CC BY
345
Journal of Stress Physiology & Biochemistry, Vol. 8 No. 4 2012, pp. 199-217 ISSN 1997-0838 Original Text Copyright © 2012 by Abdel Haleem, Mohamed, Zaki, Mogazy
ORIGINAL ARTICLE
Pre-exposure to gamma rays alleviates the harmful effect of salinity on cowpea plants
Abdel Haleem M.A. Mohammed, Heba I. Mohamed*, Laila M. Zaki, Asmaa M. Mogazy
Biological and Geological Sciences Department, Facutlty of Education, Ain Shams University, El Makres St. Roxy, Cairo, Egypt (ARE) Post code: 1575
*E-Mail: hebaibrahim [email protected]
Received August 25, 2012
Soil salinity is one of the most severe factors limiting growth and physiological response in cowpea plants. In this study, the low concentrations of NaCl (25mM) increased plant growth, photosynthetic pigments content, total soluble protein content, nucleic acids contents (DNA and RNA), lipid peroxidation, non enzymatic antioxidants (anthocyanin, ascorbic acids and a-tocopherol), number of legumes per plant, number of seeds per legume, number of seeds per plants, legume length, fresh and dry weight of legumes and weight of 1000 seeds and total soluble proteins and carbohydrate contents in harvested seeds as compared to control. On the other hand, the high concentrations of NaCl (50, 100 and 200 mM) caused reduction in plant growth, photosynthetic pigments content, total soluble protein content, nucleic acids contents (DNA and RNA), all yield attributes and harvested seeds components but increased lipid peroxidation and non enzymatic antioxidants (anthocyanin, ascorbic acids and a-tocopherol). Electrophoretic studies of proteins showed three types of modifications are observed in the protein patterns of cowpea seeds, some protein bands were disappeared, other proteins were selectively increased and synthesis of new set of protein was induced. Some of these responses were observed under gamma rays and salinity treatments, while others were induced by either gamma rays or salinity. Seeds irradiation with gamma rays alleviates the adverse effect of salt stress compared to non irradiated seeds.
Key words: lipid peroxidation, non enzymatic antioxidants, nucleic acids, protein electrophoresis, yield.
ORIGINAL ARTICLE
Pre-exposure to gamma rays alleviates the harmful effect of salinity on cowpea plants
Abdel Haleem M.A. Mohammed, Heba I. Mohamed*, Laila M. Zaki, Asmaa M. Mogazy
Biological and Geological Sciences Department, Facutlty of Education, Ain Shams University, El Makres St. Roxy, Cairo, Egypt (ARE) Post code: 1575
*E-Mail: hebaibrahim [email protected]
Received August 25, 2012
Soil salinity is one of the most severe factors limiting growth and physiological response in cowpea plants. In this study, the low concentrations of NaCl (25mM) increased plant growth, photosynthetic pigments content, total soluble protein content, nucleic acids contents (DNA and RNA), lipid peroxidation, non enzymatic antioxidants (anthocyanin, ascorbic acids and a-tocopherol), number of legumes per plant, number of seeds per legume, number of seeds per plants, legume length, fresh and dry weight of legumes and weight of 1000 seeds and total soluble proteins and carbohydrate contents in harvested seeds as compared to control. On the other hand, the high concentrations of NaCl (50, 100 and 200 mM) caused reduction in plant growth, photosynthetic pigments content, total soluble protein content, nucleic acids contents (DNA and RNA), all yield attributes and harvested seeds components but increased lipid peroxidation and non enzymatic antioxidants (anthocyanin, ascorbic acids and a-tocopherol). Electrophoretic studies of proteins showed three types of modifications are observed in the protein patterns of cowpea seeds, some protein bands were disappeared, other proteins were selectively increased and synthesis of new set of protein was induced. Some of these responses were observed under gamma rays and salinity treatments, while others were induced by either gamma rays or salinity. Seeds irradiation with gamma rays alleviates the adverse effect of salt stress compared to non irradiated seeds.
Key words: lipid peroxidation, non enzymatic antioxidants, nucleic acids, protein electrophoresis, yield.
Salinity in soil or water is one of the major salinity on plant growth are associated with (1) low
stresses especially in arid and semi-arid regions, can osmotic potential of soil solution (water stress), (2)
severely limit plant growth and productivity nutritional imbalance, (3) specific ion effect (salt
(Mahajan and Tuteja, 2005). According to FAO, stress), or (4) a combination of these factors
more than 800 million hectares of world's (Ashraf, 1994).
agricultural land are seriously affected by salinity Salinity is often associated with a reduction in
(Munns and Tester, 2008).The deleterious effects of photosynthetic capacity (Parida and Das, 2005).
Photosynthesis may decrease due to stomatal closure or by the direct effect of salt on the photosynthetic apparatus. Malondialdehyde (MDA) content, a product of lipid peroxidation, has been considered an indicator of oxidative damage. Salt stress, like other abiotic stresses, can also lead to oxidative stress through the increase in reactive oxygen species (ROS), such as superoxide (O2 -), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), which are highly reactive and may cause cellular damage through oxidation of lipids, proteins and nucleic acids (Ben Amor, et al., 2007). ROS cause chlorophyll degradation and membrane lipid peroxidation, reducing membrane fluidity and selectivity. Chlorophyll loss, lipid peroxidation measured as MDA (malondialdehyde, a product of lipid peroxidation) content, and electrolyte leakage are considered to be indicators of oxidative damage (Wise and Naylor, 1987).
To minimize the effects of oxidative stress, plants must possess efficient antioxidant system. Plant cells have evolved a complex antioxidant system, which is composed of low molecular mass antioxidants (glutathione, ascorbic acids, a-tocopherol, phenolic compounds and carotenoids) as well as ROS-scavenging enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione reductase (GR) (Piotr and Klobus, 2005).
