1. Annals of West University of Timişoara, ser. Biology, 2013, vol XVI(2), pp.67-72
67
EFFECTS OF SALICYLIC ACID ON THE ANTIOXIDANT
ENZYMES ACTIVITY IN SUNFLOWER
Mohammad SEDGHI*, Hajar Khani BASIRI, Raof Seyed SHARIFI
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
ABSTRACT
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.
KEY WORDS: salicylic acid, sunflower, antioxidant enzyme.
INTRODUCTION
Sunflower (Helianthus annuus L.) is a diploid (2n = 34) and annual plant, and
is one of the most important oil-producing 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 enzyme-mediated
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; El-Tayeb, 2005) like fungi
and viruses (Al-Hakimi, 2008) cold (Senaratna, 1998,2003) and drought (Senaratna,
2003). Application of SA on rapeseed increased the concentration of glucosinolate in

2. SEDGHI et al: Effects of salicylic acid on the antioxidant enzymes activity in sunflower
68
the leaves. It is a thio-glucoside 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
re-grow (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 cyanide-resistant 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.
MATERIALS AND METHODS
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 semi-
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 L-1
which sprayed twice before flowering.
Seeds of sunflower (hybrid Aline 122/1 × R-14) were planted in plots with 65
cm inter-row spacing and intra-row spacing was 30 cm. There were 5 rows in each

3. Annals of West University of Timişoara, ser. Biology, 2013, vol XVI(2), pp.67-72
69
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 -80 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 L of water, 50 L of
buffer Tris-HCl (1 M and pH= 8), 5 mM EDTA, 900 L of hydrogen peroxide (10
mM) was added to 20 L 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 g leaf protein and 20 L of
guaiacol (200 mM) as reducing agent was used. Then, reaction mixture added to
cuvette and before assay 10 L 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.
RESULTS AND DISCUSSIONS
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
(20.03) at the concentration of 0.2 g L-1
(table 2) followed by SOD (16.63).

4. SEDGHI et al: Effects of salicylic acid on the antioxidant enzymes activity in sunflower
70
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 (El-Beltagi et al, 2011).
Peroxidase belongs to oxido-reductase enzymes and contributes in oxidation-
reduction reactions. Oxygen is one of the precursors for oxidase enzymes (Sedghi,
2010).
Table 1. Analysis of variance for the effect of salicylic acid spraying on the antioxidant enzymes of sunflower
leaves
Mean of squares
CATPOXSODDFS.O.V
0.5677NS
0.0144NS
0.2177NS
2BLOCK
39.2744**
45.3077**
35.9744**
2SA
0.01940.43270.11944ERORR
* 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.
Table 2. Comparison of means for the effect of salicylic acid on anti-oxidant enzymes activity in sunflower
leaves.
Mean of squares
CATPOXSODTRATE
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.
CONCLUSION
Spraying of SA increased the activity of anti-oxidant enzymes and the
concentration of 0.2 g/l was more effective than others. It is concluded that spraying of

5. Annals of West University of Timişoara, ser. Biology, 2013, vol XVI(2), pp.67-72
71
SA can induce the tolerance or resistance mechanisms facing environmental conditions
and prepares the plant for avoiding oxidative damage.
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