Influence of ionizing irradiation on the antioxidant enzymes of Vicia faba L .

288 SOD, but for GST enzyme at a dose level 10.0 kGy. For HeNe laser, with or without different doses (2.5, 5.0, 10.0 and 20.0 kGy) of γ-irradiation, enzyme induction was significantly stimulated and positively correlated with the dose levels of γ-irradiation in combination with the He-Ne treatment. There was a significant increase in the concentration of MDA and this increase was more pronounced at dose level 20.0 kGy (38.2 μmol/g d.w) compared to the control (3.9 μmol/g d.w). Laser treatment by He–Ne laser or diode laser only caused a slight increase (P<0.05) in MDA content (4.4 and 5.08 μmol/g d.w respectively) compared to the control (3.9 μmol/g d.w). H2O2 content significantly increased in all treatments and this increase reached its maximum at dose level 20.0 kGy (36.3 μmol/g d.w) compared to the control (2.3 μmol/g d.w.). On the other hand, He-Ne or diode laser treatments combined with γ-irradiation significantly decreased in comparison with γ-irradiation treatments alone. In the case of glutathione content, there were significant increases by γ-irradiation at dose levels 2.5, 5.0, 10.0 and 20.0 kGy. Furthermore, with a combination of γ-irradiation and He-Ne or diode laser, a marked increase in glutathione content was found and was more pronounced than that of gamma irradiation alone.


INTRODUCTION
Under natural conditions of growth and development, plants are inevitably exposed to different types of stress, which may cause an increased production of active oxygen species (ROS) (Smirnoff, 1993).These include superoxide radicals (O 2 .-),singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (OH .), which cause tissue injury.
Plants have evolved various protective mechanisms to eliminate or reduce ROS.In plant cells, one such protective mechanism is an antioxidant system, composed of both non-enzymatic and enzymatic antioxidants (Foyer et al., 1994b).The capacity of the antioxidant defense system is often increased under stress conditions (Gressel and Galun, 1994), but in most situations the response is moderate (Foyer et al., 1994a).ROS are highly reactive in the absence of any protective mechanism, they can seriously disrupt normal metabolism through oxidative damage to membrane lipids, proteins, pigments and nucleic acids.These ROS are detoxified by the sequential and INFLUENCE OF IONIZING IRRADIATION ON THE ANTIOXIDANT ENZYMES OF VICIA FABA L irradiation and laser irradiation on Vicia faba L. seeds concerning the behavior of antioxidant enzymes as well as lipid peroxidation, hydrogen peroxide and reduced glutathione.

Irradiation treatments
Seed samples of Vicia faba L. Giza 843 were selected for uniform size, packed in polyethylene high density bags and radiated at different dose levels of γ-irradiation (2.5, 5.0, 10.0 and 20.0 kGy) at room temperature (25 ± 1ºC).γ-Irradiation was performed using a Gamma cell 200 apparatus equipped with a 6o Co γ source (dose rate, 6.5 kGy/h) at the National Center for Irradiation Research and Technology, Cairo, Egypt.Packed seed samples without irradiation served as the control.

Laser treatment
After γ-irradiation treatments at different dose levels, seeds were exposed to laser He-Ne (wavelength 632.8 nm, power density 30.0 mW, beam diameter 1.0 mm model Griat, U.S.A) and diode laser (wave length 650.0 nm, power density 30.0 mW, beam diameter 3.0 mm model NILES , A.R.E.), for 5 min, respectively.Laser applications were carried out at in the National Institute of Laser Enhanced Sciences (NILES), Laser Technology Center, Cairo Univ., Giza, Egypt.

