Impaginato 409 Adv. Hort. Sci., 2019 33(3): 409-416 DOI: 10.13128/ahs-24335 Alleviation of salinity stress by hydrogen peroxide and nitric oxide in tomato plants B. Hajivar, M.R. Zare-Bavani (*) Department of Horticultural Science, College of Agriculture, Agricultural Sciences and Natural Resources University of Khuzestan, Mollasani, Khuzestan, Iran. Key words: membrane stability, photosynthetic attributes, relative water con- tent, salt tolerance. Abstract: Salinity is one of the major abiotic stress factors limiting plant growth and productivity, particularly in arid and semi-arid climates. Hydrogen Peroxide (H2O2) and Nitric Oxide (NO) are important signaling molecules in plant response to abiotic stress. In this research the effects of foliar sprays with H2O2 (10 mM) and NO (0.1 mM sodium nitroprusside, as a NO donor) on alleviation of Salinity stress (0, 25, 50 and 100 mM NaCl) were investigated in Tomato (Solanum lycopersicum L. cv. Falat). Photosynthetic attributes, plant-water rela- tions, membrane stability index and growth parameters were decreased by NaCl treatments. Exogenous H2O2 and NO application enhanced salt stress tol- erance in tomato plants by improving the photosynthetic efficiency and plant water status as measured by relative water content and membrane stability index. These results were positively reflected by the increase in plant growth under salinity stress conditions. The results of this study described that under the adverse conditions of salinity stress, H2O2 and NO could activate the photo- synthetic system and improve the physiological attributes in plant growth. 1. Introduction Salinity in soil or water is a major problem affecting growth and pro- ductivity of many crops, especially under arid and semi-arid conditions. It was estimated that about 20% of the world’s cultivated land area and 50% of all irrigated land are salt-affected (Hayat et al., 2013). But, the area of soils with restrictions for vegetable crop production is certainly greater than the area of salinized soils, since a saline soil is generally defined as showing an electrical conductivity (EC) value of the saturation extract (ECe) in the root zone that exceeding 4 dS m-1, while the majority of vegetable crops have a salinity threshold that is 2.5 dS m-1 (Machado and Serralheiro, 2017). Salinity negatively affects plants growth and development through: low osmotic potential of soil solution (water stress), nutritional imbal- ance, specific ion effect (salt stress) or a combination of these factors (Ashraf, 2004). All of these factors cause morphological, physiological and metabolic modifications in plants, such as a decrease in seed germination, shoot and root length, leaf area, cell membranes stability, inhibition of (*) Corresponding author: mzarebavany@gmail.com Citation: HAJIVAR B., ZARE-BAVANI M.R., 2019 - Alleviation of salinity stress by hydrogen peroxide and nitric oxide in tomato plants. - Adv. Hort. Sci., 33(3): 409-416 Copyright: © 2019 Hajivar B., Zare-Bavani M.R. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 18 December 2018 Accepted for publication 15 May 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(3): 413-416 410 different enzymatic activities and photosynthesis attributes (Sairam and Tyagi, 2004; Parida and Das, 2005). Photosynthesis is one of the physiological processes that is affected by salinity stress (Munns et al., 2006; Chaves et al., 2009). Salinity stress may reduce the photosynthesis rate by decreasing in stomatal factors such as stomatal conductance (Bethke and Drew, 1992; Parida et al., 2004), internal CO2 partial pressure (Bethke and Drew, 1992; Iyenger and Reddy, 1996) and non-stomatal factors such as inhibition and degradation of photosynthetic pig- ments (Lee et al., 2004; Chaves et al., 2009), photo- synthetic electron transport reactions, quenching ability of excessive energy through chlorophyll fluo- rescence (Lee et al., 2004), efficiency of Rubisco for carbon fixation (Liu et al., 2011), and photophospho- rylation (Stoeva and Kaymakanova, 2008). Adverse effects of salinity on plant growth may also result from impairment of photosynthetic apparatus (Ashraf, 2004). Hydrogen peroxide and Nitric oxide are bioactive molecule involved in the signaling process within plants (Leshem, 2000; Uchida et al., 2002; Azevedo- Neto et al., 2005; Hung et al., 2005; Li et al., 2011). Researches have shown that hydrogen peroxide and nitroxide at low concentrations, play an important role as signaling molecules (Gechev and Hille, 2005; Quan et al., 2008). Studies have shown that hydrogen peroxide and Nitric oxide are involved in acclamatory signaling triggering tolerance against salt