect of salinity could primarily arise from toxic ion accumulation (Na and Cl) and osmotic potential (Ashraf 2009). Furthermore, toxic ion accumulation results in oxidative stress, which is defined by the overproduction of reactive oxygen species (ROS) such as hydroxyl radical, superoxide radical and hydrogen peroxide. ROS can damage lipids, proteins and DNA, thereby disordering normal cellular functions (Gadelha et al. 2017). Defense systems such as enzymatic and non-enzymatic components are developed by plant cells to overcome the oxidative stress (Noctor and Foyer 2016). Catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) are enzymatic compounds for ROS-scavenging. The non-enzymatic compounds include glutathione, tocopherols and carotenoids (Zagorchev et al. 2013).

NO is a reactive nitrogen species as a signaling molecule, which plays a key role in many biological functions in plants. Additionally, it has been reported that NO can be effective in maintaining tolerance and resistance to abiotic stress such as UV, heavy metals, salinity and drought (Ahmad et al. 2018a; Boogar et al. 2014; Hasanuzzaman et al. 2018; Shams et al. 2018; Yadu et al. 2017). The regulation of enzymatic and non-enzymatic components by NO might ameliorate the plant tolerance to salinity (Shi et al. 2005).

Pepper is widely consumed for human food because of its richness in proteins, carbohydrate, vitamins, fats, minerals, taste and aroma. The salinity can negatively affect the pepper plant growth especially when the salinity of irrigation water is above EC 2 dS m−1 which can lead to a significant decrease in pepper yield (Semiz et al. 2014). It is also reported earlier that the pepper seedling stage is more sensitive than the seed germination stage (Yildirim and Güvenç 2006).

Pepper response to salt stress has been widely investigated. However, the impact of NO on plant growth, physiological, nutrient uptake and biochemical properties under salt stress still needs to be studied. Therefore, in this study, we evaluated the effect of external NO treatment on the morphological, biochemical and the enzymatic response of pepper under salinity stress during the early stage of plant growth.

Materials and methods

Plant material and experimental set up

Pepper (Capsicum annuum L. cv. Yalova) seedlings were grown in multi-celled trays containing peat. The seedlings were transplanted to pots (50 × 15 × 20 cm) 35 days after sowing. Each pot had four seedlings. The pots were filled with a ratio of 2:1:1 (v:v:v) of soil: sand: manure having around 1.30 g cm3 bulk density. The experiment was conducted in the greenhouse, temperature ranged from 17.1 to 32.9 °C, and the humidity was 65%.

The irrigation was done with four levels of NaCl (0, 50, 100 and 150 mM) and their electrical conductivities were measured as 0.52, 5.08, 7.82 and 11.47 dS m−1 respectively. Salinity treatments were initiated 2 days after transplanting of seedlings, and NaCl were added in increments of 50 mM NaCl to avoid osmotic shock to the plants. SNP, a NO donor, was used as the NO treatment. NO treatments were prepared at 4 levels of 0 (control), 50, 100 and 150 µM SNP in ultrapure water with Tween 20 (Sigma Chemicals). The solutions were sprayed onto upper side and under side of leaves a day before transplanting, and then every week regularly until harvest (15 ml per plant). After 45 days of the transplantation, chlorophyll reading value (SPAD), photosynthesis parameters were measured and LRWC and EL were determined from each pot, then plants were harvested and sampled for the measurements of leaf area, root dry weight and shoot dry weight and the other chemical analysis.

Leaf chlorophyll reading value (SPAD)

Leaf chlorophyll content was determined as leaf chlorophyll reading value (SPAD) with a chlorophyll meter. The measurements were carried out at six different spot on leaves of each plant; two on each side of fully expanded leaves, and then the average were used for analyses.

Photosynthetic parameters

Photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr), and intercellular CO2 concentration (Ci) were measured on the third fully expanded upper leaves. The measurements were done by using a portable Li-Cor 6400 Photosynthesis System (LI-COR, Lincoln, USA) between 10:00 and 1:00 pm on a sunny day (Ors et al. 2016).

