Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings

ORIGINAL PAPER Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings Sakineh Saeidi-Sar • Hossein Abbaspour • Hossein Afshari • Saeed Reza Yaghoobi Received: 20 May 2012 / Revised: 8 September 2012 / Accepted: 11 September 2012 � Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2012 Abstract Salinity, a severe environmental factor, has limited the growth and productivity of crops. Many com- pounds have been applied to minimize the harmful effects of salt stress on plant growth. An experiment was con- ducted to investigate the interactive effects of exogenous ascorbic acid (AsA) and gibberellic acid (GA3) on common bean (Phaseolus vulgaris L. cv. Naz) seedlings under salt stress. The changes of growth parameters, photosynthetic and non-photosynthetic pigments and potassium content showed that the addition of 1 mM AsA and/or 0.05 mM GA3 considerably decreased the oxidative damage in common bean plants treated with 200 mM NaCl. The NaCl-stressed seedlings exposed to AsA or GA3, specifi- cally in their combination, exhibited an improvement in sodium accumulation in both roots and shoots, as compared to NaCl-treated plants. NaCl treatment increased hydrogen peroxide (H2O2) content and lipid peroxidation indicated by accumulation of malondialdehyde (MDA), whereas the interaction of AsA with GA3 decreased the amounts of MDA and H2O2. In the meantime, interactive effect of these substances enhanced protein content and the activity of the antioxidant enzyme, guaiacol peroxidase, in com- mon bean plants under salt stress. It was concluded that synergistic interaction between AsA and GA3 could alleviate the adverse effects of salinity on P. vulgaris seedlings. Keywords Antioxidants � Ascorbic acid � Gibberellin � Phaseolus vulgaris L. � Salt stress Abbreviations ABA Abscisic acid AsA Ascorbic acid GA3 Gibberellic acid GPOX Guaiacol peroxidase IAA Indole-3-acetic acid MDA Malondialdehyde ROS Reactive oxygen species Introduction Salinity is one of the worldwide environmental constraints to crop production. Seven percent of the land’s surface and 5 % of cultivated lands are affected by salinity, and it is an important factor which can limit the growth and produc- tivity of plants (Flowers et al. 1977). The inhibitory effect of salinity stress is largely due to the ionic and osmotic stress (Magome et al. 2008). Salt stress influences some basic metabolic processes in plants such as photosynthesis, lipid metabolism and protein synthesis (Parida and Das 2005). Decrease of photosynthetic rates in plants under salt stress is mainly due to the reduction in photosynthetic pigments (Dubey 2005), as well as limitations in photo- synthetic electron transport and partial stomatal closure (Zhang et al. 2010). Additionally, the production of reac- tive oxygen species (ROS), including superoxide radical (O2 -), hydroxyl radical (OH�), singlet oxygen (1O2) and hydrogen peroxide (H2O2), are the characteristics of bio- chemical changes during salt stress. The excess production of ROS during salinity stress results from impaired electron Communicated by P. K. Nagar. S. Saeidi-Sar (&) � H. Abbaspour � H. Afshari � S. R. Yaghoobi Department of Biology, Faculty of Science, Damghan Branch, Islamic Azad University, Damghan, Iran e-mail: [email protected] 123 Acta Physiol Plant DOI 10.1007/s11738-012-1107-7 transport processes in chloroplast and mitochondria as well as from pathways such as photorespiration (Zhu et al. 2004). When plants are subjected to stress, the balance between the production of ROS and the quenching activity of the antioxidant becomes upset, often resulting in oxi- dative stress (Smirnoff 1993). A sudden and dramatic increase in cellular ROS production disrupts normal metabolism through oxidative damage to photosynthetic pigments, proteins, nucleic acids and lipids (Zhu et al. 2004). Accordingly, malondialdehyde (MDA) as a product of lipid peroxidation is accumulated in tissues when plants are exposed to salinity stress (Shalata and Neumann 2001). Scavenging of ROS in plant cells is occurred by endogenous protective mechanisms involving antioxidant molecules and enzymes. Plants use a diverse array of enzymes such as superoxide dismutases, catalases and peroxidases as well as low molecular mass antioxidants such as ascorbate, carotenoids and reduced glutathione to scavenge different types of ROS (Zhu et al. 2004). Plants with high levels of antioxidants, both enzymatic and non- enzymatic, either constitutive or induced, have greater resistance to this oxidative damage in plant cells by avoiding lipid and protein peroxidation (Younis et al. 2010). Ascorbic acid (AsA) is an important antioxidant in plants which accumulates in plants as an adaptive mecha- nism to environmental stress such as salinity. AsA regu- lates stress response as a result of a complex sequence of biochemical