Gamma radiation can be useful for the alteration of physiological characters (Kiong et al., 2008). The biological effect of gamma rays is based on the interaction with atoms or molecules in the cell, particularly water, to produce free radicals (Kovacs and Keresztes, 2002). These radicals can damage or modify important components of plant cells and have been reported to affect differentially
the morphology, anatomy, biochemistry and physiology of plants depending on the radiation dose (Ashraf et al., 2003). These effects include changes in the plant cellular structure and metabolism e.g., dilation of thylakoid membranes, alteration in photosynthesis, modulation of the antioxidative system and accumulation of phenolic compounds (Ashraf, 2009).The relatively low-doses ionizing irradiation on plants and photosynthetic microorganisms are manifested as accelerated cell proliferation, germination rate, cell growth, enzyme activity, stress resistance and crop yields (Chakravarty and Sen, 2001). On the other hand, the irradiation of seeds with high doses of gamma rays disturbs the synthesis of protein, hormone balance, leaf gas-exchange, water exchange and enzyme activity (Hameed et al., 2008).
Cowpea is one of the most important food legume crops in the semi-arid tropics and contains high level of protein. Cowpea is a multipurpose crop and is grown as a grain legume mainly for dry beans and green pods and also as forage, green manure and cover crop.
The objective of this work was to investigate whether pre-treatment of dry seeds of cowpea (Vigna sinensis) plants with a low dose of gamma rays (5 Krad) before planting could act as a protective agent to nullify the influence of salt stress.
MATERIALS AND METHODS
Seeds of cowpea plants (Vigna sinensis) were obtained from the Agriculture Research Center, Ministry of Agriculture, Giza, Egypt. The seeds were thoroughly washed with continuous current of tap water for one hour. The washed seeds were sown in pots (25 cm in diameter and 25 cm in depth) containing 3.5 kg of homogeneous loamy clay soil.
Ten seeds were sown in each pot. Pots were divided into four groups. The seeds of the first group did not have any treatment to serve as control, while the seeds of the second group were irrigated with NaCl at concentrations (25, 50, 100 and 200 mM) to raise the pots at 80% of soil water holding capacity. The seeds of the third group were irradiated with gamma rays (5 Krad) at the Middle Eastern regional radioisotopes center for the Arab countries (Dokki, Cairo) and the fourth group was combination between gamma rays and NaCl treatments. The pots were irrigated with the water holding capacity of the soil (80%) to severe as control and the other groups were irrigated with NaCl concentrations. This experiment was conducted under natural conditions (day length 1214 hrs, temperature 25-27 °C and humidity 70%). At 130 days from sowing the plants were collected to determine the growth parameters (shoot length, root length, number of lateral roots, number of leaves, number of internodes, number of flowers, fresh and dry weights of shoots and roots and assimilating area) in addition to determination of photosynthetic pigments, total soluble protein, nucleic acids (DNA and RNA), non enzymatic antioxidants (anthocyanin, ascorbic acid, a-tocopherol) and lipid peroxidation. The seeds were collected when the fruits become mature at 160 days to determine morphological criteria in addition to determination of total carbohydrate content, total soluble protein, mineral contents and protein electrophoresis.
Determination of Photosynthetic Pigments
Chlorophyll a, Chlorophyll b and Carotenoids were determined in cowpea leaves. The spectrophotometric method recommended by Vernon and Seely (1966) was used. The pigment
contents were calculated as mg g-1 fresh weight of leaves.
Determination of Lipid Peroxidation
Lipid peroxidation was determined by estimating the malondialdehyde content following the method of Heath and Packer (1968). The absorbance of the resulting supernatant was recorded at 532 nm and 600 nm. The non-specific absorbance at 600 nm was subtracted from the 532 nm absorbance. The absorbance coefficient of malondialdehyde was calculated by using the extinction coefficient of 155mM-1 cm-1.
Quantitative Estimation of Nucleic Acids
The method used for the extraction of total RNA and DNA were determined according to the method described by Morse and Carter (1949). Ribonucleic acid (RNA) was estimated colourmetrically by the orcinol reaction as described by Dische (1953). While, deoxyribonucleic acid (DNA) was estimated by DPA (diphenylamine) colour reaction described by Burton (1956). The nucleic acids were determination by Spekol Spectrocolourimeter VEB Carl Zeiss.
Assay of Non Enzymatic Antioxidant
Shoots of cowpea plants were used for the estimation of anthocyanin contents according to Neff and Chory (1998). Ascorbic acid content was measured in roots and shoots of cowpea plants according to the method described by Mukherjee and Choudhuri (1983). The a-Tocopherols were determined using the method of Philip et al. (1954). Determination of Total Soluble Protein
Root, shoot and seeds of cowpea plants were grinded in sodium phosphate buffer at pH 6.5. The total soluble protein content in the supernatant was determined according to Lowry et al. (1951).
Determination of Total Carbohydrates
Total carbohydrates in seeds of cowpea plants were determined using the colorimetric method described by Dubois et al. (1956).
Protein Electrophoresis
For SDS-PAGE, seeds of cowpea plants were ground to powder under liquid nitrogen and melted in ice-cold extraction buffer (0.5 M Tris-HCl, pH 6.5, 1% SDS, 5% 2-mercaptoethanol, 20% sucrose, 0.4% bromophenol blue), followed by centrifugation at 10.000 xg at 4 °C for 15 min. Extracts were stored at -20°C until used. Gel electrophoresis SDS-PAGE was carried out in 12.5% separating gel and 5% stacking gel according to the method of Laemmli (1970). Gels were run at ambient temperature (20 °C) for 6 h at constant power of 130V until the tracking dye reached the end of the gel. After electrophoresis, the gel was stained with Coomassie blue R-250. Relative molecular weight of each protein was determined using a standard protein marker. The gel was scanned using Gel pro-Analyzer.
Statistical Analysis
The data were statistically analyzed using F-test and LSD at 5% and 1% levels of probability according to SAS-Programme (1982).
