Roghayyeh Babri-Bonab 1, Sara Saadatmand 1 *, Hossein Nazemiyeh 2, and Alireza Iran-Bakhsh 1. Abstract. Introduction

Transcription

1 2517 The effet of different onentrations of exogenous nitri oxide on several physiologial and biohemial parameters in NaCl-stressed oriander (Coriandrum sativum L.) Roghayyeh Babri-Bonab 1, Sara Saadatmand 1 *, Hossein Nazemiyeh 2, and Alireza Iran-Bakhsh 1 1. Department of Biology, Researh and Siene Branh, Islami Azad University, Tehran, Iran 2. Researh Center for Pharmaeutial Nanotehnology, Faulty of Pharmay, Tabriz University of Medial Sienes, Tabriz, Iran Abstrat In this study the influene of sodium nitroprusside (SNP, the donor of NO) was investigated on several physiologial and biohemial parameters in Coriandrum sativum L. grown in saline and non-saline onditions. Fifteen-day-old oriander seedlings were treated with 50 and 100 mm NaCl and 50, 75, and 100 μm sodium nitroprusside during 3 months. Then, arotenoids, arbohydrate, and soluble protein ontents and proline aumulation were measured. Results indiated that NaCl-indued ioni toxiity led to a derease in arotenoids amount. Under NaCl salinity, arbohydrate ontent inreased sharply as ompared with ontrol plants. The protein ontent of plants did not follow a determined pattern. Furthermore, results showed that NaCl-indued ioni toxiity led to a signifiant inrease in proline aumulation. Appliation of 50 μm SNP ould improve arotenoids ontent of oriander. The use of different onentrations of SNP showed various effets on arbohydrate ontent. Appliation of 50 µm of SNP signifiantly enhaned the total protein ontent and proline aumulation; appliation of 75 and 100 µm SNP had variable effets on all measured parameters. These results suggested that 50 μm of SNP is suitable for reduing damage assoiated with salt stress. Keywords: NaCl salinity; nitri oxide; biohemial parameters; physiologial parameters; Coriandrum sativum L. Babri-Bonab, R., S. Saadatmand, H. Nazemiyeh, and A. Iran-Bakhsh 'The effet of different onentrations of exogenous nitri oxide on several physiologial and biohemial parameters in NaClstressed oriander (Coriandrum sativum L.)'. Iranian Journal of Plant Physiology 8 (4), Introdution Coriander (Coriandrum sativum L.), a ulinary and mediinal plant of Apiaeae (Umbelliferae) family, is an annual and *Corresponding author address: s_saadatmand@srbiau.a.ir Reeived: February, 2018 Aepted: August, 2018 herbaeous plant originated from the Mediterranean and Middle Eastern regions (Veena and Manu, 2014). All parts of this herbal mediine were used as traditional remedies for the treatment of different disorders in the folk mediine systems of different ivilizations around the world (Sahib et al., 2013). Plants are usually exposed to different environmental stresses

2 2518 Iranian Journal of Plant Physiology, Vol (8), No (4) during their development whih ause many ommon reations suh as limiting their growth and produtivity and having a substantial impat on agriultural prodution worldwide (Shao et al., 2008). One of the most important environmental stresses is salt stress, whih imposes ion toxiity and osmoti stress on plants, leading to nutrition disorder and oxidative stress. In many ases, the primary soure of salt stress-indued oxidative bursts is the unontrolled ROS aumulation, provoking damage to maromoleules, or even to the antioxidative system (Manaa et al., 2013). Plants have evolved a speifi antioxidant protetive system onsisting of enzymes suh as superoxide dismutase (SOD), atalase (CAT), and non-enzymati onstituents suh as asorbate and glutathione, whih are responsible for savenging exessively aumulated ROS in plants under stress onditions (Jung et al., 2000). Aordingly, the regulation of the antioxidant onstituents through exogenous substanes might enhane the plant tolerane to salt stress (Shi et al. 2007). Reent plant moleular studies have shown that Nitri oxide (NO), as a versatile moleule, is involved in a wide spetrum of physiologial proesses from bateria to human. NO has an important role in growth and development of higher plants, inluding seed germination, senesene, defense response, and abioti stresses. It is also a key signaling moleule in different intraellular proesses (Corpas et