See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/263279203
Article · February 2014
CITATIONS
3
6 authors , including:
Mohamed Mahmoud Aboul Fotouh
Ain Shams University
3 PUBLICATIONS 3 CITATIONS
SEE PROFILE
READS
295
Mamdouh Tag El-Din
Ain Shams University
7 PUBLICATIONS 4 CITATIONS
SEE PROFILE
All content following this page was uploaded by Mamdouh Tag El-Din on 22 June 2014.
The user has requested enhancement of the downloaded file.

Aboul Fotouh, M.M., F.G. Moawad, H.A. El-
Naggar, M.A. Tag El-Din, H.A. Sharaf Eldeen
J. Biol. Chem.
Environ. Sci., 2014,
Vol. 9 (2): 391- 414 www.acepsag.org
Agricultural Biochemistry Dept., Fac. Agric., Ain Shams
Univ., Shoubra Elkhema Cairo, Egypt
In the present investigation, germinated green bean seeds were exposed to UV-C (254 nm) for periods of 7, 15, 30 and 60 min, then grown plants were subjected to saline stress (50 mM NaCl) as a challenge. Tolerance to saline stress was evaluated by determination of fresh weight and dry weight of both shoots and roots. Also, biochemical changes associated with UV-C induced resistance were investigated by determination of lipid peroxidation, proline concentration and antioxidant enzymes activities (SOD, CAT, G-POD and APX) in leaves and roots.
Results showed that plants grown from UV treated seeds were less affected by saline stress which was obvious in increased fresh and dry weights of shoots and roots as compared with control. However, MDA showed higher levels in leaves and roots of plants grown from UV treated seeds. Also, UV seed treatments led to significant increases in proline concentration in leaves and roots of grown plants under saline or non saline conditions which contributed to protection from osmotic shock.
Antioxidant enzymes (SOD, CAT, G-POD and APX) exhibited higher activities in leaves and roots of plants grown from UV treated seeds. Such effect was more pronounced in roots than leaves. Generally, it could be concluded that treatment of green bean seeds with UV-C induced plant tolerance to saline stress via activation of antioxidant system.
Keywords: UV radiation, Saline stress, Lipid peroxidation, Green bean,
Antioxidant system.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
Abbreviations: ROS, Reactive oxygen species; MDA, Malodialdehyde;
SOD, Super oxide dismutase; CAT, Catalase; G-POD,
Guaiacol peroxidase; APX, Ascorbate peroxides, PVP,
Polyvinyl pyrrolidone.
Due to stratospheric ozone depletion which led to a significant increase in UV radiation reaches the Earth’s surface, many studies have focused on the deleterious effect of UV-B on plant growth, development and productivity Bancroft et al.
, (2007); Mahdavian et al.
, (2008) . However, a little number of investigations considered the possible effect of UV on induced resistance in plant against biotic and abiotic stress Teklemariam and Blake , (2003); Stevens et al
.,
(2004 ) . Also, little information is available on the effect of UV-C seed treatment on the enzymatic antioxidant system in plant and its impact on plant stress tolerance. Hence, Challenging plants grown from UV-
C treated seeds with saline stress as an environmental stress was the focal point of our investigation.
Saline stress is one of the most limiting factors on agricultural production. Approximately 20% of the world’s cultivated land and nearly half of all irrigated lands are affected by salinity Zhu, (2001).
When plants are subjected to saline stress, they suffer from both water deficit and ionic toxicity causing deficiency in other nutrients Munns,
(2002).
Saline stress leads to accumulation of ROS (O
2
˙ , superoxide radicals; OH ˙ , hydroxyl radical; H
2
O
2
, hydrogen peroxide and 1 O
2
, singlet oxygen), causing an oxidative stress. ROS are mainly generated in chloroplast and mitochondria due to electron transport processes Chernyak et al
., (2006) ; Schwarzländer et al
., (2009) ;
Xing et al
., (2013).
In normal conditions, ROS are produced as byproducts in metabolic pathways; however there is a balance between ROS and the antioxidant system in the cell. When plants suffer from biotic or abiotic stress such as salinity, ROS are excessively produced which unbalances the cellular redox in favor of oxidized forms, thereby creating oxidative stress that can damage DNA, inactivates enzymes and causes lipid peroxidation Gill and Tuteja, (2010).

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 393
Tolerant plants have evolved mechanisms to overcome these harmful alterations. The results of most studies have shown that resistance to abiotic stresses is usually correlated with a more efficient antioxidant system Meratan et al
., (2008).
Also, proline accumulation is considered as a biomarker for plant tolerance to drought and salt stresses due to its osmoprotective function Huang et al.
, (2009).
Therefore, majority of studies concerned about improvement of saline stress tolerance by activation of the antioxidant system in plant
Gholizadeh and Kohnehrouz, (2010).
Therefore, the present investigation aimed:
1. To evaluate the ability of UV-C seed treatment to induce saline stress tolerance in green bean as a model for salt sensitive plants.
2. To study the effect of different doses of UV-C on the antioxidant systems in green bean and correlate between the biochemical changes and the level of saline stress tolerance.
1. Plant material
Green bean seeds ( Phaseolus vulgaris , L. cv. paulista, dwarf
French bean) were obtained from Bakker brothers, Holland.
2. UV-C Treatment
Green bean seeds were firstly germinated in petri dishes using distilled water, then germinated seeds were exposed to 254 nm UV-C radiation from an artificial source (lamp TUV 15W G158T8 UV-C long life, Holland Philips special) which was situated at 20 cm over the seeds. Durations of exposure time were 7, 15, 30 and 60 min.
After treatment, petri dishes containing the germinated seeds were covered with aluminum foil until sowing to minimize any possible photoreactivation processes Stevens et al ., (1998) and Liu et al .,
(1993) .
3. Growth conditions
Treated seeds were sown in polyethylene bags (4×8×13 cm) containing 700 g acid washed sandy soil (2 seeds/bag) irrigated with
Hoagland nutrient solution for 15 days. After the emergence of the first trifoliate leaf, plants of each group of UV treatments were divided into two subgroups: the first subgroup was irrigated with Hoagland

