147 Effects of Salicylic acid on some physiological

International Journal of AgriScience Vol. 4(2): 147-152, February 2014 ISSN: 2228-6322© International Academic Journals

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Effects of Salicylic acid on some physiological and biochemical parameters of Brassica napus L. (Canola) under salt stress Baghizadeh A.1*, Salarizadeh M.R.2, Abaasi F.2 1 Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman-Iran. * Author for correspondence (email: [email protected]) 2 Department of Biology, Islamic Azad University, Mashhad Branch, Iran

Received December 2013; accepted in revised form January 2014

ABSTRACT Saline soil and water has a detrimental effect on agricultural production by reducing plant growth and yield. Much research has reported changing levels of physiological and biochemical parameters under salinity stress. The smoolits leave fill up more dissolved sugars of glucose, fructose and proline with salicylic acid. This determines that salicylic acid (SA) has a very important role in determining plant response to stress and many studies have reported on plant modification using salicylic acid to reduce the damage caused by saline soil and water. Tests were done to evaluate the effect of salicylic acid under various concentrations of salinity (0, 4, 8 and 12 dsm -1;) as a factorial experiment in a complete randomized design (CRD) with three replications. Salicylic acid applications (0, 0.5 and 1mM) were tested under greenhouse conditions to evaluate the effects on some physiological and biochemical characteristics of Brassica napus L. Results showed that increasing levels of NaCl reduced amounts of photosynthetic pigments (Chl a, b and carotenoids); protein and soluble sugars and free amino acids including proline and MDA compared to the control plants. SA application increased photosynthetic pigments (Chl a, b and carotenoids), protein and soluble sugars, free amino acids including proline and MAD content compared to plants under salinity stress.

Keywords: salinity, salicylic acid (SA), canola

INTRODUCTION Salinity is a major environmental problem that causes a reduction in plant productivity (Irshad et al. 2002). Much research has reported on changing levels of physiological and biochemical parameters caused by salinity stress. The increasing global population is putting a strain on food production in such a way that there is now higher demand for food production and this will force the use of saline soil and water for agricultural production in the not so distant future (Babaiyan and Ziatabar 2002). The main problem with agricultural production in saline conditions is damage to plant growth caused by osmosis (Song et al. 2009). Reduced plant growth under salinity is a consequence of several physiological responses including modification of plant water status, photosynthetic efficiency and carbon allocation and utilization (Abdul Jaleel et al. 2007). Inhibited plant growth may be caused by decreased turgidity from high concentrations of salts in the soil under water deficit conditions (Kim and Lee 2001). Several studies have also mentioned that SA has an important role in modulating plant response to several types of abiotic stress (Yalpani et al. 1994). Previous studies have demonstrated that a wide range of responses might

appear after exogenous SA application, some of which are as follows: increased yield (El-Tayeb 2005, Khodary 2004, Yildirim et al. 2008); increased photosynthetic activity (Singh and Usha 2003); increased total anthocyanins (Hernandez and Vargas 1997); inhibited ethylene biosynthesis (Huang et al. 2004) and protection against biotic and abiotic stresses (Doares et al. 1995). Salicylic acid (SA) serves as a critical signaling molecule that modulates plant response to infection by pathogens. SA biosynthesis is stimulated in response to pathogen infection that induces a group of pathogenesis-related (PR) genes. These molecular events induce hypersensitive response (HR) and systemic acquired resistance (SAR) to prevent propagation of pathogens in infected plants (Durrant and Dong 2004). MATERIAL AND METHODS This study was done to investigate the effect of salicylic acid on some physiological and biochemical characteristics of Brassica napus L. under salt stress. Three germinated seeds were planted in plastic pots (vases) 10 cm in diameter and 15 cm in height, each of which was filled with perlite and kept in greenhouse conditions. In addition, for improving and accelerating

