Effect of Nitric Oxide (NO) on the Induction of Callus and Antioxidant

Journal of Applied Botany and Food Quality 91, 24 - 32 (2018), DOI:10.5073/JABFQ.2018.091.004

Department of Horticultural Sciences, Faculty of Agriculture, University of Hormozgan, Iran Effect of nitric oxide (NO) on the induction of callus and antioxidant capacity of Hyoscyamus niger under in vitro salt stress Davood Samsampour*, Fatemeh Sadeghi, Mohammad Asadi, Atiye Ebrahimzadeh (Submitted: May 31, 2017; Accepted: January 2, 2018)

Summary enzymes in plants organs is necessary for improving plant tolerance to salt stress. Therefore, increasing free radical scavenging ability Generation of reactive oxygen species (ROS) under salt stress by exogenous substance application could be a practical measure in can cause oxidative damage. Nitric oxide (NO) is considered as a the detoxification of stress-induced excessive free radical production. functional molecule in alleviating the oxidative damage of salinity Recently, rapid and economically viable approaches have been to plants through modulating antioxidant metabolism. In the present proposed to alleviate the adverse effects of salt stress (ASHRAF and study, effects of sodium nitroprusside (SNP), a NO donor, on FOOLAD, 2007). In a number of studies, it has been emphasized that induction of callus and seed germination, and antioxidant capacity exogenous application of elicitors, antioxidants, or plant growth of Hyoscyamus niger seedlings were studied under 0, 50, 100 and regulators is a meaningful approach in inducing salt tolerance in 150 mM NaCl stress. NO stimulated the germination of NaCl- crops (ASHRAF and FOOLAD, 2007). treated seeds. NaCl treatment significantly induced accumulation Nitric oxide (NO) is a small, highly diffusible gas and a ubiqui- of H O and thiobarbituric acid-reactive substances (TBARS) in 2 2 tous bioactive molecule. Its chemical properties make nitric oxide Hyoscyamus niger, and application of 100 μM SNP stimulated ROS- a versatile signal molecule that functions through interactions with scavenging enzymes and increased scavenging activity (DPPH), cellular targets via either redox or additive chemistry (LAMATTINA hydroxyl radical (OH•) scavenging activity and chelating activity of et al., 2003). NO produced by sodium nitroprusside (SNP) has re- ferrous ions, resulting in lower lipid peroxidation induced by NaCl cently been considered a new phytohormones (LETERRIER et al., stress. Therefore, it can be concluded that the increased antioxidant 2012) and reported to mediate the plants responses to both biotic and capacity by NO might be responsible for its function in alleviating abiotic stresses (CRAWFORD and GUO, 2005; DELLEDONNE, 2005); it the inhibition of Hyoscyamus niger growth and cell damage by also plays a crucial role in regulating plant growth and development, salt stress. Also the effect of SNP on the induction of callus of including germination, flowering, fruit ripening and organ senes- Hyoscyamus niger was investigated. Callus fresh weight increased cence (ARASIMOWICZ and FLORYSZAK-WIECZOREK, 2007). significantly in the combined effect of 50 μM SNP with other plant Moreover, NO is believed to be involved in two respiratory electron growth regulators (PGR) compared to control. It is evident that SNP transport pathways in mitochondria (YAMASAKI et al., 2001; ZOTTINI has a direct effect on the induction of callus by interacting with et al., 2002), where it mediates the modulation of ROS and enhances cytokinins and auxins. antioxidant defense system in plants subjected to various abiotic stresses, such as heat (UCHIDA et al., 2002), iron deficiency (SUN Key words: antioxidant capacity, callus, nitric oxide, Hyoscyamus et al., 2007), and salt and heavy metals (Singh et al., 2008). In the niger, salinity. past few years, research on function of NO in salt stress tolerance has obtained much interest (YANG et al., 2011; MOSTOFA et al., 2015). Introduction However, the information available is sometimes contradictory, de- pending on the plant species, severity and duration of the salinity It is a well-known fact that excessive accumulation of salt ions in the treatments (BEGARA-MORALES et al., 2014; MANAI et al., 2014). soil is increasingly becoming a problem for agriculture production In vitro culture of plant in the presence of high concentrations of salt (ZHU, 2003; BAATOUR et al., 2010). The effects of salt stress on provides a useful tool to study the adaptive mechanisms of plants plant physiology have been well documented. Salt stress can cause living in adverse environments. Mechanisms preventing oxidative ion toxicity, osmotic stress and reactive oxygen species (ROS) stress burst in cells exposed to high salt concentrations are crucial for cell (MITTLER, 2002). In plant cells, mitochondria are major sites for the survival. Although the antioxidant system is undoubtedly involved in generation of reactive oxygen species (ROS) in nonphotosynthetic the strategies used by the tissues to survive under high salt concentra- cells (MOLLER, 2001; FLEURY et al., 2002). As the results of oxidative tions, the variation in the degree of responses has demonstrated that stress, protein synthesis is inhibited, lipid is peroxided, enzyme is multiple mechanisms rather than a single mechanism which may be inactivated, and membrane systems are damaged (ZHU et al., 2004; responsible for the adaptation of the tissues to resist salt stress. TANOU et al., 2009). Therefore, the balance between free radical Black henbane, Hyoscyamus niger L., known as a medicinal herb generation and scavenging determines the survival of plants under belongs to the family Solanaceae and is widely distributed in Europe salt stress. To counteract the oxidative stress, plants have evolved an and Asia (SAWANT, 2004). The plant contains tropane alkaloids antioxidant system which is composed of ROS-scavenging enzymes (hyoscyamine, hyoscine and scopolamine) as well as flavonol gly- such as catalase (CAT), superoxide dismutase (SOD), guaiacol cosides (quercitin, rutin, kaempferol), which are amongst the oldest peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate drugs used in medicine (EL JABER-VAZDEKIS, 2009). Tropane alka- reductase (DHAR) and glutathione reductase (GR) (JUNG et al., loids, are widely used in medicine for their mydriatic, anticholiner- 2000). Many studies indicated that plant tolerance to salt stress was gic, antispasmodic, analgesic and sedative properties. The seeds are closely related with the expression and activities of these antioxidant used in traditional therapy for the treatment of a variety of health enzymes (ZUSHI and MATSUZOE, 2009; SECKIN et al., 2010) as problems such as asthma, cough, colic, diarrhea and genitourinary well as higher antioxidant capacity due to antioxidant metabolites complaints such as irritable bladder (GILANI, 2008). In traditional (ZUSHI and MATSUZOE, 2009). Enhancing the activity of antioxidant Chinese medicine the seeds of this plant are used as an antispas- * Corresponding author modic, sedative, and analgesic agent, as well as for the treatment Determination of antioxidant enzyme activities

