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Effect of 24-epibrassinolide on growth, yield, antioxidant system and cadmium content of bean (Phaseolus vulgaris L.) plants under salinity and cadmium stress
Scientia Horticulturae 129 (2011) 232–237
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Effect of 24-epibrassinolide on growth, yield, antioxidant system and cadmium content of bean (Phaseolus vulgaris L.) plants under salinity and cadmium stress Mostafa M. Rady Agricultural Botany Department, Faculty of Agriculture, Fayoum University, 63514 Fayoum, Egypt
a r t i c l e
i n f o
Article history: Received 25 November 2010 Received in revised form 16 March 2011 Accepted 18 March 2011 Keywords: Phaseolus vulgaris L. 24-Epibrassinolide Antioxidant system Growth Yield Salinity stress Cadmium stress
a b s t r a c t The plants of Phaseolus vulgaris L. were grown in the presence of NaCl and/or CdCl2 and were sprayed with 5 M of 24-epibrassinolide (EBL) at 15 days after transplanting (DAT) and were sampled at 30 DAT and at the end of experiment. The plants exposed to NaCl and/or CdCl2 exhibited a significant decline in growth, the level of pigment parameters, green pod yield and pod protein. However, the follow up treatment with EBL detoxified the stress generated by NaCl and/or CdCl2 and significantly improved the above parameters. The NaCl and/or CdCl2 increased electrolyte leakage, lipid peroxidation and plant Cd2+ content, and decreased the membrane stability index (MSI) and relative water content. However, the EBL treatment in absence of the stress improved the MSI and relative water content and minimized plant Cd2+ content but could not influence electrolyte leakage and lipid peroxidation. The antioxidative enzymes and the level of proline exhibited a significant increase in response to EBL as well as to NaCl and/or CdCl2 stress. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Salinity stress is one of the most serious abiotic stress factors limiting crop productivity. Salt stress affects plant physiology, both at whole plant as well as cellular levels, through osmotic and ionic stress. It generates a physiological drought or osmotic stress by affecting the water relations of the plant (Munns, 2002). The accumulation of toxic amounts of salts in the leaf apoplasm leads to dehydration and turgor loss, and death of cells and tissues. Photosynthesis is one of the most severely affected processes during salinity stress (Sudhir and Murthy, 2004), which is mediated by decreased chlorophyll pigment, inhibition of rubisco (Soussi et al., 1998) and closure of stomata, thereby, decreasing the CO2 pressure (Bethkey and Drew, 1992). The salinity stress is also reported to affect the nitrogen metabolism by affecting various enzymes (Soussi et al., 1998). All these and other alter processes lead to poor plant growth and the subsequent productivity. However, lipid peroxidation and the antioxidant system are reported to be stimulated by salt stress (Sairam et al., 2005). Heavy metal stress is another environmental problem that leads to loss in agricultural productivity and hazardous health effects. Several industries and agricultural activities contribute to heavy metal contamination of agricultural lands in peri-urban areas (Rattan et al., 2002). Among the heavy metals, cadmium is a non-essential heavy metal pollutant naturally present in the envi-
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ronment. Major anthropogenic sources of Cd2+ are Cd-containing phosphate fertilizers, sewage sludge and industrial emissions (Adriano, 1986). Mining and smelting industries also release substantial amounts of Cd2+ into the environment (Nriagu and Pacyna, 1988). Plants readily take up Cd2+ from the soil. Exposure to high levels of Cd2+ results in reduced rates of photosynthesis, chlorosis, growth inhibition, decrease in water and nutrient uptake and finally death (di Toppi and Gabbrielli, 1999). A common consequence of most abiotic stresses, including salinity (Sairam et al., 2005) and heavy metals (Cd2+ ) (Muthuchelian et al., 2001; Hayat et al., 2007), is an increased production of reactive oxygen species (ROS). These ROS, namely superoxide radical (O− ), hydrogen peroxide (H2 O2 ) and hydroxyl (HO− ) are extremely toxic to the plants that cause damage to DNA, proteins, lipids, chlorophyll, etc. (Schutzendubel and Polle, 2002). However, plants are well equipped with an antioxidant system comprised of enzymes (superoxide dismutase, peroxidase, catalase, and glutathione reductase) and metabolites (proline, tocopherols, carotenoids, glutathione, ascorbic acid, etc.) to counter the oxidative stress to protect the plants from oxidative damage (Apel and Hirt, 2004). Brassinosteroids (BRs) are a large group of steroidal plant hormones. They have been implicated in wide range of physiological responses in plants, such as, stem elongation, pollen tube growth, leaf bending and epinasty, ethylene biosynthesis, proton pump activation, vascular differentiation, the regulation of gene expression, nucleic acid and protein synthesis and photosynthesis (Sasse, 2003). The recent research has revealed that BRs also confer tolerance to plants against osmotic stress (Sairam, 1994), temperature stress (Wilen et al., 1995), salinity (Ali et al., 2007) and heavy metal
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(cadmium) stress (Hayat et al., 2007). The present research was designed with objective to evaluate the changes in antioxidant system under the influence of 24epibrassinolide in the Phaseolus vulgaris L. plants, exposed to NaCl and/or CdCl2 and to establish a relationship between the changes in antioxidant system and the degree of tolerance, in terms of improvement in growth and yield. The hypothesis tested is that 24-epibrassinolide will elevate the level of antioxidant system that will protect the stress generated by salinity and cadmium. 2. Materials and methods 2.1. Plant material and growth conditions The seeds of common bean (P. vulgaris L.) cv. Bronco were obtained from Agricultural Research Center, Egypt. The healthy seeds were sown in peatmoss trays under unheated greenhouse conditions. At the fourth leaf stage, the seedlings were transplanted in plastic pots, 20 cm in diameter. Each pot was equally filled with acid washed sand, moistened with deionized water and contained two plants. The pots were transplanted to a growth chamber adjusted to 30/24 ◦ C, 85/60% R.H. day/night and light intensity approximately 3500 lx for a period 12 h a day. After 48 h of transplanting, the seedlings were supplemented with NaCl (150 mM) and/or CdCl2 (1 mM Cd2+ ) along with the nutrient solution. At 15 days after transplanting (DAT), the seedlings were sprayed with deionized water (control) or 5 M 24-epibrasinolide (EBL) (the stock solution of EBL was prepared by dissolving the hormone in 1 ml ethanol and final volume was maintained by double distilled water). The selection of the concentration was based on preliminary study (data not shown). The concentration of 24-epibrassinolide (5 M) used generated the best response. Therefore, it was selected for the experiment. With regard to the concentrations of NaCl and Cd2+ , the concentrations above 150 mM proved lethal. Therefore, the concentration below the lethal concentration (i.e. 150 mM) was used in the experiment. Samples were collected at 30 DAT to assess chlorophylls and carotenoids of leaves, proline of roots and leaves, electrolyte leakage, relative water content, membrane stability index, lipid peroxidation and antioxidant enzymes of plant leaves and Cd2+ content of roots, leaves and pods. The other parameters were determined at the end of experiment (after green pods harvest). 2.2. Plant growth and Cd2+ content analyses and green pod yield The plants were removed from the pots 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. Number of leaves was counted. The leaves area was recorded by using a digital leaf meter (LI-3000 Portable Area meter Produced by LI-COR Lincoln, NE, USA). The plants were then placed in an oven run at 80 ◦ C for 24 h. These dried plants were weighed to record the plant dry mass. The powdery dried plant parts (i.e. roots, leaves and pods) were used to determine their content of cadmium by using a Perkin-Elmer, Model 3300, atomic absorption spectrophotometer (Chapman and Pratt, 1961). Protein percentage of pods was assessed according to Chapman and Pratt (1961). At the end of experiment, pods were collected, counted and weighed. 2.3. Photosynthetic pigments determination Leaf chlorophyll and carotenoids (mg g−1 fresh matter) were determined using a colorimetric Arnon‘s (1949) method.