Ascorbate peroxidase
. -(Asada, 1992 andWillekens et al., 1997).In biological systems, reduced glutathione (GSH) appears to be one of the most important antioxidants (Noctor and Foyer, 1998).Application of irradiation has revolutionized current research in the field of agricultural science (Bari et al., 2003).
There is compelling evidence which shows that the activities of enzymes involved in reactive oxygen species (ROS) scavenging were altered by several environmental stresses, including gamma irradiation.
The activity and isozyme patterns of POD in Nicotiana debneyi and Nicotiana tabacum, SOD in Nicotiana debneyi, and CAT in Nicotiana tabacum increased in response to gamma irradiation treatment (Wada et al., 1998).Moreover, Chaomei and Yanlin (1993) reported an increase in the activity of POD and CAT with a corresponding decline in the growth of Triticum aestivum plants under higher irradiation doses.Singh et al. (1993) reported that ascorbate peroxidase (APOX; E.C. 1.11. 1. 11) activity in two sugar cane varieties grown under γ-irradiation was increased.Lasers are divided into pulsed and continuous wave lasers described by Markolf (1996).Previous studies showed that He-Ne laser and CO 2 laser (continuous wave) had a positive role in accelerating the plant growth and metabolism (Cai et al., 2000& Qi et al., 2000).In agriculture, forestry and food technology, He-Ne lasers are used which emit red light with a wavelength of 630-650 nm.With these lasers it is possible to treat seeds, seedlings, plants and fields.Laser pretreatments have apparently induced the enzymatic activities, changed thermodynamic parameters, accelerated physiological and biochemistry metabolism and enhanced the growth of seedlings according to Chen et al. (2005).Laser irradiation significantly changed enzyme activities in faba bean seeds, particularly at the initial stage of their germination and led to a rise in the activity of amylolitic enzymes (Podleoeny, 2002).Broad bean (Vicia faba L.) is one of the important legumes in the Middle Eastern countries.It can be used as a dietary item alone or can serve as a potential supplement to cereal diets, especially for the preparation of inexpensive proteinrich food for children (Al-Kaisey et al., 2002).GSH, a disulfide reductant that protects thiols of enzymes, regenerates ascorbate (As) and reacts with singlet oxygen, hydrogen peroxide and hydroxyl radicals.Therefore, Kunert and Foyer (1994) found that, GSH plays a central role in protecting plants from the active oxygen species.Therefore, the objective of this study was to elucidate the relationship between gamma AMINA A. ALY AND HOSSAM E. S. EL-BELTAGI volume of TCA (10%) containing 0.5% (w/v) TBARS or no TBARS as blank, and heated at 95°C for 30 min and then cooled in ice.The reaction product was centrifuged at 12,000×g for 15 min and the supernatant absorbance was measured at 532 and 600 nm.After subtracting the non-specific absorbance (600 nm), the MDA concentration was determined by its molar extinction coefficient of 155 mM -1 cm -1 and the results are expressed as µmol/g d.w.

Assay of hydrogen peroxide content
Hydrogen peroxide was measured by the method described by Capaldi and Taylor (1983) with a slight modification.The ground seeds were extracted by 5% TCA (2.5 ml per 0.5 g powder) with 50 mg active charcoal at 0°C, and centrifuged for 10 min at 15,000xg.The supernatant was collected, neutralized with 4 N KOH to pH 3.6 and used for H 2 O 2 assay.The reaction mixture contained 200 µl of seed extract, 100 µl of 3.4 mM 3-methylbenzothiazoline hydrazone (MBTH).The reaction was initiated by adding 500 µl of horseradish peroxidase solution (90 U per 100 ml) in 0.2 M sodium acetate (pH 3.6).Two minutes later 1400 µl of 1 N HCl was added and the absorbance was read at 630 nm after 15 min.

Determination of reduced glutathione (GSH)
Samples powder were extracted in 6% m-phosphoric acid (pH 2.8) containing 1 mM EDTA.The GSH content of sample extracts was measured by reaction with 5, 5`dithiobis -2-nitrobenzoic (DTNB) reagent to give a compound that absorbed at 412 nm.The GSH concentration was determined from a standard curve according to Silber et al. (1992).

Determination of soluble proteins
Soluble proteins were measured by the Bio-Rad micro assay modification of the Bradford (1976) procedure using crystalline bovine serum albumin as a reference.

Statistical analysis
All analysis were performed in triplicate (n=3).Statistical analysis was done using SPSS (version 10) program.Mean and standard error were descriptive measures of quantitative data using the analysis of variance test (ANOVA) for independent samples.P-values <0.05 were considered significant.

RESULTS AND DISCUSSION
Plants are well known to possess effective enzymatic and non-enzymatic detoxifying systems continually involved in the cellular protection against reactive oxygen species (ROS) coming from both the environment and cell metabolism.Exposure of reaction mixture consisted of enzymatic extract, 50 mmol L -1 sodium phosphate buffer (cold), pH 7, 0.5 mmol L -1 ascorbate, 0.1 mmol L -1 hydrogen peroxide and 0.1 mmol L -1 EDTA, in a 0.3 mL final volume.The reaction started after the hydrogen peroxide addition.The molar extinction coefficient 2.8 mmol -1 cm -1 was used to calculate ascorbate peroxidase activity.Enzyme activity was expressed as unit's mg -1 protein.One unit of enzyme was the amount necessary to decompose 1 µmol of substrate per minute at 25 o C.