reactions such as activation or suppression of key enzymatic reactions, induction of stress responsive proteins synthesis, and the production of various chemical defense compounds (Khan et al. 2011). The protective role of AsA in plant cells from the adverse effects of salt stress was described by Athar et al. (2009) and Younis et al. (2010). On the other hand, the role of plant hormones under salinity stress is critical in modulating physiological responses that will eventually lead to adaptation to an unfavorable environment. Abiotic stresses alter the levels of plant hormones and decrease plant growth. Additionally, the complex regulation of plant responses to stress implies the existence of a special signal transduction chain between stress signals and responses, in which plant hormones are an integral part of the stress-controlling mechanism in plants (Zeevaart and Creelman 1988). Gibberellins (GAs) are a class of phytohormones that control many aspects of plant growth and development, including seed germination, leaf expansion, stem elongation, flower initiation and development, sex determination, and fruit development (Li et al. 2010). Previous studies have suggested the possible involvement of GA in stress adaptation in some plants (Achard et al. 2006; Rodriguez et al. 2006; Magome et al. 2008; Maggio et al. 2010). Reduced plant growth under stress conditions can result from an altered hormonal bal- ance, and hormone application provides an attractive approach to cope with stress (Saeidi-Sar et al. 2007). Our previous studies revealed that supplying low levels of AsA and gibberellic acid (GA3) could alleviate the poisonous effects of Ni on soybean plants (Saeidi-Sar et al. 2007). However, none of these studies have focused on the plant species in the presence of NaCl stress. The major objective of this study was to investigate the effects of AsA and/or gibberellic acid (GA3) on the number of physio- logical aspects of common bean plants under saline con- ditions, and to determine the extent to which synergistic effects of AsA and GA3 can ameliorate the adverse effects of salt stress on this plant. Materials and methods Plant materials and treatments Seeds of common bean (Phaseolus vulgaris L. cv. Naz) were surface-sterilized with 20 % NaClO3 for 5 min and then thoroughly rinsed with distilled water. Seeds were germinated on several layers of wet tissue paper at 24 �C in darkness. Five-day-old seedlings were transferred to 300 cm3 containers with half strength Hoagland’s solution (three seedlings per container). Then, 10-day-old seedlings were transferred to fresh half strength Hoagland’s solution, supplemented with sodium chloride (0, 200 mM) either with or without AsA (1 mM) or GA3 (0.05 mM), or AsA (1 mM) ? GA3 (0.05 mM). The solutions were continu- ously aerated and refreshed every day. Solution-pH was daily adjusted to 6.5. Plants were grown in growth chamber with a 16/8 light/dark photoperiod at 175 lmol m-2 s-1 PPFD, a day/night temperature cycle of 26/22 �C and 65 ± 5 % relative humidity. After 10 days, the plants were removed and washed with deionized distilled water. The samples for estimation of plant dry matter and ion analysis were dried at 70 �C for 48 h. Fresh plant materials were frozen in liquid nitrogen and stored at -70 �C. Determination of pigments The levels of Chlorophyll a, Chlorophyll b and carotenoid were measured in acetone extracts according to Arnon (1949). The concentrations of Chlorophyll a and Chloro- phyll b were calculated from equations derived by Hendry and Grime (1993). Chlorophyll a (mg g-1 FW) = [(12.7 A663 – 2.69 A645)/ 1,000 9 FW] 9 V Chlorophyll b (mg g-1 FW) = [(22.9 A645 – 4.68 A663)/ 1,000 9 FW] 9 V Acta Physiol Plant 123 Carotenoid content was determined by the equation of Price and Hendry (1991) Carotenoid (lmol g-1 FW) = [(A480 ? 0.114 A663) - (0.638 A663) 9 V/112.5 9 FW] where V is volume of the sample (mL), A is absorbance and FW is fresh weight (g). Anthocyanin concentration of leaves was determined by the method of Mancinelli et al. (1988) and anthocyanin content was expressed to A530 g -1 FW. Determination of K and Na Root and shoot samples were wet digested with a HNO3 and HClO4 acid mixture (5:1, v/v), and analyzed by a flame photometer (Flame Photometer, model 410, Sherwood Company), as described by Williams and Twine (1960). Determination of H2O2 content Content of hydrogen peroxide was measured according to the procedure of Velikova et al. (2000). Fresh samples (0.1 g) were homogenized with 3 mL 0.1 % (w/v) trichloroacetic acid (TCA) in ice bath and the homogenate was centrifuged at 12,000g for 15 min. Then 0.5 mL of 10 mM phosphate buffer (pH 7.0) and 1 mL of 1 M KI were added to 0.5 mL of the supernatant. The absorbance of supernatant was read at 390 nm. The amount of H2O2 was calculated using the extinction coefficient and expressed as lmol g-1 FW. Determination