stress (Hayat et al., 2013; Semida, 2016). Azevedo-Neto et al. (2005) reported that the pretreatment with H2O2 in nutrient solution induces acclimation to salinity stress in maize. Semida (2016) observed that exoge- nous H2O2 application enhanced salt stress tolerance in onion plants by improving the photosynthetic effi- ciency and plant water status as evaluated by relative water content and membrane stability index. The use of NO increased the resistance of Pinus eldarica to salinity and improved its growth characteristics (Zamani et al., 2014). Uchida et al. (2002) reported that H2O2 and NO are the important signaling mole- cules in rice for resistance to salinity stress. Tomato is one of the most important vegetable crop in the world. In Iran, the tomato also holds the number one position among vegetables, with almost 6.4 million metric tons of production (FAO, 2014). The cultivated tomato has been classified as moder- ately sensitive to salinity. Salinity affects tomato plant growth at various stages including seed germi- nation, root and shoot development and fruit pro- duction (Cuartero and Fernandez-Munoz, 1999). This research was undertaken to assess changes in plant growth, water relations, cell membrane stabili- ty and photosynthesis parameters in salt-treated tomato plants and to examine neutralizing effects of NO and H2O2 to exposure to salt. 2. Materials and Methods Plant material Tomato seeds, cv. Falat were surface-sterilized in 2.5% sodium hypochlorite for 10 min, followed by four washes with distilled water. Seeds were sown in the plastic tray filled with a silica sand in the green- house under controlled conditions (photoperiod of 16/8 h day/night, 60-65% humidity and 25-30°C tem- perature). Seeds were irrigated with tap water daily. Seedlings with 2 true leaves were transplanted to 25×25 cm pots (one plant per pot) maintained under similar conditions as the tray containing developing seedlings and fertilized alternate days with half- strength Hoagland solution (Hoagland and Arnon, 1950) until solution drainage occurred at the bottom of the pot at each fertigation. Treatment and experimental design Seven days after transplanting, uniform seedlings of tomato cultivars were sprayed to run off with dis- tilled water, 10 mM H2O2 or 0.1 mM SNP in 0.025% Tween 20 (as a surfactant) at 6:30 am and then the sprays were repeated at 7 and 14 days later. The con- centrations of H2O2 and SNP and the number and timing of sprays were based on results from a prelim- inary experiment (data not shown). After the last spraying, irrigation was done with half strength Hoagland solution supplemented with 0, 25, 50 and 75 mM of NaCl solution. The experimental proce- dures were completely randomized in 3 × 5 factorial design, with three foliar spray (sodium nitroprusside [SNP], H2O2 and distilled water) and four salt concen- trations (0, 25, 50, and 100 mM NaCl in nutrient solu- tion), performed in triplicate. The number of plants were six in each replicate. Plants were sampled at 90 day after seeding. Three samples were analyzed for each replication (9 samples in each treatment). The fully-expanded leaves were used for the determina- tion of all experimental parameters. Determination of plant growth traits Ninety-day-old tomato plants were carefully removed from each pot and the leaves, stems and roots of plants were weighed to record their fresh Hajivar and Zare‐Bavani ‐ Alleviation of salinity stress in tomato plants 411 weights and then placed in an oven at 70°C till the constant weight to record their dry weights. Determination of relative water content (RWC) RWC was estimated using 2-cm-diameter fully- expanded leaf discs, excluding midrib according to the method of Hayat et al. (2013). The discs were weighted for fresh mass (FM) and immediately float- ed on double-distilled water in Petri dishes for 24 h, in the dark, to saturate them with water. Water adhering to discs was blotted and the turgid mass (TM) was measured. The dry mass (DM) of discs was recorded after dehydrating them at 70°C until the constant weight. The RWC was then calculated using the formula: RWC = [(FM − DM)/(TM − DM)] × 100. Determination of proline content Free proline content was determined according to the method of Bates et al. (1973). Samples (0.5 g) were homogenized in 5 ml 3% sulfosalicylic acid and extracts were centrifuged at 8000 x g for 15 min. The amount of 1 ml filtrate was mixed with equal vol- umes of acetic acid and ninhydrin reagent (1.25 g ninhydrin, 30 ml of glacial acetic acid, 20 ml 6 M H3PO4) and incubated for 1 h at 100°C. The reaction was stopped by placing the test tubes in ice cold water. The samples were vigorously