Superoxide dismutase (SOD; EC 1.15.1.1)
Superoxide dismutase activity was measured by the photochemical method as described by Beauchamp and Fridovich (1971).One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of nitro blue tetrazolium (NBT) reduction at 560 nm in the presence of riboflavin and light.The reaction mixture contained 45 mM potassium phosphate buffer, pH 7.0, containing 0.1 mM EDTA and 13 mM methionine, 0.17 mM NBT in ethanol, 0.007 mM riboflavin and enzyme aliquot.Blanks were kept in the dark and the others were illuminated for 15 min.One unit of SOD is the amount of extract that gives 50% inhibition to the rate of NBT reduction.

Determination of lipid peroxidation
The lipid peroxidation products were estimated by the formation of thiobarbaturic acid reactive substances (TBARS) and quantified in terms of malondialdhyde (MDA) as described by Haraguchi et al. (1995).200 mg ground seeds were homogenized in 2 ml of 0.1% (w/v) trichloroacetic acid (TCA), followed by centrifugation at 12,000×g for 20 min.The supernatant (1 ml) obtained was mixed with an equal INFLUENCE OF IONIZING IRRADIATION ON THE ANTIOXIDANT ENZYMES OF VICIA FABA L hydrogen peroxide to water and oxygen (Cakmak and Horst, 1991).Moreover Cho et al. (2000) found that the expression patterns of GST, SOD, POD and CAT genes exhibited increased transcripts with γ-irradiation of Nicotiana tabacum.Slooten et al. (1995) reported that the stimulation in SOD activity in response to stresses is possibly attributed to the de-novo synthesis of the enzymatic protein.Laser pretreatment can accelerate the physiological and biochemical metabolism and accordingly enhance the growth of seedlings.Antioxidative enzymes, non-enzymatic antioxidant and lipid peroxidation showed that proper laser pretreatment of wheat seeds may provide protection for wheat from drought stress damage as mentioned by Qi et al. (2008).Furthermore Cai et al. (1994) and Qi et al. (2000) reported that laser irradiation was able to build up the activities of SOD, APOX and CAT.The results obtained in the present study also demonstrate clearly that catalase and SOD protect plant cells from the destructive effects of reactive oxygen species and constitute key components of the cellular antioxidant defense systems.In this concern, Xiong et al. (2009) found that SOD first converts superoxide anions into hydrogen peroxide, which is then removed by catalase.The changes in the enzymatic activity of some winter wheat varieties were also observed by Galova (1996) after the irradiation of kernels by He-Ne laser light.Thus the results of this study suggested Vicia faba L. seeds to different sources of ionizing irradiation had different effects on antioxidant enzymes, peroxidase, ascorbate peroxidase, catalase, superoxide dismutase and glutathione-S-transferase as well as MDA, H 2 O 2 and GSH contents as shown in (Tables 1-2).

Enzymes activities
The activities of POD, APOX, CAT, SOD and GST enzymes were significantly (P<0.05)stimulated and this stimulation reached its maximum at a dose level of 5.0 kGy for enzymes POD, APOX, CAT and SOD, but for GST enzyme at a dose level 10.0 kGy.For He-Ne laser without or with different doses (2.5, 5.0, 10.0 and 20.0 kGy) of γ-irradiation, enzyme induction was significantly (P<0.05)stimulated and positively correlated with the dose levels of γ-irradiation in combination with He-Ne treatment.Significant (P<0.05)stimulation in POD, APOX, CAT, SOD and GST activities were observed and they were more pronounced than in the case of using irradiation alone.The same trend was obtained with the diode laser.The antioxidant enzymes POD, CAT and SOD function as effective quenchers for ROS and their level may also determine the sensitivity of plants to lipid peroxidation (Smirnoff, 1993).Superoxide dismutase is an essential component (coenzyme) in plants and represents an antioxidative defense system since it transforms sample irradiated by gamma irradiation 30.0 kGy.Laser irradiation of Vicia faba L. seeds could raise SOD, POD and CAT activities and could eliminate the accumulation of poisonous free radicals and prevent lipid peroxidation.Qi et al. (2000) reported that the use of the laser pretreatment could be used to protect the cells of the broad bean from UV-B-induced damage.The reduction of MDA concentration, which proceeds through different ways, such as increasing SOD, POD and CAT activity, is very important for plants.