RESULTS
Changes in Growth Parameter
In response to salinity stress, the lowest concentration of NaCl (25 mM) caused significant increase in the shoot and root lengths at fruiting stage. On the other hand, the higher concentrations of NaCl (50, 100 and 200 mM) caused highly significant decrease or significant decrease as being compared with the control. In response to gamma radiation, shoot and root length showed a highly significant increase with different salinity concentrations (25, 50 and 100 mM) as compared
with control except (200 mM) which showed a highly significant decrease (Table 1). The other growth parameters such as number of lateral roots showed highly significant increase at all treatments. Number of bacterial nodules of cowpea plants showed variable changes. The low concentrations of NaCl (25 and 50 mM) alone or in combination with gamma ray (5 Krad) caused highly significant increase in the number of bacterial nodules of cowpea plants. High concentrations of NaCl (100 and 200 mM) alone or in combination with gamma rays showed variable effects in the same parameter as compared with control. Although number of leaves, leaf area, fresh weight of shoots and roots and dry weight of shoots and roots were significantly inhibited with increasing
concentrations of NaCl expect at the low
concentrations of NaCl (25 mM) which showed high significant increase in all the above parameters. Changes in Some Metabolites
As illustrated in table (2), chlorophyll a and b, carotenoids and total photosynthetic pigment contents in leaves of cowpea plants showed highly significant increase above the control value in response to treatment with the lower
concentrations of NaCl (25 and 50 mM). On the other hand, the other concentrations of NaCl (100 and 200 mM) caused high significant decrease in the same contents except at 100 mM of NaCl which showed high significant increased in chlorophyll b content. Irradiating seeds with gamma rays caused enhancement in photosynthetic pigment contents under salt stress except the high concentration of NaCl (200 mM).
Data showed in table (2) that the thiobarbituric acid reactive substances (TBARS) content increased progressively in shoots and roots of NaCl treated and untreated cowpea plants. Such increase was
highly significant in NaCl treated cowpea plants as compared with the untreated controls. In addition, salinized cowpea plants with gamma rays reduced the levels of TBARS in their shoots and roots as compared with salinized plants alone.
The results in table (2) showed that the total soluble protein content in shoots and roots of cowpea plants was highly significantly increase at the lowest concentrations of NaCl (25 mM) but it showed significant increased in roots of cowpea plant and no significant effect in shoots. On the other hand, the high concentrations of NaCl (1QQ and 2QQ mM) caused highly significantly decreases in the same contents as compared with control plants.
Data in table (З) revealed that the lowest concentration of NaCl (25 mM) causes a highly significant increase in the DNA and RNA contents of shoots and roots of cowpea plants. While the other concentrations of NaCl (5Q, 1QQ and 2QQ mM) caused a high significant decrease or significant decrease in the same content in shoots and roots of cowpea plants.
In addition, The effect of gamma radiation is shown to induce a highly significant increase or significant increase in the DNA and RNA contents of shoots and roots of cowpea plant under (25, 5Q and 1QQ mM) of NaCl except 2QQ mM which induce a highly significant decrease or significant decrease in the same contents of both shoots of cowpea plants as compared with control.
Changes in Non Enzymatic Antioxidants
It is demonstrated in table (З) that salinity at different concentrations (25, 5Q, 1QQ and 2QQ mM) stimulated the accumulation of non enzymatic antioxidants (anthocyanin, ascorbic acids and a-tocopherol) in shoots and roots of cowpea plants as
compared with that of the unsalinized control. Generally, gamma radiation seems to induce a highly significant increase in non enzymatic antioxidants compounds in shoots and roots of cowpea plant under all salinity levels (25, 50, 100 and 200 mM). In addition, salinized cowpea plants with gamma rays increased the levels of non enzymatic antioxidants in their shoots and roots as compared with salinized plants alone.
Changes in Yield Components
Results in table (4) revealed that the lowest salinity concentration (25 mM) induce a highly significant increase in number of legumes per plant, number of seeds per legume, number of seeds per plants, legume length, fresh and dry weight of legumes and weight of 1000 seeds. Also, 50 mM of NaCl induce a significant increase or highly significant increase in all the above parameter except number of legumes per plant which showed high significant decreased. In addition, 100 mM of NaCl induce a highly significant decrease in all the above parameter as compared with control plants. Moreover, radiation with 5 Krad induces a highly significant increase with different salinity concentrations (25, 50 and 100 mM) as compared with control in the same parameter.
Changes in Chemical Composition of harvested Seeds
Total carbohydrates and total soluble protein contents in seeds
The lowest concentration of NaCl (25 mM) induces highly significant increase in total carbohydrate and total soluble protein contents in seeds of cowpea plants. On the other hand the high concentrations of NaCl (50 and 100 mM) induce highly significant decrease in the same contents. In addition radiation with gamma ray (5Krad) induces
highly significant increase in total carbohydrate and total soluble proteins contents in seeds under different salinity concentrations (25, 50 and 100 mM) compared to those of the corresponding controls (table 4).
Changes in Protein Electrophoresis in Harvested Seeds
In the present work (Table 5 and Figure 1), three types of modifications are observed in the protein patterns of cowpea seeds, some protein bands were disappeared, other proteins were selectively increased and synthesis of new set of protein was induced. Some of these responses were observed under gamma rays and salinity treatments, while others were induced by either gamma rays or salinity.
19 protein bands of molecular weight ranged between 203 and 22 KDa were observed in cowpea
seeds. The protein patterns of cowpea seeds under all treatments comprise eight major bands (common bands) having molecular weights of (203, 158, 139, 115, 108, 34, 30 and 27 KDa). One protein band of molecular weight (180 kDa) was de novo synthesized in cowpea seeds irradiated with 100 mM NaCl alone and with combination with gamma rays (5 Krad). Generally the results of SDS-PAGE analysis could reveal two different genetic mechanisms i.e., salt stress resulted in the overexpression of some gene and / or de novo induction of gene expression.
The total number of bands not effected under all treatments but some bands appeared and the other bands disappeared. The total number of protein bands was increased from 18 bands in the untreated (controlled) plants to 19 bands under plants irradiated with gamma rays alone (5 Krad).
Figure 1. Electrophoretic banding patterns of cowpea seeds in response to treatment with different concentrations of NaCl alone or in combination with gamma rays.