of MDA content Lipid peroxidation was determined by estimating the malondialdehyde (MDA) content according to Kramer et al. (1991). Frozen samples (0.5 g) mixed with 5 mL 50 mM phosphate buffer (pH 7.8) was crushed into a fine powder in a mortar and pestle under liquid nitrogen. The homogenate was centrifuged at 10,000g for 20 min at 4 �C, with the supernatant being used for MDA determination. A mixture of 1 mL extracts (MDA) and 2 mL 0.6 % thio- barbituric acid (TBA) was produced, boiled for 15 min, cooled and centrifuged for 10 min (4,000g). The concen- tration of MDA was calculated from the absorbance at 600, 532 and 450 nm, and MDA contents were determined using the following formula: MDA (lmol g-1 FW) = [6.45 9 (D532 - D600) - 0.56 D450] 9 V/W, where D532, D600 and D450 are the absorbance at 600, 532 and 450 nm, respectively, and V is the volume of extraction, W is the fresh weight of sample. Enzyme extraction and protein content Frozen plant materials (roots or shoots) were ground in 100 mM ice-cold phosphate buffer (pH 7.0), containing 0.1 mM EDTA, 1 % (w/v) insoluble polyvinylpolypyrr- olidone, and 0.2 mM AsA (Kang et al. 2003). The homogenates were purified by centrifugation at 12,000g in 4 �C for 60 min. Protein content was measured by the method of Bradford (1976), with BSA as a standard. Determination of peroxidase activity The activity of guaiacol peroxidase (GPOX, EC 1.11.1.7) was determined in a 3-mL reaction mixture containing 50 mM potassium phosphate, pH 7.0, 0.1 mM Na2EDTA, 5 mM H2O2, and 30 mM guaiacol (Fielding and Hall 1978). The increase in absorbance, due to tetraguaiacol formation, was recorded at 470 nm. The GPOX activity was expressed in units per mg protein. One unit of enzyme activity was defined as the amount necessary to decompose 1 lmol of substrate per min. All spectrophotometric anal- yses were conducted at 25 �C with a Shimadzu UV-2 101 PC spectrophotometer (Japan). Statistical analysis All experimental data in the present study were subjected to an analysis of variance (ANOVA) using the SAS sta- tistical software. Furthermore, significant differences among the mean values of treatments were compared by the least significant difference (LSD) test method at P B 0.05 using the MSTAT-C computer program. Results ANOVA results (Table 1) showed that the salinity stress had significant effects on all parameters except root fresh weight, root length, leaf area, root MDA content and root H2O2 content. Interaction of NaCl with GA3 and/or AsA was significant on some traits, but under salinity stress, interactive effects of NaCl and GA3 were significant on more parameters than influences of other combined- treatments. The effects of NaCl, AsA and GA3 on seedling growth expressed as fresh and dry weights of roots and shoots, plant height, root length, leaf area and leaf dry weight are shown in Tables 2 and 3. Salinity caused a significant reduction of 39 and 37 % in fresh and dry weight of shoot, respectively, as compared to control plants. However, this inhibition was alleviated in the presence of AsA ? GA3, the fresh and dry weight of shoot in common bean plants in combination treatment of NaCl, AsA and GA3 decreased to 17 and 8 %, respectively. The supplied NaCl reduced fresh and dry weight of root, plant height and root length by 18, 24, 30, and 7 %, respectively, as compared with the controls. Plants treated Acta Physiol Plant 123 with AsA or GA3, specifically AsA ? GA3 ,were less affected and almost showed no growth reduction. The leaf area and leaf dry weight were reduced under salinity stress, but the reduction of leaf area was not sig- nificant. Under salinity stress, exogenous application of AsA or GA3 increased leaf area. Also, the interaction of AsA and GA3 had no significant effects on leaf area. In saline condition, treatment with AsA and GA3, alone but not combined, significantly increased leaf dry weight (Table 3). The results of photosynthetic and non-photosynthetic pigments are presented in Table 4. Salinity stress led to a significant decrease of chlorophylls (Chl a, Chl b) and carotenoid contents, whereas plants treated with AsA and GA3 were less affected. In the other hand, NaCl caused a significant decrease in anthocyanin content. In saline and non-saline conditions, exogenous application of GA3 and AsA, alone or combined, significantly enhanced anthocy- anin content (Table 4). Treatment of the common bean seedlings with NaCl resulted in significant accumulation of sodium in both organs, however in the roots it was more pronounced (Table 5). Both in the roots and shoots, Na content mark- edly decreased in response to GA3 or AsA application. The lowest content of sodium in aboveground parts of NaCl- treated plants was observed in the simultaneous application of GA3 and AsA (Table 5). In plants exposed to salinity stress, potassium concentration strongly decreased in both