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2.4. Root and leaf proline contents determination Proline content in bean roots and leaves was measured by rapid colorimetric method as suggested by Bates et al. (1973). Proline was extracted from 0.5 g of dry root or leaf samples by grinding in 10 ml of 3% sulphosalicylic acid and the mixture was then centrifuged at 10,000 × g for 10 min. 2 ml of the supernatant was added into test tubes to which 2 ml of freshly prepared acid–ninhydrin solution was added. Tubes were incubated in a water bath at 90 ◦ C for 30 min. The reaction was terminated in ice-bath. The reaction mixture was extracted with 5 ml of toluene and vortexed for 15 s. The tubes were allowed to stand at least for 20 min in darkness at room temperature to allow the separation of toluene and aqueous phase. The toluene phase was then carefully collected into test tubes and toluene fraction was read at 520 nm. The proline concentration in the sample was determined from a standard curve using analytical grade proline and calculated on dry weight basis. 2.5. Determination of membrane stability index The membrane stability index (MSI) was estimated by taking 200 mg leaf material, in two sets, in test tubes containing 10 cm3 of double distilled water. One set was heated at 40 ◦ C for 30 min in a water bath, and the electrical conductivity of the solution was recorded on conductivity bridge (C1 ). Second set was boiled as 100 ◦ C on a boiling water bath for 10 min, and conductivity was measured on conductivity bridge (C2 ). MSI was calculated by the formula: MSI % = [1 − (C1 /C2 )] × 100. 2.6. Electrolyte leakage determination The total inorganic ions leaked out in the leaves were estimated by the method of Sullivan and Ross (1979). Twenty leaf discs were taken in a boiling tube containing 10 ml of deionized water and electrical conductivity (ECa ) was measured. The content was heated at 45 ◦ C and 55 ◦ C for 30 min each in a water bath and electrical conductivity (ECb ) was measured. Later the content was again boiled at 100 ◦ C for 10 min and electrical conductivity (ECc ) was again recorded. The electrolyte leakage was calculated by using the formula: Electrolyte leakage (%) =
ECb − ECa × 100 ECc
2.7. Water use efficiency (WUE) determination WUE values as g pods L−1 of applied water were calculated for different treatments after harvest according to the following equation (Jensen, 1983): WUE =
Pod yield(g/pot) Water applied(L/pot)
2.8. Determination of lipid peroxidation Lipid peroxidation rates were estimated by measuring the malondialdehyde equivalents according to Hodges et al. (1999). 0.5 g of the leaf was homogenized in a mortar with 80% ethanol. The homogenate was centrifuged at 3000 × g for 10 min at 4 ◦ C. The pallet was extracted twice with the same solvent. The supernatants were pooled and 1 ml of this sample was added to a test tube with an equal volume of the solution comprised of 20% trichloroacetic acid, 0.01% butylated hydroxy toluene and 0.65% thiobarbituric acid. Samples were heated at 95 ◦ C for 25 min and cooled to room temperature. Absorbance of the samples was recorded at 440, 532
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and 600 nm. Lipid peroxidation rates equivalent (nmol malondialdehyde ml−1 ) were calculated by using the formula given by Hodges et al. (1999). 2.9. Determination of relative water content The relative water content (RWC) was determined in fresh leaf discs of 2 cm2 diameter, excluding midrib. Discs were weighed quickly and immediately floated on DDW in Petri dishes to saturate them with water for the next 24 h, in dark. The adhering water of the discs was blotted and turgor mass was noted. Dry mass of the discs was recorded after dehydrating them at 70 ◦ C for 48 h. RWC was calculated by placing the values in the following formula (Hayat et al., 2007): Fresh mass − dry mass RWC = × 100 Turgor mass − dry mass
enzyme activity was expressed as number of absorbance units g−1 fresh weight of leaves. Glutathione reductase was assayed as per the method of Smith et al. (1988). The reaction mixture contained, 66.67 mM potassium phosphate buffer (pH 7.5), 0.33 mM EDTA, 0.5 mM 5,5-dithiobis-(2nitrobenzoic acid) in 0.01 M potassium phosphate buffer (pH 7.5), 66.67 mM NADPH, and 66.67 mM oxidized glutathione and 0.1 ml enzyme extract. The reaction was started by adding oxidized glutathione and the increase in absorbance at 412 nm was recorded spectrophotometrically. 2.11. Statistical analysis The values for the parameters were subjected to statistical analysis, following the standard procedure described by Gomez and Gomez (1984). The ‘F’ test was applied to assess the significance of the treatment, at 5% level of probability.
2.10. Enzyme assays 3. Results Leaves of bean plants were excised rapidly weighed (1.0 g fresh weight) and ground with a pestle in an ice-cold mortar with 10 ml 50 mM phosphate buffer (pH 7.0). The homogenates were centrifuged at 20,000 × g for 30 min at 4 ◦ C. The supernatant filtered through two layers of cheese-cloth was used for the assays of enzymatic activities. The SOD activity was determined according to the method of Fridovich (1975). One enzyme unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition in the rate of nitro-blue-tetrazolium (NBT) at 560 nm. The reaction mixture (3 ml) contained 50 mM phosphate buffer (pH 7.0), 200 mM methionine, 1.125 mM NBT, 1.5 mM EDTA, 75 M riboflavin, and 10–40 l of enzyme extract. Riboflavin was added as the last component. The tubes were shaken and placed 30 cm below a light bank consisting of two 15-W fluorescent tubes. The reaction was started by switching on the light and allowed to run for 10 min, and switching the light off stopped the reaction. The tubes were then immediately covered with black cloth and the absorbance was spectrophotometrically measured at 560 nm. The non-irradiated reaction mixture had zero absorbance (log A560 ), which was plotted as a function of the volume of the enzyme extract in the reaction mixture. The volume of the enzyme extract producing 50% inhibition of the reaction was read from the resultant graph. The CAT activity in leaves of 9-week-old plants was determined by employing the method suggested by Luck (1975). CAT activity was assayed by estimating the residual H2 O2 by oxidation with KMnO4 titrimetrically. The enzyme extraction was done in a similar way to SOD extraction. The reaction mixture consisted of 3 ml of phosphate buffer (0.1 M, pH 7.0), 30 l of H2 O2 (5 mM) and 1 ml of enzyme extract. It was then incubated in a test tube at 20 ◦ C for 1 min, reaction stopped by adding 10 ml of 0.35 M H2 SO4 and the residual H2 O2 estimated by titrating the reaction mixture against 0.01 M KMnO4 . The end-point for the titration was a faint purple colour which persisted for at least 15 s. A blank was prepared by adding enzyme extract to an acidified solution of reaction mixture at zero time. The enzyme activity was expressed as moles of H2 O2 10 min−1 g−1 of fresh weight of leaves. The POD activity in leaves was estimated using the method of Thomas et al. (1981). POD was assayed using guaiacol as the substrate. The enzyme extract was prepared in a similar way to the one used for the extraction of SOD and CAT. The reaction mixture was consisted of 3 ml of phosphate buffer (0.1 M, pH 7.0), 30 l of H2 O2 (20 mM), 50 l of enzyme extract and 50 l of guaiacol (20 mM). The reaction mixture was incubated in a cuvette for 10 min at room temperature. The optical density was measured at 436 nm. The
The presence of NaCl and/or CdCl2 significantly reduced the growth traits (lengths of shoot and root, no. of leaves plant−1 , leaf area plant−1 and plant dry weight) in bean plants (Table 1). The combined effect of these two stress factors was more toxic compared to their individual ones. The growth of the root was inhibited more drastically compared to that of the shoot. Compared to the control, the above mentioned parameters were decreased by 28.23%, 49.50%, 40.67%, 40.70% and 63.19%, respectively. However, the treatment of the plants with EBL, in absence of NaCl and/or NiCl2 stress stimulated the growth that was significantly higher than the control (p = 0.05). EBL also improved the growth of the plants grown under NaCl and/or CdCl2 and the values were significantly higher than those of the plants grown under stress alone. The stress generated by CdCl2 and/or NaCl resulted in a significant decreased the levels of chlorophyll fractions (chlorophyll a, b and total) and that of carotenoids (Table 2). The combination of these two treatments was more toxic compared to their individual ones. However, these attributes were improved by EBL, both in presence as well as in absence of NaCl and/or CdCl2 . The EBL treatment significantly overcame the toxicity generated by NaCl-stress alone and almost leveled the values with those of control. However, the response generated under stress-free condition was significantly higher than that generated under the stress. The level of proline exhibited an increase in response to salinity and/or cadmium stress, both in the roots and leaves, compared to the control (Table 2). The quantity of proline was higher in roots than the leaves. The spray of EBL on unstressed plant could not bring about a significant change in the level of proline. However, in association with NaCl and/or CdCl2 , it elevated the quantity of proline, both in leaves and roots. The maximum quantity of proline was found in the plants which were subjected to both NaCl and CdCl2 stress and subsequently sprayed with EBL. The presence of NaCl and/or CdCl2 significantly reduced WUE, pods number pot−1 and pods yield pot−1 (Table 3). The combined effect of NaCl and CdCl2 stress was more toxic compared to their individual ones. The treatment of the plants with EBL, in absence of NaCl and/or CdCl2 stress stimulated the WUE and yield and its parameters which were significantly higher than the control (p = 0.05). EBL also improved the mentioned parameters recorded under NaCl and/or CdCl2 and the values were significantly higher than those of the plants grown under stress alone. The RWC (Table 3) was significantly increased by the EBL treatment, in stress-free plants. However, the NaCl and CdCl2 stress, both singly and in combination drastically decreased the RWC. Nevertheless, the follow up treatment of the stressed plants with EBL