1-Control; 2-5 Krad; 3-25 mM NaCl; 4- 25 mM NaCl+5 Krad; 5- 50 mM NaCl; 6- 50 mM NaCl+5 Krad;
7- 100 mM NaCl; 8- 100 mM NaCl+5 Krad
in in roots and shoots of cowpea
Table 1. Effect of gamma rays on growth parameters of cowpea plants under salt stress. Each value is the mean of ten replicate
Total soluble protein § lllg'g Rod? Shoot
>
10.-g 26.75 ч
ЇІЗЩ 32.26a
to
11.Л 27.S4C
l/l
7.S-S 20,41і
Ifl
7.00 17.S4b
Г"1
12.5$ 29.9 і
16.33? 37.08а
O n Я M
14.3gf
H
5
12.6$
00
g:
8.0І
to
О
to
0,65
0.94
33.8і
30.24а
22.9Ь
1.30
1.87
Treatments Shoot length (cm) Root length (cm) No of lateral roots No of bacterial nodules No of leaves Area of leaves (Cm2) Fresh weight of shoot (g) Dry weight of shoot (g) Fresh weight of root (g) Dry weight of root (g)
0.0 mM NaCl 25.3 26 6 15.20 4,4 8.0 151.8 7.91 1.06 2.22 0.28
25 mM NaCl 35.5a 30.51 37.20“ 8.8s 9.0 s 198.0s 11.31a 1.87a 3.30s 0.33s
50 mM NaCl 21.84b 25.3b 34.803 5.8s 7.0b 122,6b 6.98b 0.95's l,98b 0.22b
100 mM NaCl 19.4b 24.5b 26.403 3.6b 6.2b 114,3b 6.81b 0,88b l,90b 0.20b
200 mM NaCl 15.6b 21.6b 18.40J 2.8b 5.2b 103,7b 4.35b 0,74b l,72b 0.17b
5 Krad 38.4a 30.9 s 32.00s 6.0s 8.6s 232.1s 12.92a 1.92a 3.26s 0.39s
25 mM NaCl + 5 Krad 41.a 35.71 23.403 6.2s 11.8s 255.8a 13.35a 1.993 3.71a 0.37a
50 mM NaCl + 5 Krad 34.283 31.81 49.20“ 10.6a 9.2s 208,6a 11.38a 1.76a 2,78s 0.34a
100 mM NaCl + 5 Krad 29.2s 29.1s 22.40s 7.8s 8.9s 205 7 а 10.34a 1.59a 2.66s 0.33s
200 mM NaCl + 5 Krad 20.2b 23.04b 20.40s 4.0C 6.7b 126.5b 6.71b 0.80b 1.90b 0.21b
L.S.Dat5% 1.92 0.95 2.22 0.53 0.40 11.67 0.65 0.11 0.15 0.017
1% 2.77 1.37 3.19 0.76 0.58 16.79 0.94 0.16 0.21 0.025
high significant increase b high significant decrease c non significant effect
206 Pre-exposure to gamma rays...
JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 8 No. 4 2012
Table 2. Effect of gamma rays on photosynthetic pigment contents in leaves, lipid peroxidation and total soluble protein in roots and shoots of cowpea plants under salt stress. Each value is the mean of three replicate
Lipid peroxidation Total soluble protein
Treatments Chla mg/g Chib mg/g Carotenoids mg/g Total Pigments mg/g n mole/g F,\rt Root Shoot Root mg/g Shoot
O.OmMNaCl 4.48 1.45 3.31 9.24 10.0 17.5 10.41 26.75
25 mM NaCl 8.59a 2.64" 6.50a 17.73a 18.2" 22.5a 13.34" 32.26a
50 mM NaCl 5.60a 2.43a 4.83a 12.86" 19.9" 28.3* 11.2" 27.84c
100 mM NaCl 3.16b 1.86" 2.3 lb 7.33'& 23.9" 34.5a 7.84b 20.4 lb
200 mM NaCl 2,45b 0.99b l,55b 4.99b 25.5" 40.0a 7.01b 17,S4b
5 Krad 7.74a 3.01" 6.19a 16.94a 12.3" 16.1= 12.53a 29.9"
25 mM NaCl -5 Krad 9.65a 3.853 7.43a 20.93a 14.0" 20,2a 16.31a 37,08 =
50 mM NaCl -5 Krad 6.82a 3.17" 6.00a 15.99" 15.7" 20.8a 14.34" 33.8"
100 mM NaCl + 5 Krad 5.98a 2.73" 5.26a 13.97a 16.7" 22.4a 12.68a 30.24a
200 mM NaCl + 5 Krad 3,46b 1.09b 2.19b 6.74b 17.1" 24,4a 8.04b 22.9b
L.S.D at 5% 0.52 0.20 0.44 1.15 1.04 1.62 0.65 1.30
1% 0.75 0.29 0.64 1.65 1.49 2.33 0.94 1.87
a high significant increase b high significant decrease c non significant effect
O
<1
Abdel Haleem et al
falue is the mean often replicate
Table 3. Effect of gamma rays on nucleic acids contents and non enzymatic antioxidants compounds in roots and shoots of cowpea plants under salt stress. Each value is the mean of three replicate