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS nutrient solution, the second subgroup was irrigated with the nutrient solution containing 50 mM NaCl as a source of saline stress. Plants
(45 days-old) were collected and separated to shoots and roots for determination of growth measurement parameters. Also, other plants were separated to leaves and roots and stored at -20 ºC for biochemical analyses.
4. Plant growth measurements
Shoots and roots of plants were separated and analyzed for fresh weight. Also, dry weight of shoots and roots were determined by drying at 70 ºC to a constant weight and values were calculated as gram per plant.
5. Biochemical analysis
5.1. Lipid peroxidation (LPO)
Lipid peroxidation was estimated by measuring the concentration of malondialdehyde (MDA) according to Heath and
Packer (1968).
The MDA concentration was expressed as nmol
MDA.g
-1 FW using the extinction coefficient of ε =155 mM -1 cm -1 .
5.2 Determination of proline concentration
Proline concentration was measured using a ninhydrin colorimetric method of Troll and Lindsley (1955) as modified by
Petters et al . (1997) . Proline concentration was expressed as μ g proline.g
-1 Fresh weight (FW).
5.3. Enzymes assays
5.3.1. Enzyme extraction
Frozen tissues of roots and leaves were ground using cold mortar and pestle and homogenized with cold sodium phosphate buffer
(100mM, pH= 7) containing 1% (w/v) polyvinylpyrrolidone (PVP) and 0.1 mM EDTA. The extraction ratio was 4 ml extraction buffer for each one gram of plant tissues. The homogenate was centrifuged at
4500 rpm at 4 ºC for 15 min. The supernatant was used for measurement of guaiacol peroxidase (G-POD), catalase (CAT), polyphenol oxidase (PPO), superoxide dismutase (SOD), ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities.
Also, proteins concentration was quantified in the crude extract by the method of Lowry et al
.
(1951) using bovine serum albumin as a standard.

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 395
5.3.2. Superoxide dismutase ( SOD ) assay
Superoxide dismutase (SOD) (EC 1.15.1.1) assay was based on the method described by Beyer and Fridovich (1987).
The enzyme activity was expressed as unit.mg
-1 protein.
5.3.3. Guaiacol peroxidase (G-POD) assay
Guaiacol peroxidase (EC1.11.1.7) activity was quantified by the method of Hammerschmidt et al
.
(1982).
The enzyme activity was expressed as unit.mg
-1 protein.
5.3.4. Catalase (CAT) assay
Catalase (CAT) (EC 1.11.1.6) activity was determined according to the method of Chance and Maehly (1955) as modified by
Cakmak et al . (1993).
CAT activity was measured by monitoring the decrease in absorbance at 240 nm following the decomposition of
H
2
O
2
for 1 min using spectrophotometer (UV-Vis spectrophotometer
UV 9100 B, LabTech). The enzyme activity was expressed as unit.
Mg-1 protein.
5.3.5. Ascorbate peroxidase (APX) assay
Ascorbate peroxidase (APX) (EC 1.11.1.11) activity was measured according to method of Nakano and Asada (1981).
By monitoring the decrease of absorbance at 290 nm following the ascorbate oxidation for 3 min using spectrophotometer (UV-Vis spectrophotometer UV 9100 B, LabTech). The enzyme activity was expressed as unit.mg
-1 protein.
6. Statistical analysis
The data are presented as mean ± SE from three replicates. Data were subjected to two-way ANOVA to study the effect of seed treatment with different doses of UV-C radiation on the grown plants and the effect of saline stress on plants grown from UV-C treated seeds.
Levels of significance are represented by *
P
≤ 0.05, **
P
≤ 0.01,
***
P
≤ 0.001, and ns (not significant). The means were compared by the Duncan’s multiple-range test at P ≤ 0.05. Statistical analyses were performed using SPSS statistical software (IBM SPSS Statistics, version 20).

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
1. Plant growth measurements
Treatment of germinated seeds with UV-C for 30 and 60 min significantly enhanced shoot fresh weight of grown plants by 33.9 and
82.5% respectively (Fig. 1). Also, there was a positive relationship between UV doses and root fresh weight values. The highest root fresh weight (5.913 g) was obtained at the dose of 60 min with an increase of 94.3% over the control (Fig. 1). Under saline conditions, all UV treatments except for 60 min failed to cause a significant difference compared to control in shoot and root fresh weights.
Treatment of germinated seeds with UV-C for 60 min significantly increased shoots and roots fresh weights of grown plants by 50.1 and 86.5% respectively over the stressed control.
Analysis of variance test for dry weight showed significant variations due to UV-C seed treatments, saline stress and their interaction (Table 1). UV treatments improved shoot dry weight with increments in a range of 45.9-61.8% over the control (Fig. 1).
Similarly, root dry weight of plants grown from treated seeds significantly increased. The maximum value (0.480 g) was given at the dose of 60 min. UV treatments alleviated the deleterious effects of saline stress on shoot and root dry weights. The greatest shoot dry weight (0.485 g) was given at the dose of 30 min with an increment of
115.5% over the stressed control (Fig. 1).
Also, it is worth to mention that root dry weight of plants grown from UV treated seeds enhanced by 74% at the dose of 15 min as compared with stressed control (Fig. 1).
According to the obtained results it could be concluded that seed treatment with UV-C improvd plant growth under stressful and non stressful conditions. These results are consistent with results of
Kacharava et al
. (2009) in kidney bean where authors found that plant height, fresh weight and dry weight increased as a result of 60 and 90 min UV pre-sowing treatment for seeds.

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 397
Table 1. Level of significance of two-way ANOVA test for growth measurements of shoot and root of
Phaseolus vulgaris
at UV, salinity treatments and their interaction.
Figure 1. Effect of green bean seeds treatment with UV-C on fresh and dry weight (g) of plants grown under saline conditions (means ± SE). Different letters refer to significant differences at (P ≤ 0.05).

