Effect of Microelements and Selenium on Superoxide Dismutase Enzyme , Malondialdehyde Activity and Grain Yield Maize ( Zea mays L . ) under Water Deficit Stress

This study was carried out to investigate effects of microelements under water deficit stress at different growth stages on antioxidant enzyme alteration, chemical biomarker and grain yield of maize in the years 2007 and 2008. The experiment was conducted in a split plot factorial based on a randomized complete block design with four replications. There were three factors, water deficit stress at different stages of growth as main plot and combinations of selenium (with and without using) and microelements (with and without using) as sub plots. The result indicated that the activity of superoxide dismutase and malondialdehyde content under water deficit stress increased, but grain yield was reduced. The highest grain yield was obtained from optimum irrigation, while in the case of with water deficit stress at V8 stage it was non significant. Selenium spray increased activity of superoxide dismutase enzyme, malondialdehyde content of leaves in V8, R2 and R4 stages and also grain yield. Application of microelements increased the leaves superoxide dismutase enzyme activity and malondialdehyde content. Selenium and microelements spray under water deficit stress conditions during vegetative growth and dough stage increased grain yield in comparison to not spraying elements under water stress conditions. The present results also showed that by using selenium and microelements under water stress can obtain acceptable yield compared to not using these elements.


Introduction
Maize (Zea mays L.) is the world's most widely grown cereal and is the primary staple food in many developing countries (Morris et al., 1999).About 26% of the world's total cultivable land falls in arid and semi-arid areas (Paylore and Greenwell, 1979).The exposure of plants to environmental stresses such as drought, heat, chilling, salt and plant diseases can result in the production of reactive oxygen species (ROS) that contributes to diminished plant performance (Cheong et al., 2003).These abiotic stresses can result in the accumulation of reactive oxygen species (ROS) and other toxic compounds (Xiong et al., 2002).Production of ROS during environmental stress is one of the main causes for decreases in productivity, injury, and death that accompany these stresses in plants.ROS are produced in both unstressed and stressed cells, and in various locations (Upadhyaya and Panda, 2004).In plant cell chloroplasts, mitochondria and peroxisomes, there are important intracellular generators of ROS (Elstner, 1991).ROS play an important role in endonuclease activation and consequent DNA damage (Hagar et al., 1996).Oxidative stress can prevent photosynthetic activity, respiration process and plant growth.Plants are naturally provided by enzymatic and non-enzymatic systems to take care of active oxygen (Giang and Huang, 2001).Photosynthetic plants have a strong demand for combating oxidative stress and other abiotic stresses (Xiong et al., 2002).Plant cells respond defensively to oxidative stress by removing the ROS and maintaining antioxidant defense compounds at levels that reflect ambient environmental conditions (Scandalios, 1997).The mechanisms that act to adjust antioxidant levels afford the protection and include changes in antioxidant gene expression (Cushman et al., 2000).Some well-known antioxidants in plants include glutathione, vitamin C, vitamin E, antioxidant enzymes and carotenoids.Catalases, superoxide dismutase, peroxides, are antioxidant enzymes.Bailly et al. (2000) reported that the content of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and malondialdehyde (MDA) in sunflower seeds will increase under drought stress condition.Within a cell, superoxide dismutase (SOD) constitutes the first line of defense against ROS (Alscher et al., 2002).Malondialdehyde has been known as the end product of peroxidation of membrane lipids.Water deficit stress by increase of generation ROS is responsible for stress-dependent peroxidation of membrane lipids (Upadhyaya and Panda, 2004).
Searching for suitable ameliorants or stress alleviant is one of the tasks of plant biologists.Recent researches daily evaporation from basin pan.Daily evaporation from basin pan was calculated by equation of V = S× H V: Daily evaporation from basin pan S: Area of basin pan H: Rate of evaporated water Also from multiple coefficients of basin pan and evaporated water, the potential of evapotranspiration was obtained.Rate of entered water to every plot was calculated by the following equation (Alizadeh, 2002).
Water volume (m 3 ) = Plot area × Irrigation water efficien-cy× Maize coefficient × Potential of evapotranspiration Maize coefficient is 0.36 up to 0.58 at growth initiation, 0.71 up to 1.13 at growth meddle and 0.98 up to 0.68 at growth final (Farshi et al., 1997).Irrigation water efficiency of 80% was considered.Irrigation type was a siphoning system with polyethylene tubes that was controlled by tube regulator tap.Total of consumptive water during season of corn for every treatment was calculated (Tab.2).