mixed with 3 ml toluene. After 50 min, the light absorption of the toluene phase was estimated at 520 nm on a UV-VIS spectrophotometer. The proline concentration was determined using a standard curve. Free L- proline content was expressed as μg/g dry weight. Determination of total soluble sugar content Total soluble sugar content was determined by phenol-sulfuric acid according to the method of Dubois et al. (1956). Dry leaves sample (0.1 g) were extracted with 5 ml of 80% ethanol, by boiling the samples in glass tubes in a 95°C-water bath for 10 min. After extraction, the tubes were centrifuged at 500 x g for 5 min, and the supernatants of the extrac- tions were used for sugar analysis. One hundred ml of sample was added to 900 ml of distilled water then the mixture was vortexed. One ml of 5% phenol and 5 ml of H2SO4 were added to 1 ml of sample and the mixture was stirred. After cooling under room temperature for 15 min, the absorbance of the sam- ple was recorded at 490 nm. Determination of membrane stability index (MSI) The MSI was determined according to methods of Sairam and Srivastava (2002). Leaf disc (0.2 g) were thoroughly washed in double distilled water and thereafter placed in a test tube containing 10 ml of double distilled water in two sets. One set was heat- ed at 40°C in a water bath for 30 min and the electri- cal conductivity (EC1) of the solution was recorded using an electrical conductivity meter. Another set was boiled at 100°C for 10 min and their electrical conductivity was recorded as above (EC2). The MSI was calculated as: MSI= [1-(EC1/EC2)] ×100 Determination of leaf photosynthetic pigments Chlorophyll a, b and total chlorophyll were extracted and determined (in mg/ g FW) following the procedure is given by Lichtenthaler and Buschmann (2001). Fresh leaf samples (0.2 g) were homogenized in 50 ml acetone (80%) and then cen- trifuged at 10,000 × g for 10 min. The absorbance of the acetone extract was measured at 663, 645 and 470 nm using a UV-visible spectrometer (Shimadzu, Kyoto, Japan). Determination of leaf photosynthetic attributes Photosynthetic attributes (stomatal conductance [gs], internal CO2 concentration [Ci], transpiration rate [E], and net photosynthetic rate [Pn]) in intact leaves were measured by a infrared gas analyzer (CI- 340, Photosynthesis system, CID Bio-Science, USA) between 10:00 and 12:00 h under a clear sky. Photosynthetic Pigments and Attributes were mea- sured on three samples of leaves in each pot and three pot in each replication. Statistical analysis The experimental design was a completely ran- domized factorial, four salinity levels (0, 25, 50 and 100 mM NaCl) and two levels of H2O2 and SNP (10 and 0.1 mM respectively). All measurements were carried out in three replicates and data were subject- ed to one-way analysis of variance using SAS program (SAS 9.1; SAS Institute Inc., Cary, NC). Significant dif- ferences between means were determined by Tukey’s tests. P values less than 0.05 were consid- ered statistically significant. 3. Results Growth parameters Salinity markedly decreased fresh weight and dry weight of root, leaf and shoot (Fig. 1, A-H). However, the H2O2 and SNP spraying were able to reduce the Adv. Hort. Sci., 2019 33(3): 413-416 412 adverse effects of salt stress. Moreover, the fresh weight and dry weight of root, leaf and shoot from H2O2 and SNP-sprayed plants were higher than the ones stressed plants (Fig. 1). Relative water content (RWC) When salinity was absent, RWC was not signifi- cantly altered by H2O2 and SNP pretreated plants (Fig. 2, I). Under salinity condition, plants sprayed with SNP or H2O2 displayed higher RWC when compared to water sprayed ones. Plant pretreated with H2O2 was not significantly affected by salinity of 25 mM NaCl. Salinity did not promote any significant alter- ation in SNP pretreated plants in 25 and 50 mM NaCl stress. Under 100 mM NaCl stress conditions, the RWC was reduced in all evaluations (Fig. 2, I). Proline content The proline-specific increase in plants exposed to NaCl (Fig. 2, J). Pretreatment to either H2O2 or SNP resulted in an increase in proline levels of plants under salinity stress. Interestingly, among unstressed plants, treatment of H2O2 and SNP also increased the proline levels (Fig. 2, J). Total soluble sugar content (TSSC) Salinity stress significantly increased the TSSC (Fig. 2, K). Pretreatment of H2O2 or SNP significantly decreased the TSSC compared to water sprayed plants. The highest amount of TSSC was observed with 100 mM NaCl without H2O2 or SNP, while the lowest amount of TSSC was observed with SNP and H2O2 application without salinity (Fig. 2, K). Membrane stability index (MSI) Under