Three hundred mg fresh tissue were ground with 3 ml ice-cold 50 mM phosphate buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM ascorbate and 2% PVP. The homogenates were centrifuged at 4°C for 20 min at 12,000×g and the resulting supernatants were used for determination of antioxidant enzyme activities.

SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium following the method of STEWART and BEWLEY (1980). CAT activity was measured as the decline in absorbance at 240 nm due to the decrease of extinction of

hydrogen peroxide (H2O2) according to the method of PATRA et al. (1978). POD activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation (NICKEl and CUNNINGHAM, 1969). GR activity was measured according to Foyer and HALLIWEL (1976), which depends on the rate of decrease in the absorbance of NADPH at 340 nm.

Determination of antioxidant activity

Effect of nitric oxide on the induction of callus of HyoscyamusFor determination niger of antioxidant activities, 0.3 g25 sample was suspended in 3 ml of serine borate buffer (100 mM Tris–HCl, 10 mM borate, 5 mM serine, and 1 mM diethylenetriaminepentacetic of stomach cramps, heavy coughs, neuralgia, and manic psychosis borate, 5 mM serine,acid, and pH 1 7.0). mM Thediethylenetriaminepentacetic slurry was centrifuged at acid,5,000 × g for 10 min at 4°C and the supernatants (MACY, 2002). Hyoscyamus species is one of the four plants used in pH 7.0). The slurry was centrifuged at 5,000×g for 10 min at 4 °C Ayurveda for the treatment of Parkinson’s disease. and the supernatantswere were used used for for the the in invitro vitro antioxidant antioxidant assays. assays. All samples were placed on ice during the In previous research, sodium nitroprusside (SNP) as a NO donor All samples were placedexperiments. on ice during the experiments. was used to counteract the effect of salt stress. However not any data are available on the effect of NO as a precursor in antioxidative re- Radical scavenging power (RSP) of tissue extracts were assessed by the method of MANDA et sponses of Hyoscyamus niger against in vitro salt stress. The aim Radical scavenging power (RSP) of tissue extracts were assessed −4 of this work is designed to study the effects of NO on alleviation of by the method of al.M ANDA(2010). et 100 al. (2010).µl of the 100 extract μl of was the addedextract to was 2.9 ml of a DPPH (1×10 M) solution, and kept −4 oxidative damages induced by salt stress. Comparing these responses added to 2.9 ml of fora DPPH 30 min (1×10 at room M) temperature solution, and under kept the for darkness. 30 min The resulting color was measured at 520 nm can be useful for understanding the physiological and biochemical at room temperature under the darkness. The resulting color was mechanisms of NO in this plant that has to cope with salt stress. measured at 520 nmagainst against blanks. blanks. A Adecreasing decreasing intensity intensity of ofpurple purple color was related to a higher radical scavenging color was related toactivity a higher, which radical was scavenging calculated usingactivity, the which following was formula: calculated using the following formula: Materials and methods 30 RSP(%) = 1 ( ) × 100 Experiment 1: Seed germination and salt stress 30 Hyoscyamus niger 𝐴𝐴𝐴𝐴 Seeds of were obtained from Pakan Bazr � − � Company, Isfahan, Iran. Seeds surface were sterilized for 1 min with Where A30 is absorbanceWhere A30 of sample is absorbance 𝐵𝐵𝐵𝐵and B30 of is sample absorbance and B30 of blank is absorbance of blank at 30 min reaction time. at 30 min reaction time. 70% ethanol and for 10 min with 10% sodium hypochlorite, then Hydroxyl radical (•OH) scavenging activity was measured according to the method of MANDA rinsed several times with sterile distilled water. Sterile seeds then Hydroxyl radical (•OH) scavenging activity was measured5 according to the method of MANDA Hydroxyl et radical al. (2010). (•OH) The scavenging reaction mixture activity(2.1 was ml) measured contained ac 1- ml FeSO4 (1.5 mM), 0.7 ml H2O2 (6 were placed on half-strength MS medium (Murashige and Skoog, cording toet theal. method(2010). ofThe M andareaction et al.mixture (2010).(2.1 The ml) reaction contained mixture 1 ml FeSO4 (1.5 mM), 0.7 ml H2O2 (6 1962) containing treatment of 0, 50, 100 and 150 Mm NaCl and 0, 100 mM), 0.3 ml sodium salicylate (20 mM) and 100 µl of extracts of Hyoscyamus niger tissue. This (2.1 ml) contained 1 ml FeSO (1.5 mM), 0.7 ml H O (6 mM), and 200 μm SNP under aseptic condition. SNP ([Na2Fe(CN)5]·NO) mM), 0.3 ml sodium salicylate4 (20 mM) and 1002 2 µl of extracts of Hyoscyamus niger tissue. This was used as NO donor. The medium was supplemented with 3% 0.3 ml sodiummixture salicylate was incubated (20 mM) at and37°C 100for μ1l h,of afterextracts which of theHyos- absorbance of the solution was measured cyamus nigermixture was incubated at 37°C for 1 h, after which the absorbance of the solution was measured sucrose and solidified with 0.8% agar. The pH of the medium was at 562tissue. nm. TheThis percentage mixture was scavenging incubated effect at 37 was °C calculated for 1 h, as: adjusted to 5.8 before autoclaving. After four weeks some biochemi- after whichat 562the nm.absorbance The percentage of the solution scavenging was measuredeffect was at calculated 562 nm. as: cal and physiological parameters were measured. The percentage scavenging effect was calculated1 A2 as: Scavenging effect(%) = 1 ( 1 A2 ) × 100 Scavenging effect(%) = 1 ( 0 ) × 100 𝐴𝐴𝐴𝐴 0− Experiment 2: Induction of Callus � − � Where A0 was the absorbance 𝐴𝐴𝐴𝐴of the−𝐴𝐴𝐴𝐴 control (without extract) and A1 was the absorbance in the Callus induction was obtained from hypocotyl explants of Hyo- � − � Where A0Where was theA0 wasabsorbance the absorbance of the control of the𝐴𝐴𝐴𝐴 control(without (without extract) extract) and and A1 was the absorbance in the scyamus niger obtained from seedling germinated on MS medium A1 was thepresence absorbance of the in extract, the