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Table 1 Effect of 24-epibrassinolide (EBL) on growth traits (shoot length [cm], root length [cm], number of leaves plant−1 , leaves area plant−1 [cm2 ] and plant dry mass [g]) of bean (P. vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Shoot length
Root length
Leaves no. plant−1
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
24.8b 30.1a 21.3cd 22.0cd 17.8e 23.6bc 22.8bc 19.7de
20.0b 22.8a 15.2cd 12.5e 10.1f 16.6c 13.9de 11.8ef
8.31b 10.37a 7.13c 6.93c 4.93d 8.20b 7.77bc 6.90c
Leaves area plant−1 1108b 1383a 951c 924c 657d 1093b 1036bc 924c
Plant dry mass 7.58b 10.79a 4.43de 3.51e 2.79e 6.29bc 5.63cd 3.80e
Table 2 Effect of 24-epibrassinolide (EBL) on leaf photosynthetic pigments (chlorophyll fractions; chloro. a, chloro. b and total and carotenoids contents [mg g−1 F.W.]), root proline and leaf proline contents [mg g−1 F.W.] of bean (Phaseolus vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Chloro. (a)
Chloro. (b)
Total chlorophyll
Carotenoids
Root proline
Leaf proline
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
0.98b 1.40a 0.57de 0.55de 0.48e 0.93bc 0.82bc 0.73cd
0.39b 0.56a 0.23de 0.22de 0.19e 0.37bc 0.33bc 0.29cd
1.41b 2.02a 0.82de 0.79de 0.69e 1.34bc 1.18bc 1.05cd
0.79b 1.05a 0.63bcd 0.54cd 0.44d 0.78bc 0.69bc 0. 59bcd
12.68e 12.92e 14.13d 14.81cd 16.00b 15.43bc 15.59bc 17.29a
9.52d 10.09d 11.25c 11.63c 12.75b 13.01b 14.10a 14.85a
enhanced the RWC, more significantly in cases where the plants were exposed to CdCl2 alone. A different pattern of response was observed when electrolyte leakage, membrane stability index and lipid peroxidation were studied in EBL treated plants, in presence and absence of NaCl and/or CdCl2 (Tables 3 and 4). Under stress-free medium, EBL could not affect the electrolyte leakage and lipid peroxidation but significantly increased the membrane stability index (MSI). The presence of NaCl or CdCl2 caused a significant increase in electrolyte leakage and lipid peroxidation, compared to the control. However, when compared with each other, the effect was not significant (p = 0.05). A maximum electrolyte leakage and lipid peroxidation was recorded in the plants, exposed simultaneously to both NaCl and CdCl2 . However, the follow up treatment of the stressed plants with EBL decreased the ionic leakage and lipid peroxidation and leveled the values with those of the control. The exposure of the plants to NaCl and/or CdCl2 caused a significant decline in the membrane stability index. However, the subsequent treatment of the stressed plants with EBL partially neutralized the toxicity and caused a significant improvement in the MSI, compared to the stressed plants that were not sprayed with EBL. The antioxidative enzymes, catalase, peroxidase, superoxide dismutase and glutathione reductase exhibited an increasing trend in response to both EBL, and NaCl and/or CdCl2 treatment (Table 4). The EBL treatment caused a significant increase in the activities of all the enzymes. The plants exposed to NaCl alone also possessed an increased level of these enzymes but CdCl2 could not effect
the activity of catalase. However, in association with NaCl or EBL treatment, it also improved the activities of all the above mentioned antioxidative enzymes. Maximum activities of these enzymes were recorded in the plants exposed to combined NaCl and CdCl2 stress and subsequently received EBL treatment at 15 day stage of growth. The stress generated by CdCl2 and/or NaCl resulted in a reversed behavior of pod protein% comparable to the Cd2+ content of roots, leaves and pods (Table 5). CdCl2 stress was highly more toxic than NaCl stress. Plant roots were proved to be more influenced followed by plant leaves then the pods. The combination of these two treatments was more toxic compared to their individual ones. However, these attributes were improved by EBL, both in presence as well as in absence of NaCl and/or CdCl2 . The EBL treatment significantly overcame the toxicity generated by CdCl2 and/or NaCl stress and almost leveled the values with those of control. However, the response generated under stress-free condition was significantly higher than that generated under the stress. 4. Discussion Overproduction of reactive oxygen species (ROS) is a common consequence of different stress factors, including cadmium and salinity. To maintain metabolic functions under stress conditions, the balance between generation and degradation of ROS is required, otherwise oxidative injuries may occur. The level of ROS in plant tissues is controlled by an antioxidant system that consists of anti-
Table 3 Effect of 24-epibrassinolide (EBL) on water use efficiency (WUE) (g pods/L applied water), relative water content (RWC%), electrolyte leakage (EL%), membrane stability index (MSI%), number of pods pot−1 and pods yield pot−1 (g) of bean (Phaseolus vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
WUE b
0.77 1.10a 0.45de 0.36e 0.28e 0.64bc 0.57cd 0.39e
RWC (%) b
77.7 91.1a 52.4d 58.2cd 50.9d 64.8c 73.3b 61.6c
EL (%) d
8.1 7.9d 9.7b 9.3bc 11.1a 8.7bcd 8.6cd 9.5bc
MSI (%) bc
48.6 68.0a 41.0e 36.7f 30.2g 51.8b 45.4cd 42.1de
Pods no. pot b
10.54 14.99a 6.17de 4.87e 3.87e 8.75bc 7.83cd 5.29e
pods yield pot−1 38.45b 54.71a 22.49de 17.78e 14.13e 31.92bc 28.57cd 19.30e
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Table 4 Effect of 24-epibrassinolide (EBL) on lipid peroxidation (nmol TBARS g−1 D.W.), catalase activity (mM H2 O2 g−1 F.W.), peroxidase activity (units g−1 F.W.), superoxide dismutase activity (SOD) (units g−1 F.W.) and glutathione reductase activity (GlRase) (units g−1 F.W.) of bean (Phaseolus vulgaris L. cv. Bronco) leaves under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Lipid peroxidation
Catalase
Peroxidase
SOD
GlRase
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
119.2de 115.8e 138.1b 143.1b 150.0a 125.2cd 129.5c 125.3cd
31.78cd 32.67c 33.84b 31.32d 33.75b 34.83a 32.40c 35.37a
0.76e 0.78e 0.89d 0.94cd 1.01bc 0.87d 1.03b 1.11a
17.64f 18.60e 20.04d 20.40cd 21.84ab 21.12bc 21.72b 22.68a
12.48f 13.84e 14.88d 15.92c 17.04b 16.40bc 16.24bc 18.16a
oxidant enzymes (superoxide dismutase, catalase, peroxidase and glutathione reductase) and non-enzymatic low molecular weight antioxidants (glutathione, proline, carotenoids, tocopherols, etc.) (Schutzendubel and Polle, 2002). Therefore, it was expected that the exposure of bean plants to NaCl and/or CdCl2 could elevate the level of antioxidant enzymes as well as that of proline. However, the interesting thing that emerged in the present study is that the treatment of plants with 24-epibrassinolide (EBL) both in absence and presence of stress enhanced the activities of antioxidant enzymes as well as the level of proline (Tables 2 and 4). Therefore, maximum values were recorded in the plants subjected to simultaneous stress of NaCl and CdCl2 , followed by foliar spray of EBL. The elevation in the activities of antioxidant enzymes by BRs is a gene regulated phenomenon. Cao et al. (2005) demonstrated on the basis of molecular, physiological and genetic approaches the elevation in antioxidant enzymes was the consequence of enhanced expression of DET2 gene, which enhanced the resistance to oxidative stress in Arabidopsis. Previous reports also showed that the application of BRs modified antioxidant enzymes activities, under water stress (Li and van Staden, 1998), salinity (Ali et al., 2007) and cadmium stress (Hayat et al., 2007). Chen et al. (1997) found that application of homobrassinolide increased superoxide dismutase and peroxidase activities and decreased membrane lipid peroxidation in rice. The enzyme superoxide dismutase is the first line of defense to counter superoxide (O2 − ) radical. It catalyzes the conversion of O2 − to H2 O2 that is subsequently converted to H2 O by enzyme peroxidase (Alscher et al., 2002). Catalase scavenges H2 O2 by converting it to H2 O and finally O2 , and peroxidase reduce H2 O2 using several reductants, such as ascorbate, guaiacol and phenolic compounds (Apel and Hirt, 2004). Glutathione reductase maintains the pool of glutathione in the reduced state, which in turn reduces dehydroascorbate to ascorbate. Increased expression of glutathione reductase enhances tolerance to oxidative stress (Noctor and Foyer, 1998). Proline, under stress conditions acts as osmoprotectant (Hartzendorf and Rolletschek, 2001), membrane stabilizer (Bandurska, 2001) and ROS scavenger (Matysik et al., 2002). The acceleration of the activities of antioxidant enzymes and increased pool of proline resulted in an increase in the capacity of tolerance to NaCl and/or CdCl2 in the present study. The increased tolerance to the stress was manifested in terms of improved
growth and photosynthetic pigments (Tables 1 and 2) and the subsequent yield (Table 3). The present investigation also reveals that NaCl and/or CdCl2 stress caused a significant reduction in the chlorophyll concentration. The decrease in chlorophyll concentration may be attributed to increased activity of chlorophyll-degrading enzyme chlorophyllase, under stress conditions (Reddy and Vora, 1986). Cadmium is a highly toxic contaminant that affects many plant metabolic processes (Li et al., 2008). Cadmium can also affect root metabolism, which shows sensitivity to Cd2+ toxicity by a reduction in lateral root size (Wójcik and Tukendorf, 1999). This is due to reductions in both new cell formation and cell elongation in the extension region of the root (Prasad, 1995; Liu et al., 2004). In this study, Cd2+ decreased photosynthetic pigments content may by inhibition of their biosynthesis and consequently may disturb the photosynthetic process in Cd2+ -treated plants as well as oxidative damages (Vassilev and Yordanov, 1997). In fact, Cd2+ has a low redox potential and therefore it cannot participates in biological redox reactions, but there exists some evidence that it could perform oxidative related disturbances, including lipid peroxidation (Sandalio et al., 2001). The negative impact of Cd2+ on cell redox status is known and explained by the high affinity of Cd2+ ions to SH-groups of proteins which may affect their functional properties (Vangronsveld and Clijsters, 1994). When plant cells are not able to maintain low free Cd2+ ions in the cytosol through efficient detoxifying mechanisms, this may lead to a depletion of the cell defense network and as a consequence to oxidative damages to important molecules, including lipids (Vassilev and Yordanov, 1997). In addition, NaCl inhibits the activities of the key enzymes of photosynthesis namely rubisco and PEP carboxylase (Soussi et al., 1998). Moreover, both NaCl and Cd2+ induce the closure of stomata (Bethkey and Drew, 1992). Besides, salinity (Sudhir and Murthy, 2004) impairs the photosynthesis and the photosynthetic electron transport chain. All these impaired events finally culminate into a severe loss in the rate of photosynthesis. However, BRs are known to activate the enzymes rubisco (Yu et al., 2004) and carbonic anhydrase (Ali et al., 2006) and improve water relations such as increase relative water content and water uptake (Ali et al., 2005), leading to an increase in relative water content, water use efficiency, stomatal conductance and finally the photosynthetic rate resulting from the increase in photosynthetic pigments (Table 2). Furthermore, BRs