roots and shoots. Under the same conditions, exogenous application of AsA or GA3 significantly enhanced potas- sium concentration in both studied organs (Table 5). As shown in Table 6, treatment of seedlings with 200 mM of NaCl, led to lowered content of protein in the roots and shoots over the control. Otherwise in saline condition, AsA or GA3 treatment exhibited a stimulatory effect on the accumulation of proteins in both roots and shoots. Under the same conditions, maximum protein content was attained in plants treated with AsA ? GA3. Common bean seedlings treated with 200 mM NaCl had higher level of MDA in both roots and shoots than the control or those with other treatment. The application of AsA, compared with GA3, led to a higher decrease in the shoot MDA content under normal and stress conditions. In the presence of 200 mM NaCl, application of AsA ? GA3 Table 1 Analysis of variance (ANOVA) about the influences of NaCl, GA3, AsA and their interactions on growth parameters, pigments, ions, proteins, MDA and H2O2 contents, and GPOX activity of Phaseolus vulgaris L. The denoted symbols indicate significant difference at the 0.001 (***), 0.01 (**), and 0.05 (*) levels ns Not significant Dependent variable Independent variable NaCl GA3 AsA GA3 9 AsA NaCl 9 GA3 NaCl 9 AsA NaCl 9 GA3 9 AsA Shoot fresh weight *** *** ns ns ns ns ** Shoot dry weight *** ** * ns ** ** * Root fresh weight ns ** ** ns ns ns * Root dry weight *** *** *** ns *** ** ** Root length ns ns * ns ns ** ns Plant height ** ** ns ns ns ns ns Leaf area ns ns ns ns ns ns ns Leaf dry weight * ** ns ** ** ** ns Chlorophyll a content *** ** ns ns * ** ns Chlorophyll b content *** ** * ns ns ns * Carotenoid content *** *** * ns ** *** ns Anthocyanin content *** *** *** ** ** *** *** Root Na content *** *** *** *** *** ns ** Shoot Na content *** ** *** ** ** *** ** Root K content *** *** ** ns *** ns *** Shoot K content *** *** *** *** *** *** *** Root protein content *** *** *** ns * ** *** Shoot protein content * *** ** ns *** ns ns Root MDA content ns ns ns ns ns ns ns Shoot MDA content *** *** *** *** ** *** *** Root H2O2 content ns ns ns ns ns ns ns Shoot H2O2 content ** ns * ns ns ns ns Root GPOX activity *** ns ** ns ** * ns Shoot GPOX activity ** ns ** ns ** ns ns Acta Physiol Plant 123 decreased MDA content in both roots and shoots. Also, combination of AsA and GA3 induced maximum decrease of MDA content in the roots under salinity stress (Table 6). Our data in Table 7 showed that hydrogen peroxide (H2O2) content was increased in common bean plants which were treated with salinity, but this increment was only significant in the aboveground parts. Also, H2O2 contents in the roots of the controlled and treated seedlings were considerably higher when compared to those for the shoots. In seedlings treated with 200 mM NaCl, the application of AsA or AsA ? GA3 could decline concen- tration of hydrogen peroxide in both organs, while inter- active influences of NaCl and GA3 had no significant role in reduction of H2O2 content (Table 7). Table 2 Effects of sodium chloride, gibberellic acid and ascorbic acid on fresh weights of root (FWROOT) and shoot (FWSHOOT), dry weights of root (DWROOT) and shoot (DWSHOOT), in Phaseolus vulgaris L. NaCl (mM) AsA (mM) GA3 (mM) FWROOT (g plant -1) FWSHOOT (g plant -1) DWROOT (g plant -1) DWSHOOT (g plant -1) 0 0 0 0.57 ± 0.03cd 1.58 ± 0.04b 0.054 ± 0.001c 0.192 ± 0.007ab 0 1 0 0.53 ± 0.06cd 1.35 ± 0.08c 0.051 ± 0.002c 0.172 ± 0.006cd 0 0 0.05 0.69 ± 0.04bc 1.77 ± 0.04ab 0.065 ± 0.002b 0.183 ± 0.002abc 0 1 0.05 0.90 ± 0.01a 1.87 ± 0.09a 0.079 ± 0.004a 0.194 ± 0.005a 200 0 0 0.47 ± 0.04d 0.96 ± 0.09d 0.041 ± 0.001d 0.121 ± 0.006f 200 1 0 0.91 ± 0.07a 1.27 ± 0.11c 0.064 ± 0.002b 0.152 ± 0.006e 200 0 0.05 0.66 ± 0.05bcd 1.33 ± 0.01c 0.051 ± 0.002c 0.160 ± 0.008de 200 1 0.05 0.85 ± 0.15ab 1.30 ± 0.03c 0.062 ± 0.002b 0.178 ± 0.003bc LSD(0.05) 0.2048 0.2120 0.0054 0.0154 The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test Table 3 Effects of sodium chloride, gibberellic acid and ascorbic acid on root length (RL), plant height (PH), leaf area (LA) and leaf dry weight (DWLEAF) in Phaseolus vulgaris L. NaCl (mM) AsA (mM) GA3 (mM) RL (cm plant -1) PH (cm plant-1) LA (cm2 plant-1) DWLEAF (g plant -1) 0 0 0 10.03 ± 0.75cd 12.70 ± 1.10bc 28.10 ± 2.48a 0.076 ± 0.002a 0 1 0 9.50 ± 1.00d 11.20 ± 0.81cd 25.10 ± 3.02a 0.058 ± 0.002bc 0 0 0.05 11.73 ± 0.43abc 15.74 ± 1.11a 25.60 ± 0.90a 0.063 ± 0.002abc 0 1 0.05 10.67 ± 0.17bcd 14.50 ± 0.76ab 28.00 ± 2.77a 0.069 ± 0.003ab 200 0 0 9.40 ± 0.56d 8.93 ± 0.75d 23.20 ± 5.32a 0.049 ± 0.001c 200 1 0 13.40 ± 0.45a 11.50 ± 0.80cd 26.20 ± 4.76a 0.058 ± 0.003bc 200 0 0.05 9.67 ± 1.09cd 12.63 ± 0.91bc 25.10 ± 4.14a 0.057 ± 0.005bc 200 1 0.05 12.33 ± 0.73ab 11.83 ± 1.17bc 27.40 ± 1.66a 0.079 ± 0.005a LSD(0.05) 2.125 2.822 9.988 0.0173 The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test Table 4 