Common bean (Phaseolus vulgaris L.) seedlings overcome NaCl stress as a result of presoaking in Moringa oleifera leaf extract

Scientia Horticulturae 162 (2013) 63–70

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Common bean (Phaseolus vulgaris L.) seedlings overcome NaCl stress as a result of presoaking in Moringa oleifera leaf extract Mostafa M. Rady a,∗ , Bhavya Varma C. b , Saad M. Howladar c a

Botany Department, Faculty of Agriculture, Fayoum University, 63514 Fayoum, Egypt Fr. Cecil J. Saldanha Centre for Experimental Research in Bioscience, St. Joseph’s College Research Center, Post Box. 27094, 46, Langford Road, Bangalore 560027, India c Biology Department, Faculty of Sciences, Al-Baha University, Al-Baha, Saudi Arabia b

a r t i c l e

i n f o

Article history: Received 10 April 2012 Received in revised form 25 June 2013 Accepted 31 July 2013 Keywords: Seed soaking Moringa oleifera leaf extract Salinity Growth Yield Antioxidant system

a b s t r a c t Bean seed soaking in Moringa oleifera leaf extract to overcome NaCl stress and its effect on growth, yield, osmoprotectants and antioxidant system of bean plants were investigated. Plants exposed to NaCl exhibited a significant decline in growth, yield, leaf photosynthetic pigments and K+ contents and K+ /Na+ ratio, while showed a significant increase in the contents of osmoprotectants and Na+ , and the activity of enzymatic and non-enzymatic antioxidants. However, the presoaking treatment improved growth, yield and antioxidant system, and detoxified the stress generated by 100 mM NaCl. The combined treatment of NaCl + presoaking in Moringa oleifera leaf extract overcame the adverse effects of NaCl stress by the increase in the content of osmoprotectants, the activity of enzymatic and non-enzymatic antioxidants and the ratio of K+ /Na+ . Therefore, we recommend using Moringa oleifera leaf extract as a soaking solution for bean seeds before sowing in a saline soil. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Oceans cover about 70% of the Earth’s surface. The oceans of Earth serve many purposes, especially on the weather and temperature. The saltiest water in the Red Sea laying between Africa and Asia has a salinity of about 40% (due to very high evaporation rates and low fresh water influx). The least salty seas are in the Polar Regions, where both melting polar ice and a lot of rain dilute the salinity. In addition, irrigation with poor quality water is one of the main factors that leads to salt accumulation and the resulting decrease in agricultural productivity. Excess salinity in the soil effects total growth and yield, the extent depends on the degree of salinity. The primary effect of excess salinity is that it renders less water absorption by plants. Although the amount of salt affected land is about 9003106 ha, its extent is sufficient to pose a threat to agriculture (Flowers and Yeo, 1995; Munns, 2002). Crop plants will not grow in high concentrations of salt: only halophytes grow in concentrations of sodium chloride higher than about 400 mM. The physiological and molecular mechanisms of tolerance to osmotic and ionic components of salinity stress are reviewed at the

∗ Corresponding author. Tel.: +2 01007302668/01092392038; fax: +20 2846343970/2846334964. E-mail addresses: [email protected], [email protected], [email protected] (M.M. Rady). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.07.046

cellular, organ, and whole-plant level. Plant growth responds to salinity in two phases: a rapid, osmotic phase that inhibits growth of young leaves, and a slower, ionic phase that accelerates biological aging of mature leaves. Plant adaptations to salinity are of three distinct types: osmotic stress tolerance, Na+ or Cl− exclusion, and the tolerance of tissue to accumulated Na+ or Cl− . Salinity considers one of the major factors affecting the agricultural productivity worldwide. In the arid and semiarid regions, soil salinization may be caused by poor irrigation water which contains considerable amounts of salts, salt accumulation in the soil surface layer due to over-irrigation, proximity to the sea and/or the capillarity rise of salts from underground water into the root zone due to the excessive evaporation (Gama et al., 2007). In addition, low rainfall, high evaporation rate and poor water management may also cause salinity related problems in these regions. Salinity reduces the ability of plants to utilize water and causes a reduction in the growth and yield, and changes in the plant metabolic processes (Munns, 1993, 2002). Plants grown under saline conditions are stressed basically in three ways; water deficit caused by reduced water potential in the rhizosphere, Na+ and Cl− ions phytotoxicity and nutrient imbalance by the reduction in the uptake and/or shoot transport (Munns and Termaat, 1986; Marschner, 1995). Moringa oleifera is a highly nutritive multipurpose plant grown for fresh vegetable, livestock fodder, green manure, biogas, biopesticide, seed production (Fuglie, 1999). In various parts of the world Moringa oleifera termed as miracle tree that used in medicine