International Journal of AgriScience Vol. 4(2): 147-152, February 2014

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plant growth, the Hoagland food (nutrient) solution at the recommended dilution was added to the pots (vases) daily. After one month, when four fully expended leaves had appeared, applications of salicylic acid (0, 0.5, 1 mM) were sprayed on to the leaves. After the salicylic acid treatment, treatments were maintained in salt concentrations of (0, 4, 8 and 12) dsm-1 for ten days. The experiment was done as factorial in a completely random design (CRD) with 3 replications (36 pots). It should be noted that light conditions were 14 h light and 10 h darkness, and laboratory humidity was maintained at 30% to 40% and at the temperature of 25°C. Measurements were taken for amounts of chlorophylls, carotenoid, protein, sugar, proline and MDA after exercising the treatments. Comparisons of means were made the using LSD test using SPSS 14.0 software. Excel 2003 software was used to draw a graph. Chlorophyll and carotenoid contents: The photosynthetic pigments were extracted from 0.1 g of freshly collected leaves by 80% acetone. Chlorophyll and carotenoid (xanthophylls and carotenes) contents were determined according to the formulae described by Lichtenthaler, as follows: Chlorophyll a (mg/ml) = Ca =12.25 A663.2 – 2.79 A646.8 Chlorophyll b (mg/ml) = Cb =21.50 A646.8 – 5.10 A663.2 Carotenoids (mg/ml) =(1000 A470 –1.8 Ca – 85.02 Cb)/198 ( Lichtenthaler 1987). Total soluble protein determination: The total soluble protein content in fresh leaves was determined according to the method of Bradford (Bradford et al 1976). One gram of fresh leaf was completely ground in a chain mortar to extract protein from the leaf, with 5 ml buffer trace, Hcl 0.05 Molar with PH=7.5. The solution was then separated as follows; the obtained computable solution was transferred to a centrifuge pipe and samples were centrifuged by a refrigerator centrifuge. Inertia was then allowed for 10 minutes, 25 min in 1000 g and at the temperature of 4°C at the final term, then the pipe centrifuge was taken out of the system, the upper solution was distributed in each test pipe. The obtained extraction was then used to measure concentrations of protein solution. This was done by taking an amount of protein extraction 0.1ml, to which 5 ml biore reagent was added to the test pipe and stirred quickly to a vortex. Readings were taken for adsorption after 25 min by a spectrophotometer system at the wavelength of 595 nanometers. For the purpose of biore regent preparation, 0.1 g comasi brilliant blue G250 was dissolved in 50 ml 95% ethanol for at least one hour and then added to 100 ml phosphoric acid 85% drop by drop, the total capacity was increased to one liter with distilled water and then the obtained solution was filtered with Watman filter

paper. The protein value was measured and presented by the relevant standards curve and based on mg/g fw. Total soluble sugar determination: The total soluble sugar content in fresh leaf was determined according to the Somogy method (Somogy 1952). A sample of fresh tissue leaf (0.05g) was weighed by laboratory subtle scale (satrius) BP211D model with 0.0001g accuracy. Each sample was ground with 10 ml distilled water in a china mortar and content of the mortar was transferred to a small container and put on a heater to boil. The content was then filtered by Watman filter paper (number 1), for plant extraction. 2ml of each extraction was transferred to a test pipe and 2 ml copper sulfate solution was added to each pipe, then the pipe caps were closed with cotton. Each of these pipes was kept in a warm water bath at 100o C temperature. In this term, CU2+ was reduced to CU2O by mono sac arid aldeid. Here a brick red color was observed at the bottom of the test pipe. After cooling the pipes, 2 ml phosphomolibdic acid solution was added so that after a moment a blue color appeared, the test pipe was well shaken to spread the color within the test pipe. Observation of the solution was made by testing at 600-nanometer wavelength determined by the spectrophotometer system, and then sugar concentration was measured using the standard curve. For the spectrophotometer setting, a solution was used instead of the plant extraction, which included distilled water, and the rest of the solution was measured and presented as evidence of sugar values by using a relevant standard curve based on (mg/g. fw). MAD content: The measurement of MDA concentration was taken according to the Health and Packer method (Health and Packer 1968). 0.2 g of leaf fresh tissue was weighed and abraded in a china mortar of 5mL Trichloroacetic acid (TCA) 0.1%. The obtained juice was poured into a centrifuge device in 10000 G for 10 min. About 5.4 mL TCA solution, at 20% and that contained 0.5% Thiobarbituric acid (TBA) was added to 1ml of the above solution from the centrifuge. The obtained compound was heated in a hot bath at 950C for 30 min. Then it was immediately allowed to cool on ice and then centrifuged in 10000 G once again for 10 min. The absorption intensity of this solution was read and registered by a spectrophotometer at the wavelength of 532 nm. The silence coefficient was used to calculate concentrations of MDA and the measurements were calculated and presented according to milligrams/liter. Proline content: Proline was extracted and determined by the method of Bates (Bates et al. 1973). 0.02 g of leaf fresh tissue was ground with 19 ml, 3% sulfo salicylic solution, the obtained extraction was centrifuged using the