Microelements fertilizer (Biomin 212) treatments were in two levels without and with application.Foliar application at early vegetative stage (V 6 ) and a week before tassling stage at the rate of 2 liter ha -1 .Biomin 212 fertilizer content (%wt/wt) was 2.0 Fe, 2.0 Zn, 0.5 Cu, 1.0 Mn, 0.025 B and 0.5 Mg.
Selenium treatments were at two levels, as well.The first one was selenium application with 20 g ha -1 sodium selenite at early vegetative stage (V 6 ) and second one was control.
have identified several beneficial effects of selenium (Se) in plants although Se is not considered to be required by higher plants.Positive effects of Se on plants mainly exhibited: promoting plant growth, alleviating UV-induced (Yao et al., 2009).Selenium is an element whose deficiency causes the decrease in defense mechanisms of living organisms.Earlier studies have indicated that selenium (Se) maintains antioxidative defence systems and enhances sugar and starch accumulation.Among naturally existing elements seven micro elements including i.e., Fe, Zn, Cu, Mn, B, Mo and chlorine are needed for plant growth.Ionic proms of Fe, Zn, Cu, Mn and Mg as co-factors exist in many antioxidant enzymes.Under deficiency of microelementss antioxidant enzymes activity is reduced, therefore plants sensitivity to environmental stresses increases (Cakmak, 2000).Application of microelement fertilizers can enhance plants resistance to environmental stresses such as drought and salinity (Maleckuti and Sepehri, 2001;Movahed Dehnaviet et al., 2002).Malan et al (1990) reported that drought-tolerant and intolerant maize inbred correlated with Cu/Zn SOD and glutathione reductase activities although higher levels of one enzyme alone apparently did not confer drought tolerance.The relative tolerance of a genotype to water stress as reflected by its comparatively lower lipid peroxidation and higher membrane stability index, chlorophyll and carotenoid contents was closely associated with its antioxidant enzyme system (SOD, APO, GR, CAT).Results of experiments indicated that micronutrient application reduces the effects of environmental stresses such as drought stress and salt stress (Wang et al., 2004).
The objective of this research was to investigate the effects of selenium and microelements spraying under deficit stress on maize yield and biochemical characteristics at different growth stages.

Materials and methods
This study was conducted under water deficit stress with maize (Zea mays L. 'S.C 704').These experiments were carried out in the Agricultural Research Station of Islamic Azad University, Arak Branch, Iran, during the growing season of 2007-2008 and 2008-2009.This site is located at 34˚30'N latitude, 40˚41'E longitude, with the altitude of 1779 m above sea level in Markazi Province in the center of Iran.This region has a semi-arid climate on the base of metrological data in Arak, Iran in 2007-2008and 2008-2009 (Tab. 1) (Tab. 1).
Experimental treatments were irrigation levels in the main plots including full watering as control, water stress in vegetative stage (V 8 ), seed formation or blister stage (R 2 ) and grain pre maturity or dough stage (R 4 ).The sub plots included mix levels of selenium and microelements fertilizer.Water stress was executed by temporary stopping of irrigation at each mentioned stage.Full irrigation (control) was arranged by crop water requirement according to Before seed sowing, multiple soil samples were collected for determination of their physical and chemical properties (Tab.3).According to soil testing the amounts of fertilization were applied including 375 kgha -1 urea, 150 kgha -1 triple super phosphates and 150 kgha -1 potassium sulphate fertilizers.30 percentages of nitrogen (N) and all of phosphorous (P) and potassium (K) fertilizers were applied at planting time.The remaining N fertilizer was broadcasted twice during the vegetative growth as top-dress fertilizer at six-leaf stage and two weeks before tassling stage.
Each experimental plot included four 60 cm distanced rows with 20 cm spacing between plants in rows.Land preparation, including ploughing, was conducted in fall and perpendicular disking in May The length of each row was 6 m and two rows were left uncultivated between the adjacent plots.The hybrid maize was 'S.C 704' .The seeds were sown at 50-60 mm depth on 18 th May, 2007(first year) and 14 th May, 2008 (second year).