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because it is rich in amino acids, K, Ca, Fe, ascorbate, and growth regulating hormones like zeatin (Makkar and Becker, 1996; Basra et al., 2009a,b). Zeatin promotes cell division and cell elongation (Taiz and Zeiger, 2010). It also enhances the antioxidant properties of many enzymes and protects the cells from aging effects of reactive oxygen species (Yasmeen et al., 2013). Zeatin concentration in Moringa leaves gathered from various parts of the world was found to be very high, equal thousands of times more concentrated (from 5 mcg to 200 mcg g−1 leaf material) than most plants (from 0.00002 mcg g−1 to 0.02 mcg g−1 leaf material) studied so far (Foidl et al., 2001). Such plant growth promoters influence plant growth in several ways and also promote defense mechanisms against abiotic stresses by harmonizing the plant growth regulator’s endogenous concentration. Plant growth promoters are usually used as a foliar application or as a seed-priming agent. Foliar spray accelerates the growth of young plants. As a result plants are firmer and more resistant to biotic and abiotic stresses and improved longer lifespan. Such plants produce more and larger fruit and higher yield (20–35%) at harvest time. Soaking the seed of many crops in MLE promoted seed germination, growth and productivity under normal (Phiri and Mbewe, 2010; Basra et al., 2011; Nouman et al., 2013) and stress conditions (Afzal et al., 2012; Nouman et al., 2012; Yasmeen et al., 2012, 2013). Common bean (Phaseolus vulgaris L.) is one of the most important vegetable crops that belong to Fabaceae. Food legumes, including beans, are an important component of the agricultural sectors of developing countries due to their capacity to produce significant quantities of protein-rich seed for human nutrition. Common bean is classified as a salt-sensitive plant (Maas and Hoffman, 1977). Approximately, 20–30% of the produced bean in the Middle East, and 50–10% in Latin America is affected by soil salinity (CIAT, 1992). The effect of dissolved salts on plant growth depends on their concentration in the soil. It is extremely difficult to measure salinity in the soil at the usual field moisture contents due to sampling problems. Therefore, the present study was planned to determine the effect of bean seed soaking in Moringa oliefera leaf extract (MLE) on growth, yield and antioxidant system of bean plants when growing under the adverse effects of 100 mM NaCl-salinity. In addition, MLE was used to help in improving the bean performance better than synthetic growth stimulator. 2. Materials and methods 2.1. Crude extract of Moringa oliefera Five hundreds g of Moringa oliefera plant leaves was weighed and dried in shade for 7 days. Dried leaves were ground into fine coarse powder and then mixed with 1 l ethanol and kept aside for 72 h with occasional stirring (Nikkon et al., 2003). After stirring process, solution was filtered twice through whatman No. 1 filter paper and with non-absorbent cotton. Filtered solution was evaporated with the help of vacuum rotary evaporator. The crude extract was dissolved in 2 l distilled water and stored in the refrigerator at 4 ◦ C until use.

8, 10, 12, 14 and 16 h, in a preliminary study, under 25 ◦ C and aeration conditions, to select the appropriate soaking period that generate the best germination and growth, and then air-dried overnight. These treated seeds were sown in Petri dishes, and then germination percentage and growth were determined. The soaking period of 8 h was found to be the most effective in this concern (data not shown). For the main experiment, 8 h-soaked seed in Moringa oleifera leaf extract (MLE) (for the single MLE and combined NaCl + MLE treatments) and in distilled water (for the control and single NaCl treatments) were sown in plastic pots (3 seeds pot−1 ) containing 6 kg acid washed sand and moistened with deionized water for each. The soil of each pot of saline treatments was supplied with 600 ml of 100 mM NaCl with the nutrient solution (Hoagland solution). The supply with NaCl solution was done once a week; however the irrigation was applied twice a week. Pots were arranged in randomized blocks design with 20 replicates for each treatment, in the glass greenhouse temperature that was adjusted to 30/24 ◦ C, 85/60% R.H. day/night and light intensity approximately at 3500 lux for a period of 12 h a day. Plants were sampled for chemical analyses at 30 days after sowing and were growing until the green yield. 2.3. Estimation of plant growth and green yield Thirty-day-old plants were removed from six pots from each treatment along with the sand and were dipped in a bucket filled with water. The plants were moved smoothly to remove the adhering sand particles and the lengths of root and shoot were measured by using a meter scale. The plants were then placed in an oven run at 70 ◦ C to a constant weight. The dried plants were weighed to record the plant dry mass. The powdery dried leaves of six plants were used to determine the concentrations of glycine betaine and elements. The fresh leaves of another six plants were used to assess other parameters. At the end of experiment, green pods were collected, counted and weighed to obtain green pod yield pot−1 . 2.4. Photosynthetic pigments determination Total chlorophyll and carotenoids contents (mg g−1 FW) were estimated adopting the procedure given by Arnon (1949). Leaf discs (0.2 g) of 30-day-old plants were homogenized with 50 ml 80% acetone. The slurry was strained through a cheese cloth and the extract was centrifuged at 15,000 × g for 10 min. the optical density of the acetone extract was measured at 663, 645 and 470 nm using a UV160A UV Visible Recording Spectrometer, Shimadzu, Japan. 2.5. Total soluble sugars determination Total soluble sugars content was assessed as follows: 0.2 g leaves were washed with 5 ml 70% ethanol and homogenized with 5 ml 96% ethanol. The extract was centrifuged at 3500 × g for 10 min. The supernatant was collected and stored at 4 ◦ C (Irigoyen et al., 1992). Freshly prepared anthrone (3 ml) was added to 0.1 ml supernatant. This mixture was incubated in hot water bath for 10 min. The absorbance was recorded at 625 nm with a Bausch and Lomb2000 Spectronic Spectrophotometer. 2.6. Proline determination

