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University of Mohaghegh Ardabili, Faculty of Agricultural Sciences, Department of Agronomy
and Plant Breeding, Ardabil, Iran
*Corresponding author’s email address:mosedghi2003@yahoo.com
Received 16 July 2013; accepted 9 November 2013
The experiment was conducted to determine the effect of salicylic acid (SA) on the antioxidant enzymes activity in sunflower as a randomized complete block design with three replications. Treatments were three levels of salicylic acid including 0, 0.1 and
0.2 g L$1. Salicylic acid was sprayed twice on sunflower leaves in all treatments except
the control before flowering. The last leaves of plants were sampled to determine the activity of catalase, peroxidase, and super oxide dismutase and stored in the
laboratory at $80 ◦C. The results showed that all three enzymes activity increased by
SA spraying compared to control. The concentration of 0.2 g L$1 of SA was more
effective on these enzymes. Also among the enzymes, peroxidase showed a greater increase than the rest.
salicylic acid, sunflower, antioxidant enzyme.
Sunflower (Helianthus annuus L.) is a diploid (2n = 34) and annual plant, and
is one of the most important oil3producing plants in the world (Khajepor, 2007). Kernels due to accumulation of fats and lipids are in the risk of reactive oxygen species (ROS), lipid peroxidation and oxidative stress. At least four types of damages present in the cells by oxygen radicals including: 1) disrupt the normal function of mitochondria (Apel & Hirt, 2004; Smirnoff, 2005) 2) Turn off the enzyme3mediated degradation and disruption of enzyme synthesis in the ribosomes 3) damage to the cell membrane and increasing electrolyte leakage (Mc Donald, 1999) 4) genetic damage through the destruction of the nuclear membrane and the misalignment of the DNA (Elder & Osborne, 1993).
Salicylic acid (SA) is a phenolic compound with antioxidant properties, and
involved in the regulation of physiological processes in plants (Mehrabian et al, 2011).
SA affects on catalase and peroxidase enzymes and osmotic regulators such as proline, glycine and betaine and ameliorates the effect of drought stress, heavy metals, heat,
cold and salinity in corn and tomatoes (Janda et al, 1999; Senaratna, 2002; Hussein et
al, 2007). SA is a messenger molecule and a growth regulator in the induction of
tolerance under biotic and abiotic stresses (Li et al, 1998; El3Tayeb, 2005) like fungi
the leaves. It is a thio3glucoside which found in the leaves of Brassicaceae family. When tissues are damaged, glucosinolates were hydrolysed and release some different
compounds that protect plants against pathogens and pests (Popova et al, 1997).
Noreen et al (2009) reported that SA induces the growth in sunflower lines. SA stimulates the antioxidant capacity, so that the leaf peroxidase activity has increased. Positive correlation between leaf peroxidase and super oxide dismutase
activities were observed in sunflower lines with root fresh weight and CO2 exchange
system.
SA increases abscissic acid (ABA) content under stress and maintains the reduction of harmful effects of stress on the plant (Ianovici, 2011) and causes plants to
re3grow (Sakhabutdinova et al, 2000).
The plants treated with salicylic acid increased the activity of enzymes such as catalase, peroxidase, superoxide dismutase, ascorbate peroxidase, glutathione reductase etc (Manochehrifar, 2010).
Popova et al (1997) observed that SA increased anthocyanin and chlorophyll
content in Spirodella polyrriza.
SA affected the processes related to seed quality including protein biosynthesis, seed primary metabolism, antioxidant enzyme production and transport
of seed storage proteins which were increased like seed vigour (Rajjou et al, 2006).
Wen & Liang (1994) reported that SA increased cyanide3resistant respiration and had positive effect on the mitochondrial respiration in potatoes.
Leon et al. (1995) demonstrated that H2O2 decreased the accumulation of SA
and free benzoic acids in tobacco leaves. They suggested that H2O2 triggers the SA
biosynthesis.
Studies show that SA reduces the oxidative damage by maintaining super oxide dismutase activity for the removal of O2 (Rao et al, 1997).
This experiment was designed to investigate the effect of exogenous application of SA on the antioxidant enzymes activity in the leaves of sunflower.
This study was conducted at the research station of the University of Mohaghegh Ardabili with 38° 15' N and 48° 15' E. Climate of area classified as semi3 arid, cold and mean annual precipitation is 400 mm. Soil texture is Silty loam.
Maximum and minimum temperatures were 27.7 and 6.6°C respectively, during the
growing season.
The experiment arranged as randomized complete block design with three replications. Treatments consisted of three levels of salicylic acid including 0, 0.1 and
0.2 g L31 which sprayed twice before flowering.
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plot. Average planting depth was 5 cm. Also, 4 seeds per hole considered to achieve the desired planting density and to adjust the density, thinning operation were performed when the seedlings have two true leaves. The last leaf of a plant in each plot selected randomly to evaluate the antioxidant enzyme activity. Leaf samples were frozen at 380 ºC and catalase, peroxidase and super oxide dismutase activities were measured.
