Assessment of Antioxidant Enzyme Activity and Mineral Nutrients in Response to NaCl Stress and its Amelioration Through Glutathione in Chickpea Vinay Shankar, Dinesh Kumar & Veena Agrawal
Applied Biochemistry and Biotechnology Part A: Enzyme Engineering and Biotechnology ISSN 0273-2289 Appl Biochem Biotechnol DOI 10.1007/s12010-015-1870-1
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Author's personal copy Appl Biochem Biotechnol DOI 10.1007/s12010-015-1870-1
Assessment of Antioxidant Enzyme Activity and Mineral Nutrients in Response to NaCl Stress and its Amelioration Through Glutathione in Chickpea Vinay Shankar 1 & Dinesh Kumar 1 & Veena Agrawal 1
Received: 8 June 2015 / Accepted: 23 September 2015 # Springer Science+Business Media New York 2015
Abstract Salinity stress has been reckoned as one of the major threat towards crop productivity as it causes significant decline in the yield. The impact of NaCl stress (0, 1, 10, 50, 100 and 200 mg L−1) as well as glutathione (10 mg L−1) either alone or in combination has been evaluated on the induction of multiple shoots, antioxidant enzymes’ activity, lipid peroxidation, relative permeability, concentration of nutrients, photosynthetic pigments, protein and proline content of nodal segments of chickpea after 14 days of culture. The antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were found to be increased under salt stress as well as glutathione-supplemented medium. A significant decrease in the concentrations of chlorophylls a, b, total chlorophyll and carotenoid was observed under salt stress. Concentrations of nitrogen, phosphorus, potassium, calcium, carbon, magnesium and sulphur showed an initial increase up to 10 mg L−1 NaCl, but a decline was seen at higher NaCl levels. Proline content and malondialdehyde concentration were found to be increased under salt stress. Three isoforms of SOD, one of CAT and four of GPX were expressed during native polyacrylamide gel electrophoresis (PAGE) analysis. However, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the stressed nodal explants revealed the over-expression of several polypeptide bands related to NaCl stress. These findings for the first time suggest that glutathione (GSH) helps in ameliorating NaCl stress in nodal explants of chickpea by manipulating various biochemical and physiological responses of plants. Keywords Antioxidant enzymes . Cicer arietinum L. . Ion leakage . Lipid peroxidation . Proline . Reactive oxygen species
* Veena Agrawal
[email protected] 1
Department of Botany, University of Delhi, Delhi 110 007, India
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Introduction Salinity has been reckoned as a challenging problem in recent time causing severe damage to crop productivity. Presently, over 800 million ha of land around the world is affected by salinity. Out of the existing 230 million ha of irrigated land, 45 million ha is affected by salt [1]. The problem is further compounded by rapidly growing population of the world which is to be increased from 6.1 billion in 2001 to 9.3 billion in 2050 [2]. Plants exposed to salt stress show stunted growth, experience osmotic imbalance, nutritional deficiency and ion toxicity [3]. Besides disorganization of cellular and subcellular membranes [4, 5], increase in reactive oxygen species (ROS) level [6, 7], inhibition of enzymatic activities [8, 9] and metabolic toxicity are other important changes that occur during salt stress. Increase in ROS causes damage to cell membranes and other essential macromolecules such as photosynthetic pigments, proteins, nucleic acids and lipids [10, 11]. In order to overcome the detrimental effects of salt stress, plants have evolved various biochemical and physiological strategies, such as selective exclusion of ions, control of ion uptake by roots, their transport into leaves, ion compartmentalization, synthesis of compatible osmolytes, alteration in photosynthetic pathway, changes in membrane structure, induction of antioxidant enzymes and stimulation of phytohormones [12, 13]. To tolerate the excess of NaCl, plants also utilize certain basic strategies such as production of low molecular weight thiols like glutathione (GSH), cysteine [14] and various chelating agents like ascorbic acid, carotenoids or phytochelatins (PCs). Among salt binding ligands in plant cells, PCs are the best-characterized peptides synthesized from GSH and catalyzed by phytochelatin synthase activity [15]. They form complexes with toxic ions in the cytosol and subsequently transport them into the vacuole [16]. Glutathione is a tripeptide formed of Glu-Cys-Gly and is considered as one of the most important intracellular antioxidant defences against ROS-induced oxidative damage. It occurs abundantly in reduced form (GSH) in plant tissues and is localized in all cell compartments like cytosol, endoplasmic reticulum, vacuole, mitochondria, chloroplasts, peroxisomes as well as in apoplast and plays a key role in several physiological processes, including detoxification of salts and the expression of stress-responsive genes. The present investigation highlights the impact of salt concentrations and glutathione on the multiple shoot productions, photosynthetic pigments concentrations, antioxidant enzyme metabolism, protein content, lipid peroxidation, membrane permeability, proline concentration and 12 different nutrients concentration in nodal cultures of chickpea, an important green legume. This is our first report of depicting the beneficial effect of glutathione on the nodal culture reared under different concentrations of NaCl salt stress.