Electrolyte leakage (EL)

Ten leaf discs were placed in 25 mL glass test tubes for EL. The tubes were full of ultrapure water, and then the samples were put in an incubator at room temperature for 24 h. The EC (EC1) of the water in the tubes was measured. For heating, tubes were autoclaved for 20 min at 95 °C and then refrigerated to 25 °C and the EC (EC2) was determined. The percentage of ratio between EC1 and EC2 were considered as EL.

Leaf relative water content (LRWC)

LRWC was determined according to Esringu et al. (2011). To determine the fresh weight (FW) of leaves, firstly four leaves (young fully expanded one) were weighed to measure the FW of samples. The samples were kept in ultrapure water to obtain turgid weight (TW). Then, they were kept in an oven for 48 h at 70 °C to obtain DW at the end of the imbibitions. LRWC (%) of leaves was calculated by using following equation: RWC = [(FW − DW)/(TW − DW)] × 100.

Antioxidant enzyme activity

The frozen pepper sample was homogenized in 100 mM phosphate buffer (5 ml, pH 7.0) containing 1% (w/v) polyvinyl polypyrrolidone (PVPP). The centrifugation was carried out at 15,000 × g for 15 min and the supernatant fraction was immediately analyzed for CAT, SOD and POD activities.

The CAT activity was determined according to Liu et al. (2014) with some modification. The CAT activity was measured based on the rate of H2O2 decomposition. The reaction mixture contained 10 mM H2O2, 50 mM phosphate buffer (pH 7.0) and 100 μL of plant extract. The oxidation extinction coefficient was 39.4 mM−1 cm−1 for H2O2. The POD activity was determined according to Liu et al. (2014) which was based on its ability to convert guaiacol to tetraguaiacol at 436 nm. The activity of SOD was assayed in line with the method by Liu et al. (2014). The total SOD activity was determined by scanning the ability of enzyme extract to prevent the photochemical reduction of P-nitro-blue tetrazolium chloride (NBT).

MDA and H2O2

Lipid peroxidation was determined as the content of MDA with a method given by Liu et al. (2014). The absorbance of the supernatant was read at 532 and 600 nm. Extinction coefficient of 155 mM−1 cm−1 was used to assess the content of MDA from the absorbance. For assessment of H2O2 concentration, 200 mg of leaf tissues was ground into a fine powder and the homogenization was immediately done in 2 mL of 0.1% (w/v) TCA solution on ice and the other process was done according to Liu et al. (2014).

Sucrose

The sucrose content was obtained by using the method of Wu et al. (2011). Dry sample (0.1 g) was incubated for 24 h in phosphate buffer (10 mL, 0.1 M and pH 5.4) at 22 °C. 0.2 mL of supernatant samples were mixed with 1.0 mL invertase (10 U/mL) and distilled water, then incubated for 1 h at 50 °C in a water bath. The sucrose content was computed according to difference in reducing sugar content of the incubation solution without and with invertase.

Proline

Liquid nitrogen and a pestle and mortar were used to powder 50 mg of frozen leaf and the homogenization was done in 4.5 mL of 5-sulfosalicylic acid 3% on ice. The homogenates were centrifuged for 15 min at 12,000 × g at 4 °C. Proline concentration of the samples was determined at 520 nm by a spectrophotometer (Man et al. 2011).

Mineral analysis

The leaves of the pepper were dried at 68 °C for 48 h and grounded. P, K, Ca, Mg, Fe, Zn, Cl, B and Na content was analyzed by a coupled plasma spectrophotometer (Optima 2100 DV; Perkin-Elmer, Shelton, CT) (Helrich 1990).

Statistical analysis

A completely randomized experimental design following a factorial plan with four replications was used in this study; in each replication, we had 12 plants. The SPSS 18 was used for data analysis [ANOVA and Duncan’s multiple range test (DMRT)] at a significance level of 0.01. The presented data as % were subjected to angular transformation before variance analysis.