Measurement of catalase (CAT) activity
Catalase (EC: 1.11.1.6) activity assayed according to Chance & Maehly procedure (1955). A 1.5 ml of reaction mixture containing 30 IL of water, 50 IL of buffer Tris3HCl (1 M and pH= 8), 5 mM EDTA, 900 IL of hydrogen peroxide (10 mM) was added to 20 IL supernatant. Absorption was recorded at 240 nm by spectrophotometer for 60 seconds. Catalase activity was measured as absorbance per minute per mg protein.
Measurement of peroxidase (POX) activity
Peroxidase (EC: 1.11.1.7) activity was measured according to the method of
MacAdam et al (1992). In this method, 3 ml of the reaction mixture containing 2.5 ml
sodium phosphate buffer (0.05 mM and pH=7), 30 Ig leaf protein and 20 IL of guaiacol (200 mM) as reducing agent was used. Then, reaction mixture added to cuvette and before assay 10 IL of hydrogen peroxide (30%) as the electron acceptor was added to the reaction mixture. The reaction mixture without hydrogen peroxide was used as a control to calibrate the spectrophotometer to zero. The absorbance was measured at 475 nm for 60 seconds at 25 ° C and enzyme activity was presented as mg protein absorption per minute.
Measurement of super oxide dismutase (SOD) activity
Superoxide dismutase (EC: 1.15.1.1) activity was assayed according to Sen
Gupta et al (1993) method. A 3 ml of reaction mixture containing 0.1 ml of
methionine (200 mM), 0.01 ml of nitroblue tetrazolium (NBT, 2.25 M), 0.1 mM EDTA 3 mM, 1.5 ml of potassium buffer (100 mM), 1 mM of distilled water and 0.05 ml of enzyme extract were poured in a test tube. Reaction began by adding 0.1 ml of riboflavin (60 mM) under fluorescent lamps (two 15 watts for 15 minutes). Reaction stopped by changing the light and covering tubes with black cloth. Absorbance
recorded at 560 nm and the activity of the enzyme reported as units per mg protein.
Data were analysed by SAS9.1 statistical software after normality test and
means were compared using Duncan's multiple range test.
The results showed that the effect of salicylic acid on the antioxidant enzyme
activity was significant (table 1).The highest enzyme activity observed for peroxidase
It is believed that exogenous SA application produces signals that inform the plant is under stress conditions and leads to increase in production of antioxidant enzymes.
Enzymes are biological catalysts that speed up chemical reactions and are even called as vital factors. A cell has thousands of enzymes that are responsible for cell functions. Super oxide dismutase, peroxidase and catalase are antioxidant enzymes. These enzymes interfere during lipid peroxidation by ROS in plants and seeds and inhibit reduction of quality of plants.
Antioxidants can act at different stages of a cascade of oxidative stress and with the ways below reduce ROS damages (Gutterdge & Halliwell, 1990): 1) elimination or reduction of internal oxygen concentration 2) Removal of reactive metal ions 3) deletion of key radicals such as superoxide and hydrogen peroxide 4) Cleaning of the primary free radicals such as hydroxyl, alchoxyl and proxyl 5) Breaking the chain of reactions related to oxidative stress in the early stages 6) Turn off or removal of singlet oxygen.
Super oxide dismutase is the first line of cellular defence against free radicals and converts superoxide radicals into hydrogen peroxide. Catalase converts hydrogen
peroxide to water and oxygen (El3Beltagi et al, 2011).
Peroxidase belongs to oxido3reductase enzymes and contributes in oxidation3 reduction reactions. Oxygen is one of the precursors for oxidase enzymes (Sedghi, 2010).
!"! #$ % &" ' $#& () $$ '( #$ ! "' "' '"* !+& " , # () ("#-"* ( . / ! #$ !0 $ #1 & % !
Mean of squares
CAT POX SOD DF S.O.V 0.5677NS 0.0144NS 0.2177NS 2 BLOCK 39.2744** 45.3077** 35.9744** 2 SA 0.0194 0.4327 0.1194 4 ERORR
* and ** indicating the significant differences at 5 and 1 percent probability levels. DF: Degree of freedom. SA salicylic acid, SOD: Super Oxide Dismutase; POX: Peroxidase; CAT: Catalase.
2 #/+ &"!# #$ / ! $#& () $$ '( #$ ! "' "' '"* # ("3#-"* ( . / ! '("%"( " !0 $ #1 & % !
Mean of squares
CAT POX SOD TRATE 5.8667c 12.3333c 9.7333c CONTROL 9.6667b 15.2667b 13.7000b
0.1 g/l SA
13.1000a
20.0333a
16.6333a
0.2 g/l SA
In each column, means with the same letter are not different significantly at 5% probability level.
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SA can induce the tolerance or resistance mechanisms facing environmental conditions and prepares the plant for avoiding oxidative damage.
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