presence A2 was of the theabsorbance extract, A2 without was thesodium salicylate. with a combination of SNP (0 and 50 μm) and 2 concentrations absorbancepresence without of sodium the extract, salicylate. A2 was the absorbance without sodium salicylate. -1 (0.5 and 1 mg L ) of α 6-benzyl aminopurine (BAP), kinetin (kin), Fe2+-chelating activity was measured according to the method of MANDA et al. (2010). The and 2,4-dichlorophenoxy acetic acid (2,4-D). Before autoclaving at Fe2+-chelating activity was measured according to the method of MANDA et al. (2010). The reaction mixture (2.0 ml) contained 100 µl of Hyoscyamus niger tissue, 100 µl FeCl2 (0.6 mM), 121 °C for 15 min, pH of the media was adjusted to 5.8. Fe2+-chelating activity was measured according to the method reaction mixture (2.0 ml) contained 100 µl of Hyoscyamus niger tissue, 100 µl FeCl2 (0.6 mM), of Mandaand et 1.7al. (2010).ml deionised The reactionwater. The mixture mixture (2.0 was ml) shaken contained vigorously and left at room temperature for 100 μl ofand Hyoscyamus1.7 ml deionised niger water.tissue, The 100 mixture μl FeCl was (0.6 shaken mM), vigorously and and left at room temperature for Seed germination percentage 5 min; 100 µl of ferrozine (5 mM in methanol)2 were then added, mixed, and left for another 5 1.7 ml deionised5 min; 100 water. µl Theof ferrozine mixture (5was mM shaken in methanol) vigorously were and then left added, mixed, and left for another 5 Since all swollen seeds germinated, seed germination percentage at room temperaturemin to complex for 5 min;was measured100 μl of ferrozineat 562 nm (5 against mM in ametha blank.- Disodium ethylenediaminetetraacetic was calculated by the following formula (thus differentiating swollen min to complex was measured at 562 nm against a blank. Disodium ethylenediaminetetraacetic nol) were thenacid (EDTAadded, -mixed,Na2) was and used left asfor the another control. 5 min to complex from unswollen seeds): was measured at 562 nm against a blank. Disodium ethylenediamine- = acid (EDTA-Na2) was used as the control. Seed germination percentage Number of seeds showing swelling tetraacetic acid (EDTA-Na ) was used as the control. of the embryo/Total number of seeds The chelating activity2 of the extract for Fe2+ was calculated as: The chelatingThe chelating activity of activity the extract of the for extract Fe2+ forwas Fe2+ calculated was calculated as: as: 1 A2 Chelating effect(%) = 1 ( ) × 100 Determination of antioxidant enzyme activities 1 A2 Chelating effect(%) = 1 ( 0 ) × 100 Three hundred mg fresh tissue were ground with 3 ml ice-cold 𝐴𝐴𝐴𝐴 0− Where A0 was the absorbance of� the− control𝐴𝐴𝐴𝐴 − (without� extract) and 50 mM phosphate buffer (pH 7.8) containing 0.2 mM EDTA, 2 mM Where A0 was the absorbance� − of𝐴𝐴𝐴𝐴 the control� (without extract) and A1 was the absorbance in the ascorbate and 2% PVP. The homogenates were centrifuged at 4 °C A1 was theWhere absorbanceA0 was thein the absorbance presence of of𝐴𝐴𝐴𝐴 the the control extract, (without A2 was extract) the and A1 was the absorbance in the presence of the extract, A2 was the absorbance without ferrozine. for 20 min at 12,000×g and the resulting supernatants were used for absorbance without ferrozine. presence of the extract, A2 was the absorbance without ferrozine. determination of antioxidant enzyme activities. SOD activity was assayed by measuring its ability to inhibit the Determination of H2O2 concentration Determination of H2O2 concentration photochemical reduction of nitroblue tetrazolium following the Determination of H2O2 concentration method of STEWART and BEWLEY (1980). CAT activity was mea- The H2O2 Theconcentration H2O2 concentration was determined was determined according according to Patter tos onPATTERSON et al. (1984). The assay was The H2O2 concentration was determined according to PATTERSON et al. (1984). The assay was sured as the decline in absorbance at 240 nm due to the decrease et al. (1984).based The on assay the absorbance was based changeon the absorbanceof the titanium change peroxide of the complex at 415 nm. Absorbance values of extinction of hydrogen peroxide (H2O2) according to the method titanium peroxidebased on complexthe absorbance at 415 nm.change Absorbance of the titanium values peroxidewere quan complex- at 415 nm. Absorbance values were quantified using standard curve generated from known concentrations of H2O2. of PATRA et al. (1978). POD activity was measured by the increase tified using standard curve generated from known concentrations of were quantified using standard curve generated from known concentrations of H2O2. in absorbance at 470 nm due to guaiacol oxidation (NICKEL and H2O2. CUNNINGHAM, 1969). GR activity was measured according to Foyer Determination of lipid peroxidation and HALLIWEL (1976), which depends on the rate of decrease in the Determination of lipid peroxidation absorbance of NADPH at 340 nm. DeterminationLipid ofperoxidat lipid peroxidationion was determined in terms of thiobarbituric acid-reactive substances (TBARS) Lipid peroxidationLipid peroxidat was determinedion was determined in terms inof terms thiobarbituric of thiobarbituric acid- acid-reactive substances (TBARS) concentration according to the method of CAVALCANTI et al. (2004). Three hundred mg of fresh reactive substances (TBARS) concentration according to the method concentration according to the method of CAVALCANTI et al. (2004). Three hundred mg of fresh Determination of antioxidant activity of Cavalcsampleanti et wasal. (2004). homogenized Three hundred in 3 ml mg1.0% of (w/v)fresh sampleTCA at was4 ◦C. The homogenate was centrifuged at For determination of antioxidant activities, 0.3 g sample was sus- homogenizedsample in 3was ml homogenized1.0% (w/v) TCA in 3 at ml 4 °C.1.0% The (w/v) homogenate TCA at 4was ◦C. The homogenate was centrifuged at pended in 3 ml of serine borate buffer (100 mM Tris-HCl, 10 mM centrifuged at 12,000×g for 20 min and 1 ml of the supernatant6 was 6