Table 5 Effect of 24-epibrassinolide (EBL) on cadmium contents (ppm) of roots, leaves and yielded pods and pod protein (%) of bean (Phaseolus vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Root Cd
Leaf Cd
pod Cd
Pod protein%
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
4.6e 2.1f 6.8d 39.4b 48.2a 3.0f 5.1e 8.9c
2.8e – 4.3d 21.4b 29.5a – 2.4e 7.0c
1.7e – 2.6d 15.3b 20.7a – – 3.2c
17.60b 22.29a 10.24de 9.86de 8.61e 16.73bc 14.73bc 13.11cd
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also modify the membrane structure/stability under stress conditions (Hamada, 1986), therefore, the plants treated with EBL, both in presence and absence of stress had higher membrane stability index and decreased peroxidation of membrane lipids (Table 4). Sasse (2003) suggested that BRs affect the synthesis of proteins or enzymes by involving the specific gene expression that improves the overall metabolic activities of plants. They also modify the plasma membrane (Hamada, 1986), increase the nutrient uptake and assimilation (Mai et al., 1989) and facilitate the translocation of photosynthates to the sink (Petzold et al., 1992), besides improving metabolic activities, under stress conditions (Ali et al., 2007). The healthy metabolic state of the stressed plants treated with EBL resulted in the healthy plant growth, in terms of increased root and shoot lengths, leaf area and total plant dry weight (Table 1). 5. Conclusion 24-Epibrassinolide enhanced the level of antioxidant system (superoxide dismutase, catalase, peroxidase and glutathione reductase, and proline), both under stress and stress-free conditions. The influence of EBL on antioxidant system was more pronounced under stress situation, suggesting that the elevated level of antioxidant system, at least in part, increased the tolerance of bean plants to NaCl and/or CdCl2 stress, thus protected the photosynthetic machinery and the plant growth. References Adriano, D.C., 1986. Trace Elements in the Terrestrial Environment. Springer-Verlag, Berlin/Heidelberg/New York. Ali, B., Hayat, S., Ahmad, A., 2005. Response of germinating seeds of Cicer arietinum to 28-homobrassinolide and/or potassium. Gen. Appl. Plant Physiol. 31, 55–63. Ali, B., Hayat, S., Hasan, S.A., Ahmad, A., 2006. Effect of root applied 28 homobrassinolide on the performance of Lycopersicon esculentum. Sci. Hort. 110, 267–273. Ali, B., Hayat, S., Ahmad, A., 2007. 28-Homobrassinolide ameliorates the saline stress in chickpea (Cicer arietinum L.). Environ. Exp. Bot. 59, 217–223. Alscher, P.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plant. J. Exp. Bot. 53, 1331–1341. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism oxidatives and signal transduction. Ann. Rev. Plant Physiol. Plant Mol. Biol. 55, 373–399. Arnon, D.I., 1949. Copper enzymes in isolated chloroplast. Polyphenol-oxidase in Beta vulgaris L. Plant Physiol. 24, 1–5. Bandurska, H., 2001. Does proline accumulated in the leaves of water deficit stressed barley plants confine cell membrane injuries? II. Proline accumulation during hardening and its involvement in reducing membrane injuries in leaves subjected to severe osmotic stress. Acta Physiol. Plant. 23, 483–490. Bates, L.S., Waldeen, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Bethkey, P.C., Drew, M.C., 1992. Stomatal and non-stomatal components to inhibition of photosynthesis in leaves of Capsicum annum during progressive exposure to NaCl salinity. Plant Physiol. 99, 219–226. Cao, S., Xu, Q., Cao, Y., Qian, K., An, K., Zhu, Y., Binzeng, H., Zhao, H., Kuai, B., 2005. Lossof-function mutations in DET2 gene lead to an enhanced resistance to oxidative stress in Arabidopsis. Physiol. Plant 123, 57–66. Chapman, H.D., Pratt, P.F., 1961. Methods of Analysis for Soil. Plants and Water. Univ. Calif., D.V., Agric. Sci., USA. Chen, S.N., Li, J.N., You, H.L., Zhu, H.J., Qin, Z.B., Hong, G.M., Shen, Y.G., 1997. The effect of a compound inducing cold resistance and homobrassinolide on the chilling resistance of plateau rice. Acta Bot. Yunn. 19, 184–190. di Toppi, L.S., Gabbrielli, R., 1999. Response to cadmium in higher plants. Environ. Exp. Bot. 41, 105–130. Fridovich, I., 1975. Superoxide dismutase. Ann. Rev. Biochem. 44, 147–159. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research, 2nd ed. John Wiley and Sons, New York. Hamada, K., 1986. Brassinolide in crop cultivation. In: Macgregor, P. (Ed.), Plant Growth Regulators in Agriculture. Food Fertility Technology, Central Asia Pacific Region. , pp. 190–196. Hartzendorf, T., Rolletschek, H., 2001. Effect of NaCl-salinity on amino acid and carbohydrate contents of Phragmites australis. Aquat. Bot. 69, 195–208.
237
Hayat, S., Ali, B., Hasan, S.A., Ahmad, A., 2007. Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea. Environ. Exp. Bot. 60, 33–41. Hodges, M.D., DeLong, J.M., Forney, C.F., Prange, R.K., 1999. Improving the thiobarbituric acid-reactive-substances assay for lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604–611. Jensen, M.E., 1983. Design and Operation of Farm Irrigation Systems. ASAE, Michigan, USA, P827. Li, L., van Staden, J., 1998. Effects of plant growth regulators on the antioxidant system in callus of two maize cultivars subjected to water stress. Plant Growth Regul. 24, 55–66. Li, M., Zhang, L.J., Tao, L., Li, W., 2008. Ecophysiological responses of Jussiaea rapens to cadmium exposure. Aquat. Bot. 88 (4), 347–352. Liu, D., Jiang, W., Gao, X., 2004. Effects of cadmium on root growth, cell division and nucleoli in root tips of garlic. Physiologia Plantarum 47, 79–83. Luck, H., 1975. In: Bergmeyer, H.U. (Ed.), Methods in Enzymatic Analysis: Catalase Estimation, vol. 2. Academic Press, New York, p. 885. Mai, Y., Lin, S., Zeng, X., Ran, R., 1989. Effect of brassinolide on nitrate reductase activity in rice seedlings. Plant Physiol. Commun. 2, 50–52. Matysik, J., Alia, B.B., Mohanty, P., 2002. Molecular mechanism of quenching of reactive oxygen species by proline under stress in plants. Curr. Sci. 82, 525–532. Munns, R., 2002. Comparative physiology of salt and water stress. Plant Cell Environ. 25, 239–250. Muthuchelian, K., Bertamini, M., Nedunchezhian, N., 2001. Triacontanol can protect Erythrina variegate from cadmium toxicity. J. Plant Physiol. 158, 1487–1490. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of world wide contamination of air, water and soil by trace metals. Nature 333, 134–139. Petzold, U., Peschel, S., Dahse, T., Adams, G., 1992. Stimulation of 14C-sucrose export in Vicia faba plants by brassinosteroids, GA3 and IAA. Acta Bot. Neer. 41, 469–479. Prasad, M.N.V., 1995. Cadmium toxicity and tolerance in vascular plants. Environ. Exp. Bot. 35, 525–545. Rattan, R.K., Datta, S.P., Chandra, S., Saharan, N., 2002. Heavy metals and environmental quality. Fertil. News 47, 21–26, and 29–40. Reddy, M.P., Vora, A.B., 1986. Changes in pigment composition, hill reaction activity and saccharide metabolism in bajra (Pennisetum typhoides S&H) leaves under NaCl salinity. Photosynthetica 20, 50–55. Sairam, R.K., 1994. Effect of homobrassinolide application on plant metabolism and grain yield under irrigated and moisture stress condition of two wheat varieties. Plant Growth Regul. 14, 173–181. Sairam, R.K., Srivastava, G.C., Agarwal, S., Meena, R.C., 2005. Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes. Biol. Plant 49, 85–91. Sandalio, L.M., Dalurzo, H.C., Gomez, M., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 25, 2115–2126. Sasse, J.M., 2003. Physiological actions of brassinosteroids: an update. J. Plant Growth Regul. 22, 276–288. Schutzendubel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal induced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53, 1351–1365. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5 -dithiobis (2-nitrobenzoic acid). Ann. Biochem. 175, 408–413. Soussi, M., Ocand, A., Lluch, C., 1998. Effect of salt stress on growth, photosynthesis and nitrogen fixation in chickpea (Cicer arietinum L.). J. Exp. Bot. 49, 1329–1337. Sudhir, P., Murthy, S.D.S., 2004. Effect of salt stress on basic process of photosynthesis. Photosynthetica 42, 481–486. Sullivan, C.Y., Ross, W.M., 1979. Selecting the drought and heat resistance in grain sorghum. In: Mussel, H., Staples, R.C. (Eds.), Stress Physiology in Crop Plants. John Wiley & Sons, New York, pp. 263–281. Thomas, R.L., Jen, J.J., Morr, C.V., 1981. Changes in soluble and bound peroxidase, IAA oxidase during tomato fruit development. J. Food Sci. 47, 158–161. Vangronsveld, J., Clijsters, H., 1994. Toxic effects of metals. In: Farago, M.E. (Ed.), Plants and the Chemical Elements. Biochemistry, Uptake, Tolerance and Toxicity. VCH Publishers, Weinheim, Germany, pp. 150–177. Vassilev, A., Yordanov, I., 1997. Reductive analysis of factors limiting growth of Cdexposed plants: a review. Bulg. J. Plant Physiol. 23, 114–133. Wilen, R.W., Sacco, M., Gusta, L.V., Krishna, P., 1995. Effects of 24-epibrassinolide on freezing and thermotolerance of bromegrass (Bromus inermis) cell cultures. Physiol. Plant 95, 195–202. Wójcik, M., Tukendorf, A., 1999. Cd-tolerance of maize, rye and wheat seedlings. Acta Physiologia Plantarum 21, 99–107. Yu, J.Q., Huag, L.F., Hu, W.H., Zhou, Y.H., Mao, W.H., Ye, S.F., Nogues, S., 2004. A role of brassinosteroids in the regulation of photosynthesis in Cucumis sativus. J. Exp. Bot. 55, 1135–1143.