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
Also, similar results were observed in cabbage Brown et el
.,
(2001) , where the treatment of dry seeds with a hormetic dose of UV-
C resulted in the heaviest cabbage heads as compared to their control.
These results also appear to support the UV hormesis suggested in the model of Luckey (1980) , who proposed that application of the optimal doses of UV-C should cause repairable damage or DNA lesions and this slight shock should activate the repair mechanisms for radiation induced DNA damage.
Such effect may stimulate vital processes inside the cells like over compensation of normal metabolic processes, stimulation of the basic physiological functions that had previously been repressed and directing the homeostasis of the plant to a positive change which leads to growth augmentation.
2. Biochemical analysis
2.1. Lipid peroxidation (LPO)
Under non saline conditions, MDA concentration in leaves of green bean was not significantly affected by UV treatment for seeds.
Among all doses, 15 and 30 min UV treatments elevated MDA level in leaves with increments of 19.5 and 48.1% over the control respectively (Fig. 2). However, it seems that the growth of plants in this group was not affected by the oxidative damage caused by UV treatment. Also, no significant increase in root MDA levels was detected as a result of seed irradiation with UV-C except for the dose of 15 min. The increment was 57.7% over the control (Fig. 2).
In addition, it is noteworthy that MDA concentration was significantly reduced in roots at the dose of 7 min (Fig. 2).
Interestingly, MDA levels in roots and leaves contrasted with fresh and dry weight results in plants at the dose of 30 min for leaves and dose of 15 min for roots. This observation was previously found in leaves of Arabidopsis challenged with the fungus Botrytis when MDA levels were correlated with the survival of tissues. It was suggested that trienoic fatty acids (the major precursors of MDA) contribute to
ROS control via non enzymatic oxidation Mène-Saffranè et al
.,
(2009).
Furthermore, MDA is not necessarily generated as a result of lipid peroxidation. It was observed that Arabidopsis mutants lacking trienoic fatty acids (TFAs) contained 25% of MDA pool present in wild type plants, referring to existence of other origins for MDA
Weber et al ., (2004).

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 399
Under saline conditions, no significant increase in leaf MDA concentration was detected in almost all treatments as a result of salt stress. However, plants grown from UV treated seeds for 60 min showed a considerable elevation in leaf MDA levels as compared to unstressed plants of the same treatment. On the contrary, MDA levels were significantly affected by UV seed treatment (Table 2 and Fig. 2). A gradual increase was observed in leaves of plants grown from UV treated seeds as compared with stressed control.
Contrary to leaves, the effect of saline stress on MDA concentration was more pronounced in roots. Saline stress resulted in significant increases in MDA levels as compared to unstressed plants (Table 2 and
Fig. 2). Also, treatment of germinated seeds with doses of 30 and 60 min significantly increased MDA concentration as compared with stressed control with increments of 44.2 and 40.9% respectively (Fig. 2).
Table 2. Level of significance of two-way ANOVA test for Lipid peroxidation (LPO) and proline concentration in leaves and roots of
Phaseolus vulgaris at UV, salinity treatments and their interaction.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
The results together indicated that treatment of seeds with UV-C did not reduce MDA concentration in leaves and roots of green bean under saline conditions. However, rise in MDA levels did not reflect the enhanced growth of plants grown from UV treated seeds under saline conditions.
MDA is a secondary product of lipid peroxidation, therefore it can be used as an index for the oxidative damage accompanied by biotic and abiotic stress in plant cells Hodges et al
., (1999).
In addition, MDA is a highly reactive molecule and reacts rapidly with
DNA and proteins, causing modification to their structures
Refsgaard, et al
., 2000; Marnett, (2002).
However, it was found that the expression of 26 genes was altered as a response to application of exogenous MDA. The expression of these genes was directed towards the genes that have roles in abiotic stress like SODFE gene which encodes for SOD Weber et al
., (2004).
Such response suggests an important role that MDA plays under environmental stresses.
Moreover, it was stated by more than evidence that MDA exists also in healthy tissues. For instance, Mène-Saffranè et al
.
(2007) found that part of MDA pool was concentrated in meristematic cells
(cell division region) of the root of Arabidopsis seedlings and the majority of this MDA was not derived from TFAs. Thus, the enhanced growth in spite of high MDA levels in the present investigation may be attributed to the vital role of MDA in directing cells to induce survival programs.
2.2. Proline concentration
Treatment of green bean seeds with UV-C did not affect markedly proline levels in leaves (Fig. 3). Also, proline levels in roots were not influnced by treatment of germinated seeds with UV-C.
Under salt stress, foliar proline concentration increased in all treatments as compared to unstressed plants in the same group. For instance, the increment in stressed control plants was 51.7% over the unstressed plants (Fig. 3).
Plants grown from UV treated seeds exhibited higher proline concentrations as compared with stressed controls. All UV treatments except for 60 min attained a significant increase over the stressed control. The maximum increment (47.5 %) was observed at the dose of 15 followed by doses of 7 and 30 min with increments of 46.2 and
43.0 % respectively over the stressed control (Fig. 3).

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 401
Similarly, roots responded to salt stress by increasing proline concentration in control and all treatments. Nevertheless, UV seed treatment at the doses of 15 and 60 min drastically elevated proline concentration as compared with stressed control plants. The increments were 130.4 and 107.5% respectively (Fig. 3).
Generally, it could be concluded that UV seed treatment nonsignificantly increased proline levels in most cases in both leaves and roots. Individual treatment of saline stress led to a significant increase in proline in leaves and roots. The effect of interaction between UV seed treatment and saline stress on increasing proline levels was higher than individual treatment of both. These results are in agreement with Rajabbeigi et al
.
(2013) as they found that UV treated plants showed no significant change in proline contents, while proline levels dramatically increased as a result of drought stress. Moreover, the combined effect of UV and drought stresses revealed a pronounced increase in proline content more than the individual treatments of both stresses. In conclusion, seed treatment with UV enhanced plant tolerance against saline stress by increasing proline synthesis in leaves and roots.
Apart of being an osmoregulator Ambikapathy et al
., (2002), proline also protects proteins from damage and enhances enzymes activities Sharma and Dubey, (2005); Mishra and Dubey, (2006);
Huang et al ., (2009) . Moreover, proline was found to act as a ROS scavenger Siripornadulsil et al., (2002).
In addition, high levels of proline synthesis maintains NADP + /NADPH ratio in the cell as it is in the normal conditions Hare and Cress, (1997).