Materials and Methods Plant Materials and Generation of Nodal Explants Seeds of Cicer arietinum L. (variety BG 2019) were procured from Pulse Seed Laboratory, Genetic Division, Indian Agricultural Research Institute, Pusa, New Delhi, India. The seeds were surface-sterilized after thorough washing under running tap water for 20 min and treating
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with them Teepol (3 %) for 10–15 min and subsequently immersing in 1 % citric acid followed by 1 % Bavistin for 10 min; at each step, seeds were rinsed with autoclaved distilled water. The seeds were finally sterilized with 0.1 % HgCl2 for 2 min and rinsed with autoclaved distilled water. The sterilized seeds were grown in Murashige and Skoog’s medium having 3 % sucrose pH 5.8 and solidified with 0.8 % agar [17]. Nodal explants were harvested from 28-day-old seedlings and were used for culture.
Experimental Design, NaCl Treatment and Culture Conditions The experiment has a 2 × 6 factorial design with two glutathione conditions: supplementation of the growth medium with or without 10 mg L−1 glutathione (optimized, data not shown) and six levels of NaCl (0, 1, 10, 50, 100 and 200 mg L−1). The nodal explants obtained from 28-day-old seedlings were transferred to two sets of treatments. One set of treatment has MS+1 μM BA+10 mg L−1 glutathione amended with different concentrations (0, 1, 10, 50, 100 and 200 mg L−1) of NaCl. The other set did not contain glutathione in their treatments. The explants were grown for 2 weeks and harvested for various analyses. The cultures were incubated in continuous light of 400–450 μW cm−2 emitted by cool day light fluorescent incandescent tubes (40 W Philips, CFL tubes), and the temperature was maintained at 25±2 °C and relative humidity at 55±10 %.
Harvest and Measurement of Parameters The plants were harvested after 14 days of treatment and analyzed for various parameters. Plant growth was evaluated in terms of the average number of shoots developed per explant and the average shoot length. The fresh weight (FW) of the nodal cultures was determined. The nodal cultures were dried in oven at 70 °C for 72 h, and the dry weight (DW) was determined. The concentrations of K+, Ca2+, Mg2+, Na+, Cu2+, Fe+, Mn3+ and Zn2+ ions were determined according to Allen [18], using an atomic absorption spectrophotometer (AA-6300 Shimadzu). Phosphorus concentration was determined by ammonium molybdate blue method. The concentration of C, N and S was determined in a CHNS analyzer (Elementar Analysensysteme GmbH vario EL III). Pigment concentrations (chlorophyll and carotenoids) were estimated using the method described by Arnon [19]. Membrane permeability was measured according to Zwiazek and Blake [20] by an ELEINS conductivity metre and lipid peroxidation as described by Heath and Packer [21]. Proline concentration was estimated according to Bates [22]. Proteins were extracted following Zivy [23]. Fresh tissue (1 g) of each sample was powdered in chilled pestle and mortar using liquid nitrogen and homogenized in 4 mL of chilled Zivy’s extraction buffer (1:4). The homogenate was centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was used to determine protein concentration at 595 nm using bovine serum albumin as the standard. Amount of 0.1 mL of the supernatant was reacted with 5 mL of Bradford’s reagent and kept in the dark for 15 min. The intensity of blue colour developed in the reaction was measured spectrophotometrically at 595 nm in a UV spectrophotometer DU®730. The protein concentration was calculated from the standard BSA and expressed as microgramme per gramme fresh weight.