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Effect of 24-epibrassinolide on growth, yield, antioxidant system and cadmium content of bean (Phaseolus vulgaris L.) plants under salinity and cadmium stress Mostafa M. Rady Agricultural Botany Department, Faculty of Agriculture, Fayoum University, 63514 Fayoum, Egypt
a r t i c l e
i n f o
Article history: Received 25 November 2010 Received in revised form 16 March 2011 Accepted 18 March 2011 Keywords: Phaseolus vulgaris L. 24-Epibrassinolide Antioxidant system Growth Yield Salinity stress Cadmium stress
a b s t r a c t The plants of Phaseolus vulgaris L. were grown in the presence of NaCl and/or CdCl2 and were sprayed with 5 M of 24-epibrassinolide (EBL) at 15 days after transplanting (DAT) and were sampled at 30 DAT and at the end of experiment. The plants exposed to NaCl and/or CdCl2 exhibited a significant decline in growth, the level of pigment parameters, green pod yield and pod protein. However, the follow up treatment with EBL detoxified the stress generated by NaCl and/or CdCl2 and significantly improved the above parameters. The NaCl and/or CdCl2 increased electrolyte leakage, lipid peroxidation and plant Cd2+ content, and decreased the membrane stability index (MSI) and relative water content. However, the EBL treatment in absence of the stress improved the MSI and relative water content and minimized plant Cd2+ content but could not influence electrolyte leakage and lipid peroxidation. The antioxidative enzymes and the level of proline exhibited a significant increase in response to EBL as well as to NaCl and/or CdCl2 stress. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Salinity stress is one of the most serious abiotic stress factors limiting crop productivity. Salt stress affects plant physiology, both at whole plant as well as cellular levels, through osmotic and ionic stress. It generates a physiological drought or osmotic stress by affecting the water relations of the plant (Munns, 2002). The accumulation of toxic amounts of salts in the leaf apoplasm leads to dehydration and turgor loss, and death of cells and tissues. Photosynthesis is one of the most severely affected processes during salinity stress (Sudhir and Murthy, 2004), which is mediated by decreased chlorophyll pigment, inhibition of rubisco (Soussi et al., 1998) and closure of stomata, thereby, decreasing the CO2 pressure (Bethkey and Drew, 1992). The salinity stress is also reported to affect the nitrogen metabolism by affecting various enzymes (Soussi et al., 1998). All these and other alter processes lead to poor plant growth and the subsequent productivity. However, lipid peroxidation and the antioxidant system are reported to be stimulated by salt stress (Sairam et al., 2005). Heavy metal stress is another environmental problem that leads to loss in agricultural productivity and hazardous health effects. Several industries and agricultural activities contribute to heavy metal contamination of agricultural lands in peri-urban areas (Rattan et al., 2002). Among the heavy metals, cadmium is a non-essential heavy metal pollutant naturally present in the envi-
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ronment. Major anthropogenic sources of Cd2+ are Cd-containing phosphate fertilizers, sewage sludge and industrial emissions (Adriano, 1986). Mining and smelting industries also release substantial amounts of Cd2+ into the environment (Nriagu and Pacyna, 1988). Plants readily take up Cd2+ from the soil. Exposure to high levels of Cd2+ results in reduced rates of photosynthesis, chlorosis, growth inhibition, decrease in water and nutrient uptake and finally death (di Toppi and Gabbrielli, 1999). A common consequence of most abiotic stresses, including salinity (Sairam et al., 2005) and heavy metals (Cd2+ ) (Muthuchelian et al., 2001; Hayat et al., 2007), is an increased production of reactive oxygen species (ROS). These ROS, namely superoxide radical (O− ), hydrogen peroxide (H2 O2 ) and hydroxyl (HO− ) are extremely toxic to the plants that cause damage to DNA, proteins, lipids, chlorophyll, etc. (Schutzendubel and Polle, 2002). However, plants are well equipped with an antioxidant system comprised of enzymes (superoxide dismutase, peroxidase, catalase, and glutathione reductase) and metabolites (proline, tocopherols, carotenoids, glutathione, ascorbic acid, etc.) to counter the oxidative stress to protect the plants from oxidative damage (Apel and Hirt, 2004). Brassinosteroids (BRs) are a large group of steroidal plant hormones. They have been implicated in wide range of physiological responses in plants, such as, stem elongation, pollen tube growth, leaf bending and epinasty, ethylene biosynthesis, proton pump activation, vascular differentiation, the regulation of gene expression, nucleic acid and protein synthesis and photosynthesis (Sasse, 2003). The recent research has revealed that BRs also confer tolerance to plants against osmotic stress (Sairam, 1994), temperature stress (Wilen et al., 1995), salinity (Ali et al., 2007) and heavy metal
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(cadmium) stress (Hayat et al., 2007). The present research was designed with objective to evaluate the changes in antioxidant system under the influence of 24epibrassinolide in the Phaseolus vulgaris L. plants, exposed to NaCl and/or CdCl2 and to establish a relationship between the changes in antioxidant system and the degree of tolerance, in terms of improvement in growth and yield. The hypothesis tested is that 24-epibrassinolide will elevate the level of antioxidant system that will protect the stress generated by salinity and cadmium. 2. Materials and methods 2.1. Plant material and growth conditions The seeds of common bean (P. vulgaris L.) cv. Bronco were obtained from Agricultural Research Center, Egypt. The healthy seeds were sown in peatmoss trays under unheated greenhouse conditions. At the fourth leaf stage, the seedlings were transplanted in plastic pots, 20 cm in diameter. Each pot was equally filled with acid washed sand, moistened with deionized water and contained two plants. The pots were transplanted to a growth chamber adjusted to 30/24 ◦ C, 85/60% R.H. day/night and light intensity approximately 3500 lx for a period 12 h a day. After 48 h of transplanting, the seedlings were supplemented with NaCl (150 mM) and/or CdCl2 (1 mM Cd2+ ) along with the nutrient solution. At 15 days after transplanting (DAT), the seedlings were sprayed with deionized water (control) or 5 M 24-epibrasinolide (EBL) (the stock solution of EBL was prepared by dissolving the hormone in 1 ml ethanol and final volume was maintained by double distilled water). The selection of the concentration was based on preliminary study (data not shown). The concentration of 24-epibrassinolide (5 M) used generated the best response. Therefore, it was selected for the experiment. With regard to the concentrations of NaCl and Cd2+ , the concentrations above 150 mM proved lethal. Therefore, the concentration below the lethal concentration (i.e. 150 mM) was used in the experiment. Samples were collected at 30 DAT to assess chlorophylls and carotenoids of leaves, proline of roots and leaves, electrolyte leakage, relative water content, membrane stability index, lipid peroxidation and antioxidant enzymes of plant leaves and Cd2+ content of roots, leaves and pods. The other parameters were determined at the end of experiment (after green pods harvest). 2.2. Plant growth and Cd2+ content analyses and green pod yield The plants were removed from the pots 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. Number of leaves was counted. The leaves area was recorded by using a digital leaf meter (LI-3000 Portable Area meter Produced by LI-COR Lincoln, NE, USA). The plants were then placed in an oven run at 80 ◦ C for 24 h. These dried plants were weighed to record the plant dry mass. The powdery dried plant parts (i.e. roots, leaves and pods) were used to determine their content of cadmium by using a Perkin-Elmer, Model 3300, atomic absorption spectrophotometer (Chapman and Pratt, 1961). Protein percentage of pods was assessed according to Chapman and Pratt (1961). At the end of experiment, pods were collected, counted and weighed. 2.3. Photosynthetic pigments determination Leaf chlorophyll and carotenoids (mg g−1 fresh matter) were determined using a colorimetric Arnon‘s (1949) method.
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2.4. Root and leaf proline contents determination Proline content in bean roots and leaves was measured by rapid colorimetric method as suggested by Bates et al. (1973). Proline was extracted from 0.5 g of dry root or leaf samples by grinding in 10 ml of 3% sulphosalicylic acid and the mixture was then centrifuged at 10,000 × g for 10 min. 2 ml of the supernatant was added into test tubes to which 2 ml of freshly prepared acid–ninhydrin solution was added. Tubes were incubated in a water bath at 90 ◦ C for 30 min. The reaction was terminated in ice-bath. The reaction mixture was extracted with 5 ml of toluene and vortexed for 15 s. The tubes were allowed to stand at least for 20 min in darkness at room temperature to allow the separation of toluene and aqueous phase. The toluene phase was then carefully collected into test tubes and toluene fraction was read at 520 nm. The proline concentration in the sample was determined from a standard curve using analytical grade proline and calculated on dry weight basis. 2.5. Determination of membrane stability index The membrane stability index (MSI) was estimated by taking 200 mg leaf material, in two sets, in test tubes containing 10 cm3 of double distilled water. One set was heated at 40 ◦ C for 30 min in a water bath, and the electrical conductivity of the solution was recorded on conductivity bridge (C1 ). Second set was boiled as 100 ◦ C on a boiling water bath for 10 min, and conductivity was measured on conductivity bridge (C2 ). MSI was calculated by the formula: MSI % = [1 − (C1 /C2 )] × 100. 2.6. Electrolyte leakage determination The total inorganic ions leaked out in the leaves were estimated by the method of Sullivan and Ross (1979). Twenty leaf discs were taken in a boiling tube containing 10 ml of deionized water and electrical conductivity (ECa ) was measured. The content was heated at 45 ◦ C and 55 ◦ C for 30 min each in a water bath and electrical conductivity (ECb ) was measured. Later the content was again boiled at 100 ◦ C for 10 min and electrical conductivity (ECc ) was again recorded. The electrolyte leakage was calculated by using the formula: Electrolyte leakage (%) =
ECb − ECa × 100 ECc
2.7. Water use efficiency (WUE) determination WUE values as g pods L−1 of applied water were calculated for different treatments after harvest according to the following equation (Jensen, 1983): WUE =
Pod yield(g/pot) Water applied(L/pot)
2.8. Determination of lipid peroxidation Lipid peroxidation rates were estimated by measuring the malondialdehyde equivalents according to Hodges et al. (1999). 0.5 g of the leaf was homogenized in a mortar with 80% ethanol. The homogenate was centrifuged at 3000 × g for 10 min at 4 ◦ C. The pallet was extracted twice with the same solvent. The supernatants were pooled and 1 ml of this sample was added to a test tube with an equal volume of the solution comprised of 20% trichloroacetic acid, 0.01% butylated hydroxy toluene and 0.65% thiobarbituric acid. Samples were heated at 95 ◦ C for 25 min and cooled to room temperature. Absorbance of the samples was recorded at 440, 532
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and 600 nm. Lipid peroxidation rates equivalent (nmol malondialdehyde ml−1 ) were calculated by using the formula given by Hodges et al. (1999). 2.9. Determination of relative water content The relative water content (RWC) was determined in fresh leaf discs of 2 cm2 diameter, excluding midrib. Discs were weighed quickly and immediately floated on DDW in Petri dishes to saturate them with water for the next 24 h, in dark. The adhering water of the discs was blotted and turgor mass was noted. Dry mass of the discs was recorded after dehydrating them at 70 ◦ C for 48 h. RWC was calculated by placing the values in the following formula (Hayat et al., 2007): Fresh mass − dry mass RWC = × 100 Turgor mass − dry mass
enzyme activity was expressed as number of absorbance units g−1 fresh weight of leaves. Glutathione reductase was assayed as per the method of Smith et al. (1988). The reaction mixture contained, 66.67 mM potassium phosphate buffer (pH 7.5), 0.33 mM EDTA, 0.5 mM 5,5-dithiobis-(2nitrobenzoic acid) in 0.01 M potassium phosphate buffer (pH 7.5), 66.67 mM NADPH, and 66.67 mM oxidized glutathione and 0.1 ml enzyme extract. The reaction was started by adding oxidized glutathione and the increase in absorbance at 412 nm was recorded spectrophotometrically. 2.11. Statistical analysis The values for the parameters were subjected to statistical analysis, following the standard procedure described by Gomez and Gomez (1984). The ‘F’ test was applied to assess the significance of the treatment, at 5% level of probability.