26 D. Samsampour, F. Sadeghi, M. Asadi, A. Ebrahimzadeh added to 3 ml 20% TCA containing 0.5% (w/v) thiobarbituric acid 120 a (TBA). The mixture was incubated at 95 °C for 30 min and the reac- b 100 bc c tion was stopped by quickly placing in an ice bath. The cooled mix- d d d d e e ture was centrifuged at 10,000×g for 10 min, and the absorbance of 80 f g 0 snp the supernatant at 532 and 600 nm was read. After substracting the 60 non-specific absorbance at 600 nm, the TBARS concentration was 100 snp −1 −1 40 determined by its extinction coefficient of 155 mM cm . % Germina9on 200 snp 20 0 Glycine betaine content 0 50 100 150 The amount of glycine betaine was estimated according to the me- salinity (mM) thod of GRIEVE and GRATTAN (1983). The plants tissues were groun- ded with 20 ml of deionised water for 24 h at 25 ºC. TheFig. samples 1. Effects Fig.of SNP 1: Effectson seed of germination SNP on seed of germination Hyoscyamus of Hyoscyamusniger under nigerNaCl under stress. Means with NaCl stress. Means with different letters are significantly different were then filtered and filtrates were diluted to 1:1 with 2 N H2SO4. Aliquots were kept in centrifuge tubes and cooled in icedifferent water for letters are significantly(P<0.05). different (P<0.05). 1 h. Cold KI-I2 reagent was added, and the reactants were gently stirred with a vortex mixture. The tubes were stored at 4 ºC for 16 h Tab. 1: Salt*SNP effect separate by salt for germination. *, **, ***, and then centrifuged at 10,000×g for 15 min at 4 ºC. The supernatant significant at 0.05, 0.01, and 0.001 levels, respectively. was carefully aspirated with a new glass tube. The periodide crystals Table 1. Salt*SNP effect separate by salt for germination. *, **, ***, significant at 0.05, were dissolved in 9 ml of 1,2-dichloroethane. After 2 h, the absorbance was measured at 365 nm using glycine betaine as a0.01, standard and 0.001 levels,Salt respectively (mM) df Mean Square −1 and expressed in mg g DW. 0 2 234.10*** Salt (mM) 50 df 2 143.44*** Mean Square 100 2 74.08*** Total soluble protein content 0 2 234.10*** Total soluble protein content was measured according to Bradford 150 2 115.96*** (1976) using bovine serum albumin (BSA) as a protein standard. 50 2 143.44*** Fresh leaf samples (1 g) were homogenized with 4 mL Na-phosphate buffer (pH 7.2) and then centrifuged at 4 °C. Supernatants and dye 100 tration in Hyoscyamus niger2 tissue (P < 0.05) (Fig. 2). In 50, 100 74.08*** were pipetting in spectrophotometer cuvettes and absorbance was and 150 mM NaCl when seedlings were treated with 100 μM SNP, measured using a spectrophotometer at 595 nm. TBARS concentration was significantly lower than non-treated plant. 150 2 115.96*** In our experiment, it appears that NO has protective effect on membrane damage Hyoscyamus niger tissue. Statistical analysis: Comparison of SNP different levels in each level of NaCl showed The experiment was carried out as bi-factorial in a completely ran- that SNP effect was statistically significant for H O and TBARS H O concentrations and lipid peroxidation 2 2 domized design (4 salinity level × 3 SNP level) with 3 replications.2 2 concentration except in control (Tab. 2). Analysis of variance (ANOVA) for different responses parameters were evaluated by SAS 9.4 software package. Least significanceCompared dif- to control, salt stress significantly induced accumulation of H2O2 in Hyoscyamus niger ference (LSD) test was used to determine the significant difference Antioxidant enzyme activities tissue (P < 0.05), and application of exogenous NO dramatically reduced concentration of H2O2 among the mean values at the 0.05 level. As shown in Fig. 3, salt stress significantly increased the activities of (P < 0.05) (Fig.SOD, 2), CAT,and effectPOD andof saltGR instress Hyoscyamus was blocked niger by than addition those of NO the incon medium- . Under trol groups, which may be a reflection of the oxidative stress induced normal conditions, exogenous NO did not significantly affect H O in Hyoscyamus niger tissue. Results by salt stress. With increasing NaCl in the medium,2 2 POD activities In vitro seed germination: Control of dormancy release and ger- increased until 100 mM and then decreased (Fig. 3). Application of mination of plant seeds TBARS was usedSNP toincreased indicate activities lipid peroxidation, of all antioxidant similar enzymes with the under change salt stress.of H2 O2, salt stress It was observed that the seeds started germination in thesignificantly five days increasedUnder normal TBARS conditions, concentration activities in Hyoscyamus of POD and niger GR antioxidant tissue (P < en0.05)- (Fig. 2). In after placing on the medium. Complete seedlings with two leaves zymes were not significantly influenced by application of exogenous appeared in the 2rd week. When salinity was applied singly, seed SNP. Treatment of plants with SNP9 increased the activity of CAT and germination percentage was low compared to control. In all levels of APX enzymes in control, and stress conditions. salinity, Medium supplemented with 100 μM SNP significantly in- Interaction effect of salinity* SNP showed that SNP different levels creased seed germination (Fig. 1). Medium supplemented with SNP in each level of salinity was significant except for POD and GR en- 100 μM in control resulted in 100% seed germination. Interaction zymes in the control (Tab. 3). effect of salinity* SNP showed that SNP different levels in each level of salinity was significant (Tab. 1). Antioxidant capacity DPPH scavenging capacity, •OH scavenging capacity and metal H2O2 concentrations and lipid peroxidation chelating capacity were usually used to express antioxidant capac- Compared to control, salt stress significantly induced accumulation ity of plant tissues. Compared to the control, salt stress dramatically of H2O2 in Hyoscyamus niger tissue (P < 0.05), and application of decreased antioxidant capacity of Hyoscyamus niger tissue, and exogenous NO dramatically reduced concentration of H2O2 (P < reduced DPPH scavenging capacity, •OH scavenging capacity and 0.05) (Fig. 2), and effect of salt stress was blocked by addition NO metal chelating capacity (Fig. 4). in medium. Under normal conditions, exogenous NO did not signifi- While application of exogenous SNP significantly alleviated the in- cantly affect H2O2 in Hyoscyamus niger tissue. hibition of antioxidant capacity by salt stress, and increased DPPH TBARS was used to indicate lipid peroxidation, similar with the scavenging capacity, •OH scavenging capacity and metal chelating change of H2O2, salt stress significantly increased TBARS concen- capacity compared to the salt stress. Under normal conditions, ap- 50, 100 and 150 mM NaCl when seedlings were treated with 100 µM SNP, TBARS concentration was significantly lower than non-treated plant. In our experiment, it appears that NO has protective effect on membrane damage Hyoscyamus niger tissue.