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
2.3. Enzymes assays
2.3.1. SOD activity
No significant change was observed between SOD activities in leaves of control and plants grown from UV-C treated seeds at the dose of 7 min. Also, treatment of germinated seeds with the dose of 15 min was effective to enhance SOD activity by 20.6%. In contrast, doses of 30 and 60 min significantly reduced SOD activity by 23.4 and 58% respectively (Fig. 4). In roots, exposing germinated seeds to
UV-C did not affect SOD levels at the doses of 7,30 and 60 min, whereas 15 min treatment led to a considerable decrease by 21.2%
(Fig. 4).
Under saline conditions, SOD activities exhibited a gradual decrease by increasing UV doses in leaves. Also, Fig. 4 shows that saline stress had an inhibitory effect on SOD in roots of control plants and plants grown from UV treated seeds. Furthermore, SOD levels in roots of plants grown from UV treated seeds were lower than control except for plants grown from treated seeds at the dose of 7 as there was no significant change between this treatment and control. Also, it is worth mentioning that similar to leaves, SOD activity in roots of plants grown from treated seeds decreased gradually with increasing doses of UV under saline conditions (Fig. 4). Such observation may explain the elevation of MDA levels in plants at higher doses of UV-
C.
Moreover, according to to Rybus-Zaj ą c and Kubi ś (2010), it was expected that plants grown from UV treated seeds should elevate
SOD in leaves and roots of grown plants. However, the effect of UV-
C treatment on SOD activity varied (from inhibition to induction) with different doses. Also, the impact of UV treatment on SOD levels varied in different organs. Therefore, it could be observed that SOD activity in leaves was higher than control at the dose of 15 min, while there were no significant differences between SOD activities in control roots and the roots of plants grown from UV treated seeds at the doses of 7, 30 and 60 min. In addition, UV treatment at the dose of
60 min suppressed SOD in leaves of plants grown from germinated a seed which was not observed in the roots.

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 403
The effect of UV on SOD activity in plants was inspected by many investigators and the results were different. Strid et al
.
(1994) noticed that chloroplastic SOD transcripts were reduced as a result of exposure of pea seedlings to UV. Also, Chen (2009) demonstrated that elevated UV enhanced MDA concentrations and reduced SOD activities in
Isatis indigotica
seedlings. On the other hand, SOD levels were increased in
Abelmoschus esculentus
L. (Okra) plants as after exposure to UV-B Kumari et al ., (2009).
Other studies showed that UV treatment did not change SOD activity Vyšniauskien ė and Ran č elien ė , (2014).
Moreover, factors like temperature and the seedlings age should be taken in consideration in interpreting the effect of UV on SOD activity in plants Takeuchi et al
., (1996).
Table 3. Level of significance of two-way ANOVA test for antioxidant enzymes activities in leaves and roots of Phaseolus vulgaris
at UV, salinity treatments and their interaction.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
2.3.2. CAT activity
Treatment of green bean seeds with UV-C led to a significant reduction in CAT activity in leaves of plants at all treatments. The reductions were in a range of 11.6% at the dose of 15 min treatment for germinated seeds to 43.5% at the dose of 60 min (Fig. 5).
In contrast, opposite results were observed in roots. UV-C seed treatment induced CAT in roots of grown plants at the doses of 7, 15 and 60 min. The greatest increment (3.5-fold increase) was observed at the dose of 60 min followed by dose of 15 min which led to a 1.6fold increase over the control (Fig. 5).
Saline stress stimulated CAT activity in control leaves with increment of 23.2% over unstressed plants. Nevertheless, it was observed that CAT activity showed a noticeable increase over stressed control at the doses of 15 and 30 min under saline conditions. The increments were 49.4 and 42.4% for doses of 15 and 30 min respectively (Fig. 5). Similarly, all UV treatments attained a significant increase over stressed control in CAT activity in roots under saline conditions. The increments were in a range of 58.3 to
258.3%.
According to the obtained results, it could be concluded that the effect of UV-C treatment on CAT induction was more pronounced in roots. In addition, CAT was repressed in leaves of plants grown from
UV treated seeds. These results are concomitant with decreased SOD

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 405 activities. The balance between SOD and CAT or APX activity is crucial for conserving steady state levels of H
2
O
2
Mittler, (2002).
Also, it should be noticed that there is integration between APX and CAT activities particularly at the dose of 7 min as APX compensated for the CAT inhibition. Such observation was previously reported in Trigonella after exposure to irradiation stress Al-Rumaih and Al-Rumaih, (2008).
In the presnt study, seed treatment with UV-C led to a dramatic increase in CAT activity under saline conditions in roots. Yasar et al .
(2008) reported that CAT activity increased in salt-tolerant cultivar of
Phaseolus vulgaris
under salt stress conditions with a rate greater than salt-sensitive cultivar. Thus, increased CAT activity in roots of plants grown from UV treated seeds provided better protection from salt stress.
2.3.3. G-POD activity
Treatment of germinated seeds led to significant reductions in leaves of grown plants under non saline conditions at the doses of 7,
15, 30 min. By contrast, UV treatment at the dose of 60 min significantly increased G-POD activity by 59.1% over the control
(Fig. 6). Furthermore, G-POD showed 1.1-fold increase in roots of plants grown from UV treated seeds at the dose of 60 min when compared with control (Fig. 6).
Under saline conditions, G-POD activities in leaves were elevated at all treatments except for dose of 60 min. Moreover, G-
POD activity fluctuated with increasing doses of UV and the highest activities were observed at the doses of 7 and 30 min with increments of 22.6 and 8.6% respectively (Fig. 6).
Also, saline stress elevated G-POD in roots of plants at all treatments. Similarly, UV treatment of germinated seeds raised G-
POD in roots at all doses when compared with stressed control. The increments ranged from 31.9% at the dose of 7 min to 384.1% at the dose of 15 min (Fig. 6).