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centrifuge nap co 2038R model for 50 min in 1000 g then 2 ml of upper liquid mixed with 2 mg ninehydrin reagent and 2 ml pure acetic acid and then kept in a hot water bath at 100o C for one hour. After that, reactions were stopped by allowing the pipes to cool in an ice bath, then a quantity of 4 ml toluene was added and the pipes were shaken well for 15-20 seconds to separate the layers formed by fixing the pipes. The uppermost, colored layers of toluene and proline were used for taking measurements of proline concentration. The absorption of some specific colored material was determined through 520nanometer wavelength, and the proline content of each sample was obtained using a standard curve, based on mg/g fw. RESULTS AND DISCUSSION It has been proposed that SA is one of the agents to act as a signal transducer in plant response to salt stress (Molina et al. 2002). Research has shown that SA accumulation below the threshold required for PR1 induction is a frequently used marker for SA accumulation (Borsani et al. 2001). That report emphasized that exogenous application of SA resulted in a significant increase in plant growth both in saline

and non- saline conditions. Plants produce proteins in response to abiotic and biotic stress and many of these proteins are induced by phytohormones such as ABA (Jin et al. 2000) and salicylic acid (Hoyos et al. 2000). Salicylic acid is an endogenous growth regulator of phenolic nature, which influences a range of diverse processes in plants, such as seed germination (Cutt et al. 1992); stomatal closure (Larque-Saavedra A 1979); ion uptake and transport; membrane permeability (Barkosky et al. 1993) and rate of photosynthesis and growth (Khan et al. 2003). The resultant amount of pigment change in leaves Brassica napus under salinity and SA treatments tested in this research and shown in figure (1) demonstrates that increasing NaCl levels significantly reduces amounts of chlorophyll a, chlorophyll b and carotenoid reduced compared to the control. SA with and without salinity increased the amount of pigment in the tested Brassica napus. Amounts of chlorophylls and carotenoid were significantly reduced in maize tested under salt stress (khodary 2004). SA, by increasing pigments and rubisco activity, increased the amount of photosynthesis in tests on soybean (Zhao et al. 1995).

Figure 1. Effects of application of SA on the amount of chlorophyll.a (1a), chlorophyll.b (1b) and carotenoids (1c) in Brassica leave under saline and non-saline conditions. Results are shown as Mean ± SE The results related to changes in amounts of sugar and protein in Brassica napus leaves under SA and salinity treatments are shown in figure (2). Chlorophyll and protein contents were reduced (Elstner EF 1982) in conditions of salt stress that induced production of reactive oxygen species that cause deterioration of membrane per oxidation lipids. The reason for reduced

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amount of protein recorded in wheat, bean and pea under salt stress is due to the prevention of nitrate reductase activity and a decrease in activity of bonding ammonium to amino acids (Undovenko et al. 1971). Other tests reported that amounts of protein decreased because of a decrease in nitrate reductase activity in maize under salt stress (Abd-EI baki 2000). SA


induced the formation of defensive types of proteins such as protein kinase and rubisco indicating that SA application induced a protein synthesis inhibitor of plant proteases (Raskin I 1992). In barley plants under

SA treatment, a high quantity of protein was produced that included PR proteins that play an active role against the effects of salt stress (Afzal et al. 2006).