Comparison of SNP different levels in each level of NaCl showed that SNP effect was Effect of nitric oxide on the induction of callus of Hyoscyamus niger 27 statistically significant for H2O2 and TBARS concentration except in control (Table 2).

160 a 4 b a 140 b b 3,5 c b b 120 cd 0 snp FW) bc 0 snp FW) de 3 e -1 cd

-1 de 100 f 100 snp 2,5 f ef 100 snp g g 80 g g 200 snp 2 200 snp

(nmol g 60 1,5 h 2 h h O Antioxidant2 40 enzyme activities 1 H

20 TBARS (nmol g 0,5 0 As shown0 in Fig. 3, salt stress significantly increased the activities of SOD, CAT, POD and GR 0 50 100 150 0 50 100 150 salinity (mM) salinity (mM) in Hyoscyamus niger than those of the control groups, which may be a reflection of the oxidative

Fig. 2: Effects of SNPstressFig. on the2 induced. Effectsconcentrations by of saltSNP of stress. Hon2O 2the (A)With concentrations and increasing TBARS (B) NaCl ofof Hyoscyamus H inO the (A) medium, andniger TBARS under POD NaCl (B)activities stress. of Means increased with different until letters are significantly different (P<0.05). 2 2 Hyoscyamus niger 100under mM NaCl and stress.then decreased Means with (Figure different 3). Application letters are significantly of SNP increased different activities (P<0.05) of all antioxidant

Tab. 2: Salt*SNP effectenzymes by salt underfor H2O 2salt and stress.TBARS concentration.Under normal *, **,conditions, Tab. 3: Salt*SNPactivities effect of PODby salt and for POD,GR antioxidantCAT, SOD and GR. *, **, ***, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non- significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non- significant. enzymes were not significantly influenced by applicationsignificant. of exogenous SNP. Treatment of plants

withTable SNP 2. Salt*SNPincreased theeffect activity by salt of CATfor H and2O2 andAPX TBARS enzymes concentration in control, and. *, stress**, ***, conditions. significant at Mean Square Salt (mM) df POD CAT SOD GR 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. ns ns Salt (mM) Interactiondf effect ofH salinity*2O2 SNP TBARSshowed that SNP different0 levels2 in8.38 each level73.50** of salinity 2918.11** was 0.0032 0 2 15.19ns 0.016ns 50 2 129.53*** 273.79*** 661.44*** 0.036*** significant except for POD and GR enzymes inMean the control Square (T able 3). 50 2 584.91*** 0.178* 100 2 245.29*** 346.77*** 2550.33*** 0.240*** 100 2 587.32*** 1.50*** 150 2 85.20** 43.11*** 868.11** 0.163*** Salt df H2O2 TBARS 150 ** 2 155.39 (mM) 0.75***