2.2. Seed treatment and growth conditions The common bean (Phaseolus vulgaris L. cv. Bronco) was selected for this experiment. This selection is based on our preliminary studies which showed that this cultivar was most salt-sensitive among all common bean cultivars cultivated in Middle East, using the NaCl concentrations less than 100 mM (data not shown). Seed were soaked in the crude extract of Moringa oleifera leaf for 4, 6,

Proline content in bean leaves was measured following the rapid colorimetric method of Bates et al. (1973). Proline was extracted from 0.5 g of dry leaf samples by grinding in 10 ml of 3% sulphosalicylic acid. The mixture was then centrifuged at 10,000 × g for 10 min. Two ml of the supernatant was added into test tubes and 2 ml of freshly prepared acid-ninhydrin solution was also added. Tubes were incubated in a water bath at 90 ◦ C for 30 min. The

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reaction was terminated in ice-bath. The reaction mixture was extracted with 5 ml of toluene and the vortex process was done for 15 s. The tubes were allowed to stand at least for 20 min in the dark at room temperature to allow the toluene and aqueous phases to be separated. The toluene phase was then carefully collected into test tubes and toluene fraction was read at 520 nm using a UV-160A UV Visible Recording Spectrometer, Shimadzu, Japan. The proline content in the sample was determined from a standard curve using analytical grade proline.

EDTA (10 mM), NADPH (10 mM), DTNB (12 mM) and 20 U ml−1 glutathione reductase enzyme. The results were expressed as mmol total GSH g−1 FW.

2.7. Glycine betaine determination

2.9.4. O2 •− Bean leaves (100 mg) were cut into 1 mm × 1 mm fragments and immersed for 1 h at room temperature in 10 mM K-phosphate buffer, pH 7.8, 0.05% NBT and 10 mM NaN3 . Two ml of immersed solution was heated at 85 ◦ C for 15 min and cooled rapidly. Optical density was measured colorimetrically at 580 nm and the O2 •− content was expressed as A580 g−1 FW (Kubis, 2008).

Glycine betaine (GB) assessment was carried out using the method described by Grive and Gratton (1983). The extraction of GB was made using 0.2 g of leaf material in 10 ml of distilled water under stirring process back and forth for 24 h. Extract was then filtered and 2 ml of 2 N H2 SO4 was added. The solution was incubated 16 h at 4 ◦ C and then centrifuged at 9200 × g for 15 min at 0 ◦ C. The pellet obtained by centrifugation was re-suspended in 1,2 dichloroethane. After 2 h was quantified at an absorbance of 365 nm against a standard curve of GB. The result of GB content was expressed as ␮g g−1 dry weight. 2.8. Leaf mineral determinations The concentrations of Na+ and K+ were determined as follows: 0.2 g of dried leaf was digested with sulphuric acid in the presence of H2 O2 (Wolf, 1982). The mixture was then diluted with distilled water. The total leaf concentrations of Na+ and K+ were measured directly using Flame Spectrophotometry (Lachica et al., 1973). The leaf Cl− concentration was analyzed by an aqueous extraction of 0.2 g of dry plant material in 20 ml of distilled water. Cl− concentration was measured according to Diatloff and Rengel (2001). The results were calculated and expressed as mg g−1 dry weight. 2.9. Ascorbate, glutathione, malondialdehyde, O2 •− and H2 O2 determinations 2.9.1. Ascorbate (AsA) The method of Okamura (1980) was followed to determine AsA with the modification of Law et al. (1992). Four hundred ␮l chlorophyll (250–350 ␮g) was taken into a test tube with 200 ␮l trichloroacetic acid (10%) was added. The mixture was mixed in a vortex and cooled by keeping it in an ice for 5 min. To this solution, 10 ␮l NaOH (5 M) was added and centrifuged for 2 min in a Microfuge. Supernatant was collected. In one test tube, 200 ␮l supernatant was taken and 200 ␮1 of 150 mM-NaH2 PO4 buffer, pH 7.4, also 200 ␮1 of distilled water were added. In another test tube, 200 ␮l supernatant was taken to which 200 ␮l buffer, 100 ␮l of dithiothreitol (l0 mM) were added and incubated at room temperature for 15 min. After incubation, 100 ␮l N-ethylmaleimide (0.5%) was added. 400 ␮l trichloroacetic acid (10%), 400 ␮l H3 PO4 (44%), 400 ␮l bipyridyl (4%), 70% ethanol and 200 ␮l FeCl3 (3%) were added to both samples. Samples were incubated at 37 ◦ C for 60 min and Optical density was recorded at A525 . A standard curve in the range 0–40 nmol of AsA was used for calibration. The results were expressed as mmol total AsA g−1 FW. 2.9.2. Glutathione Total glutathione was determined according to the method of Gossett et al. (1994). A weight of 0.5 g leaves was homogenized in 10 ml HCl (0.2 N) and centrifuged at 16,000 × g for 10 min. Supernatant solution was collected. 500 ␮l supernatant was taken into a test tube and neutralized with sodium phosphate buffer (0.2 M), pH 5.6. After neutralization, the extract was added to the reaction mixture consisting of sodium phosphate buffer (0.2 M), pH 7.5,