Results

Growth

NaCl drastically suppressed the measured growth parameters in this experiment (Table 1). The highest application dose of NaCl (150 mM) had the highest negative effect, and caused a significant decrease in root and shoot dry weight, shoot length and leaf area by 64, 67, 30 and 44%, respectively, as compared to the control. To investigate the proactive role of NO on the NaCl toxicity, we sprayed 50, 100 and 150 µM of SNP and we found that NO treatment mitigated the adverse effect of NaCl on growth parameters of pepper seedlings. Simultaneous application of NaCl (150 mM) and NO (100 µM SNP) increased shoot length, leaf area, shoot dry weight and root dry weight by 17, 24, 62 and 41%, respectively, as compared to the plants treated only with 150 mM of NaCl, without NO (Table 1). In addition, the results of this study highlighted that NO treatment increased the leaf area, root and shoot dry weight in the absence of salinity.

Table 1.

The effect of salinity and NO treatments on the shoot length, root dry weight, shoot dry weight and leaf area in pepper

Treatments Shoot length (cm) Root D.W (g) Shoot D.W (g) Leaf area (cm2/plant)
NaCl (mM) NO (µM SNP)
0 0 21.18 abc 0.34 c 2.39 c 28.84 b
50 21.83 ab 0.48 b 2.70 b 29.12 b
100 21.96 ab 0.48 b 2.91 ab 33.69 a
150 22.46 a 0.52 a 3.08 a 33.27 a
50 0 21.17 abc 0.26 d 1.66 de 25.05 cd
50 20.96 bc 0.31 c 1.70 de 24.19 d
100 18.87 d 0.31 c 1.90 d 26.50 c
150 20.12 c 0.26 d 1.63 e 24.92 cd
100 0 17.25 efg 0.16 f 0.89 h 19.09 gh
50 18.34 de 0.23 de 1.34 f 21.08 ef
100 18.25 de 0.21 e 1.37 f 22.58 e
150 16.67 fg 0.17 f 0.98 h 17.73 h
150 0 14.88 h 0.12 g 0.77 h 16.03 i
50 15.96 gh 0.16 f 1.02 gh 18.55 gh
100 17.42 ef 0.17 f 1.25 fg 20.01 fg
150 16.67 fg 0.14 fg 0.97 h 17.51 hi
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Data followed by a different letter in column were significantly different (p ≤ 0.01) according to the DMRT

Chlorophyll reading value (SPAD), LRWC and EL

The impact of salinity on EL, LRWC and SPAD are given in the Fig. 1. Salinity had a significant effect on the investigated parameters and caused a significant decrease in SPAD values and LRWC, but it increased EL. In this regard, 150 mM of NaCl had the greatest negative impact on SPAD values and LRWC, but it had an incremental impact on the EL as compared to the untreated one. However, in the presence of 150 mM of NaCl, the NO treatment (100 µM of SNP) caused an increase in SPAD and LRWC, and a decrease in EL in pepper seedlings (Fig. 1). In the absence of salt stress, the NO treatment caused a decrease in LRWC in pepper as compared to the untreated one.

Fig. 1.

Fig. 1

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Chlorophyll content (a), leaf relative water content (LRWC) (b) and Electrolyte leakage (EL) (c) of pepper seedlings in response to NO (SNP) application under salt stress. Different letters on top bars indicate differences (Duncan Multiple Range Test, p < 0.01 at each treatment). Vertical bars indicate the mean ± standard error

Mineral uptake

Salinity treatments reduced the P, K, Ca, Mg, B, Zn and Fe content in the leaves of pepper (Figs. 2, 3, 4). However, application of NO (150 µM SNP) at 100 mM of NaCl increased the content of Ca, K, Mg, P, Zn and Fe in plant as compared to the plants at 150 mM NaCl without NO. The application of NO (150 µM SNP) also increased the B and K content in the leaves of pepper treated with 150 mM of NaCl and without NO. As expected, the pepper seedlings showed relatively higher Na and Cl content in the salt treated plants as compared to the control. However, lower Na and Cl content in pepper plants were obtained from NO applications.

Fig. 2.

Fig. 2

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Calcium (Ca) (a), phosphorous (P) (b) and magnesium (Mg) (c) concentration in leaves of pepper seedlings in response to NO (SNP) application under salt stress. Different letters on top bars indicate differences (Duncan Multiple Range Test, p < 0.01 at each treatment). Vertical bars indicate the mean ± standard error

Fig. 3.

