Effect of Potassium Levels on Antioxidant Enzymes and Malondialdehyde Content under Drought Stress in Sunflower (Helianthus annuus L.)

Problem statement: Drought stress as a major adverse factor can lower leaf water potential, leading to reduced turgor and some other responses and ultimately lower crop productivity in arid and semi arid zones. Sunflower is one of the main oil s eed crops in Iran, where drought stress is the most limiting factor. Drought stress tolerance requires the activation of complex metabolic activities including antioxidative pathways, especially Activa ed Oxygen Species (AOS) and scavenging systems within the cells which can contribute to co ntinued growth under drought stress. Approach: To evaluate the effect of limited irrigation systems a nd potassium fertilizer on grain yield, antioxidant enzymes and lipid peroxidation biomarker (MDA), the crop was sown in the Research Farm of College of Agriculture, Islamic Azad University, Karaj Bran ch in 2009. The experimental treatments were arranged as split plots based on a Randomized Compl ete Block Design with three replications. The main plots were allocated to three different irriga t on regimes. The irrigation regimes comprised of: Full Irrigation (IR1), Moderate drought stress (IR 5) and Severe drought stress (IR 2). The subplots were allocated to four potassium chemical fertilizer (Po tassium nitrate) consisting of K 1 = 25, K2 = 50, K3 = 75 and K4 = 100% recommended. Results: Plants under drought stress and potassium levels s howed a significant increase and decrease, respectively, in SOD, CAT and GPX activity and MDA in compared to control plants. In this context, plants with higher levels of potassium showed higher resistance to drought stress conditions and higher yield and dry matter allocation to grain filling process i.e. harvest index. Results of this study s uggested that drought stress leads to production of oxygen radicals, which results in increased lipid p eroxidation (MDA biomarker) and oxidative stress in the plant. Conclusion: The scavenging of AOS by the scavenging system esp ecially by SOD, CAT and GPX was done well and damage to membranes or MD A was controlled at higher levels of potassium.


INTRODUCTION
Adequate water and nutrient supply are important factors affecting optimal plant growth and successful crop production. Water stress is one of the severe limitations of crop growth especially in arid and semiarid regions of the world as it has a vital role in plant growth and development at all growth stages (Shamim et al., 2009).
Nitrogen, phosphorous and potassium are major elements essential for plant growth and development. To date use of chemical fertilizers has been confined mainly to the application of nitrogen and phosphorous and due attention has not been paid to the potassium. Its role is well documented in photosynthesis, increasing enzyme activity, improving synthesis of protein, carbohydrates and fats, translocation of photosynthetic, enabling their ability to resist pests and diseases. Potassium also plays key role in increasing crop yield and improving the quality of produce (Tisdale et al., 1985).
The limitation of CO 2 assimilation in drought stressed plants causes the over-reduction of photosynthetic electron chain. This access of reducing power determines a redirection of photon energy into processes that favor the production of Activated Oxygen Species (AOS), mainly in the photosynthetic (Asada, 1999) and mitochondrial electron transport chains (Moller, 2001).
The aim of this study was to investigate the influence of drought stress and different levels of potassium on antioxidant enzymes activities and on MDA level in sunflower. We hypothesize that potassium could minimize the oxidative effect of the damage following a period of drought stress.

MATERIALS AND METHODS
The experiment was initiated in Research Farm of College of Agriculture, Islamic Azad University, Karaj Branch located in Karaj/Iran during summer 2009. Karaj is classified among the temperate climatic regions in the country with average rainfall of 256 mm per year. The soil physical and chemical characteristic of the experimental site is presented in Table 1.
The experimental treatments were arranged as split plots based on a Randomized Complete Block Design with three replications. The main plots were allocated to three different irrigation regimes. The irrigation regimes comprised of:

Full irrigation (IR 1 ) (control):
The plots in this treatment were irrigated at weekly intervals up to the end of the growing period.

Moderate drought stress (IR 5 ):
The plots in this treatment were irrigated at weekly intervals up to the start of the R5 stage, after this stage irrigation was cut off.