2.9.3. Malondialdehyde (MDA) Leaf tissue (0.1 g) was homogenized with 5 ml 0.07% NaH2 PO4 ·2H2 O and 1.6% Na2 HPO4 ·12H2 O (50 mM) and centrifuged at 20,000 × g for 25 min. The results of MDA were expressed as A532–600 g−1 FW (Heath and Packer, 1968).

2.9.5. H2 O2 Hydrogen peroxide was determined using the method of Mukherje and Choudhuri (1983). Plant tissue was extracted in acetone. Titanium reagent and ammonium were added to the extract and dissolved in sulphuric acid (1 M). Absorbance of the supernatant was measured at 415 nm. The results of H2 O2 concentration were expressed as ␮mol g−1 FW. 2.10. Enzymatic antioxidants activities Superoxide dismutase (SOD; EC 1.15.1.1) activity was assessed by monitoring the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT) (Giannopilitis and Ries, 1977; Beyer and Fridovicht, 1987; Yu et al., 1998). One unit of SOD activity was defined as the amount of enzyme required for the reduction of 50% NBT. SOD activity was expressed as A564 min−1 g−1 protein. Catalase (CAT; EC 1.11.1.6) activity was determined by measuring the consumption of H2 O2 (Nakano and Asada, 1981). The reaction mixture consisted of 25 mM Tris-acetate buffer, pH 7.0, 0.8 mM Na-EDTA and 20 mM H2 O2 . The enzyme assay was performed at 25 ◦ C. CAT activity was expressed as A290 min−1 g−1 protein. Ascorbate peroxidase (APX; EC 1.11.1.11) activity was determined following the method described by Rao et al. (1996) by recording the optical density at 290 nm and the activity was expressed as A290 min−1 g−1 protein. Glutathione reductase (GR; EC 1.6.4.1) activity was measured after monitoring the oxidation of NADPH for 3 absorbance was taken at340 nm activity expressed as A340 min−1 mg−1 protein (Rao et al., 1996). Protein was estimated in crude enzyme extracts by dye binding assay (Bradford, 1976). 2.11. Statistical analysis: Data were analyzed by a simple variance analysis (ANOVA) and differences between the means were compared by Fisher’s least-significant difference test (LSD) at a probability level of 95%. Significance levels were expressed as P = 0.05, data are significant when P = 0.05. 3. Results Moringa leaf extract (MLE), as shown in Table 1, is rich in many minerals such as calcium, magnesium, potassium, phosphorus, iron, manganese, zinc and copper. It is also rich in some antioxidants, i.e. proline, phenols, carotenoids and ascorbic acid, besides

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Table 1 Some chemical constituents of moringa leaf extract (on dry weight basis). Component

Value (mg g−1 DW)

Amino acids Proline Total sugars Ash Calcium Magnesium Potassium Phosphorus Sodium Iron Manganese Zinc Copper Total phenols Total carotenoids Total chlorophyll Ascorbic acid (mg 100 g−1 FW)

398.6 31.24 347.8 121.0 16.33 3.892 14.62 3.684 0.845 0.394 0.076 0.048 0.035 1.542 1.656 4.592 810.2

Phytohormones (␮g g−1 DW): Indole-3-acetic acid Gibberellins Cytokinins Abscisic acid

0.82 0.74 0.96 0.34

some osmoprotectants, i.e. amino acids, soluble sugars and K. In addition, it is rich in phytohormones such as indole-3-acetic acid, gibberellins and cytokinins.