Fig. 3

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Boron (B) (a), iron (Fe) (b) and zinc (Zn) (c) concentration in leaves of pepper seedlings in response to NO (SNP) application under salt stress. Different letters on top bars indicate differences (Duncan Multiple Range Test, p < 0.01 at each treatment). Vertical bars indicate the mean ± standard error

Fig. 4.

Fig. 4

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Potassium (K) (a), chloride (Cl) (b) and sodium (Na) (c) concentration in leaves of pepper seedlings in response to NO (SNP) application under salt stress. Different letters on top bars indicate differences (Duncan Multiple Range Test, p < 0.01 at each treatment). Vertical bars indicate the mean ± standard error

Photosynthetic parameters

Under severe salt stress (150 mM NaCl), Pn, gs, Ci and Tr of the pepper plants decreased by 65, 43, 29 and 30% respectively as compared to the control treatment. However, the application of NO (100 µM SNP) at 150 mM NaCl caused an increase in Pn, gs, Ci and Tr by 28, 55, 16 and 16%, respectively, as compared to the plants at 150 mM NaCl without NO application (Table 2). The application of NO (without NaCl) significantly decreased Ci and Tr but no significant effect has been observed in Pn and gs as compared to the control treatment.

Table 2.

The effect of salinity and NO treatments on the photosynthetic rate (Pn), stomatal conductance (gs), intercellular CO2 concentration (Ci) and transpiration rate (Tr) in pepper

Treatments Tr (µmol m−2s−1) Ci (µmol mol−1) gs (mol m−2s−1) Pn (mmol m−2s−1)
NaCl (mM) NO (µM SNP)
0 0 3.73 a 206.00 bc 0.07 a 7.23 a
50 2.53 h 192.33 d 0.03 c 6.14 cd
100 2.47 h 203.33 c 0.07 a 6.66 b
150 3.16 de 195.00 d 0.07 a 7.44 a
50 0 3.11ef 172.00 ef 0.05 b 5.10 f
50 3.42 bc 212.33 b 0.08 a 5.80 de
100 3.37 bc 223.00 a 0.05 b 4.03 gh
150 2.96 fg 213.33 b 0.07 a 6.44 bc
100 0 2.92 g 162.00 g 0.04 bc 3.78 hi
50 3.31 cd 199.67 cd 0.08 a 5.59 e
100 3.52 b 220.67 a 0.08 a 4.91 f
150 2.97 fg 207.00 bc 0.05 b 4.87 f
150 0 2.62 h 146.33 h 0.04 bc 2.53 j
50 3.36 bc 165.33 fg 0.08 a 2.81 j
100 3.13 ef 174.33 e 0.09 a 3.54 i
150 2.82 g 168.67 efg 0.05 b 4.40 g
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Data followed by a different letter in column were significantly different (p ≤ 0.01) according to the DMRT

Sucrose and proline

The effects of salinity on proline and sucrose content are shown in Table 3. The sucrose and proline content elevated markedly in saline conditions. In this regard, 150 mM NaCl had the highest positive effect on the proline and sucrose content. NO applications increased proline and sucrose content in fresh water treated plants. In this respect, NO (150 µM SNP) had the highest effect on their content (Table 3). The highest accumulation of sucrose and proline was recorded in case of the seedlings that were subjected to 150 mM NaCl and NO (150 µM of SNP).

Table 3.

The effect of salinity and NO treatments on the H2O2, MDA, proline and sucrose content in pepper

Treatments Sucrose (%) Proline (mmol kg−1) MDA (mmol kg−1) H2O2 (mmol kg−1)
NaCl (mM) NO (µM SNP)
0 0 1.02 j 0.076 h 5.91 j 13.96 j
50 1.05 i 0.081 ef 7.07 i 15.99 hi
100 1.07 ghi 0.080 efg 8.42 g 16.96 gh
150 1.11 f 0.091 a 9.62 c 19.90 cd
50 0 1.06 hi 0.078 gh 9.55 d 19.77 cde
50 1.09 f 0.087 b 7.39 hi 17.02 gh
100 1.12 cd 0.080 efg 7.71 h 19.04 c–f
150 1.12 cd 0.077 gh 9.14 e 18.53 f
100 0 1.07 ghi 0.080 efg 10.53 b 22.11 b
50 1.11 de 0.086 b 9.22 de 15.53 i
100 1.14 ab 0.082 cd 8.67 fg 19.15 c–f
150 1.15 a 0.084 cd 10.21 b 18.68 ef
150 0 1.09 f 0.085 bc 11.8 a 26.13 a
50 1.08 fg 0.077 gh 9.13 e 20.10 c
100 1.13 bc 0.082 def 8.92 ef 17.11 g
150 1.15 a 0.092 a 9.08 e 18.80 def
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Data followed by a different letter in column were significantly different (p ≤ 0.01) according to the DMRT