Severe drought stress (IR 2 ):
The plots in this treatment were irrigated at weekly intervals up to the start of the R2 stage, after this stage irrigation was cut off.
Seed bed preparation was done in early autumn. The cultivation rows were 60 cm apart in each plot (at 10 plants m 2 density). Weeds were removed by hand and plots were irrigated as required through the growing season. Sampling: After drought stress treatment, three leaves of each plant were removed. The samples were washed and then frozen in liquid N2 and then stored at -80°C pending biochemical analysis.
Preparation of extracts: Leaf sample was homogenized in a mortar and pestle with 3 mL ice-cold extraction buffer (25 mM sodium phosphate, pH 7.8).
The homogenate was centrifuged at 18000 g for 30 min at 48°C and then supernatant was filtered through paper. The supernatant fraction was used as a crude extract for the assay of enzyme activity. All operations were carried out at 48°C.
Assay of antioxidant enzymes: Catalase activity was estimated by the method of Cakmak and Horst (1991). The reaction mixture contained 100 crude enzyme extract, 500 µL 10 mM H2O2 and 1400 µL 25 mM sodium phosphate buffer. The decrease in the absorbance at 240 nm was recorded for 1 min by spectrophotometer, model Cintra 6 GBC (GBC Scientific Equipment, Dandenong, Victoria, Australia). CAT activity of the extract was expressed as CAT units per milligram of PROT. Superoxide dismutase activity was determined with the reaction mixture contained 100 µL 1 µM riboflavin, 100 µL 12 mM L-methionine, 100 µL 0.1 mM EDTA (pH 7.8), 100 µL 50 mM Na2 CO3 (pH 10.2) and 100 µL 75 µM Nitroblue Tetrazolium (NBT) in 2300 µL 25 mM sodium phosphate buffer (pH 6.8), 200 µL crude enzyme extract in a final volume of 3 mL. SOD activity was assayed by measuring the ability of the enzyme extract to inhibit the photochemical reduction of NBT glass test tubes containing the mixture were illuminated with a fluorescent lamp (120 W); identical tubes that were not illuminated served as blanks. After illumination for 15 min, the absorbance was measured at 560 nm. One unit of SOD was defined as the amount of enzyme activity that was able to inhibit by 50% the photo reduction of NBT to blue Formosan. The SOD activity of the extract was expressed as SOD units per milligram of PROT. Peroxides activity was determined by the oxidation of guaiacol in the presence of H 2 O 2 . The increase in absorbance was recorded at 470 nm (Hernandez et al., 2000). The reaction mixture contained 100 µL crude enzyme, 500 µL H 2 O 2 5 mM, 500 µL guaiacol 28 mM and 1900 µL potassium phosphate buffer 60 mM (pH 6.1). POX activity of the extract was expressed as POX units per mg. Malondialdehyde (MDA) was measured by colorimetric method (Stewart and Bewley, 1980). About 0.5 g of leaf samples were homogenized in 5ml of distilled water. An equal volume of 0.5% Thiobarbituric Acid (TBA) in 20% trichloroacetic acid solution was added and the sample incubated at 95°C for 30 min. The reaction stopped by putting the reaction tubes in the ice bath. The samples then centrifuged at 10000×g for 30 min. The supernatant removed, absorption read at 532 nm and the amount of nonspecific absorption at 600 nm read and subtracted from this value. The amount of MDA present calculated from the extinction coefficient of 155 mM −1 cm −1 .
Enzyme activity and MDA content of samples were recorded with duplication.
Statistical analysis: Data were subjected to analysis of variance. Mean comparison was conducted using the Duncan's Multiple Range Test (DMRT) at 5% level of probability.