3.1. Growth and green yield of bean plants as affected by Moringa oleifera leaf extract (MLE), NaCl and their combination Data in Table 2 show a significant reduction in growth termed as shoot and root lengths and plant dry mass which resulted in a significant loss in green yield of bean plants under the treatment of 100 mM NaCl when compared to the control. However, bean presoaking in Moringa oleifera leaf extract (MLE) for 8 h significantly increased these parameters. The combined treatment of MLE + NaCl (100 mM) alleviated the adverse effects of NaCl-salinity and kept the growth traits and bean green yield to be in the same rate with those in the control.

3.2. Leaf photosynthetic pigments and osmoprotectants as affected by Moringa oleifera leaf extract (MLE), NaCl and their combination Total chlorophyll and total carotenoids contents were significantly reduced; however the contents of total soluble sugars, free proline and glycine betaine were significantly increased in bean leaves subjected to 100 mM NaCl compared to in the control plants (Table 3). MLE treatment significantly increased the contents of leaf pigments and total soluble sugars, but did not significantly affect the contents of proline and glycine betaine. The combination between MLE and 100 mM NaCl was found to be overcame the undesirable effects of NaCl-salinity. It significantly increased total soluble sugars and proline contents, however did not significantly affect the contents of leaf photosynthetic pigments and glycine betaine. 3.3. Leaf minerals as affected by Moringa oleifera leaf extract (MLE), NaCl and their combination A significant increase in the contents of Na+ and Cl− ions was recorded as a result in 100 mM NaCl treatment compared to the control (Table 4). In contrast, K+ ion content and K+ /Na+ ratio were reduced. The significant increase in the content of K+ ions and insignificant increase in the Na+ and Cl− ions content and in the K+ /Na+ ratio were noted under the treatment of MLE when compared to those in the control. The combined treatment of MLE + 100 mM NaCl showed a significant increase in K+ , Na+ and Cl− ions content as compared to those in the control, however Na+ and Cl− ions content was significantly reduced when compared with the saline (100 mM NaCl) treatment. Although the ratio of K+ /Na+ was reduced under the combined treatment as compared to the control and MLE treatments, it significantly increased when compared to the saline treatment. 3.4. Ascorbate, glutathione, malondialdehyde, O2 •− and H2 O2 as affected by Moringa oleifera leaf extract (MLE), NaCl and their combination Table 5 shows that the contents of ascorbate, glutathione, malondialdehyde, O2 •− and H2 O2 were significantly increased as a result in subjecting bean plants to 100 mM NaCl as compared to those in the control plants. When bean seeds soaked in MLE, plants exhibited a significant increase in the contents of ascorbate and

Table 2 Effect of seed soaking (8 h) in Moringa oleifera leaf extract (MLE) on the growth traits (shoot length, root length and plant dry mass) and yield of bean (Phaseolus vulgaris L. cv. Bronco) plants grown under 100 mM NaCl stress. Treatments

Shoot length (cm)

Control MLE NaCl NaCl + MLE

23.8 28.1 13.3 22.3

± ± ± ±

Root length (cm)

1.2b 1.4a 0.5c 0.9b

19.0 22.8 10.2 18.1

± ± ± ±

1.0b 0.8a 0.7c 1.2b

Plant dry mass (g) 6.58 7.79 2.43 6.29

± ± ± ±

0.23b 0.33a 0.10c 0.31b

No. of pods pot−1 8.04 9.79 2.17 7.75

± ± ± ±

0.22b 0.33a 0.20c 0.27b

Green pod yield pot−1 (g) 30.45 38.71 12.49 28.92

± ± ± ±

1.26b 2.04a 0.72c 1.44b

Values are means ± SE (n = 6) and differences between means were compared by Fisher’s least-significant difference test (LSD; P = 0.05). Mean pairs followed by different letters are significantly different.

Table 3 Effect of seed soaking (8 h) in Moringa oleifera leaf extract (MLE) on the accumulation of total chlorophyll, total carotenoids, total soluble sugars, free proline and glycine betaine in the leaf of bean (Phaseolus vulgaris L. cv. Bronco) plants grown under 100 mM NaCl stress. Treatments

T. chlorophyll (mg g−1 FW)

Control MLE NaCl NaCl + MLE

1.41 1.72 0.82 1.30

± ± ± ±

0.07b 0.06a 0.03c 0.07b

T. carotenoids (mg g−1 FW) 0.79 0.95 0.63 0.75

± ± ± ±

0.03b 0.05a 0.05c 0.04b

T. S. Sugars (mg g−1 FW) 10.17 13.21 29.10 35.28

± ± ± ±

0.82d 0.91c 1.08b 1.29a

F. Proline (␮g g−1 DW) 42.98 44.62 84.13 65.43

± ± ± ±

2.11c 2.02c 3.44a 3.03b

Glycine betaine (␮g g−1 DW) 79.52 83.39 96.25 80.01

± ± ± ±

4.21b 3.96b 5.04a 4.12b

Values are means ± SE (n = 6) and differences between means were compared by Fisher’s least-significant difference test (LSD; P = 0.05). Mean pairs followed by different letters are significantly different.