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Antioxidant Enzyme Activities The nodal explant extracts for determining the activities of antioxidant enzymes were prepared according to Elavarthi and Martin [24]. One gramme of frozen sample was ground to a fine powder with liquid nitrogen and was extracted in 2 mL of ice-cold 0.2 M potassium phosphate buffer (pH 7.8 with 0.1 mM EDTA). The homogenates was centrifuged at 15,000 × g for 20 min at 4 °C, and the supernatant was used for assay of enzyme activities. All the steps were performed at 4 °C. An aliquot of the supernatant was used to determine protein concentration by Bradford’s [25] method using bovine serum albumin as the standard. All the spectrophotometric readings were recorded using UV spectrophotometer (Beckman Coulter DU®730). Superoxide dismutase (SOD) (EC 1.15.1.1) activity was assayed spectrophotometrically at 560 nm using modified nitro blue tetrazolium (NBT) method [26]. The activity was expressed as units per milligramme protein. One unit of SOD activity is defined as the amount of enzyme that inhibits reduction of NBT by 50 %. Catalase (CAT) (EC 1.11.1.6) activity was determined by monitoring the rate of decomposition of H2O2 as measured by the decrease of absorbance at 240 nm for 3 min. According to Aebi [27], CAT activity was expressed as millimole H2O2 decomposed min−1 mg−1 protein. Ascorbate peroxidase (APX) (EC 1.11.1.11) activity was determined from the decrease in absorbance at 290 nm due to oxidation of ascorbate in the reaction following the protocol of Nakano and Asada [28]. One millilitre of reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM H2O2 and 25 μL of the enzyme extract. H2O2 was added last to initiate the reaction, and the decreased in absorbance was recorded for 3 min. One unit of APX activity is defined as millimole ascorbate oxidized min−1 mg−1 protein. Guaiacol peroxidase (GPX) (EC 1.11.1.7) activity was determined in a reaction mixture containing 65 mM phosphate buffer pH 6.0, 11 mM H2O2, 2.25 mM guaiacol and 50 μL enzyme sources following the protocol of Thimmaiah [29]. The enzyme activities were expressed as millimole tetraguaiacol−1 min−1 mg−1 protein. Glutathione reductase (GR) (EC 1.6.4.2) activity was assayed in 1 mL assay mixture containing 0.75 mM DTNB, 0.1 mM NADPH, 1 mM GSSG and 25 μL of sample extract following the protocol of Smith [30]. GSSG was added last to initiate the reaction and the increase in absorbance was recorded for 3 min. The reduction of DTNB to TNB by GSH in the reaction was monitored spectrophotometrically at 412 nm, and the enzyme activity was expressed as millimole TNB min−1 mg−1 protein.
Antioxidant Enzyme Activity Gel Analysis Equal amounts (15 μg) of protein were loaded on each well in a discontinuous native polyacrylamide gel electrophoresis (PAGE) (10 %) as described by Laemmli [31] without SDS and β-mercaptoethanol. Electrophoretic separation was performed at 4 °C a constant voltage of 80 V. SOD gel activity was assayed following Beauchamp and Fridovich’s [32] protocol. After completion of electrophoresis, the gel was incubated in a solution containing 2.45 mmol NBT for 20 min, followed by incubation in 50 mmol potassium phosphate buffer (pH 7.8) containing 28 μmol riboflavin and 28 mmol TEMED under dark condition. SOD expression was observed after light exposure for 10 to 20 min at room temperature. For identification of individual SOD isoforms, the gels were treated
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with either 3 mmol KCN or 5 mmol H2O2 in 50 mmol potassium phosphate buffer (pH 7.8) for 30 min. CAT gel activity was detected following Jebara’s method [33]. After the electrophoretic separation of protein, the gel was incubated in 50 mmol potassium phosphate buffer (pH 7.0) containing 3.27 mmol H2O2 for 20 min under dark conditions. Thereafter, the gel was rinsed with distilled water and stained in a solution containing 1 % potassium ferricyanide and ferric chloride. The enzymatic zone appears as achromatic band over the green-stained background. GPX activity in gel was identified following Thimmaiah [29]. After the completion of electrophoresis, the gel was incubated in 200 mmol sodium acetate-acetic acid buffer containing 1.3 mM benzidine and 1.3 mM H2O2 under dark conditions for 20 min. The enzymatic zone appeared as a brown band.