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of chlorophyll a (Chl a), chlorophyll b (Chl b), carotenoid (Car) and anthocyanin (Ant) in leaves of Phaseolus vulgaris L. NaCl (mM) AsA (mM) GA3 (mM) Chl a (mg g -1 FW) Chl b (mg g-1 FW) Car (lmol g-1 FW) Ant (Abs530 g -1 FW) 0 0 0 1.685 ± 0.123ab 0.578 ± 0.057a 0.157 ± 0.007a 0.0208 ± 0.0004fg 0 1 0 1.404 ± 0.049c 0.412 ± 0.028cd 0.114 ± 0.004cd 0.0230 ± 0.0010ef 0 0 0.05 1.760 ± 0.020a 0.558 ± 0.059a 0.166 ± 0.013a 0.0344 ± 0.0011d 0 1 0.05 1.525 ± 0.119abc 0.534 ± 0.012ab 0.126 ± 0.004bc 0.0565 ± 0.0021a 200 0 0 0.856 ± 0.046e 0.249 ± 0.014e 0.077 ± 0.008e 0.0186 ± 0.0002 g 200 1 0 1.050 ± 0.115de 0.242 ± 0.029e 0.103 ± 0.005d 0.0245 ± 0.0003e 200 0 0.05 1.267 ± 0.084cd 0.418 ± 0.025bc 0.123 ± 0.004bc 0.0406 ± 0.0004b 200 1 0.05 1.423 ± 0.124bc 0.269 ± 0.053de 0.138 ± 0.001b 0.0374 ± 0.0013c LSD(0.05) 0.2791 0.1161 0.0173 0.0029 The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test Acta Physiol Plant 123 The activity of antioxidant enzyme, GPOX, was always lower in the shoots than in the roots in controls as well as in other treatments (Table 7). The activity of this enzyme was slightly increased in response to NaCl alone; but in the combination with AsA or GA3, enzyme activity was much higher than when NaCl was given alone. The plants treated with NaCl and AsA ? GA3 exhibited the highest values for GPOX activity in both roots and shoots (Table 7). Discussion In the present study, salt stress caused significant inhibition in growth of P. vulgaris seedlings. Similar results were reported by Tejera et al. (2004, 2005), who indicated that growth of common bean plants considerably decreased by salt stress. Salinity can inhibit plant growth by altering the water potential, increasing the ion toxicity, inhibiting the cell division and cell expansion, or causing an ion imbal- ance (Arshi et al. 2005). In this context, Younis et al. (2010) reported that the growth reduction caused by salinity stress is due to inhibited apical growth in plants as well as endogenous hormonal imbalance. In both cases, reduction could have been caused by the toxic effects of ions (Na? and Cl-) on metabolism or from adverse water relations. In addition, a secondary aspect of salinity stress in plants is the stress-induced production of ROS (Manchanda and Garg 2008). The enhanced production of ROS during salinity stress lead to the progressive oxidative damage and ultimately cell death and growth suppression (Ruiz-Lozano et al. 2012). The growth characteristics of common bean seedlings under salinity stress were effectively improved with GA3 ? AsA supplement, implying that this treatment can alleviate the deleterious effects of salt stress. Gibberellic acid has been reported to be helpful in enhancing growth of wheat, maize and tomato under saline conditions (Ashraf et al. 2002; Kaya et al. 2006; Maggio et al. 2010). Salt stress was strong enough to inhibit plant growth due to reduction in gibberellin production (Kaya et al. 2006). Table 5 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of sodium in root (NaROOT) and shoot (NaSHOOT), and potassium in root (KROOT) and shoot (KSHOOT) in Phaseolus vulgaris L. NaCl (mM) AsA (mM) GA3 (mM) NaROOT (lmol g-1 DW) NaSHOOT (lmol g-1 DW) KROOT (lmol g-1 DW) KSHOOT (lmol g-1 DW) 0 0 0 73.33 ± 4.41e 36.11 ± 1.47d 247.22 ± 4.75a 196.67 ± 2.55c 0 1 0 49.44 ± 2g 36.67 ± 0.96d 234.44 ± 3.89b 181.67 ± 2.89d 0 0 0.05 62.22 ± 1.47ef 34.44 ± 1.47d 191.11 ± 1.11c 180.56 ± 1.47d 0 1 0.05 59.44 ± 2.42fg 34.44 ± 2.94d 226.67 ± 3.85b 189.44 ± 2cd 200 0 0 372.22 ± 7.35a 93.33 ± 2.89a 81.67 ± 3.85g 163.33 ± 2.89e 200 1 0 324.44 ± 4.01b 61.11 ± 3.64bc 122.78 ± 1.47d 255.00 ± 6.74ab 200 0 0.05 295.00 ± 1.67d 67.78 ± 2.94b 109.44 ± 2.42e 249.44 ± 2b 200 1 0.05 307.22 ± 3.09c 57.78 ± 2c 93.33 ± 5.09f 263.33 ± 0.96a LSD(0.05) 11.310 7.340 10.730 9.457 The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test Table 6 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of protein in root (ProROOT) and shoot (ProSHOOT), and malondialdehyde in root (MDAROOT) and shoot (MDASHOOT) in Phaseolus vulgaris L. NaCl (mM) AsA (mM) GA3 (mM) ProROOT (mg g-1 FW) ProSHOOT (mg g-1 FW) MDAROOT (lmol g-1 FW) MDASHOOT (lmol g-1 FW) 0 0 0 1.58 ± 0.01c 1.21 ± 0.13bc 19.33 ± 4.89a 4.73 ± 0.33bc 0 1 0 1.94 ± 0.05b 1.23 ± 0.07c 14.51 ± 2.13a 2.75 ± 0.21de 0 0 0.05 2.15 ± 0.11a 0.94 ± 0.05d 18.14 ± 6.36a 3.14 ± 0.15de 0 1 0.05 2.09 ± 0.05ab 1.27 ± 0.01bc 15.90 ± 2.98a 2.01 ± 0.14e 200 0 0 0.59 ± 0.06e 0.62 ± 0.07e 24.28 ± 7.43a 13.41 ± 1.15a 200 1 0 0.87 ± 0.01d 1.06 ± 0.03 cd 20.31 ± 10.46a 3.29 ± 0.11cde 200 0 0.05 0.92 ± 0.04d 1.55 ± 0.06b 25.45 ± 6.87a 5.71 ± 0.59b 200 1 0.05 1.66 ± 0.05c 2.02 ± 0.21a 19.88 ± 4.80a 3.68 ± 0.20cd LSD(0.05) 0.1624 0.2984 18.740 1.4660 The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to LSD test Acta Physiol Plant 123 Therefore, addition of exogenous