MDA and H2O2

The pepper seedlings under salt stress showed a remarkable increase in H2O2 and MDA content, but together with the application of NO, the H2O2 and MDA content were decreased (Table 3). In this regard, the 150 mM of NaCl drastically increased the H2O2 and MDA content by 89 and 100%, respectively, as compared to the control plants. However, the combined treatments of NaCl (150 mM) and NO (150 µM SNP) decreased the H2O2 and MDA by 39 and 30%, respectively, as compared to the plants treated with 150 mM of NaCl, without NO. In the absence of NaCl, the H2O2 and MDA content decreased with increasing the levels of NO. In this aspect, the application of NO (150 µM SNP) had the highest negative impact on H2O2 and MDA content as compared to the untreated ones.

Antioxidant enzyme activity

Pepper seedlings response to NaCl and NO application usually occurred with a modification in the activities of antioxidant enzymes. We evaluated the activity of SOD, POD and CAT in leaves of pepper. The 150 µM NaCl caused a decrease in the SOD and POD activities. However, the combined application of NaCl (150 µM) and NO (100 µM SNP) increased further their activities as compared to the plants treated with 150 mM of NaCl. The NaCl had no significant impact on CAT activity, but the combined treatment of NaCl and NO had a significant impact on the CAT activity as compared to the control (Fig. 5). For instance, 100 mM NaCl treatment had no effect on the CAT activity but simultaneous application of NaCl (100 mM) and NO (50 µM SNP) increased the CAT activity (Fig. 5).

Fig. 5.

Fig. 5

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POD (a), CAT (b) and SOD (c) activities in leaves of pepper seedlings in response to NO (SNP) application under salt stress. Different letters on top bars indicate differences (Duncan Multiple Range Test, p < 0.01 at each treatment). Vertical bars indicate the mean ± standard error

Discussion

The present study revealed negative impact of salinity on pepper growth. The root and shoot dry weight, shoot length and leaf area were decreased by salinity stress (Table 1). Decrease in those parameters due to salinity stress has also been reported formerly for a number of crops such as wheat, lettuce, strawberry and pepper (Yildirim and Güvenç 2006; Yildirim et al. 2009; Shams et al. 2016; Ahanger and Agarwal 2017; Ahmad et al. 2018b). The reduction in the investigated parameters can be related to the toxic and osmotic effect of Na+ since deleterious salt impacts can arise firstly from lessened osmotic potential and toxic ion accumulation. Secondary, inordinate ion accumulation might lead to oxidative stress, specified by the over production of ROS which can end in constant metabolic dysfunction, causing a lower growth rate and plant death (Chaves et al. 2009). In addition, it is well known that salinity can cause the cell wall thicker and harder and hence diminishes cell growth and leaf expansion by reducing its elasticity (Munns et al. 1988). However, we found that NO application reduced the deleterious impact of salt stress and improved the shoot length, root dry weight, shoot dry weight and leaf area. NO increased the growth of rice, maize and Calendula officinalis under salt stress (Bai et al. 2011; Hayat et al. 2011; Jabbarzadeh et al. 2017). In this study, treatment of NO might have a beneficial impact on growth in pepper against salinity stress by regulating mineral nutrient intake, antioxidant enzyme activity, osmolite accumulation, LRWC and photosynthetic activity.

As expected, increased salinity level from 0 to 150 mM of NaCl, raised the uptake of Na and Cl, but decreased the Zn, Fe, B, K, Ca and Mg content (Figs. 2, 3, 4). This nutritional disorders of plants were attributed to one or more mechanisms, including osmotic effects of salinity and competitive interactions among ions, thus producing extreme ratios of Na/K, Na/Ca, and Na/Mg (Grattan and Grieve 1999; Tester and Davenport 2003).