Biochemical analysis was based on plant sampling 24 h before irrigation times from five leaves of each plant.The harvested leaves were frizzed and kept at -80°C for further biochemical analysis.Leaf samples (0.2 g) were homogenized in a mortar and pestle with 3 ml ice-cold extraction buffer (25 mM sodium phosphate, pH 7.8).The homogenate plant material was centrifuged by18000 rm -1 for 30 minutes, and then the supernatant was filtered through filter paper.The supernatant fraction was used as a crude extract for the assay of enzyme activity.The biochemical activity was measured based on Misra and Fridorich (1979).The ability to inhibit free radical chain-propagating radical and the auto oxidation of epinephrine (0.25 mM) were determined.Furthermore, SOD standard was used for calibration of activity and lipid peroxidation was determined by estimating the malondialdehyde (MDA) content in 1g fresh leaf material according to Madhava Rao and Sresty (2000).Determination of MDA that is a product of lipid peroxidation was determined by thiobarbituric acid reaction.The concentration of MDA was calculated from the spectroscopy absorbance at 532 nm (correction was done by subtracting the absorbance at 600 nm for unspecific turbidity).
Two years data were combined and the analysis of variance was performed using SAS (SAS Institute Inc, 1997).Each treatment was analyzed in four replications.The means comparisons were estimated by Tukey's multiple tests at %5 probability level.

Results and discussion
According to the combined mean comparison results, the years significantly affected superoxide dismutase enzyme activity and malondialdehyde content.The superoxide dismutase enzyme activity and malondialdehyde leaves content in the second year in all three plant growth and development stages were higher than in the first year (Tab.4, Fig. 1 and 2).
In this study, the highest and lowest superoxide dismutase enzyme activity and malondialdehyde content of leaves was observed in water deficit stress for V 8 and R 4 stage respectively.Under water deficit condition, the ROS generation and antioxidant enzymes activity increased at V 8 and R 4 growth and development stages.In other words, water deficit condition in V 8 , R 2 and R 4 in maize growth and development stages could be increase malondialdehyde level by 21, 23 and 26% respectively compared to control (Tab.4).The simultaneous increase in these enzymes activity contributes to a decrease of injurious effects of H 2 O 2 under drought stress.As plants produce new organs and have high metabolism at V 8 stage, there upon ROS generation will increased under water deficit stress condition.Upadhyaya and Panda (2004)   malondialdehyde has been known as the end product of peroxidation of membrane lipids.Water deficit stress by the increase of generation ROS is responsible for stressdependent peroxidation of membrane lipids.It seems that the enhancement in produce ROS can increase substrate for superoxide dismutase reaction.Increased superoxide dismutase and catalase activities in response to water deficit stress have been reported (Halliwell and Gutteridge, 1989).
Our results showed that drought stress in maize farms could make the significant changes in antioxidant enzymes activity in leave, similar to those reported by Ghorbanli et Tab. 4 Malan et al. (1990), Bailly et al. (2000), Giang and Huang (2001) and Habibi et al. (2004) in sunflower.
Selenium and microelement fertilizers could also increase superoxide dismutase enzyme activity and malondialdehyde content in leaves in V 8 , R 2 and R 4 stages.Selenium role in plants under water stress could increase antioxidant enzymes activities by reducing oxidative conditions and free radicals which have a determinate effect on plant cells.
According to means comparison microelements application was increased antioxidant activities of superoxide dismotase in V 8 , R 2 and R 4 plant growth and development stages by 16.7%, 18.9% and 7.6%.The malondialdehyde content of leaves was increased by 24%, 26.7% and 9.7% respectively compared to control (Tab.4).Rahimizade et al. (2007) reported that SOD activity was increased by 31%, under drought stress in sunflower.Hacisalihoglu et al. (2003) reported that under Zn deficiency stress, activity of Cu/Zn SOD decreases as Zn is directly involved in both gene expression and protein synthesis.Cakmak (2000) reported that Zn deficiency stress may inhibit the activities of a number of antioxidant enzyme.Similarly, Rahmati et al. (2004) found that the activity of SOD, CAT and APX (ascorbate peroxidase) in excess Mn treated cells increased compared to control treatment.
Using selenium in water deficit stress condition increased superoxide dismutase enzyme activity and malondialdehyde content as compared to treatments of not using selenium.The highest amounts of measured traits under water deficit were found in V 8 and R 4 stages (Tab.5).
The effects of three way factors interactions on superoxide dismutase enzyme activity and malondialdehyde V 8 and R 2 stages were significant but, in R 4 stages not significant.In water deficit stress condition in each three stage, the highest malondialdehyde content were observed from treatments of without microelements and without selenium.This might indicate plant sensitivity due to no protection factor under water stress.