RESULTS
The statistical analysis of data showed that there was a significant difference in grain yield production and harvest index due to different irrigation regimes ( Table 2). The highest grain yield of 3.477 t/ha was obtained from control plots while the lowest grain yield of 1.449 t/ha was produced in cut off irrigation in R 2 stage. Alza and Fernandez-Martinez (1997) explained that the significant difference in grain sunflower yield at different limited irrigation regimes was due to different irrigation intervals. The severe reduction of grain yield in irrigation regimes of IR 5 and IR 2 indicated the plant sensitivity to drought stress at different phonological stages. grain production decreased about 36 and 59% in IR 5 and IR 2 treatments compared to control, respectively.
There was significant difference among potassium fertilizer levels on harvest index (not grain yield).
Plants under drought stress showed significant increase in SOD, CAT, GPX activity and MDA in leaves compared to control plants. With increasing of potassium levels at all irrigation regimes, plants decreased the antioxidant enzymes activity and MDA biomarker. In this context, plants with higher levels of antioxidants, either constitutive or induced, have been reported to possess SOD eater resistance to these stress conditions and higher yield and dry matter allocation to filling process i.e., harvest index (Table 3). H 2 O 2 can be removed using the ascorbateglutathion cycle ascorbic acid (ASA)-GSH cycle which APX and SOD are key enzymes in this cycle (Pasternak et al., 2005). In this study, drought stress and low potassium levels led to a significant increase in the GPX compared to the respective control (Table 3).  3.700 ns, * and **: Non significant and significant at the 5 and 1% levels of probability, respectively The diverse responses of GPX, CAT and SOD enzyme activities in the plants subjected to drought conditions suggest that oxidative stress is an important of drought stress (Turk et al., 1980). These results are in agreement with those of Tohidi Moghadam et al. (2009) who have propounded that GPX, SOD and CAT action suggests that the more active ascorbateglutathione cycle may be related to the development of relatively higher drought tolerance in canola. These results may point out that low potassium level provokes antioxidant enzyme responses (Table 3).

DISCUSSION
The result indicated that there was a negative relationship between SOD, CAT and GPX activity and lipid peroxidation or MDA content. In this study, SOD activity increased with increasing drought stress and decreased with increasing potassium levels. When SOD activity was high, AOS, especially superoxide radical, scavenging was done properly and thus, damage to membranes and oxidative stress decreased, leading to the increase of tolerance to oxidative stress. Drought stress increased the superoxide level in cells. If this radical is not scavenged by SOD, it disturbs vital bimolecular (Mittler, 2002). Moreover, it inactivates antioxidant enzymes which are very important for H 2 O 2 scavenging such as catalases (Kono and Fridovich, 1983) and peroxides (Esfandiari et al., 2007). Candan and Tarhan (2003); Martinez et al. (2001); Scandalios (1993); Sen Gupta et al. (1993); Zhao et al. (2006) and Esfandiari et al. (2007) had similar findings and expressed that the increase in SOD activity and decrease in oxidative damage were closely related.
A-biotic stress, such as drought stress cause molecular damage to plant cells either directly or indirectly through the formation of AOS. In the present study, the plants exposed to drought showed a significant increase in CAT and GPX activity and a significant decrease in CAT and GPX activity with increase of potassium levels. The enzymes assayed are scavengers of free radical species. Hydrogen peroxide is converted to oxygen and water by CAT and GPX, which use ascorbate as the hydrogen donor. In conclusion, the results of the present study clearly showed that there was scavenging enzymes in sunflower under different drought stress and high potassium levels.
MDA is regarded as a biomarker for evaluation of lipid peroxidation or damage to plasmalemma and organelle membranes that increases with environmental stresses. Lipid per oxidation is linked to the activity of antioxidant enzymes e.g., with the increase of SOD, APX, GPX and CAT. Oxidative stress tolerance is enhanced and MDA is decreased. In this study, the amount of MDA in plants increased with the increase of drought stress, but it was decreased with increasing of potassium levels. According to this experiment data, the increase in the concentration of MDA in higher drought stress and lower potassium levels due to the low activity of SOD and GPX or CAT was a critical factor for the damage of oxidative stress.

CONCLUSION
The sum of the above results showed that AOS plays a key role in the functionality of sunflower plants subjected to drought stress conditions. For successful scavenging of AOS by a scavenging system, some antioxidant enzymes must cooperate with each other. Moreover, there was a positive relationship between antioxidant enzymes activity such as SOD, CAT and GPX and MDA. The repairing of damage due to oxidative stress, generated by drought stress, was associated with a different antioxidant response in plants grown in optimum potassium conditions.