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
In the present investigation, although plants grown from UV treated seeds exhibited lower G-POD activities under non saline conditions in leaves and roots, their response to salt stress with regard to G-POD activity was higher than control. Such effect may be attributed to priming effect which makes plants response to abiotic stress more rapid and stronger.
One of the hypotheses proposed to explain priming is accumulation of inactive proteins that play important roles in signal amplification then challenging with abiotic stress should activate these dormant signaling proteins, leading to signal amplification initiation more rapidly and robustly than unprimed plants Conrath, (2011).
In this context, Kubi ś and Rybus-Zaj ą c (2008) reported that UVpretreated cucumber seedlings exhibited higher antioxidant enzyme activities under drought stress and suggested that one of the stresses reduced the adverse effects of the other. Also, results show that roots were affected by UV seed irradiation more than leaves under saline stress.
2.3.4. APX activity
Exposure of germinated seeds to UV-C for 7 min led to a significant increase in APX activity in leaves of grown plants with an increment of 7.8% over the control. By contrast, APX activity decreased gradually with increasing UV doses, exhibiting lower activities than control (Fig. 7). In roots, opposite results were observed as APX exhibited higher activities at the doses of 7, 30 and 60 min

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 407 when compared with control. The increments were 69.2, 19.5 and
73.4% over the control respectively (Fig.7).
Also, APX showed higher activity in leaves as response to saline stress in control plants. By contrast, APX activity was negatively affected by saline stress in leaves of plants grown from UV treated seeds at the doses of 7 and 15 min, whereas opposite results were noticed at the doses of 30 and 60 min. Generally, treatment of seeds with UV led to a gradual decline in APX activity in leaves under saline conditions when compared with control (Fig. 7).
In roots, saline stress resulted in elevating APX activity in control and most treatments. Also, application of UV-C on germinated seeds was effective in inducing APX activity in roots under saline conditions. The increments were significant in comparison with stressed control at the doses of 15, 30 and 60 min. The maximum activity (0.328 Unit.mg
-1 protein) was given at the dose of 15 min with an increment of 43.2% over the control (Fig. 7).
Induction of APX as a result of UV treatment in leaves at the dose of 7 min and in roots at almost all doses under non saline conditions is consistent with results of Zacchini and de Agazio
(2004) who reported that exposure of tobacco callus to UV-C led to an enhancement in antioxidant enzymes (APX and G-POD) after 24 h from the treatment. On the contrary, Nottaris et al.
(1997) reported that APX activity decreased in sugar beet calli after exposure to UV-C which agrees with the observed results of APX activity at doses higher than 7 min under saline or non saline conditions.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
In this context, Feng et al.
(2009) declared that plant responses to enhanced UV with regard to APX levels depend on genetic backgrounds and developmental stage.
Under saline conditions, APX levels increased in both control and plants grown from UV treated seeds. However, the increment was higher in control leaves, whereas roots of plants grown from UV treated seeds exhibited greater APX levels. However, it was found that
CAT and G-POD activities compensated for APX activity decrements in leaves under saline conditions.
According to the obtained results, it could be concluded that the exposure of green bean seeds to UV-C enhanced their tolerance to saline stress via activation of the antioxidant system and accumulation of proline in leaves and roots which lessened the effect of osmotic stress. However, this effect varied among the applied doses. Also, it could be concluded that the optimal dose for green bean seeds was at
60 min which achieved the best growth in shoots and roots of grown plants under saline or non saline conditions.
Al-Rumaih, M. M. and M. M. Al-Rumaih (2008). Influence of ionizing radiation on antioxidant enzymes in three species of
Trigonella. American J. Environ. Sci. 4 (2), 151-156.
Ambikapathy, J.; J. S. Marshall; C. H. Hocart and A. R. Hardahm
(2002). The role of proline in osmoregulation in Phytophthora nicotianae. Fungal genetics and biology 35(3), 287-299.
Bancroft, B. A.; N. J. Baker and A. R. Blaustein (2007). Effects of
UVB radiation on marine and freshwater organisms: a synthesis through meta-analysis.
Ecol Lett. 10(4), 332-45.
Beyer, W.F. and I. Fridovich (1987) . Assaying for superoxide dismutase activity: some large consequences of minor changes in condition. Anal. Biochem. 161, 559–566.
Brown, J. E.; T. Y. Lu, C. Stevens, V. A. Khan, J.Y. Lu, C.L.
Wilson, D.J. Collins, M. A. Wilson, E.C.K. Igwegbe, E. Chalutz and S. Droby (2001). The effect of low dose ultraviolet light-C seed treatment on induced resistance in cabbage to black rot
(
Xanthomonas campestris pv. campestris
). Crop Protection 20,
873-883.