"fetal
carbc^yd rates
m^lOOg
о
§6
я
Ш1
Л
4 к
3$)3b
Х/1
^9Ь
О
■$з3
Я
4&8а
о
и
м
§
ЩГ
%
<
IM28
0Л84
Total soluble protein
g/ 100g
13.28 14.04= 12,67b 12,48b 14.04=
16.89Hs 15.61=
14.28 =
0.281
0.404
Treatments
0.0 mM NaCI 25 mM NaCI 50 mM NaCI 100 mM NaCI 200 mM NaCI 5 Krad 25 mM NaCI +
5 Krad 50 mM NaCI +
5 Krad 100 mM NaCI +
5 Krad 200 mM NaCI +
5 Krad L.S.D at 5%
1%
a high significant increase
DNA RNA Anthocyanin Ascorbic acid Tocopherol
Mg/g Pg/g g/100 ml pb/b Pg/g
Root Shoot Root Shoot Shoot Root Shoot Root Shoot
58.5 139.5 866 826 42.38 4.54 7.58 11.87 12.05
85.5“ 153.3= 954= 933= 65.13“ 5.59= 8.74“ 15.52= 17.41“
47.5b 101.9b 780b 787b's 55.55= 5.65= 9.80 = 15.72= 20.04 =
40.3b 75.7b 699b 629b 58.24= 6.01= 10.75= 16.15= 23.03“
26.4b 4Э.9Ь 610b 563b 51.09“ 7.25= 11.82= 17.90= 25.94“
75.2“ 156.4a 906= 932= 69.13“ 7.68= 9.55“ 18.39= 24.61“
139 4= 179.2= 1214= 967= 82.73“ 8.05= 13.27 = 20.07= 26.66 =
100.4“ 159.6= 1042= 933= 69.02= 8,82Hs 13.67= 20.61= 32.39“
89.4a 151.7a 947= 883= 53.94“ 9.54= 14.13= 21.12= 38.88“
49.0b 83.7b 656b 665b 48.33“ 12.08“ 16.18= 21.55= 45.12“
7.23 9.36 39.98 31.21 2.99 0.48 0.58 0.66 2.12
10.39 13.46 57.49 44.88 4.31 0.69 0.84 0.95 3.05
ise b high significant decrease с non significant effect
208 Pre-exposure to gamma rays...
Table 5. Effect of gamma rays on the protein patterns separated by SDS- PAGE in seeds of cowpea plants under salt stress
Treatments
o ^ N M.Wt KDa 203 Control + 5 Krad + 25 mM NaCl + Z..J IIIIVI NaCl+5 Krad + 50 mM NaCl + JU IIIIVI NaCl+5 Krad + 100 mM NaCl +
2 180 — — — — — — +
3 175 + + + + + + —
4 158 + + + + + + +
5 139 + + + + + + +
6 115 + + + + + + +
7 108 + + + + + + +
8 102 + + + + + +
9 93 + + + + + +
10 85 + + — — — + +
11 76 + + + + + — —
12 67 + + + +
13 62 + + +
14 57 + + + + + +
15 54 + + — — — — —
16 49 + + + + + +
17 44 + — — — + + +
18 41 + + + + — —
19 37 + + + + + +
20 34 + + + + + + +
21 30 + + + + + + +
22 27 + + + + + + +
23 24 + — — — — +
24 22 + + + + + +
Total No. of bands 18 19 18 18 18 18 18
DISCUSSION
Salinity stress caused reduction in cowpea plant growth at the high concentrations (50, 100 and 200 mM). These results are similar to Dolatabadian et al. (2011) who found that salinity stress was significantly decreased shoot and root weight either fresh weight or dry weight, in addition, total plant weight, plant height and leaf number were decreased due to salinity stress. Interestingly, leaf
area was not affected by salinity stress. It has been reported that the plants had the reduction in their fresh weights because of the proportional increase in Na+ concentration, which could imply that an ionic effect was being manifested. It is also assumed that in addition to toxic effects of NaCl, higher concentration of salt reduces the water potential in the medium which hinders water absorption and thus reduces plant growth.
Table 5. Effect of gamma rays on the protein patterns separated by SDS- PAGE in seeds of cowpea plants under salt stress
NO. M.Wt KDa Control
1 203 +
2 180 —
3 175 +
4 158 +
5 139 +
6 115 +
7 108 +
8 102
9 93 +
10 85 +
11 76 +
12 67
13 62 +
14 57
15 54 +
16 49
17 44 +
18 41 +
19 37
20 34 +
21 30 +
22 27 +
23 24 +
24 22 +
5 Krad
+
+
+
+
+
19
Treatments
25 mM NaCl +
+
+
+
+
+
+
+
+
+
+
+
18
25 mM NaCl+5 Krad +
+
+
+
+
+
18
50 mM NaCl +
+
+
+
+
+
+
+
+
+
+
+
+
+
18
50 mM NaCl+5 Krad +
100 mM NaCl
18
+
+
+
+
+
+
+
18
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
DISCUSSION
Salinity stress caused reduction in cowpea plant growth at the high concentrations (50, 100 and 200 mM). These results are similar to Dolatabadian et al. (2011) who found that salinity stress was significantly decreased shoot and root weight either fresh weight or dry weight, in addition, total plant weight, plant height and leaf number were decreased due to salinity stress. Interestingly, leaf area was not affected by salinity stress. It has been reported that the plants had the reduction in their
fresh weights because of the proportional increase in Na+ concentration, which could imply that an ionic effect was being manifested. It is also assumed that in addition to toxic effects of NaCl, higher concentration of salt reduces the water potential in the medium which hinders water absorption and thus reduces plant growth. Reduction in growth attributes especially plant height may be due to changes in plant-water relationships under salt stress, which suppress meristem activity as well as cell elongation
(Dorgham, 1991).
Stimulating effect of low salinity level on plant growth may be resulted from the beneficial effect of low concentration of chloride on many physiological processes such as osmoregulators, photosynthesis and enzyme activities, chloride acts a cofactor of an NH2OH sensitive, Mn-containing, O2-evolving enzyme. Moreover, Critchley (1983) suggested that chloride facilitates electron transport by reversible ionic binding to the O2" evolving complex or to the thylakoid membranes. In addition, some enzymes are known to be stimulated by Cl" such as ATPase and alpha-amylase hydrolysize starch to sugars requires chloride for activation. On the other hand, salinity stress can affect seed germination through reduction of water uptake leading to moisture stress (osmotic effect), by ion toxicity and/or ionic imbalance, or by the accumulation of Na+ and Cl- ions and inhibition of the uptake of several essential nutrients such as K+ causing nutritional imbalance in the plants or accumulation of these factors (Taamalli et al., 2004) and/or decreasing the activity of certain enzymes by either decreasing the rate of transcription or translation, which lead to decreasing both cell division and cell elongation (Dodd and Donovan,
1999).
The irradiated seeds of cowpea plants with gamma rays under salt stress caused highly significantly increase in all the above growth parameters as compared with control (H2O) except the high concentration of NaCl (200 mM). Irradiation with gamma rays will induce the growth stimulation by changing the hormonal signaling network in plant cells or by increasing the anti oxidative capacity of the cells to easily overcome daily stress factors such as fluctuations of light
intensity and temperature in the growth condition (Wi et al., 2007).
The severe reduction in photosynthetic pigments in cowpea leaves in response to salinity treatment might be ascribed to the toxic action of NaCl on the biosynthesis of pigments, increasing their degradation and / or due to damage of the chloroplast thylakoid. The observed severe reduction in Mg+2 ions in salt treated plants which are essential for chlorophyll biosynthesis reinforced the view that salinity decreased chlorophyll biosynthesis. These results are similar to Helaly and El-Hosieny (2011) who found that the interaction treatments (salinity and gamma) indicated that irradiation counteracted the inhibiting effects of salinity on chlorophylls concentration in Citrus limon even at the highest level of salinity. The effect of irradiation on increasing chlorophylls may be due to its effects on stimulation chlorophyll its biosynthesis and/or delaying of its degradation.