It was found in this study that superoxide dismutase enzyme had positive and significant correlation with malondialdehyde content under water deficit stress in vegetative stage (V 8 ), in blister stage (R 2 ) and water deficit stress in dough stage (R 4 ) (Tab. 7).
Tab. 6. Mean comparison of threefold interaction effects of traits An antagonistic interaction was found for selenium and microelements application for grain yield.This may be due to antagonistic interaction between microelements and selenium (Tab.5 and Fig. 4).Interactions effect had also a significant result on grain yield.The highest grain yield up to 9519 kgha -1 was obtained from optimum irrigation conditions, no selenium and microelements application treatment (Tab.6).Selenium and microelements application in vegetative growth stage and dough grain development stages in water deficit conditions could increase maize grain yield significantly (Tab.6).It seems that in the end of the growth and development stage in increased water stress conditions, selenium and microelements find biological activity roles in plant cells.Plant tissue health and protection of cell membranes were formed as a permanent function.In general, by selenium and microelements application under water stress, higher grain yield was obtained.These study results also showed that there is a negative and significant correlation between grain yield with superoxide dismutase enzyme and malondialdehyde content under water deficit in the vegetative stage, blister stage and water deficit in dough grain development stages (Tab.7).This was observed as the decrease of grain yield.

Conclusions
Microelements and selenium application on maize farms could not only increased superoxide dismutase enzyme activity and malondialdehyde content in leaf tissues under normal irrigation but also, under water deficit in V 8 , R 2 and R 4 stages.Grain yield by selenium application was increased.The role of selenium in plants under stress was to increase antioxidant enzyme activities to reduce oxidative conditions or free radical injures which have a determinate effect on plant cells.The effect of microelements acts as a component of superoxide dismutase, catalase, peroxidase and nitrate reductase.Therefore, when plants are deficient in these elements, activities of antioxidant enzymes decrease, thus imposing and increased sensitivity to environmental stresses.Results showed that with microelements in water optimum conditions, and selenium spray under water deficit stress condition an optimum grain yield can be obtained.
The result showed that grain yield was higher in the first year than the second year (Tab.4 and Fig. 3).This seems to be related to better climate condition in the first year (Tab.1).The effect of water deficit stress on grain yield was significant.The highest grain yield was obtained from normal irrigation and water deficit at V 8 stage without significant difference (Tab.4).It seems that under drought stress in maize the antioxidative defence system enhances sugar and starch accumulation in cells.
Grain yield of selenium sprayed-plants was increased as compared with control (Tab.4).It may be due to enhancement of photosynthesis and decrease in leaf senescence, increases assimilate production and transport towards seeds and as a result seed yield (Xue and Hartikaine 2001).
Grain yield of microelements sprayed-plants was decreased by 5% as compared to no microelements application.This may be related to antagonistic interaction of microelements with each other.These results are similar to those reported by Himayatullah and Khan (1998).They reported that in maize, copper application alone and with iron and manganese decreased kernel number per ear, 1000 grain weight and grain yield.
Results of combined analysis two way interactions of water deficit stress and selenium on grain yield was significant.The highest grain yield was obtained from control treatment (without stress and without selenium) which showed significant differences compared to other treatments.Least grain yield under water deficit was obtained from treatment of water deficit stress in grain filling stage and without selenium application (Tab.5).This might indicate plant sensitivity due to the lack of a protection factor under water stress.Xue et al. (2001) reported that selenium has antioxidant properties and under conditions of environmental stress, especially water stress, it can scavenge reactive oxygen.Seppanen et al. (2003) reported that selenium prevents chlorophyll degradation under water stress.
Microelement applications under optimum irrigation increased grain yield compared to non microelement application.But in water deficit stress conditions, grain yield was decreased in all growth and development stages.It seems that, in water deficit conditions due to disorder transmission and increase concentration of microelements plant toxicity was created.

Fig. 4 .
Fig. 3. Effect irrigation and year on grain yield reported that Tab. 3. Chemical and physical properties of farm soil . Mean comparisons for traits at different treatment levels Means followed by similar letters in each column are not significantly different at the 5% level of probability according to Tukey's Test.L 1 : Optimum condition (control) L 2 : Water limitation in V 8 stage.L 3 : Water limitation in blister stage.L 4 : Water limitation in dough stage.Se0: Without selenium.Se1: With selenium.M0: Without microelements.M1: With microelements.SOD: Superoxid dismutase.MAD: Malondialdehyde al. (2004),