2.10. Enzyme assays 3. Results Leaves of bean plants were excised rapidly weighed (1.0 g fresh weight) and ground with a pestle in an ice-cold mortar with 10 ml 50 mM phosphate buffer (pH 7.0). The homogenates were centrifuged at 20,000 × g for 30 min at 4 ◦ C. The supernatant filtered through two layers of cheese-cloth was used for the assays of enzymatic activities. The SOD activity was determined according to the method of Fridovich (1975). One enzyme unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition in the rate of nitro-blue-tetrazolium (NBT) at 560 nm. The reaction mixture (3 ml) contained 50 mM phosphate buffer (pH 7.0), 200 mM methionine, 1.125 mM NBT, 1.5 mM EDTA, 75 M riboflavin, and 10–40 l of enzyme extract. Riboflavin was added as the last component. The tubes were shaken and placed 30 cm below a light bank consisting of two 15-W fluorescent tubes. The reaction was started by switching on the light and allowed to run for 10 min, and switching the light off stopped the reaction. The tubes were then immediately covered with black cloth and the absorbance was spectrophotometrically measured at 560 nm. The non-irradiated reaction mixture had zero absorbance (log A560 ), which was plotted as a function of the volume of the enzyme extract in the reaction mixture. The volume of the enzyme extract producing 50% inhibition of the reaction was read from the resultant graph. The CAT activity in leaves of 9-week-old plants was determined by employing the method suggested by Luck (1975). CAT activity was assayed by estimating the residual H2 O2 by oxidation with KMnO4 titrimetrically. The enzyme extraction was done in a similar way to SOD extraction. The reaction mixture consisted of 3 ml of phosphate buffer (0.1 M, pH 7.0), 30 l of H2 O2 (5 mM) and 1 ml of enzyme extract. It was then incubated in a test tube at 20 ◦ C for 1 min, reaction stopped by adding 10 ml of 0.35 M H2 SO4 and the residual H2 O2 estimated by titrating the reaction mixture against 0.01 M KMnO4 . The end-point for the titration was a faint purple colour which persisted for at least 15 s. A blank was prepared by adding enzyme extract to an acidified solution of reaction mixture at zero time. The enzyme activity was expressed as moles of H2 O2 10 min−1 g−1 of fresh weight of leaves. The POD activity in leaves was estimated using the method of Thomas et al. (1981). POD was assayed using guaiacol as the substrate. The enzyme extract was prepared in a similar way to the one used for the extraction of SOD and CAT. The reaction mixture was consisted of 3 ml of phosphate buffer (0.1 M, pH 7.0), 30 l of H2 O2 (20 mM), 50 l of enzyme extract and 50 l of guaiacol (20 mM). The reaction mixture was incubated in a cuvette for 10 min at room temperature. The optical density was measured at 436 nm. The
The presence of NaCl and/or CdCl2 significantly reduced the growth traits (lengths of shoot and root, no. of leaves plant−1 , leaf area plant−1 and plant dry weight) in bean plants (Table 1). The combined effect of these two stress factors was more toxic compared to their individual ones. The growth of the root was inhibited more drastically compared to that of the shoot. Compared to the control, the above mentioned parameters were decreased by 28.23%, 49.50%, 40.67%, 40.70% and 63.19%, respectively. However, the treatment of the plants with EBL, in absence of NaCl and/or NiCl2 stress stimulated the growth that was significantly higher than the control (p = 0.05). EBL also improved the growth of the plants grown under NaCl and/or CdCl2 and the values were significantly higher than those of the plants grown under stress alone. The stress generated by CdCl2 and/or NaCl resulted in a significant decreased the levels of chlorophyll fractions (chlorophyll a, b and total) and that of carotenoids (Table 2). The combination of these two treatments was more toxic compared to their individual ones. However, these attributes were improved by EBL, both in presence as well as in absence of NaCl and/or CdCl2 . The EBL treatment significantly overcame the toxicity generated by NaCl-stress alone and almost leveled the values with those of control. However, the response generated under stress-free condition was significantly higher than that generated under the stress. The level of proline exhibited an increase in response to salinity and/or cadmium stress, both in the roots and leaves, compared to the control (Table 2). The quantity of proline was higher in roots than the leaves. The spray of EBL on unstressed plant could not bring about a significant change in the level of proline. However, in association with NaCl and/or CdCl2 , it elevated the quantity of proline, both in leaves and roots. The maximum quantity of proline was found in the plants which were subjected to both NaCl and CdCl2 stress and subsequently sprayed with EBL. The presence of NaCl and/or CdCl2 significantly reduced WUE, pods number pot−1 and pods yield pot−1 (Table 3). The combined effect of NaCl and CdCl2 stress was more toxic compared to their individual ones. The treatment of the plants with EBL, in absence of NaCl and/or CdCl2 stress stimulated the WUE and yield and its parameters which were significantly higher than the control (p = 0.05). EBL also improved the mentioned parameters recorded under NaCl and/or CdCl2 and the values were significantly higher than those of the plants grown under stress alone. The RWC (Table 3) was significantly increased by the EBL treatment, in stress-free plants. However, the NaCl and CdCl2 stress, both singly and in combination drastically decreased the RWC. Nevertheless, the follow up treatment of the stressed plants with EBL
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Table 1 Effect of 24-epibrassinolide (EBL) on growth traits (shoot length [cm], root length [cm], number of leaves plant−1 , leaves area plant−1 [cm2 ] and plant dry mass [g]) of bean (P. vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Shoot length
Root length
Leaves no. plant−1
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
24.8b 30.1a 21.3cd 22.0cd 17.8e 23.6bc 22.8bc 19.7de
20.0b 22.8a 15.2cd 12.5e 10.1f 16.6c 13.9de 11.8ef
8.31b 10.37a 7.13c 6.93c 4.93d 8.20b 7.77bc 6.90c
Leaves area plant−1 1108b 1383a 951c 924c 657d 1093b 1036bc 924c
Plant dry mass 7.58b 10.79a 4.43de 3.51e 2.79e 6.29bc 5.63cd 3.80e
Table 2 Effect of 24-epibrassinolide (EBL) on leaf photosynthetic pigments (chlorophyll fractions; chloro. a, chloro. b and total and carotenoids contents [mg g−1 F.W.]), root proline and leaf proline contents [mg g−1 F.W.] of bean (Phaseolus vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Chloro. (a)
Chloro. (b)
Total chlorophyll
Carotenoids
Root proline
Leaf proline
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
0.98b 1.40a 0.57de 0.55de 0.48e 0.93bc 0.82bc 0.73cd
0.39b 0.56a 0.23de 0.22de 0.19e 0.37bc 0.33bc 0.29cd
1.41b 2.02a 0.82de 0.79de 0.69e 1.34bc 1.18bc 1.05cd
0.79b 1.05a 0.63bcd 0.54cd 0.44d 0.78bc 0.69bc 0. 59bcd
12.68e 12.92e 14.13d 14.81cd 16.00b 15.43bc 15.59bc 17.29a
9.52d 10.09d 11.25c 11.63c 12.75b 13.01b 14.10a 14.85a
enhanced the RWC, more significantly in cases where the plants were exposed to CdCl2 alone. A different pattern of response was observed when electrolyte leakage, membrane stability index and lipid peroxidation were studied in EBL treated plants, in presence and absence of NaCl and/or CdCl2 (Tables 3 and 4). Under stress-free medium, EBL could not affect the electrolyte leakage and lipid peroxidation but significantly increased the membrane stability index (MSI). The presence of NaCl or CdCl2 caused a significant increase in electrolyte leakage and lipid peroxidation, compared to the control. However, when compared with each other, the effect was not significant (p = 0.05). A maximum electrolyte leakage and lipid peroxidation was recorded in the plants, exposed simultaneously to both NaCl and CdCl2 . However, the follow up treatment of the stressed plants with EBL decreased the ionic leakage and lipid peroxidation and leveled the values with those of the control. The exposure of the plants to NaCl and/or CdCl2 caused a significant decline in the membrane stability index. However, the subsequent treatment of the stressed plants with EBL partially neutralized the toxicity and caused a significant improvement in the MSI, compared to the stressed plants that were not sprayed with EBL. The antioxidative enzymes, catalase, peroxidase, superoxide dismutase and glutathione reductase exhibited an increasing trend in response to both EBL, and NaCl and/or CdCl2 treatment (Table 4). The EBL treatment caused a significant increase in the activities of all the enzymes. The plants exposed to NaCl alone also possessed an increased level of these enzymes but CdCl2 could not effect