Gamma rays caused reduction in lipid peroxidation in cowpea plants. Similar results obtained by Moussa (2011) who found that gamma irradiation decreased the malondialdehyde concentration of the leaves of soybean plants under drought stress. Also, Hameed et al. (2008) reported that lipid peroxidation, which have an important role in abiotic stresses decreased significantly from 100-200 Gy radiation dose in chickpea. Similarly, Baek et al. (2006) found that gamma irradiation lowered the MDA content in rice plants under salt stress.
The total soluble protein contents in cowpea plants increased at lowest concentrations of salinity but decrease at the high concentrations of salinity. These results are in accordance with Astorga and Melendez (2010) who found that protein content increase at the lowest concentration of NaCl (20
mM) but decreased at the high concentrations (40, 60 and 80 mM) of NaCl in Paulownia imperialis plants. One characteristic of saline stress is the removal of potassium ions by plant roots, which causes physiological imbalance because potassium is necessary for protein synthesis. Potassium loss causes diminished plant growth and development (Chen et al., 2007). If the stress is prolonged, it could affect protein synthesis and eventually cause it to decline (Caplan et al., 1990). A decrease in the protein level in salt-stressed plants may be attributed to a decrease in protein synthesis; the decreased availability of amino acid and the denaturation of enzyme involved in amino acid and protein synthesis.
Treatment with gamma rays caused highly significantly increase under all salinity treatments (25, 50 and 100 mM) except at 200 mM of NaCl which caused high significant decrease in the same content. These results are in accordance with Moussa (2011) who stated the total soluble protein content of soybean plants pre-exposed to a low dose of gamma rays (20 Gy) was 11% higher than that of plants subjected to drought stress. Owing to altered gene expression under gamma stress, qualitative and quantitative changes in total soluble protein content were observed (Corthals et al.,
2000). These proteins might play a role in signal transduction, anti-oxidative defense, anti-freezing, heat shock, metal binding, anti-pathogenesis or osmolyte synthesis, which are essential to a plant's function and growth (Gygi, et al., 1999). Also, Ling et al. (2008) reported that a low dose of gamma irradiation (30 Gy) enhanced protein synthesis in Citrus sinensis.
Nucleic acids content was in parallel with growth parameters. These results are in accordance with Zeid (2009 a) who found that leaf content of
DNA and RNA of maize seedlings decreased under salinity stress. Growth which is the integral of cell division and cell elongation was negatively affected by salinity which reduced the cellular water content and decreased content of nucleic acids required for anabolic processes. Also, Zeid (2009 b) suggested that the decrease in nucleic acids content in bean concurrently with the increase in RNase activity caused by the increase in salinity level might be involved in inhibiting nucleic acids biosynthsis and/or stimulating their degradation. In addition, Kabanov and Aziyashvili (1976) attributed the depression of nucleic acid metabolism to the cation imbalance (in particular to the abundance of Na+ cations in the cell) and also to intensify the activity of cytoplasmic RNase or to the reduction of phosphorus incorporation into nucleic acids.
The effect of gamma radiation is shown to induce a highly significant increase or significant increase in the DNA and RNA contents of shoots and roots of cowpea plant under (25, 50 and 100 mM) of NaCl except 200 mM which induce a highly significant decrease or significant decrease in the same contents of both shoots of cowpea plants as compared with control. These results are accordance with Khodary (2004) who found that treatment with gamma rays increased nucleic acids contents in salinized lupine plants. The mitigating effect of y - rays on the nucleic acids level in those plants treated with NaCl was also reported by Mostafa et al. (2000), who observed a stimulation in RNA synthesis of embryos of castor oil plants and maize grains irradiated with y - doses that activated plant growth and development.
Salinity caused accumulation in non enzymatic antioxidants in cowpea plants. Thus, these results seem to support the hypothesis that tolerance to oxidative stress plays an important role in adapting
plants to adverse environmental conditions (Smirnoff 1998). The same trend also was reported by YU and LIU (2003) in wild soybean. The increased ascorbic acid (AsA) content is a stress-protecting mechanism of plants under salinity conditions (Shalata et al., 2001). A high level of endogenous AsA is essential for maintaining the non-enzymatic scavenging system that protects plants from oxidative damage due to salinity stress (Shigeoka et al., 2002). In addition, a- tocopherols are groups of compounds that play different rols in plant metabolism and can play important roles in amelioration of biotic and abiotic stress (Hussein et al., 2007).
The high concentrations of NaCl (200 mM) affect on yield of cowpea plants and don't produce legumes. Enhancement of seed yield by using gamma rays was recorded by (Sundaravadivelu et al., 2006) in cotton, (El Sherif et al., 2011) in Hibiscus Sabdariffa.
The magnitude of salt-induced yield losses is dependent on different physiological and biochemical factors governed at different developmental stages of plants. One of the most important factors under saline environment may be the overall control mechanism of salt uptake through root and its subsequent distribution to shoot. The other factor is the maintenance of mineral nutrients such as K and Ca which are essentially required for the activities of enzymes, proteins synthesis and integrity of cell wall and plasma membrane (Taiz and Zeiger, 2006).
Gamma rays and salinity stress induced a considerable variation in the protein patterns of cowpea seeds. This variation has been manifested as the novel expression of some polypeptide, the absence of others and over expression of a third class polypeptides.
The protein bands which have molecular weights (102, 57, 49 and 37 kDa) were de novo synthesized in salinized cowpea seeds alone or in combination with gamma rays. It has been suggested that these proteins have an osmoprotection function or protected cellular structures. In this respect, Eman et al. (1992) showed the appearance of excess bands in leaf samples of Zea mays under salt stress. These findings indicated that these bands were salt inducible and could be involved in plant adaptation for growth under stress condition. Also, salt stress lead to difference in gene expressions where alterations in protein could be due to alteration in regulation of transcription, mRNA processing or due to altered rates of protein degradation. Also, Changes in protein synthesis under salinity and gamma rays treatments may be due to changes in the efficiency of mRNA translation or the regulation of RNA transcription transport and stability
The high concentrations of NaCl (100 mM) caused disappearance of some bands in cowpea seeds. These bands have molecular weights of 175, 67, 62, 54 and 41 kDa. One possible explanation for complete disappearance of some proteins under salt stress is that the gene (S) responsible for certain proteins might be completely suppressed as a result of stress. Therefore, the developed tissues had lost their ability to synthesize these proteins. It is also possible that the gene (S) had not been completely suppressed, but inhibited as the result of stress, and complete recovery of the inhibition was not achieved. This may apply to the protein that stained less densely under stress (Elobeidy et al., 2001).