gibberellic acid might increase seedling growth by enhancing the content of endogenous gibberellin as that mentioned by Rodriguez et al. (2006). Additionally, the enhancement of growth rate by gibberellin might result in an enlargement of leaf area, activation of cell division and/or cell elongation, stimula- tion of photosynthetic rate, modified partitioning of pho- tosynthates, or in their combination. The GA3-mediated invertase activity in elongating shoots could result in a significant accumulation of hexoses required for the pri- mary cell wall biosynthesis, thus favoring seedling growth under stress condition (Saeidi-Sar et al. 2007). On the other hand, the beneficial effect of AsA on plant growth may be attributed to the fact that AsA is involved in the regulation of root elongation, cell vacuolation and cell expansion (Smirnoff 1996). AsA-induced increase in growth under non-saline conditions may have been due to a double action of AsA on cell growth by modifying the cell cycle and stimulating quiescent cells to divide, and by accelerating cell elongation. In addition, ascorbate is a co-factor for prolyl-hydroxylase that post-translationally hydroxylates proline residues in cell wall hydroxyproline- rich glycoproteins required for cell division and expansion (Smirnoff and Wheeler 2000). Moreover, AsA increases the content of IAA, which stimulates cell division and/or cell enlargement and this, in turn, improves plant growth (Khan et al. 2011). Measurement of photosynthetic pigments under salinity conditions in P. vulgaris seedlings explains that NaCl salinity has the negative effects on these pigments. The adverse effects of salt stress on chlorophylls and carote- noids were counteracted by exogenous application of GA3 and AsA. The high salinity caused a disturbed chloroplast structure, number and size, which affected chlorophyll content and/or caused disruption of chloroplasts by oxi- dative stress that causes a decrease in chlorophyll content (Rahman et al. 2000). Also, reduction in chlorophyll con- tent under salinity stress is attributed to salt-induced acceleration of chlorophyll enzymes degradation and the instability of pigment–protein complex (Hernandez and Almansa 2002). Among the positive effects of AsA in the counteraction of the adverse effects of salt stress are the stabilization and protection of the photosynthetic pigments and the photo- synthetic apparatus from oxidization (Khan et al. 2011). Ascorbic acid can mitigate the adverse effects of salinity through increasing the content of IAA and GA3 and decreasing ABA level (Khan et al. 2011), which may be involved in protecting the photosynthetic apparatus and consequently increasing the photosynthetic pigments. AsA has a major role in photosynthesis, acting in the Mehler peroxidase reaction with ascorbate peroxidase to regulate the redox state of photosynthetic electron carriers and as a co-factor for violaxanthin de-epoxidase, an enzyme involved in xanthophyll cycle-mediated photoprotection (Smirnoff and Wheeler 2000). Consequently, in AsA- treated plants, high level of carotenoids can synergistically function with ascorbic acid to provide an effective barrier against oxidation under salinity stress. On the other hand, GA3 also plays a vital role in toler- ance to salt stress by improving plant growth and chloro- phyll synthesis (Maggio et al. 2010). In addition, the inhibitory effect of GA3 on chlorophyll catabolism might be partly due to the down regulation of the activities of enzymes involved in chlorophyll catabolism and the alle- viation of oxidative chlorophyll bleaching (Li et al. 2010). The concentration of anthocyanin was appreciably increased due to exogenous application of GA3 and AsA under both stressful and non-stressful conditions. AsA functions as an enzyme co-factor for anthocyanins syn- thesis (Smirnoff and Wheeler 2000). Zhang et al. (2012) indicated that leaves containing anthocyanins had a greater Table 7 Effects of sodium chloride, gibberellic acid and ascorbic acid on content of hydrogen peroxide in root (H2O2 ROOT) and shoot (H2O2 SHOOT), and guaiacol peroxidase activity in root (GPOXROOT) and shoot (GPOXSHOOT) in Phaseolus vulgaris L. NaCl (mM) AsA (mM) GA3 (mM) H2O2 ROOT (lmol g-1 FW) H2O2 SHOOT (lmol g-1 FW) GPOXROOT (U mg-1 pro) GPOXSHOOT (U mg-1 pro) 0 0 0 7.31 ± 2.70a 2.09 ± 0.36bc 67.3 ± 5.9cd 41.6 ± 5.2bc 0 1 0 8.98 ± 3.86a 1.62 ± 0.19c 68.5 ± 8.1dc 54.4 ± 8.1ab 0 0 0.05 7.42 ± 2.10a 1.52 ± 0.35c 56.7 ± 4.4d 33.5 ± 3.8c 0 1 0.05 6.36 ± 1.73a 1.56 ± 0.29c 60.8 ± 5.2d 34.0 ± 4.6c 200 0 0 12.56 ± 4.81a 3.33 ± 0.60a 68.7 ± 2.6cd 42.0 ± 1.2bc 200 1 0 10.94 ± 5.69a 3.00 ± 0.58ab 89.3 ± 5.2b 54.0 ± 3.8ab 200 0 0.05 13.26 ± 5.84a 3.53 ± 0.29a 81.2 ± 6.7bc 46.3 ± 8.6bc 200 1 0.05 9.66 ± 3.48a 1.78 ± 0.39c 108.0 ± 6.0a 68.0 ± 4.2a LSD(0.05) 9.996 1.207 17.160 16.240 The values (mean ± SE) with different letter within columns are