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 409
Cakmak, I.; D. Strbac and H. Marchner (1993) . Activities of hydrogen peroxide-scavenging enzymes in germinated wheat seeds. J. Exp. Bot. 44, 127-132.
Chance, B. and A.C. Maehly (1955) . Assay of catalase and peroxidases. Method Enzymol. 2, 764-775.
Chen, Y. (2009). Response of antioxidant defense system to laser radiation apical meristem of Isatis indigotica seedlings exposed to
UV-B.
Plant Signaling and Behavior 4(7), 571-573.
Chernyak, B. V.; D. S. Izyumov; K. G. Lyamzaev; A. A.
Pashkovskaya; O. Y. Pletjushkina; Y. N. Antonenko; D.V.
Sakharov; K. W. A. Wirtz and V.P. Skulachev (2006).
Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress . Biochim. Biophys. Acta 1757, 525-
534.
Conrath, U. (2011). Molecular aspects of defence priming. Trends
Plant Sci. 16(10), 524-531.
Feng, Y.; Y. Zhu; Y. Zu; S.C. Yang; J. J. Chen and Y. Li (2009).
Physiological differences and their genetic backgrounds of
Erigeron breviscapus populations under enhanced UV-B radiation.
Yingyong Shengtai Xuebao 20(12), 2935-2942.
Gholizadeh, A. and B.B. Kohnehrouz (2010). Activation of phenylalanine ammonia lyase as a key component of the antioxidative system of salt-challenged maize leaves. Braz. J. Plant
Physiol. 22(4), 217-223.
Gill, S.S. and N. Tuteja (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants.
Plant Physiol. Biochem. 48, 909-930.
Hammerschmidt, R.; E. Nuckles and J. Kuc (1982). Association of enhanced peroxidase activity with induced systemic resistance of cucumber to
Colletotrichum lagenarium
.
Physiol. Plant Pathol. 20,
73-82.
Hare, P. D. and W. A. Cress (1997). Metabolic implications of stressinduced proline accumulation in plants. Plant Growth Regul.
21:
79–102.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
Heath, R.L. and L. Packer (1968).
Photoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiometry of fatty acids peroxidation. Arch. Biochem. Biophys. 125,189-198.
Hodges, D.M.; J.M. DeLong; C.F. Forney and R. K. Prange
(1999). Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604-
611.
Huang, Y.; Z. Bie; Z. Liu; A. Zhen and W. Wang (2009). Protective role of proline against salt stress is partially related to the improvement of water status and peroxidase enzyme activity in cucumber. Soil Science and Plant Nutrition 55, 698-704.
Kacharava, N.; S. Chanishvili, G. Badridze, E. Chkhubianishvili and N. Janukashvili (2009). Effect of seed irradiation on the content of antioxidants in leaves of Kidney bean, Cabbage and
Beet cultivars. Australian Journal of Crop Science 3(3), 137-145.
Kubi ś , J. and M. Rybus-Zaj ą c (2008). Drought and excess UV-B irradiation differentially alter the antioxidant system in cucumber leaves. Acta Biol. Cracoviensia Ser. 50(2), 35-41.
Kumari, R.; S. Singh and S. B. Agrawal (2009).
Combined effects of Psoralens and ultraviolet-B on growth, pigmentation and biochemical parameters of Abelmoschuses culentus L. Ecotoxicol.
Environ. Saf. 72, 1129–1136.
Liu, J.; C. Stevens; V. A. Khan; J. Y. Lu; C. L. Wilson; O.
Adeyeye; M. K. Kabwe; P. L. Pusey; E. Chalutz; T. Sultana and S. Droby (1993) . Application of ultraviolet-C light on storage rots and ripening of tomatoes. J. Food Prot. 56, 868-872.
Lowry D. H.; N. J. Rosebrough; A. L. Farr and R. J. Randall
(1951).
Protein measurement with the Folin phenol reagent, J. Biol.
Chem. 193, 265–275.
Luckey, T. D. (1980). Hormesis with ionizing radiation.
CRC Press,
Boca Raton.
Mahdavian, K.; M. Ghorbanli and K. M. Kalantari (2008).
The
Effects of ultraviolet radiation on the contents of chlorophyll, flavonoid, anthocyanin and proline in
Capsicum annuum
L.
Turk J.
Bot. 32, 25-33.

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 411
Marnett, L.J. (2002). Oxy radicals, lipid peroxidation and DNA damage. Toxicology 181-182, 219-222.
Mène-Saffranè, L.; C. Davoine; S. Stolz; P. Majcherczyk and E.E.
Farmer (2007). Genetic removal of tri-unsaturated fatty acids suppresses developmental and molecular phenotypes of an
Arabidopsis tocopherol deficient mutant. Whole-body mapping of malondialdehyde pools in a complex eukaryote. J. Biol. Chem. 282
(49), 35749–35756.
Mène-Saffranè, L.; L. Dubugnon; A. Chételat; S. Stolz; C.
Gouhier-Darimont and E.E. Farmer (2009). Nonenzymatic oxidation of trienoic fatty acids contributes to reactive oxygen species management in
Arabidopsis
.
J. Biol. Chem. 284(3), 1702–
1708.
Meratan, A. A.; S.M. Ghaffari and V. Niknam (2008). Effects of salinity on growth, proteins and antioxidant enzymes in three
Acanthophyllum species of different ploidy levels. JSUT 33(4),1-8.
Mishra, S. and R. S. Dubey (2006). Inhibition of ribonuclease and protease activities in arsenic exposed rice seedlings: role of proline as enzyme protectant. J. Plant Physiol. 163(9), 927-936.
Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance.
Trends Plant Sci. 7(9), 405-410.
Munns, R. (2002).
Comparative physiology of salt and water stress.
Plant Cell Environ. 25(2), 239-250.
Nakano, Y. and K. Asada (1981) . Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chlroplasts. Plant Cell
Physiol. 22, 867–880.
Nottaris, D.; P. Crespi; H. Greppin and C. Penel (1997).
Effect of
UV-C on two cell lines from sugarbeet. Arch. Sci. 50, 223–232.
Petters, W.; M. Piepenbrock; B. Lenz and J.M. Schmitt (1997).
Cytokinine as a negative effector of phosphoenolpyruvate carboxylase induction in
Mesembryanthemum crystallinum
.
J.
Plant Physiol. 151,362-367.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
Rajabbeigi, E.; I. Eichholz; N. Beesk; C. Ulrichs; L. W. Kroh; S.
Rohn and S. Huyskens-Keil (2013). Interaction of drought stress and UV-B radiation – impact on biomass production and flavonoid metabolism in lettuce (
Lactuca sativa
L.). J. Appl. Bot. Food Qual.
86, 190 – 197.
Refsgaard, H. H. F.; L. Tsai and E. R. Stadtman (2000).
Modifications of proteins by polyunsaturated fatty acid peroxidation products. PNAS 97(2), 611-616.
Rybus-Zaj ą c, M. and J. Kubi ś (2010). Effect of UV-B radiation on antioxidative enzyme activity in cucumber cotyledons.
Acta Biol.
Cracoviensia Ser. 52(2), 97-102.
Sharma, P. and R. S. Dubey (2005). Modulation of nitrate reductase activity in rice seedlings under aluminium toxicity and water stress: role of osmolytes as enzyme protectant.
J. Plant Physiol.
162(8), 854-864.
Schwarzländer, M.; M. D. Fricker and L. J. Sweetlove (2009).
Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge. Biochim. et Biophys. Acta 1787(5), 468-
475.
Siripornadulsil, S.; S. Traina; D.P.S. Verma and R.T. Sayre
(2002). Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. The Plant Cell 14,
2837–2847.
Stevens, C.; V. A. Khan; J. Y. Lu; C. L. Wilson; P. L. Pusey; M.
K. Kabwe; E. C. K. Igwegbe; E. Chalutz and S. Droby (1998) .
The germicidal and hormetic effects of UV-C light on reducing brown rot disease and yeast microflora of peaches. Crop Prot. 17,
75-84.
Stevens, C.; J. Liu; V. A. Khan; J. Y. Lu; M. K. Kabwea; C. L.
Wilson; E. C. K. Igwegbe; E. Chalutz and S. Droby (2004).
The effects of low-dose ultraviolet light-C treatment on polygalacturonase activity, delay ripening and Rhizopus soft rot development of tomatoes. Crop Protection 23, 551–554.