Beltagi et al. (2006) studied the effect of gamma rays (2 and 32 Krad) on SDS PAGE proteins in salinized common bean plants. They found that, the
low dose (2 Krad) of gamma radiation induced stability in the total number of protein bands of common bean and synthesis of certain polypeptides under the highest level (3000 ppm) of NaCl.
CONCLUSION
Soil salinity is an enormous production problem for vegetable crops as saline conditions are known to suppress plant growth under irrigation. Salt stress affects all the major processes such as growth, yields, photosynthesis, protein synthesis and lipid metabolism. To conclude the present study, gamma irradiation can use to alleviate the harmful effect of salt stress. Gamma irradiation can solve the production problems caused by high salinity.
REFERENCES
Ashraf, M. (2009) Biotechnological approach of improving plant salt tolerance using antioxidants as markers. Biotechnol. Advances, 27, 84-93.
Ashraf, M., Cheema, A. A., Rashid, M., and Qamar, Z. (2003) Effect of gamma rays on M1 generation in Basmati rice. Pak. J. of Bot., 35(5), 791-795.
Ashraf, M. (1994) Breeding for salinity tolerance in plants. Critical Reviews in Plant Sci., 13, 17-42. Astorga, G. I. A. and Melendez, L. A. (2010) Salinity effects on protein content, lipid peroxidation, pigments and proline in Paulownia imperialis (Siebold and Zuccarini) and Paulownia fortunei (Seemann and Hemsley) grown in vitro. Elect. J.Biotechnol., 13( 5), online.
Baek, M. H., Chung, B. Y., Kim, J. H., Wi, S. G., Kim, J. S. and Lee, I. J. (2006) Gamma radiation and hormone treatment as tools to reduce salt stress in rice (Oryza sativa L.). J. Plant Biol., 49
(3), 257-260.
Beltagi, M. S., Ismail, M. A. and Mohamed, F. H.
(2006) Induced salt tolerance in common bean (Phaseolus vulgaris L.) by gamma irradiation. Pak. J. Biolo. Sci., 9(6), 1143-1148.
Ben Amor, N., Jimenez, A., Megdiche, W., Lundqvist, M., Sevilla, F. and Abdelly, C. (2007) Kinetics of the anti-oxidant response to salinity in the halophyte Cakile maritime. J. Integrative Plant Biol., 49, 982-992.
Burton, K. (1956) A study of the conditions and mechanism of the diphenylamine reaction of colorimetric estimation of deoxyribonucleic acid. Bioch. J., 62, 315.
Caplan, A. B., Dekeyser, C. R. and Van Montagu, M. (1990) Salinity and drought stress in rice. In: Sangwan, R. S., Sangwan-Norrel, B. eds. The Impact of Biotechnology in Agriculture. Kluwer Academic Publishers The Netherlands, 391402.
Chakravarty, B. and Sen, S. (2001) Enhancement of regeneration potential and variability by irradiation in cultured cells of Scilla indica. Biol. Plant., 44(2), 189-193.
Chen, Z., Cuin, A., ZhoU, M., Twomey, A., Naidu, B. P. and Shabala, S. (2007) Compatible solute accumulation and stress mitigating effects in barley genotypes contrasting in their salt tolerance. J. Exp. Bot., 58(15-16), 4245-4255.
Corthals, G., GygI, S., Aebersold, R. and Patterson, S. D. (2000) Identification of proteins by mass spectrometry. Proteome Res., 2(1), 286-290.
Critchley, C. (1983) Further studies on the role of chloride in photosynthetic O2 evolution in higher plants. Biochimica et Biophysica Acta, 724, 1-5.
Dische, E. L. (1953) J American Chemistry Society
22, 3014. In Physiological studies on the herbicide "Cotorane" ROUSHDY, S. S., 1983, M. Sc. Thesis, Ain Shams Univ. Cairo Egypt.
Dodd, G. L. and Donovan, L. A. (1999) Water potential and ionic effects on germination and seeding growth of two cold desert shrubs. Amer. J. Bot., 86, 1146-1153.
Dolatabadian, A., Modarressanavy, S. A. M. and Ghanati, F. (2011) Effect of salinity on growth, xylem structure and anatomical characteristics of soybean. Notulae Scientia Biologicae, 3(1), 41-45.
Dorgham, E. A. (1991) Effect of water stress, irradiation and nitrogen fertilization on grain filling, yield and quality of certain wheat cultivars. Ph.D. Thesis, Ain Shams University of Cairo, Egypt.
Dubois, M., GilleS, K. A., Hamilton J. K., Rebers, P. A. and Smith, F. (1956) Colorimetric method for determination of carbohydrates and related substances. Analytical Chemistry, 28, 350-356.
El Sherif, F., Khattab, S., Ghoname, E., Salem, N. and Radwan, K. (2011) Effect of gamma irradiation on enhancement of some economic traits and molecular changes in Hibiscus Sabdariffa L. Life Sci. J., 8(3), 220-229.
Elobeidy, A. A., Fayek, M. A. and Mohmoud, R. A.
(2001) Quantifying salt tolerance responses of some banana genotypes in vitro.1 st international conference on: "Biotechnology Applications for the Arid Regions", Eds. Shayji, Y.A., Siolhu, J.S., Saleem, M. and Guerinik, K. State of Kuwait- Kuwait. 135-165.
Fahmy, E. M., Abdel-Tawab, F. M., Tayel, A. A., Baheildin, A. and El-Enany, M. A. (1992) Biochemical genetic markers for salt tolerance in maize (Zea mays). Annals Agricultural
Science Ain Shams University Cairo. 37, 147157.