It is also well documented that the tolerance to salinity has been related significantly with maintenance of the ratio of K/Na in cellular segments (De Souza Miranda et al. 2016). In this study, the favorable impact of NO treatment should be noticed, which caused the low accumulation of Na in pepper with a significant change in, K, Ca, Mg, P, Zn and Fe content under salt stress (Figs. 2, 3, 4). The decrease in the Na/K ratio in NO-treated plants under salinity can be due to inhibition in vacuolar Na compartmentalization or due to Na influx through the plasma membrane of root, thus partaking to increase salinity tolerance (Guo et al. 2009). Moreover, exogenously treatment of NO also play a key role as a peripheral signal in induction expression of H+-PPase and H+-ATPase, producing a powerful electrochemical gradient for enhancing the activity of Na/H exchange and sec transporters (Zhang et al. 2006).

Salinity stress significantly decreased Zn, Fe, B, K, Ca and Mg uptake while exogenous NO application improved their uptake. It is worth mentioning that increasing B content in corn ameliorated the plant’s tolerance to salinity stress by adjusting the water transport and water uptake via the functions of certain aquaporin isoforms (Del Carmen Martínez-Ballesta et al. 2008). Ca is an important component of membrane and cell wall, and it also acts as a signal that leads to stimulation of antioxidant enzymes (Agarwal et al. 2005). K channel/transporters and water channels are functionally co-regulated as a part of plant osmo-regulation to keep appropriate cytosolic osmolarity and acclimatize the plant to drought or other stresses (Liu et al. 2006). Therefore, it can be concluded that exogenous NO treatment increased the ratio of K/Na, Ca/Na, and Mg/Na by enhancing the absorption of K, Ca and Mg and declining the absorption of Na, and then it led to mitigate the deleterious effect of salinity.

In the present study, we found that NaCl negatively affected the chlorophyll content (as SPAD), and it could be attributed to the degradation of chlorophyll pigments, minimized the vulnerability of the pigment-protein complexes and chlorophyll syntheses by over accumulation of Na+ and Cl− in cells of pepper plants (Ahmad et al. 2016). However, NO application increased the SPAD values under salt stress (Fig. 1). In agreement with this, ameliorated chlorophyll fluorescence with foliar-applied NO under salt stress was reported earlier for rice seedlings by Habib et al. (2013) and for tomato by Ahmad et al. (2018b). It is well known that Fe and Mg play a role in chlorophyll structure and have a role in its synthesis (Marschner 2012). Therefore, it can be mentioned that exogenous NO treatment by increasing the Fe and Mg uptake elevated the SPAD values of pepper under salt stress and finally, it improved plant growth and increased pepper plant tolerance to salinity.

Soluble sugars perform a significant function in plant metabolism and act as an osmotic protector through the stabilization of the cell membrane and maintaining turgor pressure (Couée et al. 2006). In this study, NO application markedly increased soluble sugars in pepper seedlings exposed to NaCl (Table 3) and this results is in accordance with that of Wu et al. (2011) who found that NO treatment increased soluble sugars in tomato seedlings under salt stress.

In the present study, salt stress operated the accumulation of proline, and NO treatment markedly increased its accumulation in the presence of salinity. Our results are in agreement with those of Wu et al. (2011) who found that NO application increased the proline content in tomato under salt stress. This increase can be due to the decrease in proline oxidase activity that leads to proline degradation in plants (Misra and Saxena 2009) and might be outcome from alteration in the activities of proline synthesizing and degradation enzymes (Ahmad et al. 2010). In addition, the highest accumulation of proline in pepper seedlings subjected to 150 mM NaCl and NO (150 µM of SNP) can also be related to the regulation role of NO in proline metabolism.