statistically different (P B 0.05) according to the LSD test Acta Physiol Plant 123 antioxidant potential than did green leaves, and anthocya- nins can be used as antioxidants to extinguish ROS pro- duced in either PSI or PSII under environmental stresses. Accordingly, the powerful antioxidative capability of anthocyanins in the presence of GA3 and AsA mitigated the adverse effects of NaCl on common bean plants. The present study also revealed that the imposition of salt stress caused an increase in uptake and accumulation of Na? and a decrease in K? concentration in both roots and shoots of common bean plants, whereas improvements in amounts of these ions were observed in application of GA3 and AsA to stressful media. Salt stress is known to enhance the uptake and accumulation of toxic ions such as Na? in plant species (Sibole et al. 1998; Ashraf et al. 2002; Kaya et al. 2006; Nasir et al. 2010; Zhang et al. 2011). Higher Na? uptake by roots of common bean seedlings under salt stress conditions is possibly caused by inhibition of the Na?/H?-antiporters operating normally at the plasma membrane (Roslyakova et al. 2011). Sodium uptake is mediated by both voltage-dependent and -independent cation channels. Voltage-dependent cation channels such as K? inward rectifiers (HKT, HAK and KUP) mediate Na? uptake into root cells. Sodium competes with K? uptake through Na?–K? co-transporters and may also block the K?-specific transporters of root cells, thus high levels of Na? can induce the conformational changes in protein structure and membrane depolarization, and lead to the perception of ion toxicity in saline conditions (Manchanda and Garg 2008). Exogenous supply of AsA enhanced potassium con- centration in NaCl-stressed plants. This increase may be attributed to the positive effect of AsA on the root growth, which consequently increased the absorption of different nutrients and alleviated the harmful effects of salinity. In addition, AsA would inhibit a stress-induced increase in the leakage of essential electrolytes following peroxidative damage to plasma membranes (Khan et al. 2011). More- over, the increase of transmembrane electron transport via cytochrome b using ascorbic acid depolarizes the plasma membrane, and activates the H?-ATPase resulting in increased ion uptake such as K? (Smirnoff and Wheeler 2000). Almost all interactions between salinity and both GA3 or AsA decreased Na ? concentration and/or increased K? concentration as compared to salinized plants. These results agree with Aldesuquy (1995) who reported that GA3 reduced the accumulation of toxic ions in plant tissues under saline conditions; and Athar et al. (2008) who sug- gested that the protection of wheat plants against salt stress by an exogenous supply of AsA is caused indirectly as a result of its effect on K? uptake, which plays an essential role in many metabolic processes. GA3 and/or AsA-treated plants accumulated lower quantities of Na? in roots, which were possibly related to mobilization of defense mechanisms that restricted Na? entry or promoted the extrusion of Na? to the external medium. The preferential accumulation of sodium in roots over the shoots may be interpreted as a mechanism of tolerance in at least two ways, firstly: maintenance of a substantial potential for osmotic water uptake into the roots, secondly: restricting the spread of Na? to the shoots (Renault et al. 2001). We observed a great reduction in protein content of both shoot and root of common bean plants under NaCl stress. Metabolic toxicity of Na? is largely a result of its ability to compete with K? for binding sites of multiple enzymes in the cytoplasm that are required for cellular function. Pro- tein synthesis also requires high concentrations of K?, owing to the K? requirement for the binding of tRNA to ribosomes and probably other aspects of ribosome function (Tester and Davenport 2003). Thus, disruption of protein synthesis by elevated concentrations of Na? appears to be an important cause of damage by NaCl. Salt stress can also increase the production of ROS and cause damage to proteins. One possibility is that autophagy might be responsible for degrading oxidized proteins under salt stress (Liu et al. 2009). But in the presence of AsA, the soluble protein content increased, indicating that it may have an important role in plant adaptation to a high NaCl content (Huang et al. 2011). Possibly, the protective effect of AsA under salinity stress is more related to a reduction in ROS damages to essential proteins and/or nucleic acids (Noctor and Foyer 1998; Smirnoff and Wheeler 2000; Khan et al. 2011). It has been reported that ROS, including superoxide and hydrogen peroxide, are elevated with increasing the salin- ity, due to the imbalance in the production and destruction of ROS (Harinasut et al. 2003). In the present study, the P. vulgaris seedlings responded to NaCl