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Table 4 Effect of seed soaking (8 h) in Moringa oleifera leaf extract (MLE) on the leaf K+ , Na+ and Cl− accumulation and the ratio of K+ /Na+ in bean (Phaseolus vulgaris L. cv. Bronco) plants grown under 100 mM NaCl stress. Treatments

K+ (mg g−1 DW)

Na+ (mg g−1 DW)

Cl− (mg g−1 DW)

K+ /Na+ ratio

Control MLE NaCl NaCl + MLE

3.62 ± 0.29c 4.70 ± 0.41b 2.45 ± 0.11d 5.54 ± 0.32a

2.17 ± 0.10c 2.41 ± 0.12c 7.49 ± 0.25a 5.82 ± 0.17b

0.88 ± 0.03c 0.97 ± 0.03c 2.07 ± 0.07a 1.77 ± 0.08b

1.67 ± 0.21a 1.95 ± 0.27a 0.33 ± 0.02c 0.95 ± 0.03b

Values are means ± SE (n = 6) and differences between means were compared by Fisher’s least-significant difference test (LSD; P = 0.05). Mean pairs followed by different letters are significantly different.

Table 5 Effect of seed soaking (8 h) in Moringa oleifera leaf extract (MLE) on total ascorbic acid (AsA), total glutathione (GSH), malondialdehyde (MDA), hydrogen peroxide (H2 O2 ) and O2 •− contents in the leaf of bean (Phaseolus vulgaris L. cv. Bronco) plants grown under 100 mM NaCl stress. Treatments

AsA (mmol g−1 FW)

Control MLE NaCl NaCl + MLE

0.52 0.58 0.69 0.79

± ± ± ±

0.01d 0.03c 0.03b 0.02a

GSH (mmol g−1 FW) 3.98 4.89 7.03 8.24

± ± ± ±

0.13d 0.11c 0.21b 0.18a

MDA (A532–600 g−1 FW) 0.12 0.12 0.18 0.13

± ± ± ±

H2 O2 (␮mol g−1 FW)

0.009b 0.007b 0.011a 0.007b

1.24 1.21 2.33 1.32

± ± ± ±

O2 •− (A580 g−1 FW)

0.05b 0.06b 0.09a 0.07b

0.43 0.42 0.59 0.46

± ± ± ±

0.03b 0.02b 0.03a 0.02b

Values are means ± SE (n = 6) and differences between means were compared by Fisher’s least-significant difference test (LSD; P = 0.05). Mean pairs followed by different letters are significantly different.

glutathione, while their contents of malondialdehyde, O2 •− and H2 O2 were unaffected compared to the control plants. The combined treatment of MLE + NaCl gave the same result of the single treatment of MLE which enabled plants to overcome the adverse influences resulted in saline treatment. 3.5. Enzymatic antioxidants activities as affected by Moringa oleifera leaf extract (MLE), NaCl and their combination Data in Table 6 show that SOD, APX and GR activities were significantly increased, but CAT activity was reduced under the treatment of 100 mM NaCl as compared to those in the control. The same result was noted with the single treatment of MLE and the combined treatment of MLE + 100 mM NaCl. However, maximum activities of SOD, APX and GR as well as minimum activities of CAT were recorded under the combined treatment followed by the saline treatment. This result helped bean plants to overcome the undesirable effects of saline treatment. 4. Discussion Salinity is one of the major factors affecting agricultural productivity worldwide. In the arid and semiarid areas, salinity could be caused by poor irrigation water which contains considerable amounts of salts, accumulation of salts in the top layer of the soil due to over-irrigation, proximity to the sea, and the capillarity rise of salts from underground water into the root zone due to excessive evaporation. Also, low rainfall, high evaporation rate and poor water management could cause salinity related problems in these areas. In this study, NaCl-salinity (100 mM) significantly reduced growth of bean, in terms of reduced shoot and root lengths and plant dry mass, and its productivity (Table 2).