Gygi, S. P., Rochon, Y., Franza, B. R. and Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Mol. Cell Biol., 19(1), 1720-1730.
Hameed, A., Naseer, S., Iqbal, T., Sayed, H. and Ahsanul, M. (2008) Effect of NaCl salinity on seedling growth, senescence, catalase and protease activities in two wheat genotypes differing in salt tolerance. Pak. J. Bot., 40(3), 1043-1051.
Heath, R. L. and Packer, L. (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Bioch. Bioph., 125(1), 189-198.
Helaly, M. N. M. and El-Hosieny, H. A. M. (2011) Effectiveness of gamma irradiated protoplasts on improving salt tolerance of lemon (Citrus limon L. Burm. f.). Amer. J. Plant Physiol., 6(4), 190 - 208.
Hussein, M. M., Balbaa, L. K. and Gadallah, M. S.
(2007) Developing a salt tolerant cowpea using alpha tocopherol. J. Appl. Sci. Res., 3(10), 1234-1239.
Kabanov, V. V. and Aziyashvili, L. N. (1976) Changes in nucleic acid metabolism of plants under salinisation conditions. Soviet. Plant Physiol., 14, 606 - 613.
Khodary, S. E. A. (2004) Effect of NaCl salinity on improvement of nitrogen metabolism and some ions uptake in lupine plants subjected to gamma irradiation. Inter. J. Agri. Biol., 6(1), 14.
Kiong, A. L. P., Grace Lai, A., Hussein, S. and Harun, A. R. (2008) Physiological responses of Orthosiphon stamineus plantlets to gamma
irradiation. Amer. Eur. J. Sustain Agri., 2(2), 135-149.
Kovacs, E. and Keresztes, A. (2002) Effect of gamma and UV-B/C radiation on plant cell. Micron, 33, 199-210.
Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of head bacteriophage T4. Nature, 227, 680-685.
Ling, A. P. K., Chia, J. Y., Hussein, S. and Harun, A. R.
(2008) Physiological responses of Citrus sinensis to gamma irradiation. World Appl. Sci. J., 5 (1), 12-19.
Lowry, O. H., Rosembrough, N. J., Farr, A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193(1), 267-275.
Mahajan, S. and TutejA, N. (2005) Cold, salinity and drought stresses. Arch. Bioch. Biophy., 444, 139-158.
Morse, M. L. and Carter, C.F. (1949) The synthesis of nucleic acid in cultures of Escherchia coli, strain B and B/R. J. Bacteriol., 58, 317.
Mostafa, I. Y., Laila, M. A., Aly, M. A. S., Kord, M. A. and Kamel, H. A. (2000) Manifestation of some biochemical changes in wheat and Beans due to salinity and /or y - irradiation. Arab J. Sci. Appl., 33, 233-41.
Moussa, H. R. (2011) Low dose of gamma irradiation enhanced drought tolerance in soybean. Acta Agronomica Hung., 59(1), 1-12.
Mukherjee, S. P. and ChoudhurI, M. A. (1983) Implications of water stress induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol. Plant., 58 (2), 166-170.
Munns, R. and TesteR, M. (2008) Mechanisms of
Salinity Tolerance. Annual Review Plant Biol., 59, 651-8.
Neff, M. M. and Chory, J. (1998) Genetic interactions between Phytochrome A, Phytochrome B, and Cryptochrome 1 during Arabidopsis development. Plant Physiol., 118, 27-36.
Parida, A. K. and Das, A. B. (2005) Salt tolerance and salinity effects on plants: a review. Ecoto. Environ. Safety, 60, 324-349
Philip, B., Bernard, L. and William, H. (1954) Vitamins and Deficiency Diseases, In, Practical Physiological Chemistry, McGraw-Hill company, INC. New York, Toronto, London, 1272-1274.
Piotr, S. and Klobus, G. (2005) Antioxidant defense in the leaves of c3 and c4 plants under salinity stress. Physiol. Plant., 125, 31-40.
SAS-Programme (1982) SAS user,s Guide Statistics SAS Institute, INC, Raleiegh. NC. 584.
Shalata, A., Mittova, V., Guy, M. andTal, M. (2001) Response of the cultivated tomato and its wild salt tolerant relative Lycopersicum pennellii to salt dependent oxidative tress: the root antioxidant system. Physiol. Plant., 112, 487494.
Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y. and Yoshimura, K.
(2002) Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot., 53, 13051319.
Smirnoff, N., (1998). Plant resistance to
environmental stress. Current Opinion in Biotechnol., 9, 214-219.
Sundaravadivelu, K., Ranjithselvi, P. and Reddy, V. R. K. (2006) Induced genetic variability in cotton (Gossypium hirsutum L.) for yield and its
components. Crop Res. Hisar, 32(3), 442-446. Taamalli, W., Abz, L., Youssef, N. B., Miled, D. D. B. and Zarrouk, M. (2004) Lipid breakdown in sunflower (Helianthus annuus L.) seeds during post germinative growth under salt-stress. Rivista Italian delle Sostanze Grasse, 81, 90-97 Taiz, L. and Zeiger, E. (2006) Plant Physiology (4th edn). Sinauer Associates, Inc., Sunderland, Massachusetts.
Vernon, L. P. and Seely, G. R. (1966) The chlorophylls. Academic Press. New York.
Wi, S. G., Chung, B. Y., Kim, J. S., Kim, J. H., Baek, M. H., Lee, J. W. and Kim, Y. S. (2007) Effects of gamma irradiation on morphological changes and biological responses in plants. Micron, 38, 553-564.
Wise, R. R. and Naylor, A. W. (1987) Chilling enhanced photo-oxidation. Plant Physiology, 83, 278 - 282.
Yu, B. J. and. Liu, Y. L. (2003) Effects of salt stress on the metabolism of active oxygen in seedlings of annual halophyte Glycine soja. Acta Botanica Boreali-Occidentalia Sinica, 23, 1822.
Zeid, I. M. (2009a) Trehalose as osmoprotectant for maize under salinity-induced stress. Res. J. Agri. Biol. Sci., 5(5), 613-622.
Zeid, I. M. (2009b) Effect of arginine and urea on polyamines content and growth of bean under salinity stress. Acta Physiol.Plant., 31, 65-70.