J. Biol. Chem. Environ. Sci., 2014, 9 (2), 391-414 413
Strid, A.; W. S. Chow and J. M. Anderson (1994).
UV-B damage and protection at the molecular level in plants. Photosynth. Res.
39, 475–489.
Takeuchi, Y.; H. Kubo; H. Kasahara and T. Sakaki (1996).
Adaptive alterations in the activities of scavengers of active oxygen in cucumber cotyledons irradiated with UV-B.
J. Plant
Physiol.
147, 589–592.
Teklemariam, T. A. and T. J. Blake (2003).
Effects of UVB preconditioning on heat tolerance of cucumber (Cucumis sativus
L.). Environ. Exp. Bot. 50, 169-182.
Troll, W. and J. Lindsley (1955). A photometric method for the determination of proline.
J. Biol. Chem. 215, 655–660.
Vyšniauskien ė , R. and V. Ran č elien ė (2014). Effect of UV-B radiation on growth and antioxidative enzymes activity in
Lithuanian potato (
Solanum tuberosum
L.) cultivars. Zemdirbyste-
Agric. 101(1), 51–56.
Weber, H.; A. Chételat; P. Reymond and E.E. Farmer (2004).
Selective and powerful stress gene expression in Arabidopsis in response to malondialdehyde. Plant J. 37, 877–888.
Xing, F.; Z. Li; A. Sun and D. Xing (2013). Reactive oxygen species promote chloroplast dysfunction and salicylic acid accumulation in fumonisin B1-induced cell death. FEBS Lett. 587, 2164-2172.
Yasar, F.; S. Ellialtioglu and K. Yildiz (2008). Effect of salt stress on antioxidant defense systems, lipid peroxidation, and chlorophyll content in green bean. Russian J. Plant Physiol.
55(6), 782-786.
Zacchini, M. and M. de Agazio (2004). Spread of oxidative damage and antioxidative response through cell layers of tobacco callus after UV-C treatment. Plant Physiol. Biochem. 42, 445-450.
Zhu, J. K. (2001).
Plant salt tolerance. Trends Plant Sci. 2, 66–71.