non saline conditions, the MSI were not affected by H2O2 and SNP spraying (Fig. 2, L). Although salinity had decreased the leaf MSI, it did not promote any significant alteration in H2O2 and SNP sprayed plants in 25 mM NaCl stress compared to non saline conditions. The MSI was significantly decreased by 50 and 100 mM NaCl stress, however, plants treated with H2O2 and SNP were less affected by salinity stress compared to water sprayed plants. The SNP-sprayed plants showed values of MSI higher than the stressed plants sprayed with water and H2O2 (Fig. 2, L). Leaf photosynthetic pigments There were significant decreases in the chloro- phyll a, b and total chlorophyll contents in salt- stressed plants. Plants treated with H2O2 and SNP Fig. 1 - Effect of salt treatment and application of exogenous H2O2 and SNP on growth parameter of tomato plants. Leaf fresh weight (A), stem fresh weight (B), root fresh weight (C), leaf dry weight (D), stem dry weight (E), root dry-weight (F), total fresh weight (G), and total dry wei- ght (H). Data shown are the mean (±SE) of three inde- pendent experiments. Significant differences among treatments were determined by Tukey's Test (P<0.05). Fig. 2 - Effect of salt treatment and application of exogenous H2O2 and SNP on relative water content (I), proline (J), total soluble sugar (K) and membrane stability index (L). Three plants were analyzed for each treatment. Data shown are the mean (±SE) of three independent experi- ments. Significant differences among treatments were determined by Tukey's Test (P<0.05). Hajivar and Zare‐Bavani ‐ Alleviation of salinity stress in tomato plants 413 4. Discussion and Conclusions Salt stress is a major abiotic stress that imposes osmotic and toxicity stress to plants and consequent- ly induces a reduction in plant photosynthesis and growth (Acosta-Motos et al., 2017). In this study, our results confirmed that salinity at the tested concen- trations inhibited the growth of tomato plants (Fig. 1, A-H). H2O2 and NO are bioactive molecules that are known as important signals not only in plant disease resistance, but also in the process of growth, devel- opment, and responses against abiotic stress (Mazid et al., 2011; Niu and Liao, 2016). Salinity stress is known detrimental effect on the overall growth and productivity of plants (Ashraf, 2004) and may inhibit plant growth due to reduction of water uptake by plants (Kaya et al., 2003). Several studies have shown the beneficial effects of H2O2 and SNP pretreatment on salt tolerance in plants (Uchida et al., 2002; Azevedo-Neto et al., 2005; Wahid et al., 2007). Potikha et al. (1999) suggested that H2O2 increases cell division and is involved in the differentiation of the cell wall. Our results are in agreement with those previously reported for maze (Azevedo-Neto et al., 2005; Gondim et al., 2013), rice (Sathiyaraj et al., 2014), cotton, cowpea and sorghum (Freitas et al., 2011). Terasaki et al. (2001) noted that SNP possibly enhances exo- and endo-β-D-glucanase activities in cell walls, where the glycosidic linkage between glu- cose units within cell walls is broken by these enzymes (Zhang et al., 2003), and growth enhance by increasing internal turgor pressure and water con- tent. Similarly to our results, growth stimulation by exogenous NO was demonstrated in tomato (Wu et al., 2010; Hayat et al., 2013). In this study H2O2 or NO resulted in higher increase of relative water content, proline and mem- brane stability index in leaf of tomato plant and decrease of total soluble sugar content (Fig. 2, I-L) which could promote plant growth under salt stress (Duan et al., 2007) and non saline conditions (Zhang et al., 2005), indicating that H2O2 and NO are involved in the intrinsic mechanism of growth under different conditions. The higher relative water content in H2O2 and SNP-sprayed stressed plants (Fig. 2, I) appears to be the result of H2O2 and NO-induced increased lev- els of compatible solutes under salt-induced osmotic stress (Tan et al., 2008; Hayat et al., 2013), which resulted in better growth of stressed plants. Proline accumulation is an essential indicator for plant response to salinity stress (Sathiyaraj et al., 2014). The H2O2 and SNP-pretreated plants showed a signifi- spray had higher values compare to the water- sprayed plants (Fig. 3, M-O). The chlorophyll content (a, and total chlorophyll) in the water-sprayed plants and plants receiving H2O2 and SNP were similar in 0 and 25 mM NaCl treatment (Fig. 3, M and O). Leaf photosynthetic attributes The H2O2 and SNP-sprayed plants showed higher Net photosynthesis (Pn), Stomatal conductance (gs) and intercellular CO2 concentration (Ci) than the