treatment with enhancement of H2O2 content, which was higher in the roots than the shoots. Hydrogen peroxide has been shown to negatively influence proliferation of cells (Santoro et al. 2005). Thus, H2O2 can play an important role in the inhi- bition of growth of NaCl-stressed plants. Salt stress is also known to result in extensive lipid peroxidation, which has often been used as indicator of salt induced oxidative damage in membranes. The observed enhanced value for MDA content in the plants raised from treatment with NaCl alone indicates that NaCl toxicity may be responsible for increasing lipid peroxidation leading to cell damage. Exogenous application of GA3, AsA and GA3 ? AsA reduced NaCl-induced increase in the contents of H2O2 and MDA. Gibberellins appear to play a key role against oxi- dative stress by decreasing accumulation of ROS and preventing lipid peroxidation, which were induced by salinity. These alleviating effects of GA3 were highly correlated with the increasing activities of antioxidant enzymes (Maggio et al. 2010). However, in this study AsA Acta Physiol Plant 123 was a more efficient antioxidant than GA3 against NaCl- induced oxidative damage to P. vulgaris plants. Ascorbate is involved in controlling the intracellular ROS level by direct scavenging or via the ascorbate–glutathione cycle. This might provide the means to protect the cell against uncontrolled oxidation and improved growth (Noctor and Foyer 1998; Smirnoff and Wheeler 2000; Bartoli et al. 2006). Plants possess efficient systems for scavenging active oxygen species that protect them from destructive oxida- tive reactions (Zhu et al. 2004). As part of this system, antioxidant enzymes are key elements in the defense mechanisms. In our study, slight increase in GPOX activity was observed in root and shoot of common bean seedlings exposed to NaCl. These results suggest that this antioxidant enzyme has possible defensive role in the removal of H2O2 under salinity stress. The increased total peroxidase activity in response to salinity has been reported (Chen et al. 1993; Sreenivasulu et al. 2000; Harinasut et al. 2003). The plants treated with NaCl in combination with GA3 and AsA exhibited much higher values for antioxidant enzyme activity than when NaCl was given alone. GA3 and AsA probably reverse the effect of NaCl stress in common bean seedlings by enhancing peroxidase activity, as well as by decreasing H2O2 production and lipid peroxidation. Accordingly, improved activity of GPOX by the combined application of GA3 and AsA resulted in an increase in the capacity of detoxification mechanism and also in an improvement in the capacity of tolerance to NaCl stress. In conclusion, results of the present study suggest that NaCl toxicity is associated with induction of oxidative stress in P. vulgaris seedlings leading to increased gener- ation of H2O2, elevated levels of lipid peroxidation and protein oxidation, and a decline in ratios of carotenoids and anthocyanins, but deleterious effect of salinity was coun- teracted by ascorbic acid and gibberellin A3. However, the mechanisms by which GA3 could induce salt tolerance in plants are not yet clear. Salinity perturbs the hormonal balance in plants. Therefore, hormonal homeostasis under salt stress might be the possible mechanism of GA3- induced plant salt tolerance. In this respect, the beneficial effects of AsA might be attributed to the increase of pho- tosynthesis activity, as well as to the role of AsA in several defense mechanisms against oxidative stress under salinity stress conditions. In order to reduce the oxidative damages caused by NaCl stress, the protective role of AsA ? GA3 was often better than applying AsA or GA3 alone. The increased tolerance to NaCl stress in the presence of AsA and GA3 was manifested in terms of enhanced growth, GPOX activity, chlorophylls and potassium contents. But further researches are required to decipher the mechanisms through which AsA in combined with GA3 acts, and how synergistically effects of them might be connected with salt tolerance. Author contribution Dr. S. Saeidi-Sar designed the experimental framework and supervised the whole work, and contributed to all the experimental process, data interpretation and discussion as well as paper writing. Dr. H. Abbaspour and Dr. H. 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Plant Sci 167:527–533. doi:10.1016/j.plantsci.2004.04.020 Acta Physiol Plant 123 Effects of ascorbic acid and gibberellin A3 on alleviation of salt stress in common bean (Phaseolus vulgaris L.) seedlings Abstract Introduction Materials and methods Plant materials and treatments Determination of pigments Determination of K and Na Determination of H2O2 content Determination of MDA content Enzyme extraction and protein content Determination of peroxidase activity Statistical analysis Results Discussion Acknowledgments References