al., 2011). In relation to abioti stresses, it was shown that the appliation of SNP (sodium nitroprusside), an NO donor, abates the harmful effets of salinity (Lopez-Carrion et al., 2008). Furthermore, as an antioxidant agent, NO an savenge ROS as a signaling moleule leading to alterations of antioxidative gene expression, and thus funtion to protet plant ells from oxidative damage (Arasimowiz and Floryszak-Wiezorek, 2007). However, studies have shown that nitri oxide is a gaseous reative nitrogen speies, and its effet on different ells is either protetive or toxi, based on its onentration and position of ation (Lamattina et al., 2003). In this work, we investigated the effet of different onentrations of nitri oxide on several physiologial and biohemial parameters of oriander in both salt and not salt stress onditions for determination of the best onentration of NO for alleviation of salt stress. Materials and Methods Plant growth and treatment Seeds of oriander (Coriandrum sativum L.) were surfae sterilized by dipping in 0.5% (v/v) sodium hypohlorite for 5 min., and then washed thoroughly with sterile distilled water for several times. Sterilized oriander seeds were plaed between two layers of filter paper in petri dishes ontaining distilled water before they were kept at 25 C under dark in an inubator. The water in petri dishes were regulated every day. After germination, 15-day-old seedlings were planted in a hydroponi system in the greenhouse (Hoagland solution (Hoagland and Arnon, 1957), a 14-h light, day/night temperatures of 25 C/20 C, and 50-60% relative humidity). Flower pots were randomly arranged in a greenhouse during the treatment period. Sodium hloride (0, 50, and 100 mm) with or without SNP (0, 50, 75, and 100 μm) was added to Hoagland solution during the growth. SNP (Merk, Germany) was used as a donor of NO. The Hoagland solution was renewed 3 times eah week, and the ph was adjusted near 6.5 (Hoagland and Arnon, 1957). At the end of the growth stage, the plants were harvested, and roots and shoots were separated and washed with deionized distilled water. To determine some parameters, fresh plant material immediately were frozen by liquid nitrogen and then stored at -20 C. For eah treatment, three repliates were onsidered. Determination of arotenoids ontent The ontent of arotenoids (xanthophylls and arotenes) were determined aording to the proedure desribed by Lihtenthaler and Wellburn (1983). The photosyntheti pigments were extrated from 0.1 g leaf fresh weight by 80% aetone. Then the homogeneous mass was filtered through Whatman No. 1 filter paper. Using a spetrophotometer, absorbane at nm (hlorophyll a), nm (Chlorophyll b) and 470 nm (arotenoids) was measured and 80% aetone

3 Carbohydrate Content (mg g -1 F.W.) Carotenoids amount (mgg -1 FW) The effet of exogenous nitri oxide on NaCl-stressed oriander (Coriandrum vum L.) 2519 was used as the ontrol to set zero absorbane spetrophotometer. For assay of arotenoids, we used the following equation: Carotenoids (x+) 1 = (1000 A Chl a Chl b 4 )/198 Where (x+): xanthophylls and arotenes, A 470 : absorbane at 470 nm, Chl a : hlorophyll a ontent, and Chl b : hlorophyll b ontent. Determination of arbohydrate amount Determination of arbohydrate ontents was arried out by Cu +2 redution test aording to Nelson (1944) and Somogyi (1952) method. Two ml of the extrat (0.05 g ground fresh leaf, 5 ml water) were mixed with 2 ml opper reagent of Somogyi. The mixtures were transferred to a boiling water bath for 20 minutes. After ooling, 2 ml Nelson s arsenomolybdatere agent was added. After 10 minutes, the absorbane of the reation mixture was measured at 600 nm using a spetrophotometer. The amounts of arbohydrates were alulated by a standard urve obtained from different onentrations of standard gluose solution. Protein extration and assay Frozen leaf samples (0.5 g) were used for protein extration aording to the method introdued by Bradford (1976). Samples were ground and lixiviated to 5 ml of 50 mm phosphate buffer (ph 7.5), using a pre-hilled mortar with a pestle. The phosphate buffer ontained 1 mm EDTA, 1 mm PMSF, and 1% PVP-40. This was followed by entrifugation at 4 C at 15,000 g for 30 min. The supernatant was used to measure