0 2 15.19ns 0.016ns 70 a 70 a a *** b b b 60 50b 2 584.9160 c 0.178* cd bc

FW) 50 de 50 e e FW) d -1 e -1 e 40 f 100 2 0 snp 587.3240 *** f f 1.50*** g 0 snp 30 g g 100 snp 30 g g 100 snp 20 200 snp 20 ** 150 2 155.39CAT(U.mg 0.75*** POD(U.mg 10 10 200 snp 0 0 0 50 100 150 0 50 100 150 salinity (mM) 10 salinity(mM)

600 2,5 a b a c b 500 d b e 2 b f 400 h gh g c c

FW) h 1,5 -1

i FW) 0 snp 300 0 snp -1 SOD de d 100 snp 1 ef 100 snp 200 f (U mg 200 snp g (U mg g g 200 snp 100 0,5

0 Glutathione reductase 0 0 50 100 150 0 50 100 150 salinity (mM) salinity (mM)

Fig. 3: Effects of SNP on the activities of SOD, CAT, POD and GR of Hyoscyamus niger under NaCl stress. Means with different letters are significantly different (P<0.05). 11 plication of exogenous. NO did not greatly change the antioxidant Glycine betaine concentration capacity of Hyoscyamus niger tissue (Fig. 4). As shown in Fig. 5, salt stress significantly induced accumulation of Comparison of SNP different levels in each level of NaCl showed glycine betaine in Hyoscyamus niger (P < 0.5), and application of that SNP effect was not statistically significant in control for DPPH SNP significantly increased glycine betaine concentration under salt scavenging and metal chelating and 150 mM NaCl for DPPH scav- stress, Like the change of other parameters, under normal conditions, enging (Tab. 4). glycine betaine concentrations in Hyoscyamus niger tissue were not Comparison of SNP different levels in each level of NaCl showed that SNP effect was not

statistically significant in control for DPPH scavenging and metal chelating and 150 mM NaCl 28 for DPPH scavenging (TableD. Samsampour, 4). F. Sadeghi, M. Asadi, A. Ebrahimzadeh

60 45 a a a a a ab 40 b b 50 bc bc Salt(mM)35 df DPPHcd cd scavengingd d OH scavenging Metal c c c de 40 30 f d d ef chelating d d 0 snp 25 0 snp 30 20 100 snp 100 snp ns ns 20 15 2 10.023 66.33*** 0.22 0200 snp 200 snp 10 DPPH scavenger % 10 5 OH scavenging capacity % 0 50 0 2 142.69*** 57.33** 54.84*** 0 50 100 150 0 50 100 150 salinity (mM) 100 2 51.30*salinity (mM) 44.77** 170.35***

100 a a a ns 150bc c c 2 6.48 44.77** 94.63*** 90 d d 80 e e f 70 g 60 0 snp 50 Glycine40 betaine concentration 100 snp 30 200 snp 20 As10 shown in Figure 5, salt stress significantly induced accumulation of glycine betaine in metal chela9ng capacity % 0 Hyoscyamus0 niger50 (P100 < 0.5),150 and application of SNP significantly increased glycine betaine salinity (mM) concentration under salt stress, Like the change of other parameters, under normal conditions, Fig. 4: Effects of SNP on DPPH scavenging activity, hydroxyl radical (•OH) scavenging activity and Fe2+ chelating activity of Hyoscyamus niger under NaCl stress. MeansFig with. 4. differentEffects letters of SNP are onsignificantly DPPH scavenging different (P<0.05). activity, hydroxyl radical (•OH) scavenging activity glycine betaine concentrations in Hyoscyamus niger tissue were not significantly changed by and Fe2+ chelating activity of Hyoscyamus niger under NaCl stress. Means with different letters application of exogenous SNP. Interaction effect of salinity* SNP showed that SNP different Tab. 4: Salt*SNP effectare significantly by salt for DPPH different scavenging, (P<0.05) OH scavenging. and higher BAP and 2,4-D had a decrease in callus fresh weight. The OH scavenging. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, minimum day until callus induction was recorded with media sup- respectively. ns, non-significant. levels in each level of salinity was significant-1 except-1 in control (Table 5). plemented with 0.5 mg 2,4-D, 0.5 mg kin and 50 μM SNP (Fig. 7).