Hydrogen peroxide content
As with lipid peroxidation, H 2 O 2 content significantly (P<0.05)increased in all the treatments (Table 2) and this increase reached its maximum at dose level 20.0 kGy (36.3 µmol/g d.w) compared to control (2.3 µmol/g d.w.).On the other hand, He-Ne or diode laser treatments combined with γ-irradiation significantly decreased (P<0.05) in comparison with γ-irradiation treatments alone.There is a correlation between MDA and H 2 O 2 contents and the scavenging efficiency of the antioxidant enzymes.The concentration of H 2 O 2 increased in various pumpkin tissues such as leaves, petioles and hypocotyls after γ-irradiation (Wi et al., 2007).Moreover Qi et al. (2002) and Han et al. (2002) suggested that He-Ne laser prevent plants from an increase in MDA and H 2 O 2 concentration.
In the present study, the decreasing in H 2 O 2 content in the seed pretreatment with gamma irradiation before laser might be due to the great that APOX, GST and POD activities along with SOD activity play an important protective role in the O 2 .
and H 2 O 2 scavenging process.

Lipid peroxidation content
There was a significant (P<0.05)increase in the concentration of MDA (Table 2), and this increase was more pronounced at dose level 20.0 kGy (38.2 µmol/g d.w) compared to control (3.9 µmol/g d.w).Meanwhile, laser treatment by He-Ne laser or diode laser alone caused a slight increase (P<0.05) in MDA content (4.4 and 5.08 µmol/g d.w respectively) compared to control (3.9 µmol/g d.w).However, γ-irradiation followed by laser (He-Ne laser or diode laser) showed that the increase in MDA concentration was notably lower (P<0.05)than that of γ-irradiation treatments alone (Table 2).Gamma irradiation could lead to an accumulation of free radicals such as O 2 .
and H 2 O 2 which unbalance the system of free radical elimination and thus induce lipid peroxidation.As a result, the cells of plants are injured and the MDA concentration increased (Murphy, 1990 andJoshi et al., 1991).In this concern, Štajner et al. (2007) stated that the use of γ-irradiation at a dose up to 10 kGy did not provoke significant changes in lipid peroxidation and caused a slight increase which could be the consequence of the appropriate antioxidant response.Also Horvathova et al. (2007) reported that irradiation cased a considerable increase in the TBAS values of oregano extract prepared from the increase in the activity of POD, which uses H 2 O 2 as substrate.

Reduced glutathione (GSH) content
In the case of glutathione content, there were significant (P<0.05)increases (Table 2) by γ-irradiation at dose levels 2.5, 5.0, 10.0 and 20.0 kGy.Furthermore, with a combination of γ-irradiation and He-Ne and diode laser resulted in a marked increase (p<0.05) in glutathione content which was more pronounced than that of gamma irradiation alone.GSH is involved in the maintenance of the redox status in plant cells and organs (Horemans et al., 2000).Moreover, Cotter and Sawyer (1961) found that γ-irradiation significantly increased the glutathione content of potato tubers and glutathione was dose dependent.The glutathione pool was slightly affected by solar UV-B exposure.Increased thiol content by UV-B irradiation was reported in many experiments hence, only GSSG content was decreased by UV-B irradiation (Galatro et al., 2001 andCosta et al., 2002).The formation of the reactive oxygen species (ROS) is prevented by an antioxidant system involving the activity of low molecular mass antioxidants (ascorbic acid, glutathione, tocopherols).Enzymes regenerate the reduced forms of antioxidants and antioxidant enzymes such as SOD, POD and CAT (Štajner et al., 2009).Research on the effects of γ-irradiation has been done with little information available on antioxidant systems in plants.This preliminary study may pave way for further investigation on the basis of such variability with biochemical correlation thereof to cast more light on the possible use of such irradiation in biotechnological advancement in the agricultural field.

CONCLUSIONS
In conclusion, this study showed that increases in GST, CAT, SOD and POD activities in Vicia faba L. seeds could be attributed to ionizing irradiation stress.The results may also suggest that the combination of γ-irradiation and laser irradiation is more effective for increasing the ability of the antioxidant enzymes to protect against stress and plays a central protective role in the O 2 .
and H 2 O 2 scavenging process.Indeed, the mechanisms of laser pretreatment effects on seedling physiology are not clear at present and will be investigated in the future.

Table 1 Effect of gamma and laser irradiation on POD, APOX, SOD, CAT and GST activities of Vicia faba L. Treatment POD Unit/mg protein APOX Unit/mg protein CAT (µ mol/mg protein/min) SOD Unit/mg protein GST Unit/mg protein
a,b,c,….. Means within same column followed by different letters are signifi cantly different at P<0.05.Values are means of three replicates (±SE) AMINA A. ALY AND HOSSAM E. S. EL-BELTAGI

Table 2 Effect of gamma and laser irradiation on the MDA, H 2 O 2 and GSH contents of Vicia Faba L.
a,b,c,….. Means within same column followed by different letters are signifi cantly different at P < 0.05.Values are means of three replicates (±SE)