the activity of catalase. However, in association with NaCl or EBL treatment, it also improved the activities of all the above mentioned antioxidative enzymes. Maximum activities of these enzymes were recorded in the plants exposed to combined NaCl and CdCl2 stress and subsequently received EBL treatment at 15 day stage of growth. The stress generated by CdCl2 and/or NaCl resulted in a reversed behavior of pod protein% comparable to the Cd2+ content of roots, leaves and pods (Table 5). CdCl2 stress was highly more toxic than NaCl stress. Plant roots were proved to be more influenced followed by plant leaves then the pods. The combination of these two treatments was more toxic compared to their individual ones. However, these attributes were improved by EBL, both in presence as well as in absence of NaCl and/or CdCl2 . The EBL treatment significantly overcame the toxicity generated by CdCl2 and/or NaCl stress and almost leveled the values with those of control. However, the response generated under stress-free condition was significantly higher than that generated under the stress. 4. Discussion Overproduction of reactive oxygen species (ROS) is a common consequence of different stress factors, including cadmium and salinity. To maintain metabolic functions under stress conditions, the balance between generation and degradation of ROS is required, otherwise oxidative injuries may occur. The level of ROS in plant tissues is controlled by an antioxidant system that consists of anti-
Table 3 Effect of 24-epibrassinolide (EBL) on water use efficiency (WUE) (g pods/L applied water), relative water content (RWC%), electrolyte leakage (EL%), membrane stability index (MSI%), number of pods pot−1 and pods yield pot−1 (g) of bean (Phaseolus vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
WUE b
0.77 1.10a 0.45de 0.36e 0.28e 0.64bc 0.57cd 0.39e
RWC (%) b
77.7 91.1a 52.4d 58.2cd 50.9d 64.8c 73.3b 61.6c
EL (%) d
8.1 7.9d 9.7b 9.3bc 11.1a 8.7bcd 8.6cd 9.5bc
MSI (%) bc
48.6 68.0a 41.0e 36.7f 30.2g 51.8b 45.4cd 42.1de
Pods no. pot b
10.54 14.99a 6.17de 4.87e 3.87e 8.75bc 7.83cd 5.29e
pods yield pot−1 38.45b 54.71a 22.49de 17.78e 14.13e 31.92bc 28.57cd 19.30e
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Table 4 Effect of 24-epibrassinolide (EBL) on lipid peroxidation (nmol TBARS g−1 D.W.), catalase activity (mM H2 O2 g−1 F.W.), peroxidase activity (units g−1 F.W.), superoxide dismutase activity (SOD) (units g−1 F.W.) and glutathione reductase activity (GlRase) (units g−1 F.W.) of bean (Phaseolus vulgaris L. cv. Bronco) leaves under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Lipid peroxidation
Catalase
Peroxidase
SOD
GlRase
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
119.2de 115.8e 138.1b 143.1b 150.0a 125.2cd 129.5c 125.3cd
31.78cd 32.67c 33.84b 31.32d 33.75b 34.83a 32.40c 35.37a
0.76e 0.78e 0.89d 0.94cd 1.01bc 0.87d 1.03b 1.11a
17.64f 18.60e 20.04d 20.40cd 21.84ab 21.12bc 21.72b 22.68a
12.48f 13.84e 14.88d 15.92c 17.04b 16.40bc 16.24bc 18.16a
oxidant enzymes (superoxide dismutase, catalase, peroxidase and glutathione reductase) and non-enzymatic low molecular weight antioxidants (glutathione, proline, carotenoids, tocopherols, etc.) (Schutzendubel and Polle, 2002). Therefore, it was expected that the exposure of bean plants to NaCl and/or CdCl2 could elevate the level of antioxidant enzymes as well as that of proline. However, the interesting thing that emerged in the present study is that the treatment of plants with 24-epibrassinolide (EBL) both in absence and presence of stress enhanced the activities of antioxidant enzymes as well as the level of proline (Tables 2 and 4). Therefore, maximum values were recorded in the plants subjected to simultaneous stress of NaCl and CdCl2 , followed by foliar spray of EBL. The elevation in the activities of antioxidant enzymes by BRs is a gene regulated phenomenon. Cao et al. (2005) demonstrated on the basis of molecular, physiological and genetic approaches the elevation in antioxidant enzymes was the consequence of enhanced expression of DET2 gene, which enhanced the resistance to oxidative stress in Arabidopsis. Previous reports also showed that the application of BRs modified antioxidant enzymes activities, under water stress (Li and van Staden, 1998), salinity (Ali et al., 2007) and cadmium stress (Hayat et al., 2007). Chen et al. (1997) found that application of homobrassinolide increased superoxide dismutase and peroxidase activities and decreased membrane lipid peroxidation in rice. The enzyme superoxide dismutase is the first line of defense to counter superoxide (O2 − ) radical. It catalyzes the conversion of O2 − to H2 O2 that is subsequently converted to H2 O by enzyme peroxidase (Alscher et al., 2002). Catalase scavenges H2 O2 by converting it to H2 O and finally O2 , and peroxidase reduce H2 O2 using several reductants, such as ascorbate, guaiacol and phenolic compounds (Apel and Hirt, 2004). Glutathione reductase maintains the pool of glutathione in the reduced state, which in turn reduces dehydroascorbate to ascorbate. Increased expression of glutathione reductase enhances tolerance to oxidative stress (Noctor and Foyer, 1998). Proline, under stress conditions acts as osmoprotectant (Hartzendorf and Rolletschek, 2001), membrane stabilizer (Bandurska, 2001) and ROS scavenger (Matysik et al., 2002). The acceleration of the activities of antioxidant enzymes and increased pool of proline resulted in an increase in the capacity of tolerance to NaCl and/or CdCl2 in the present study. The increased tolerance to the stress was manifested in terms of improved
growth and photosynthetic pigments (Tables 1 and 2) and the subsequent yield (Table 3). The present investigation also reveals that NaCl and/or CdCl2 stress caused a significant reduction in the chlorophyll concentration. The decrease in chlorophyll concentration may be attributed to increased activity of chlorophyll-degrading enzyme chlorophyllase, under stress conditions (Reddy and Vora, 1986). Cadmium is a highly toxic contaminant that affects many plant metabolic processes (Li et al., 2008). Cadmium can also affect root metabolism, which shows sensitivity to Cd2+ toxicity by a reduction in lateral root size (Wójcik and Tukendorf, 1999). This is due to reductions in both new cell formation and cell elongation in the extension region of the root (Prasad, 1995; Liu et al., 2004). In this study, Cd2+ decreased photosynthetic pigments content may by inhibition of their biosynthesis and consequently may disturb the photosynthetic process in Cd2+ -treated plants as well as oxidative damages (Vassilev and Yordanov, 1997). In fact, Cd2+ has a low redox potential and therefore it cannot participates in biological redox reactions, but there exists some evidence that it could perform oxidative related disturbances, including lipid peroxidation (Sandalio et al., 2001). The negative impact of Cd2+ on cell redox status is known and explained by the high affinity of Cd2+ ions to SH-groups of proteins which may affect their functional properties (Vangronsveld and Clijsters, 1994). When plant cells are not able to maintain low free Cd2+ ions in the cytosol through efficient detoxifying mechanisms, this may lead to a depletion of the cell defense network and as a consequence to oxidative damages to important molecules, including lipids (Vassilev and Yordanov, 1997). In addition, NaCl inhibits the activities of the key enzymes of photosynthesis namely rubisco and PEP carboxylase (Soussi et al., 1998). Moreover, both NaCl and Cd2+ induce the closure of stomata (Bethkey and Drew, 1992). Besides, salinity (Sudhir and Murthy, 2004) impairs the photosynthesis and the photosynthetic electron transport chain. All these impaired events finally culminate into a severe loss in the rate of photosynthesis. However, BRs are known to activate the enzymes rubisco (Yu et al., 2004) and carbonic anhydrase (Ali et al., 2006) and improve water relations such as increase relative water content and water uptake (Ali et al., 2005), leading to an increase in relative water content, water use efficiency, stomatal conductance and finally the photosynthetic rate resulting from the increase in photosynthetic pigments (Table 2). Furthermore, BRs
Table 5 Effect of 24-epibrassinolide (EBL) on cadmium contents (ppm) of roots, leaves and yielded pods and pod protein (%) of bean (Phaseolus vulgaris L. cv. Bronco) plants under NaCl and/or CdCl2 stress. Mean pairs followed by different letters are significantly different (p = 0.05); n = 5. Treatments
Root Cd
Leaf Cd
pod Cd
Pod protein%
Control EBL NaCl CdCl2 NaCl + CdCl2 NaCl + EBL CdCl2 + EBL NaCl + CdCl2 + EBL
4.6e 2.1f 6.8d 39.4b 48.2a 3.0f 5.1e 8.9c
2.8e – 4.3d 21.4b 29.5a – 2.4e 7.0c
1.7e – 2.6d 15.3b 20.7a – – 3.2c
17.60b 22.29a 10.24de 9.86de 8.61e 16.73bc 14.73bc 13.11cd