Salt (mM) df DPPH OH Metal 25 Tablescavenging 4. Salt*SNP scavengingeffect by salt forchelating DPPH scavenging, OH scavenging and OH scavenging. *, a 0 2 10.023ns 66.33*** 0.22ns **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively.20 ns, non-significant. b FW)

50 2 142.69*** 57.33** 54.84*** -1 c d d 100 2 51.30* Salt(mM)44.77** df DPPH170.35*** scavenging OH scaven15 ging Metal 0 snp 150 2 6.48ns 44.77** 94.63*** chelatinge e 10 f 100 snp g 200 snp significantly changed by application of exogenous SNP. Interaction h 13 5 i hi effect of salinity* SNP showed that SNP different levels in each level ( mg·g Glycin betaine of salinity was significant except in control (Tab. 5). 0 0 50 100 150 Total soluble proteins salinity Addition of NaCl caused a significant reduction in soluble proteins content. This reduction was more pronounced in 150 mM NaCl. In Fig. 5: Effects of SNP on Glycine betaine of Hyoscyamus niger under the lower salt concentrations (control, 50 mM),Fig .when 5. Effects plants were of SNP on GlycineNaCl stress. betaine Means with of differentHyoscyamus letters are niger significantly under different NaCl stress. Means with treated with 100 μM SNP, they showed higher amount of protein in (P<0.05). comparison with their controls. 100 μM SNP differentwas found lettersto be most are significantly different (P<0.05) effective (Fig. 6). Interaction effect of salinity*SNP showed that SNP Tab. 5: Salt*SNP effect by salt for glycine betaine. *, **, ***; significant at different levels in each level of salinity was significant (Tab. 6). 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant.

Salt (mM) df Mean Square Callus induction: The results varied with respect to plant growth regulators (PGR) and 0 142 0.57ns SNP (Tab. 7). Among eight callus induction media, media supple- 50 2 4.85*** -1 -1 mented with 0.5 mg 2,4-D, 0.5 mg BAP and 50 μM SNP was 100 2 24.023*** the most average of callus fresh weight (Tab. 7). It was significantly higher than all other media combinations. The concentration of 150 2 7.77*** Table 5. Salt*SNP effect by salt for glycine betaine. *, **, ***; significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant.

Salt (mM) df Mean Square

0 2 0.57ns

50 2 4.85***

100 2 24.023***

150 2 7.77***

Total soluble proteins

Addition of NaCl caused a significant reduction in soluble proteins content. This reduction was more pronounced in 150 mM NaCl. In the lower salt concentrations (control, 50mM), when plants were treated with 100 µM SNP, they showed higher amount of protein in comparison with their controls. 100 µM SNP was found to be most effective (Fig. 6). Interaction effect of salinity*SNP showed that SNP different levels in eachEffect level of nitricof salinity oxide on was the induction significant of callus (T ableof Hyoscyamus 6). niger 29

Tab. 7: Effects of SNP and plant growth regulators on callus induction of 80 Hyoscyamus niger hypocotyl. Means with different letters are 70 a significantly different (P<0.05).

60 b b FW)

-1 50 c c PGR SNP Callus FW Day until induction 0 snp (μM) (g) of callus 40 d d de 100 snp 30 ef f 0 0.25± 0.03 d 10± 0.57 b g g 0.5 2,4.D + 0.5 BAP 20 200 snp 50 0.54± 0.04 a 8± 0.57 c Protein(mg·g 10 0 0.19± 0.015 ef 11.66 ± 0.57 a 1 2,4-D + 1 BAP 0 50 0.2± 0.01 ef 8 cd 0 50 100 150 0 0.35± 0.035 c 10.3± 0.57 b salinity (mM) 0.5 2,4-D + 0.5 kin 50 0.43± 0.04 b 7.33± 0.57 d Fig. 6: Effects of SNP on Total soluble proteins of Hyoscyamus niger under 0 0.16± 0.03 f 11.66 ± 0.57 a Fig. 6. Effects of SNP on NaClTotal stress. soluble Means proteins with different of Hyoscyamus letters are significantly niger underdifferent NaCl 1stress. 2,4-D + Means 1 kin (P<0.05). 50 0.23± 0.01 de 11.33± 0.57 a with different letters are significantly different (P<0.05)

Tab. 6: Salt*SNP effect by salt for total protein. *, **, ***, significant at 0.05, 0.01, and 0.001 levels, respectively. ns, non-significant. Discussion Exogenous NO has a strong stimulating effect on seed germina- Salt (mM) df Mean Square tion under stress or no-stress conditions (BELIGNI and LAMATTINA, 2000). Some studies provided evidence that NO could counteract the 0 152 366.093*** inhibitory effect of salinity in the process of germination (ZHENG 50 2 271.37*** et al., 2009). In agreement with these, we observed that exogenous 100 2 18.78* NO treatment significantly stimulated seed germination under severe salt stress in Hyoscyamus niger (Fig. 1). 150 2 22.73* In this work, SNP, as an exogenous donor, significantly reduced