protein onentration. To assay total protein ontent, 1 ml of the supernatant was added to 5 ml Coomassie Brilliant Blue G-250 reagent. After 20 min, the solution absorbane was read at 595 nm, and the ativity was alulated using the standard bovine serum albumin (BSA) (Bradford, 1976). Proline ontent determination Proline ontent determination proeeded aording to Bates et al. (1973). Leaf tissue (5 g) was homogenized in 3% sulfosaliyli aid, and then filtered. To 2 ml of the filtrated solution, 2 ml of ninhydrin aid and 2 ml of glaial aeti aid were added, and the mixture was inubated for 1 h in a boiling water bath, whih was followed by an ie bath. To do this, 4 ml toluene was added and mixed vigorously. Afterward, the hromophore ontaining toluene was separated from the aqueous phase and the absorbane was measured at 520 nm. A standard urve was established using known onentrations of authenti proline, and was alulated as µm g -1 FW a,d d e b e,f f,g g,hf,g g,hh NaCl(mM) 0 NO 50 NO 75 NO 100 NO Fig. I. Effets of NO (μm) on the arotenoids ontent in oriander under saline and non-saline onditions; results are shown as mean ± SE (p<0.05), obtained from three repliates. Means that do not share a letter are signifiantly different f,g,hf,g h g,h e d,ed f a,ba b, NaCl(mM) 0 NO 50 NO 75 NO 100 NO Fig. II. Effets of NO (μm) on the arbohydrate ontent in oriander under saline and non-saline onditions; results are shown as mean ± SE (p<0.05), obtained from three repliates. Means that do not share a letter are signifiantly different.

4 Proline Content (µmol g- 1 FW) Protein Content (µgg -1 FW) 2520 Iranian Journal of Plant Physiology, Vol (8), No (4) Statistial analysis Data were analyzed using ANOVA (ompletely randomized) to determine if there were signifiant differenes among the obtained means. Dunan's multiple range tests were arried out to determine if there were signifiant differenes (P 0.05) between individual treatments (SPSS-13). Results Carotenoid ontents As shown in Fig. (I) when NaCl onentration inreases, the ontent of arotenoids signifiantly redued. Under salinity and non-salinity onditions, the amount of arotenoids inreased by 50 μm SNP. In this ondition, 75 and 100 μm SNP, dereased ontent of arotenoids. Carbohydrate ontent As shown in Fig. (II), a signifiant inrease in arbohydrate ontent was observed in response to 50 and 100 mmnacl salinity as ompared with ontrol plants. Our data showed that appliation of 50 μm SNP enhaned the arbohydrate ontent in non-saline ondition, but, it redued arbohydrate ontent in saline ondition. In all onentrations of NaCl, appliation of 75 and 100 μm SNP, enhaned the arbohydrate ontent of plants. Protein ontent Results indiated that the amount of protein does not follow a speifi pattern (Fig. III). Thus, the addition of 50 mm NaCl to the medium inreased signifiantly the amount of total protein while the addition of 100 mm NaCl dereased it. In the absene of salinity medium, the appliation of all three onentrations of nitri oxide (50, 75, and 100 µm) signifiantly inreased the amount of protein. In the presene of 50 mm NaCl, addition of 50 µm NO signifiantly inreased protein amount while the addition of 75 and 100 µm NO signifiantly dereased it. In the medium Fig. III. Effets of NO(μM) on the protein amount in oriander under salin and non-salin onditions. Results are shown as Mean ± SE (p<0.05), obtained from three repliates. Means that do not share a letter are signifiantly different Fig. IV. Effets of NO (μm) on proline ontent in oriander under salin and non-salin onditions. Results are shown as Mean ± SE (p<0.05), obtained from three repliates. Means that do not share a letter are signifiantly different. ontaining 100 mm NaCl, appliation of 50 µm NO signifiantly inreased the total protein ontent; however, both 75 and 100 µm nitri oxide signifiantly dereased the amount of protein. Proline onentration d b a b d d e g f,ge,f e,f,g Results showed that proline onentrations at 50 mm and 100 mm NaCl were enhaned up to 19.37% and %, respetively, ompared to ontrol plants. As shown in Fig. (IV), a signifiant inrease in free proline aumulation was observed in response to 50 and 100 mm NaCl salinity. Our data showed that the amount of proline was enhaned by NO under saline and non-saline onditions. The effet of nitri oxide on the inreasing proline ontent was more pronouned in the saline medium than the non-saline medium. e NaCl (mm) d d d f f b a,b a NaCl(mM) 0 NO 50 NO 75 NO 100 NO 0 NO 50 NO 75 NO 100 NO