Statistical Analysis The data means of Duncan’s deviation
were subjected to one-way analysis of variance (ANOVA) to compare the all samples. The differences between individual means were determined by multiple range test (DMRT). The values are presented as mean±standard (SD).
Results Effects of Salt Stress on Morphogenesis Exogenous addition of different concentrations (0, 1, 10, 50, 100 and 200 mg L−1) of NaCl to the medium with or without GSH showed varying toxicity to chickpea nodal cultures. The toxic effect was observed in terms of shoot growth, shoot length, shoot number, FW and DW of chickpea nodal cultures (Table 1). The shoot number decreased significantly with increasing concentration of salt, both in non-GSH and + GSH-supplemented medium. It decreased by 35.7 and 41 % at 200 mg L−1 NaCl over control, both in non-GSH and +GSH-containing medium, respectively; whereas in terms of shoot length, it decreased by 41.7 and 29 % at 200 mg L−1 NaCl over control in non-GSH and +GSH medium, respectively. Fresh weight decreased by 38.8 and 23.5 % at 200 mg L−1 NaCl over control, whereas dry weight showed significant decline over control in both non-GSH and +GSH-containing medium, respectively (Table 1).
Photosynthetic Pigments The concentration of photosynthetic pigments decreased with increasing NaCl concentration. Chlorophylls a and b showed a gradual and significant decline at all levels of NaCl. However, at all NaCl levels, the decrease in chlorophyll concentration was less in +GSH plants compared to their non-GSH plants. At 200 mg L−1 NaCl, chlorophyll a and b concentrations were less by more than 80 and 85 % of control, respectively (Fig. 1a, b). Thus, in the case of chlorophylls a and b, the concentration of total chlorophyll showed a declining trend with the increasing levels of NaCl (Fig. 1c).
2.1±0.2 d 1±0.31 a
1.7±0.31 b
1±0.22 a
1.5±0.11 b
1±0.12 a
1.4±0.21 b
+GSH
−GSH
+GSH
−GSH
+GSH
50
2.4±0.50 a
3.26±0.72 a
4.2±0.46 b
5.3±0.67 c
5.7±0.29 c
3.9±0.2 b 5.5±0.37 c
5.6±0.23 c
4.1±0.22 b
7.4±0.56 d
8.2±0.22 e
7.8±0.34 d
Average shoot length (cm)
0.21±0.32 a
0.28±0.52 b
0.35±0.32 bc
0.41±0.41 d
0.41±0.32 d
0.28±0.22 b 0.37±0.22 bc
0.31±0.27 b
0.32±0.13 b
0.54±0.32 d
0.89±0.21 f
0.72±0.23 e
FW (g)
0.016±0.002 a
0.026±0.005 b
0.025±0.001 b
0.038±0.007 c
0.033±0.002 c
0.021±0.005 b 0.022±0.003 b
0.038±0.008 c
0.027±0.002 b
0.041±0.006 d
0.039±0.004 d
0.031±0.003 c
DW (g)
2.70±0.008 b
2.65±0.008 b
3.31±0.06 d
2.97±0.01 c
5.14±0.03 g
5.41±0.03 g 4.81 v 0.06 f
5.26±0.07 g
2.70±0.02 b
2.41±0.008 a
4.14±0.03 e
4.07±0.02 e
C (%)
1.66±0.04 d
1.52±0.05 c
2.70±0.04 i
2.15±0.06 g
2.48 v 0.03 h
1.65±0.05 d 2.03±0.01 f
1.23±0.03 b
1.35±0.03 b
1.04±0.02 a
1.80±0.03 e
1.67±0.02 d
N (%)
1.42±0.03 c
1.28±0.008 b
1.56±0.02 d
1.49±0.02 cd
2.46±0.04 g
1.58±0.02 d 2.28±0.02 f
1.42±0.005 c
1.30±0.02 b
1.16±0.03 a
1.99±0.06 e
1.92±0.008 e
S (%)
Values are means of replicates±SD. Values followed by different letters are significantly different from each other at p