Fig. 7: Effects of SNP and plant growth regulators on callus induction of Hyoscyamus niger hypocotyl. A: 0.5 mg/l 2,4-D+ 0.5mg/l kinetin and 50 μM SNP; B: 1 mg/l 2,4-D + 1 mg/l kinetin; C: 0.5 mg/l 2,4-D+ 0.5 mg/l BAP; D: 0.5 mg/l 2,4-D+ 0.5 mg/l BAP and 50 μM SNP; E: 0.5 mg/l 2,4-D+ 0.5 mg/l kinetin and 50 μM SNP; F: 0.5 mg/l 2,4-D+ 0.5 mg/l BAP and 50 μM SNP. 30 D. Samsampour, F. Sadeghi, M. Asadi, A. Ebrahimzadeh oxidative stress in Hyoscyamus niger imposed by salt stress, and chinery and activate stress-related genes (CHEN and MURATA, 2008). reduced the accumulation of H2O2 and TBARS accumulation. Salt- In this study, NaCl stress induced glycine betaine accumulation in stressed Hyoscyamus niger plant accumulated higher levels of H2O2 Hyoscyamus niger, and exogenous NO increased glycine betaine and TBARS contents (Fig. 2), which might be due to membrane level under salt stress (Fig. 5). Recently, many studies have indicated destruction caused by ROS-induced oxidative damage. Exogenous that NO appears to be involved in the metabolism of glycine betaine application of NO reduced levels of TBARS and H2O2 in NaCl- in stress tolerance (LÓPEZ-CARRIÓN et al., 2008). treated Hyoscyamus niger plants (Fig. 2), which is in agreement In the present study, a similar accumulation trend of total soluble pro- with the observations of ZHENG et al. (2009) and KHAN et al. (2012). teins was recorded in Hyoscyamus niger under NaCl stress (Fig. 6). Therefore, application of NO could be an effective practice to protect Soluble proteins play a main role in osmotic adjustment under NaCl plants against oxidative injury caused by salt stress. NO alleviates stress and can provide storage form of nitrogen (SINGH et al., 1987). abiotic stress through different metabolism, and antioxidant capacity Increase in soluble protein content under stress may be the result of modulation was reported to be one of important pathways in many enhanced synthesis of specific stress-related proteins (DOGANLAR investigations (HAO et al., 2009; QIAO and FAN, 2008). et al., 2010). Thus, application of NO to salt-stressed Hyoscyamus The activities of SOD, CAT, POD and GR in the presence of SNP niger provoked a remarkable increase in levels of total soluble pro- under salt stress were also higher than those under salt stress alone teins and glycine betaine perhaps to provide a better protection to (Fig. 3). Exogenous application of NO increased activity of CAT, plants exposed to stress. The protective nature of the osmolytes under SOD, POD and APX in seashore mallow (GUO et al., 2009), mustard NO treatment corroborates with the findings of HAYAT et al. (2012), (ZENG et al., 2011), wheat (RUAN et al., 2002) and protected plants KHAN et al. (2012), KAUSAR et al. (2013), and DONG et al. (2014) from oxidative damage under salt stress. In tomato, exogenous ap- in various plants. Noticeable accumulation of glycine betaine, total plication of NO increased the activity of antioxidant enzymes SOD, soluble proteins due to NO application might enhance salt tolerance POD, CAT, APX, non-enzymatic antioxidant ascorbate and reduced of cells through osmotic regulation. glutathione under salinity stress thus helping to alleviate salt-induced Also, the results indicated that addition of 50 μM SNP led to increase oxidative damage (WU et al., 2011). NO can act (i) as a direct sca- in Callus induction compared with the control (Tab. 7). Elicitations venger of ROS, and (ii) antioxidant system inducer to enhance the are considered to be an important strategy towards improved in vi- expression of antioxidant enzyme-encoding genes (GROSS et al., tro production of secondary metabolites. Various biotic and abiotic 2013). NO applied exogenously may also induce the synthesis of en- factors added to the medium of callus production influence their dogenous NO (HAO et al., 2008; ZHAO et al., 2009; FAN and LIU, production by activating genes for de novo synthesis or by stimula- 2012), which can function as signaling molecule or ROS scavenger tion the physiological processes leading to enhanced accumulation under prolonged stress conditions by regulating/enhancing the activi- of such products. In a previous study conducted in CAB-6P, Gisela 6, ties of antioxidant enzymes (HAO et al., 2008; FAN and LIU, 2012). and M.M 14 cherry rootstocks, the callus formation percentage was Antioxidant capacity including DPPH-radical scavenging activity, 100% in all SNP (0-50 μM) + 17.6 μM BAP + 2.68 mM NAA treat- hydroxyl radical scavenging activity and the chelating activity of ments (SARROPOULOU et al., 2014). According to ΧU et al. (2009), ferrous ions were measured for further investigation of NO func- 87% callus induction frequency was obtained when Dioscorea op- tion in antioxidant capacity induction of Hyoscyamus niger under posite Thunb tuber explants were treated with 40 μM SNP. Thus, the salt stress. In the present experiment, exogenous NO reversed the effect of SNP on callus formation is genotype-dependent. inhibition of DPPH-radical scavenging activity, hydroxyl radical In conclusion, exogenous NO could significantly alleviate the growth scavenging activity and the chelating activity of ferrous ions by inhibition of Hyoscyamus niger, and protect mitochondria and cell salt stress in Hyoscyamus niger (Fig. 4), furthermore, compared to wall from damage under salt stress, which might depend on the high- the change of antioxidant enzymes, their changing degrees were er antioxidant capacity modulated by NO. much higher related with lower accumulation of H2O2 and TBARS, this was accordance with the observed results in Cakile maritima (KSOURI et al., 2007). H2O2 can also lead to the production of OH• References and is very reactive on important biomolecules or membranes in AHMAD, P., OZTURK, M., SHARMA, S., GUCEL, S., 2014: Effect of sodium plants (KARUPPANAPANDIAN et al., 2011). In this study, as suggested carbonate-induced salinity-alkalinity on some key osmoprotectants, by results in lipid peroxidation in membranes of Hyoscyamus niger protein profile, antioxidant enzymes, and lipid peroxidation in two plants, the OH• scavenging activity decreased at high-salt concen- mulberry (Morus alba L.) cultivars. J. Plant Interact. 9, 460-467. tration. However, both SNP concentrations under stress conditions ARASIMOWICZ, M., FLORYSZAK-WIECZOREK, J., 2007: Nitric oxide as a improved the capacity of OH• scavenging. Therefore, the enhanced bioactive signaling molecule in plant stress responses. Plant Sci. 172, scavenging ability of OH• inhibited ROS accumulation in plant, and 876-887. thus plants were protected from lipid peroxidation of membrane sys- ASHRAF, M., FOOLAD, M.R., 2007: Roles of glycine-betaine and proline in tems and oxidative damages. Also JASID et al. (2008) reported that improving plant abiotic stress resistance. Environ. Exp Bot. 59, 206-216. 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