5 The effet of exogenous nitri oxide on NaCl-stressed oriander (Coriandrum vum L.) 2521 Disussion Although there is a plethora of plant reations to irumvent the harmful effets of environmental onditions, a wide range of stresses, suh as high and low temperature, drought, alkalinity, salinity, UV stress, and pathogen infetion are potentially harmful to the plants (Van Breusegem et al., 2001). Salinity of soil or water is one of the major stress obstales, espeially in arid and semi-arid regions, whih an severely limit plant growth and produtivity. Coriander as a ulinary and mediinal plant is one of the most onsumed vegetables in the world. However, it is extremely sensitive to different adverse environmental onditions, inluding salinity, whih provokes a signifiant redution in rop produtivity (Neffati et al., 2011). Plants have developed a wide variety of defense strategies to ombat oxidative damage. There are many lines of evidene indiating that NO plays important roles in plant tolerane to environmental stress, inluding salt stress (Uhida et al., 2002), drought stress (Arasimowiz-Jelonek et al., 2009), heavy metal toxiity (Xu et al., 2010), et. Researh findings suggest that NO alleviates abioti stress through different metabolism, and antioxidant apaity modulation is reported to be one of the most important pathways in many investigations (Hao et al., 2009). Our results showed that salinity redued the arotenoids ontent in leaves of oriander plants. Furthermore, the effet of severe salinity is higher than low salinity. It has been observed that exogenous appliation of 50 μm SNP inreases the ontent of photosyntheti pigments under 50 mm NaCl salinity, but it had no positive effet under 100 mm NaCl salinity. Aording to our findings, high onentration of SNP has a positive effet and mitigates the damaging effets of salt. It has been suggested that exogenous NO leads to the promotion of protetive reations to the photosyntheti pigments and indues a preadaptive 25response to salt stress (El-Tayeb, 2005), whih onfirm our results. The most important moleule whih affets different physiologial responses is sugar (Crowe et al. 1990). Aumulation of arbohydrate in plants tissue under ondition of environmental stress is a result of regulation and modifiation in urrent stress (Dhanapakiam, and Ilyas, 2010). Generally, the inrease in ellular osmolality whih is ahieved from aumulation of ompatible solutes is assoiated with the influx of water into ell (Hare et al. 1998). So, inreasing sugar under environmental stress ould be the result of starh deay, sugar synthesis by non-photosynthesis pathways, non-onverting of sugar to other produtions, and derease in transportation from leaves to other parts of plant (Premahander et al. 1991). Proteins play important roles in salt stress alimation and plant ellular adjustment sine proteins perform a vast array of funtions within living organisms, inluding signaling, regulation of gene expression and protein metabolism, defense-related proteins, mehanial stressrelated proteins, and seondary metabolism (Kosova et al., 2013). In this study, total soluble protein onentration inreased in plants under 50 mm NaCl onentrations. These effets may be due to the synthesis of proteins required to protet the plant against salt stress. However, in response to inreasing salinity, harmful effets were inreased, many metaboli pathways were inhibited, and most proteins were degraded. Finally, results showed the redution of total protein ontents under high stress. A derease in protein onentration was observed in plants grown under high onentrations of NaCl (100 mm). Our results is similar to those of Agastian et al. (2000) who reported that soluble protein inreases at low salinity, and dereases at high salinity in mulberry ultivars. Proteins that aumulate in plants under saline onditions may provide a storage form of nitrogen whih is reutilized later (Singh et al., 1987), and may play a role in osmoti adjustment. They may show de novo synthesis in response to salt stress or may be present onstitutively at low onentrations, and inrease when exposed to salinity stress (Pareek et al., 1997). In the present study, exogenous 50 µm NO was able to signifiantly inrease the protein ontent at all salt onentrations, but 75 µm and 100 µm NO had a reverse effet under salt stress ondition. Zhang et al. (2010) showed that the inhibition of NO aumulation dereased soluble protein ontent in salt-stressed wild plants, whih is in ongruene with our findings. At lower