Many investigators proved that MLE is rich in zeatin, ascorbic acid, vitamin E, phenolic compounds, and minerals (Makkar and Becker, 1996; Fuglie, 1999; Foidl et al., 2001; Nagar et al., 2006; Yasmeen et al., 2012, 2013). Among growth regulators discovered in MLE, Cytokinins have critical role for the promotion of cell division, cell elongation, chlorophyll biosynthesis and modification in apical dominance in plants (Taiz and Zeiger, 2010). Cytokinin application under abiotic stressful conditions can delay the leaf biological aging directly by scavenging free radicals (Miller, 1992; Grossman and Leshem, 2006). In the present study, plants pretreated with MLE were shown to be dark green and healthy as compared to plant untreated with this extract. Seed priming with diluted MLE has been reported to effectively improve germination and seedling growth in maize (Basra et al., 2011) and sunflower (Basra et al., 2009a). Among antioxidants found in MLE, ascorbic acid has an important role in plant resistance to abiotic stresses. Foliar application of ascorbic acid has been reported to be growth- and yield-improving tool in various crops, especially under saline conditions (Jyotsna and Srivastava, 1998). We suggest that bean seed derived some of their content of ascorbic acid from MLE, which support bean seed and then seedlings against salinity. In our results, NaCl-salinity caused a significant reduction in bean growth, and a considerable loss in green yield was also noted (Table 2). These results are in agreement with those obtained by some researchers (Zhu, 2001; Parida and Das, 2005) with most plant species. Plant productivity negatively affected by the toxic effects of NaCl-salinity stress as a result in the increased accumulation of Na+ and Cl− ions in bean leaves (Table 4). Therefore, biomass production inserted to be one of the most agricultural indices used to define salt-stress tolerance (Juan et al., 2005). The increased accumulation of Na+ and Cl− ions can disturb or upset the ionic balance, inducing a nutritional imbalance due to the

Table 6 Effect of seed soaking (8 h) in Moringa oleifera leaf extract (MLE) on superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) activities in the leaf of bean (Phaseolus vulgaris L. cv. Bronco) plants grown under 100 mM NaCl stress. Treatments

SOD (A564 min−1 g−1 protein)

Control MLE NaCl NaCl + MLE

3.24 4.18 5.76 7.12

± ± ± ±

0.11d 0.10c 0.14b 0.13a

CAT (A290 min−1 g−1 protein) 88.7 81.6 72.8 64.3

± ± ± ±

3.2a 4.1b 2.6c 2.3d

APX (A290 min−1 g−1 protein) 59.2 64.1 81.2 97.6

± ± ± ±

2.6d 3.1c 3.2b 4.1a

GR (A340 min−1 g−1 protein) 27.8 33.4 44.3 61.4

± ± ± ±

0.5d 0.8c 0.8b 1.2a

Values are means ± SE (n = 6) and differences between means were compared by Fisher’s least-significant difference test (LSD; P = 0.05). Mean pairs followed by different letters are significantly different.

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blockage of other cations such as K+ , Ca2+ , Mg2+ or anions such as NO3 − and thereby the induction of nutritional deficiency symptoms (Sariam et al., 2002). In this experiment, we noted some yellowing on bean leaves treated with 100 mM NaCl-salinity as shown in the reduction of leaf chlorophyll by about 42% (Table 3). The maintenance of the ionic balance during salinity stress is prerequisite to protect the plant against the build-up of toxic ions, with K+ accumulating and Na+ reaching the minimum content in bean leaves (Table 4). Thus, the control of Na+ accumulation and therefore a high K+ /Na+ ratio may strengthen salinity tolerance (Cuartero and Fernández-Mu˜noz, 1999). Soluble sugars are also altered in plants subjected to salinity. They contribute to osmotic adjustment (Hayashi et al., 1997) and can directly or indirectly modulate the expression of genes involved in metabolic processes, storage functions, and defence (Hebers and Sonnewald, 1998). In our study, the contents of total soluble sugars increased about 3 times as compared to the treatments where 100 mM NaCl were applied. This increase was greater in bean plants that were also presoaked with MLE. Other solutes that show a positive correlation with the salinity tolerance of plants are N-rich compounds (Mansour, 2000). Glycine betaine (GB) is one of the most important of these compounds and increases its content during stress in numerous plants (Saneoka et al., 1999). In addition, the accumulation of proline is one of the most frequent changes induced by salinity or drought, although there is controversy concerning whether its accumulation is a stress resistance mechanism or a mere indicator of the existence of stress (Thakur and Sharma, 2005). In this study, the GB and proline levels in bean leaves were significantly increased during stress when compared with their values under unstressed plants (Table 3). These increased antioxidants supported the antioxidant system in bean plants to enable them to tolerate salt stress. The MLE treatments registered lower values for the proline and GB contents than in the NaCl treatment, suggesting that if proline is a stress indicator, bean plants with presoaking in MLE should have better salinity tolerance. Nevertheless, the proline accumulation, apart from being important in osmoregulation and acting as a nitrogen reserve (Kalaji and Pietkiewicz, 1993), may reduce stress in itself, acting as a substrate for respiration and generating energy that could be inverted for plant recovery from stress (Tarakcioglu and Inal, 2002). Therefore, with soaking the bean seed, the accumulation of these osmolytes, GB and proline did not increase, probably because the combined MLE + NaCl treatment maintained a better ionic balance than did the treatment 100 mM NaCl (Table 3). These reduced accumulation of GB and proline may resulted in mitigating the stress effects of salinity on bean plants. However, under stress the accumulation of these antioxidants found to be increased. This may be attributed to that GB and proline have many specific roles as antioxidants, osmoprotectants, electron carriers. During stress, CO2 fixation is reduced after the stomatal closure by abscisic acid (ABA) to avoid water imbalance within the plants. This in turn limits the oxidation of NADP, the prime acceptor of electrons during photosynthesis. Thus, when ferrodoxine is reduced in the photosystem, free oxygen radicals are generated by the Mehler reaction (Hsu and Kao, 2003). This transfer of one, two, and three electrons generates the formation of O2 •− , H2 O2 , and OH• , respectively, with grave consequences for DNA, lipids, and proteins (Mittler, 2002). In this way, the integrity of the cell membranes is affected, as are the activity of numerous enzymes and the functioning of the photosynthetic machinery (Serrano et al., 1999). In our study, the increased contents of the reactive species O2 •− and H2 O2 which noted in the NaCl-stressed plants were reduced at the same levels of the control when plants presoaked using MLE. This may be due to that MLE is rich in some antioxidants; AsA,