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also modify the membrane structure/stability under stress conditions (Hamada, 1986), therefore, the plants treated with EBL, both in presence and absence of stress had higher membrane stability index and decreased peroxidation of membrane lipids (Table 4). Sasse (2003) suggested that BRs affect the synthesis of proteins or enzymes by involving the specific gene expression that improves the overall metabolic activities of plants. They also modify the plasma membrane (Hamada, 1986), increase the nutrient uptake and assimilation (Mai et al., 1989) and facilitate the translocation of photosynthates to the sink (Petzold et al., 1992), besides improving metabolic activities, under stress conditions (Ali et al., 2007). The healthy metabolic state of the stressed plants treated with EBL resulted in the healthy plant growth, in terms of increased root and shoot lengths, leaf area and total plant dry weight (Table 1). 5. Conclusion 24-Epibrassinolide enhanced the level of antioxidant system (superoxide dismutase, catalase, peroxidase and glutathione reductase, and proline), both under stress and stress-free conditions. The influence of EBL on antioxidant system was more pronounced under stress situation, suggesting that the elevated level of antioxidant system, at least in part, increased the tolerance of bean plants to NaCl and/or CdCl2 stress, thus protected the photosynthetic machinery and the plant growth. References Adriano, D.C., 1986. Trace Elements in the Terrestrial Environment. Springer-Verlag, Berlin/Heidelberg/New York. Ali, B., Hayat, S., Ahmad, A., 2005. Response of germinating seeds of Cicer arietinum to 28-homobrassinolide and/or potassium. Gen. Appl. Plant Physiol. 31, 55–63. Ali, B., Hayat, S., Hasan, S.A., Ahmad, A., 2006. Effect of root applied 28 homobrassinolide on the performance of Lycopersicon esculentum. Sci. Hort. 110, 267–273. Ali, B., Hayat, S., Ahmad, A., 2007. 28-Homobrassinolide ameliorates the saline stress in chickpea (Cicer arietinum L.). Environ. Exp. Bot. 59, 217–223. Alscher, P.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plant. J. Exp. Bot. 53, 1331–1341. Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism oxidatives and signal transduction. Ann. Rev. Plant Physiol. Plant Mol. Biol. 55, 373–399. Arnon, D.I., 1949. Copper enzymes in isolated chloroplast. Polyphenol-oxidase in Beta vulgaris L. Plant Physiol. 24, 1–5. Bandurska, H., 2001. Does proline accumulated in the leaves of water deficit stressed barley plants confine cell membrane injuries? II. Proline accumulation during hardening and its involvement in reducing membrane injuries in leaves subjected to severe osmotic stress. Acta Physiol. Plant. 23, 483–490. Bates, L.S., Waldeen, R.P., Teare, I.D., 1973. Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207. Bethkey, P.C., Drew, M.C., 1992. Stomatal and non-stomatal components to inhibition of photosynthesis in leaves of Capsicum annum during progressive exposure to NaCl salinity. Plant Physiol. 99, 219–226. Cao, S., Xu, Q., Cao, Y., Qian, K., An, K., Zhu, Y., Binzeng, H., Zhao, H., Kuai, B., 2005. Lossof-function mutations in DET2 gene lead to an enhanced resistance to oxidative stress in Arabidopsis. Physiol. Plant 123, 57–66. Chapman, H.D., Pratt, P.F., 1961. Methods of Analysis for Soil. Plants and Water. Univ. Calif., D.V., Agric. Sci., USA. Chen, S.N., Li, J.N., You, H.L., Zhu, H.J., Qin, Z.B., Hong, G.M., Shen, Y.G., 1997. The effect of a compound inducing cold resistance and homobrassinolide on the chilling resistance of plateau rice. Acta Bot. Yunn. 19, 184–190. di Toppi, L.S., Gabbrielli, R., 1999. Response to cadmium in higher plants. Environ. Exp. Bot. 41, 105–130. Fridovich, I., 1975. Superoxide dismutase. Ann. Rev. Biochem. 44, 147–159. Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research, 2nd ed. John Wiley and Sons, New York. Hamada, K., 1986. Brassinolide in crop cultivation. In: Macgregor, P. (Ed.), Plant Growth Regulators in Agriculture. Food Fertility Technology, Central Asia Pacific Region. , pp. 190–196. Hartzendorf, T., Rolletschek, H., 2001. Effect of NaCl-salinity on amino acid and carbohydrate contents of Phragmites australis. Aquat. Bot. 69, 195–208.
237
Hayat, S., Ali, B., Hasan, S.A., Ahmad, A., 2007. Brassinosteroid enhanced the level of antioxidants under cadmium stress in Brassica juncea. Environ. Exp. Bot. 60, 33–41. Hodges, M.D., DeLong, J.M., Forney, C.F., Prange, R.K., 1999. Improving the thiobarbituric acid-reactive-substances assay for lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207, 604–611. Jensen, M.E., 1983. Design and Operation of Farm Irrigation Systems. ASAE, Michigan, USA, P827. Li, L., van Staden, J., 1998. Effects of plant growth regulators on the antioxidant system in callus of two maize cultivars subjected to water stress. Plant Growth Regul. 24, 55–66. Li, M., Zhang, L.J., Tao, L., Li, W., 2008. Ecophysiological responses of Jussiaea rapens to cadmium exposure. Aquat. Bot. 88 (4), 347–352. Liu, D., Jiang, W., Gao, X., 2004. Effects of cadmium on root growth, cell division and nucleoli in root tips of garlic. Physiologia Plantarum 47, 79–83. Luck, H., 1975. In: Bergmeyer, H.U. (Ed.), Methods in Enzymatic Analysis: Catalase Estimation, vol. 2. Academic Press, New York, p. 885. Mai, Y., Lin, S., Zeng, X., Ran, R., 1989. Effect of brassinolide on nitrate reductase activity in rice seedlings. Plant Physiol. Commun. 2, 50–52. Matysik, J., Alia, B.B., Mohanty, P., 2002. Molecular mechanism of quenching of reactive oxygen species by proline under stress in plants. Curr. Sci. 82, 525–532. Munns, R., 2002. Comparative physiology of salt and water stress. Plant Cell Environ. 25, 239–250. Muthuchelian, K., Bertamini, M., Nedunchezhian, N., 2001. Triacontanol can protect Erythrina variegate from cadmium toxicity. J. Plant Physiol. 158, 1487–1490. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. Nriagu, J.O., Pacyna, J.M., 1988. Quantitative assessment of world wide contamination of air, water and soil by trace metals. Nature 333, 134–139. Petzold, U., Peschel, S., Dahse, T., Adams, G., 1992. Stimulation of 14C-sucrose export in Vicia faba plants by brassinosteroids, GA3 and IAA. Acta Bot. Neer. 41, 469–479. Prasad, M.N.V., 1995. Cadmium toxicity and tolerance in vascular plants. Environ. Exp. Bot. 35, 525–545. Rattan, R.K., Datta, S.P., Chandra, S., Saharan, N., 2002. Heavy metals and environmental quality. Fertil. News 47, 21–26, and 29–40. Reddy, M.P., Vora, A.B., 1986. Changes in pigment composition, hill reaction activity and saccharide metabolism in bajra (Pennisetum typhoides S&H) leaves under NaCl salinity. Photosynthetica 20, 50–55. Sairam, R.K., 1994. Effect of homobrassinolide application on plant metabolism and grain yield under irrigated and moisture stress condition of two wheat varieties. Plant Growth Regul. 14, 173–181. Sairam, R.K., Srivastava, G.C., Agarwal, S., Meena, R.C., 2005. Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes. Biol. Plant 49, 85–91. Sandalio, L.M., Dalurzo, H.C., Gomez, M., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 25, 2115–2126. Sasse, J.M., 2003. Physiological actions of brassinosteroids: an update. J. Plant Growth Regul. 22, 276–288. Schutzendubel, A., Polle, A., 2002. Plant responses to abiotic stresses: heavy metal induced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53, 1351–1365. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5 -dithiobis (2-nitrobenzoic acid). Ann. Biochem. 175, 408–413. Soussi, M., Ocand, A., Lluch, C., 1998. Effect of salt stress on growth, photosynthesis and nitrogen fixation in chickpea (Cicer arietinum L.). J. Exp. Bot. 49, 1329–1337. Sudhir, P., Murthy, S.D.S., 2004. Effect of salt stress on basic process of photosynthesis. Photosynthetica 42, 481–486. Sullivan, C.Y., Ross, W.M., 1979. Selecting the drought and heat resistance in grain sorghum. In: Mussel, H., Staples, R.C. (Eds.), Stress Physiology in Crop Plants. John Wiley & Sons, New York, pp. 263–281. Thomas, R.L., Jen, J.J., Morr, C.V., 1981. Changes in soluble and bound peroxidase, IAA oxidase during tomato fruit development. J. Food Sci. 47, 158–161. Vangronsveld, J., Clijsters, H., 1994. Toxic effects of metals. In: Farago, M.E. (Ed.), Plants and the Chemical Elements. Biochemistry, Uptake, Tolerance and Toxicity. VCH Publishers, Weinheim, Germany, pp. 150–177. Vassilev, A., Yordanov, I., 1997. Reductive analysis of factors limiting growth of Cdexposed plants: a review. Bulg. J. Plant Physiol. 23, 114–133. Wilen, R.W., Sacco, M., Gusta, L.V., Krishna, P., 1995. Effects of 24-epibrassinolide on freezing and thermotolerance of bromegrass (Bromus inermis) cell cultures. Physiol. Plant 95, 195–202. Wójcik, M., Tukendorf, A., 1999. Cd-tolerance of maize, rye and wheat seedlings. Acta Physiologia Plantarum 21, 99–107. Yu, J.Q., Huag, L.F., Hu, W.H., Zhou, Y.H., Mao, W.H., Ye, S.F., Nogues, S., 2004. A role of brassinosteroids in the regulation of photosynthesis in Cucumis sativus. J. Exp. Bot. 55, 1135–1143.