6 2522 Iranian Journal of Plant Physiology, Vol (8), No (4) amounts, NO ats as signals for the ativation of defense responses; however, higher onentrations of NO produed from unontrolled ROS generation, resulted in severe damages. Proline aumulates under salt stress at as an adjustment osmolyte in plants. It an savenge ROS, elevate anti oxidation ability, stabilize the struture of maromoleule, derease the ellular aidifiation, and detoxify ammonia toxi (Ruan et al., 2002). Our results showed that proline ontent signifiantly inreased in plants exposed to NaCl stress. The results also indiated that the amount of proline was enhaned by NO under saline and non-saline onditions. The effet of nitri oxide on inreasing the amount of proline was highly pronouned in the saline medium ompared with non-saline medium. Aording to Zong et al. (2001), the role of proline in alleviating the negative effets of drought and salt stress in rie might be related to Ca 2+, and that NO signaling transdution inluded Ca 2+ signaling. They onluded that the protetive effets of NO on salt stress-indued oxidative damage in wheat leaves were related to the regulation roles in proline levels. To sum up, the present study provided evidene that exogenous 50 µm NO alleviated NaCl-indued oxidative stress in Coriandrum sativum. Moreover, results indiated that exogenous75 and 100 µm NO had no positive effet on oriander under salt stress ondition. Aknowledgments The authors would like to thank the Laboratory of Biology of Researh and Siene Branh of Tehran, Islami Azad University and Faulty of Pharmay of Tabriz University. Referenes Agastian, P., S.J. Kingsley and M. Vivekanandan, 'Effet of salinity on photosynthesis and biohemial harateristis in mulberry genotypes'. Photosynthetia, 38: Arasimowiz, M. and J. Floryszak-Wiezorek 'Nitri oxide as a bioative signaling moleule in plant stress responses'. Plant Siene, 172: Arasimowiz-Jelonek, M., J. Floryszak- Wiezorek and J. Kubis 'Involvement of nitri oxide in water stress-indued responses of uumber roots'. Plant Siene, 177 (6): Bates, L.S., R. P.Waldren and I. D. Teare 'Rapid determination of free proline for water-stress studies'. Plant and Soil, 39: Bradford, M.M 'A rapid and sensitive method for quantitation of mirogram quantities of protein utilizing the priniple of protein dye binding'. Analytial Biohemistry, 72: Corpas, F., M. Leterrier, R. Valderrama, M. Airaki, M. Chaki, M. Palma and B. Barroso 'Nitri oxide imbalane provokes a nitrosative response in plants under abioti stress'. Plant Siene, 181: Crowe, J.H., J.F. Carpenter, L. Crowe and T.J. Anhordouguy 'Are freezing and dehydration similar stress vetors? A omparison of models interation of stabilizing solutes with biomoleules'. Cryobiology, 27: Dhanapakiam, S. and M. Ilyas 'Effet of salinity on hlorophyll and arbohydrate ontents of Sesbania grandiflora seedlings'. Indian Journal of Siene and Tehnology, 3(1): El-Tayeb, M.A 'Response of barley grains to the interative effet of salinity and saliyli aid'. Plant Growth Regulation, 45(3): Hao, G., X. Du, F. Zho, R. Shi and J. Wang 'Role of nitri oxide in UV-B indued ativation of PAL and stimulation of flavonoid biosynthesis in Ginkgo biloba allus'. Plant Cell, Tissue and Organ Culture (PCTOC), 97: Hare, P.D., W.A. Cress and J. Van Staden 'Disseting the roles of osmolyte aumulation during stress'. Plant Cell Environ, 21(6): Hoagland, D.R. and D. I. Arnon 'The waterulture for growing plants without soil'. Agriultural Experiment Station Cirulation, Cirular-347. Jung, S., J.S. Kim, K.Y. Cho, G., S. Tae and B. G. Kang 'Antioxidant responses of uumber (Cuumis sativus) to photoinhibition and oxidative stress indued

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