INFLUENCE OF SEED TREATMENT WITH UV-C ON SALINE STRESS
– رﺎﺠﻨﻟا ﻰﻠﻋ ىﺪﻤﺣ – ضﻮﻌﻣ ىﺪﻨﺟ قورﺎﻓ – حﻮﺘﻔﻟا ﻮﺑأ دﻮﻤﺤﻣ ﺪﻤﺤﻣ
ﻦﻳﺪﻟا فﺮﺷ ﻖﻟﺎﺨﻟا ﺪﺒﻋ ﻰﻧﺎه – ﻦﻳﺪﻟا جﺎﺗ ﻢﻠﺴﻣ ﻮﺑأ حوﺪﻤﻣ
– ﺔﻤﻴﺨﻟا اﺮﺒﺷ – ﺲﻤﺷ ﻦﻴﻋ ﺔﻌﻣﺎﺟ – ﺔﻋارﺰﻟا ﺔﻴﻠآ – ﺔﻴﻋارﺰﻟا ﺔﻳﻮﻴﺤﻟا ءﺎﻴﻤﻴﻜﻟا ﻢﺴﻗ
ﺮﺼﻣ – ةﺮهﺎﻘﻟا
قﻮ ﻓ ﺔﻌ ﺷﻸﻟ ءاﺮﻀ ﺨﻟا ﺎﻴﻟﻮ ﺻﺎﻔﻟا تﺎ ﺒﻨﻟ ﺔﺘﺒﻨﺘﺴ ﻤﻟا روﺬ ﺒﻟا ﺾﻳﺮ ﻌﺗ ﻢ ﺗ ﺔ ﺳارﺪﻟا ﻩﺬ ه ﻰ ﻓ
فوﺮ ﻈﻟ ﺔ ﻴﻣﺎﻨﻟا تﺎ ﺗﺎﺒﻨﻟا ﺾﻳﺮ ﻌﺗ ﻢ ﺗ ﻢ ﺛ ﺔ ﻘﻴﻗد 60 ، 30 ، 15 ، 7 ةﺪ ﻤﻟ ( UV-C ) ﺔﻴﺠﺴ ﻔﻨﺒﻟا
ﻰ ﺤﻠﻤﻟا دﺎ ﻬﺟﻺﻟ تﺎ ﺗﺎﺒﻨﻟا ﻞ ﻤ ﺤﺗ ﻢﻴ ﻴﻘﺗ ﻢ ﺗ .( مﻮﻳدﻮﺼ ﻟا ﺪ ﻳرﻮﻠآ ﺮﻟﻮ ﻤﻴﻠﻠﻴﻣ 50 ) ﻰ ﺤﻠﻤﻟا دﺎﻬﺟﻹا
تاﺮ ﻴﻐﺘﻟا سﺎ ﻴﻗ ﻢﺗ ﺎﻤآ .
ﺔﻳرﺬﺠﻟا و ﺔﻳﺮﻀﺨﻟا ﻊﻴﻣﺎﺠﻤﻟا ﻦﻣ ﻞﻜﻟ فﺎﺠﻟا و جزﺎﻄﻟا نزﻮﻟا ﺮﻳﺪﻘﺘﺑ
ةﺪﺴ آأ ﺮﻳﺪ ﻘﺗ : لﻼ ﺧ ﻦ ﻣ ﺔﻴﺠﺴ ﻔﻨﺒﻟا قﻮ ﻓ ﺔﻌ ﺷﻷﺎﺑ ﺔﺜﺤﺘﺴ ﻤﻟا ﺔ ﻣوﺎﻘﻤﻠﻟ ﺔﺒﺣﺎﺼ ﻤﻟا ﺔ ﻴﺋﺎﻴﻤﻴﻜﻟا
ﺪﻴﺴ آأ ﺮﺑﻮ ﺳ ) ة ﺪﺴ آﻸﻟ ةدﺎﻀ ﻤﻟا تﺎ ﻤﻳﺰﻧﻹا طﺎﺸ ﻧ سﺎ ﻴﻗ ﻦﻴﻟوﺮ ﺒﻟا ﺰ ﻴآﺮﺗ – تاﺪ ﻴﺒﻴﻠﻟا
و قاروﻷا ﻰ ﻓ ( ﺰﻳﺪﻴﺴ آوﺮﻳ تﺎﺑرﻮﻜ ﺳأ – ﺰﻳﺪﻴﺴ آوﺮﻴﺑ لﻮ ﻜﻳاﻮﺟ – ﺰﻴﻟﺎ ﺘآ – ﺰﻴﺗﻮﻴﻤﺴ ﻳد
ﺖ ﻧﺎآ ﺔﻴﺠﺴ ﻔﻨﺒﻟا قﻮ ﻓ ﺔﻌﺷﻷﺎﺑ ﺔﻠﻣﺎﻌﻤﻟا روﺬﺒﻟا ﻦﻣ ﺔﻴﻣﺎﻨﻟا تﺎﺗﺎﺒﻨﻟا نأ ﺞﺋﺎﺘﻨﻟا ﺖﺤﺿوأ .
روﺬﺠﻟا
ﻦ ﻣ ﻞ ﻜﻟ فﺎ ﺠﻟا و جزﺎ ﻄﻟا نزﻮ ﻟا عﺎ ﻔﺗرا ﻰ ﻓ ﺎﺤ ﺿاو نﺎ آ ﻚ ﻟذ و ﻰ ﺤﻠﻤﻟا دﺎ ﻬﺟﻹﺎﺑ اﺮﺛﺄﺗ ﻞﻗأ
ىاد نﻮﻟﺎ ﻤﻟا تﺎﻳﻮﺘﺴ ﻣ نﺈ ﻓ ﻚ ﻟذ ﻊ ﻣ و .
لوﺮﺘﻨﻜﻟﺎ ﺑ ﺔ ﻧرﺎﻘﻤﻟﺎﺑ ﺔ ﻳرﺬﺠﻟا و ﺔﻳﺮﻀ ﺨﻟا ﻊﻴﻣﺎ ﺠﻤﻟا
قﻮ ﻓ ﺔﻌ ﺷﻷﺎﺑ ﺔ ﻠﻣﺎﻌﻤﻟا روﺬ ﺒﻟا ﻦ ﻣ ﺔ ﻴﻣﺎﻨﻟا تﺎ ﺗﺎﺒﻨﻟا روﺬ ﺟ و قاروأ ﻰﻓ ﺎﻋﺎﻔﺗرا تﺮﻬﻇأ ﺪﻴهﺪﻟأ
ﺰ ﻴآﺮﺗ ﻰ ﻓ ﺔ ﻳﻮﻨﻌﻣ تادﺎ ﻳز ﻰ ﻟإ ﺔﻴﺠﺴ ﻔﻨﺒﻟا قﻮ ﻓ ﺔﻌ ﺷﻷﺎﺑ روﺬ ﻟا ﺔ ﻠﻣﺎﻌﻣ تدأ ﺎﻀ ﻳأ .
ﺔﻴﺠﺴ ﻔﻨﻟا
ﻰ ﻓ ﻢﻬ ﺳأ ىﺬ ﻟا و ﺔ ﻴﺤﻠﻤﻟا ﺮ ﻴﻏ و ﺔ ﻴﺤﻠﻤﻟا فوﺮﻈﻟا ﺖﺤﺗ تﺎﺗﺎﺒﻨﻟا روﺬﺟ و قاروأ ﻰﻓ ﻦﻴﻟوﺮﺒﻟا
ﺎﻬﻃﺎﺸ ﻧ ﻰ ﻓ ﺎ ﻋﺎﻔﺗرا ةﺪﺴآﻸﻟ ةدﺎﻀﻤﻟا تﺎﻤﻳﺰﻧﻹا تﺮﻬﻇأ ﺎﻤآ .
ىزﻮﻤﺳﻷا دﺎﻬﺟﻹا ﻦﻣ ﺎﻬﺘﻳﺎﻤﺣ
اﺬ ه نﺎ آ و .
ﺔﻴﺠﺴ ﻔﻨﺒﻟا قﻮ ﻓ ﺔﻌ ﺷﻷﺎﺑ ﺔ ﻠﻣﺎﻌﻤﻟا روﺬ ﺒﻟا ﻦ ﻣ ﺔ ﻴﻣﺎﻨﻟا تﺎ ﺗﺎﺒﻨﻟا روﺬ ﺟ و قاروأ ﻰ ﻓ
روﺬ ﺑ ﺔ ﻠﻣﺎﻌﻣ نأ جﺎﺘﻨﺘ ﺳا ﻦ ﻜﻤﻳ ﻪ ﻧﺈﻓ ﺎ ﻣﻮﻤﻋ .
قاروﻷا ﻦ ﻋ روﺬ ﺠﻟا ﻰ ﻓ ﺮ ﺜآأ ﺎ ﺳﻮﻤﻠﻣ ﺮﻴﺛﺄ ﺘﻟا
ﻰ ﺤﻠﻤﻟا دﺎﻬﺟﻹا ﻞﻤﺤﺗ ﻰﻠﻋ تﺎﺗﺎﺒﻨﻟا ﺚﺣ ﻰﻟإ ىدأ ﺔﻴﺠﺴﻔﻨﺒﻟا قﻮﻓ ﺔﻌﺷﻷﺎﺑ ءاﺮﻀﺨﻟا ﺎﻴﻟﻮﺻﺎﻔﻟا
.
ﺲآﻸﻟ دﺎﻀﻤﻟا مﺎﻈﻨﻟا ﻂﻴﺸﻨﺗ لﻼ ﺧ ﻦﻣ