water-sprayed plants under non saline conditions (Fig. 1A-1C). Although the Pn, E, gs and Ci were strongly decreased by salinity stress, plants sprayed with H2O2 and SNP were less affected than the water- sprayed ones. The Pn in the plants receiving H2O2 and SNP were higher at 25 mM NaCl treatment compared with the water-sprayed ones. Plants receiving 100 mM NaCl had lower Pn, gs, Ci and E than other treat- ment. Fig. 3 - Effect of salt treatment and application of exogenous H2O2 and SNP on photosynthesis pigment and attributes of tomato plants. Chlorophyll a (M), chlorophyll b (N), total chlorophyll (O), Photosynthetic rate (P), stomatal conductance (Q), transpiration (R) and intercellular CO2 concentration (S). Data shown are the mean (±SE) of three independent experiments. Significant differences among treatments were determined by Tukey's Test (P<0.05). 414 Adv. Hort. Sci., 2019 33(3): 413-416 cantly higher amount of proline than the salt- stressed ones (Fig. 2, J). stress-induced proline accu- mulation in plants help in osmotic adjustment (Sathiyaraj et al., 2014). In addition to the role as a compatible osmolyte, proline can also increase mem- brane stability, confer enzyme protection and help in non-enzymatic free radical detoxifications (Khan et al., 2002; Sathiyaraj et al., 2014). Thus, the increase of proline may trigger tolerance to salt stress in tomato plants. The salt stressed plant showed an increase in Total soluble sugar content, but H2O2 and SNP sprayed plants showed a significantly decreased of TSSC compared with the water-sprayed plants (Fig. 2, K). The reduction in TSSC by H2O2 and NO application in this experiment (Fig. 2, K) may be attributed to the crucial role of H2O2 and NO in mitigating the negative effect of salinity stress (Semida, 2016). Similarly, TSSC reduction by exogenous H2O2 was demonstrated in onion (Semida, 2016). H2O2 and SNP sprayed plants showed a significant- ly increased of Membrane Stability Index compared with the water-sprayed ones (Fig. 2, L). The salt stressed plant showed an decreas in MSI, and the decrease in MSI reflects the extent of lipid peroxida- tion caused by active oxygen species. The rate of lipid peroxidation has been widely used as an indicator of oxidative damage (Sathiyaraj et al., 2014). Result showed exogenous H2O2 and SNP treatment are able to prevent lipid peroxidation and thus protect the cells from the damage of salinity stress. H2O2 and NO are known to enhance chlorophyll content in plant (Gondim et al., 2013; Hayat et al., 2013). In this experiment, the chlorophyll content was negatively affected by salinity (Fig. 3, M- O). Singh and Dubey (1995) showed that the loss of chlorophyll content could be related to photoinhibi- tion or oxidative damages that acts as a cellular mark- er of salinity stress. Therefore, the pretreatment with H2O2 and SNP was effective to reduce the detrimental effects of salinity in chlorophyll content (Fig. 1A). Salinity stress is also known to reduce photosyn- thesis, due to an increase in reactive oxygen species formation, water status alteration, and a decrease in chlorophyll content and CO2 diffusion through stom- atal guard cells (Chaves and Oliveira, 2004; Munns and Tester, 2008). Silva et al. (2011) reported that reduction in photosynthesis by stomatal closure occurs during early exposure to salinity stress, while biochemical limitations concern due to long-term NaCl exposure. Thus, the reduction of photosynthesis in plants was caused by reduction in stomatal con- ductance, decreasing the intercellular CO2 concentra- tion for Rubisco activity (Shahbaz et al., 2010). Some studies reported the maintenance of gas exchange correlate with salt tolerance in plants (James et al., 2006; Munns and Tester, 2008). In this experiment, results showed all gas exchange parameters were less affected by salinity in plants previously treated with H2O2 and SNP (Fig. 3, P-S). Therefore, our data indicate that H2O2 and NO-pretreatment increased stomatal conductance, which enabled high net pho- tosynthetic rate and improved growth parameters. In addition, the higher leaf MSI induced by the H2O2 and SNP pretreatment in NaCl stressed plants is an evidence that plants were able to control oxidative damages caused by ROS in the photosynthetic apparatus and maintain leaf gas exchange (Fig. 2, L). Similarly, it is observed that that the H2O2 and SNP-pretreatment caused increases in net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 con- centration in plants subjected to salinity when com- pared to non-treated seedlings (Wahid et al., 2007; Gondim et al., 2013; Hayat et al., 2013). 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