phenols and praline, and phytohormones; auxins, gibberellins and cytokinins (Table 1) that may be absorbed by bean seed and supported the antioxidant system in seeds and then in seedlings, which enable seedlings to overcome salinity stress by lowering the ROS damages. In this investigation, lipid peroxidation was measured as malondialdehyde (MDA) content, this being a biochemical indicator of stress, where it inhibits the production of biomass and reduces the plant adaptation possibilities to stress (Hernández and Almansa, 2002). MDA content in the treatment of 100 mM NaCl, like the contents of O2 •− and H2 O2 , was higher than its content of other treatments (Table 5), a fact explained by the high reactive oxygen species (ROS) concentrations generated in this treatment. This could positively affect the biomass of the plants subjected to the combined treatment of 100 mM NaCl + MLE. Several studies have indicated that the oxidative damage generated during salinity stress is due to the imbalance in production of ROS and antioxidant activity alterations (Hernández et al., 1993). To avoid the damage caused by oxidative stress, plants have developed many antioxidant systems; among enzymatic ones, SOD constitutes the first line of defence against ROS (Alscher et al., 2002) by reducing the O2 •− radical to H2 O2 . Hydrogen peroxide can serve as that substrate for numerous enzymes such as CAT which in turn though located in the peroxysomes where the H2 O2 concentration is very high, is absent in the cytosol and chloroplasts, and thus H2 O2 is eliminated by peroxidases. These include APX, which is considered one of the most important enzymes in the reduction of this reactive molecule (Feierabend, 2005; Foyer, 1996). Foyer and Noctor (2009) described the regenerating enzymes DHAR and GR as a fundamental part of the Halliwell–Asada cycle, as they formed part of the regeneration of AsA from DHA using GSH as a reducing power. In turn, the GSH consumed can be regenerated from its oxidized form (GSSG) by the reaction of GR (Foyer et al., 1991). Our data show that all the treatments boosted the activities of SOD, APX and GR compared to the control (Table 6); this being more pronounced in the case of the combined treatment of 100 mM NaCl + MLE. Because of the decline in CAT activity, the lower H2 O2 content found in this study presoaked plants using MLE under salinity stress (Table 5) could be due to the better functioning of the Halliwell–Asada cycle, reflected in an increase in the activities of SOD, APX and GR. The substrates of the Halliwell–Asada cycle, AsA and GSH also act as antioxidants in an isolated way on being involved in the direct reduction of ROS during different types of stress (Del Río et al., 2006) taking part in the control of the H2 O2 levels. This situation is reflected in the total contents of AsA and GSH in our study, which are increased both with the treatment of 100 mM NaCl, and their maximum contents were noted with the combined application of 100 mM NaCl + MLE, perhaps to overcome O2 •− accumulation with this combined treatment, since the AsA can directly eliminate O2 •− and H2 O2 in a non-enzymatic way (Foyer et al., 1991). The healthy metabolic state of the stressed bean plants pretreated with MLE resulted in the healthy plant growth, in terms of increased shoot and root lengths and total plant dry mass (Table 2). This may be attributed to that MLE is excellent source in minerals, amino acids, soluble sugars and some antioxidants (Table 1). Salt stress tolerance in bean plants, in this study, was improved with the elevated antioxidant system; antioxidant enzymes and non-enzymatic antioxidants by the application of minerals, AsA, cytokinins, gibberellins and auxins containing-MLE (Table 1). Since moringa is a rich source of zeatin (Foidl et al., 2001), minerals and other phytohormones, so the effectiveness of MLE in mitigating salinity stress by better chlorophyll, antioxidants and

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