SOD, CAT and POD are known as ROS eliminator among the antioxidant enzymes in plants. The ROS are scavenged by these enzymes until their concentration enhanced in threshold level under stress (Ahanger and Agarwal 2017). In the present study, the activity of POD and SOD was decreased by salinity stress. However, with NO application, their activities were increased (Fig. 5). These results are in agreement with those of Ahmad et al. (2018b) who found that NO treatment increased enzyme activity in tomato under salt stress. Therefore, it can be concluded that NO treatment increased the enzyme activity under saline condition. The increase could be due to role of NO in enhancing the absorption of K and increasing the K/Na ratio (Fig. 4), and this is in line with the results of Ahanger and Agarwal (2017) who reported the positive role of K on increasing the enzyme activity in wheat under salinity. NO also may act as a messenger molecule, and it activates the expression of antioxidant enzymes-related biosynthetic genes, thereby providing salt tolerance (Ahmad et al. 2016; Gadelha et al. 2017).

Reactive oxygen species such as H2O2 and O−2 are usually produced in large quantities by plants to various response of stress. The production of ROS under salinity has also a great role in inhibition of photosynthesis (Idrees et al. 2012). In this study, NaCl exposure caused over accumulation of MDA and H2O2 in leaves of pepper but NO application markedly decreased the accumulation of H2O2 and MDA in pepper seedlings. This result is in line with those of Lin et al. (2012) who reported that H2O2 and thiobarbituric acid-reactive substances (TBARS) accumulation increased in hypocotyls and root of cucumber with 100 mM of NaCl but decreased with 100 µM of SNP treatment (Lin et al. 2012). In addition, our findings corroborate with those of Ahmad et al. (2018b) who reported the role of NO treatment on decreasing the MDA and H2O2 content in tomato under salt stress. In this study, a decrease in MDA in NO-treated pepper seedlings can be attributed to the up-regulation of the antioxidant system that quickly dispels ROS including H2O2. In a system where cellular damage is predominantly from ROS, NO can act as a chain breaker and hence limits damages (Lipton et al. 1993). In addition, NO plays an important role as cellular preservatives by altering the level of ROS, and inducing expression of genes that control various metabolic processes (Siddiqui et al. 2011).

Photosynthetic performance of plants due to its sensitivity to abiotic stress is an informative indicator (Maxwell and Johnson 2000). Under stress, the plants can make modifications in the photosynthetic electron transport that can create O2−, because the molecular oxygen competes with nicotinamide adenine dinucleotide phosphate for reduction in the photosystem I (PSI) accepter side (Molinari et al. 2007). These regulations cause modifications in thylakoid membranes oxidative status and stomatal closure, as a result of a decrease in photosynthetic rate (Guerfel et al. 2009). In present study, we found that salinity decreased the Pn, gs, Tr and Ci (Table 2), but the application of NO increased photosynthetic parameters investigated under salinity conditions. Our results are supported by those of Fatma et al. (2016) who reported that exogenous NO treatment increased Pn, Ci and gs values in mustard plants under salt stress. In addition, the beneficial effect of NO on the photosynthetic parameters has also been reported in tomato under salt stress (Wu et al. 2011). This can be explained by enhanced absorption of Fe, Mg and K in the seedling treated by NO (Figs. 2, 3) and enzyme activity under salinity. Increasing the absorption of nutrient elements and reinstating of stomatal aperture by NO treatment under salinity cause to reinstate photosynthesis, chlorophyll fluorescence, gas exchange and tolerance to salt stress in pepper seedlings.

Conclusion

The findings of this study highlighted that exogenous NO application was effective in alleviating the deleterious impact of salinity in pepper seedlings. NO increased enzyme activity and osmolyte accumulation and maintained the LRWC, chlorophyll and photosynthetic capacity in salt-stressed pepper seedlings. In addition, exogenously NO treatment diminished oxidative damage by upregulating antioxidant enzymes and osmolytes, thereby ameliorating a significant decrease in ROS-induced lipid peroxidation and electrolyte leakage. NO also boosts Ca, B, Zn, Fe, P, Mg and K absorption and decreased Na and Cl accumulation in pepper seedlings exposed to salinity. Hence, manipulation of endogenous NO content in plants or the exogenous NO treatment can be an effective procedure for management of salinity problems with low quality irrigation waters.

Acknowledgements

We appreciate Atatürk University, Scientific Research Projects Foundation for generous financial support (Project Number 2015/161).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

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