Figure 2. Effects of application of SA on the amount of protein and solution sugars in Brassica leave under saline and non-saline conditions. Results are shown as Mean ± SE.

M D A c o n te n t (μm o l. g 1 FW )

In this research the amount of protein was decreased under salt stress. But SA increased the amount of protein in brassica under conditions of salinity as well as without it. Various concentrations of salinity increased the amount of soluble sugars in leaf compared to the control plants. Other research showed that the amount of soluble sugars was increased in corn and tomatoes under NaCl salinity (Gunes et al. 2007). Some plants such as tomato and maize, when exposed to salinity treatment, indicated a gradual increase of sugars in solutions, while observation showed that polysaccharide concentration was reduced

(Khodary 2004). Tests on the effect of the interaction of SA and salinity on solution sugars content in maize plants showed that levels decreased (Gunes et al. 2007). This suggests that SA may activate the consumption of metabolic solution sugars for new cell structure and growth regulation. In this study, salt stress increased the amount of solution sugars in the samples of Brassica napus. The results of changes to amounts of proline and malondealdeid in leaves Brassica napus under SA and salinity treatments are shown in figure (3).

4 3.5 3 2.5 2 1.5 1 0.5 0

0 4dsm-1 8dsm-1 12dsm-1 controle

1mM

SA treatment(mM)

Figure 3. Effects of application of SA on malondialdehyde (MDA) content (a) and proline content (b)in Brassica leaves under salinity stress and non-saline control. Results are shown as Mean ± SE.


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International Journal of AgriScience Vol. 4(2): 147-152, February 2014 ISSN: 2228-6322© International Academic Journals SA application reduced the amounts of proline and MDA in Brassica under salt stress. Tests demonstrated that SA application, by increasing the development of stress response and by stimulating the growth process after stress response, caused an increase in proline (Ashraf 1989). Tests for oxidative stress done on other plants; barley, wheat, bean and tomato demonstrated that increased proline and sugars content increased under application of SA treatment and these results are consistent with those reported in (Tan et al 2008). SA was reported to inhibit the production of the hydroxyl radical and to decrease the content of MDA. In this study, SA significantly increased the amount of proline in Brassica napus under salt stress indicating the influence of SA on decreasing the effect of salt stress. Peroxidation of lipids was increased significantly in G. max plants that were treated with salt stress from sodium chloride, especially in conditions of high salinity. REFERENCES Abd-EI Baki GK et al (2000) Nitrate reductase in Zea mays L. under salinity. Plant, Cell & Environ: 515-521. Abdul J, Gopi R, Sankar B, Manivannan P, Kishorekumar A, Sridharan R, Panneerselvam R (2007) Studies on germination, seeding vigour,lipid peroxidation and proline metabolism in Catharanthus roseus seedings under salt stress . J Bot 73: 190-195. Afzal I, Basra SMA, Farooq M, Nawaz A (2006) Alleviation of salinity stress in spring wheat by hormonal priming with ABA, salicylic acid and ascorbic acid. Int J Agric Biol 8: 23–28. Ashraf M (1989) The effect of NaCl on water relations, chlorophyll and protein and proline contents of two cultivars of blackgram (Vigna mungo L.). Plant & Soil 119: 205–210. Babaiyan JN, Ziatabar AM (2002) Plants growing in saline and arid lands, (Translation), Mazandaran Univ. Press. p. 407. Barkosky RR, Einhelling FA(1993) Effect of salicylic acid on plant water relationship. J Chem Ecol 19: 237–247. Bates LS, Waldren RP, Teare ID (1973). Rapid determination of free proline for water stress studies .Plant & soil 29: 205-207. Borsani O, Valpuesta V, Botella MA (2001) Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings. Plant Physiol 126: 1024–1030. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantites of

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