ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 5, с. 688-697
^ ЭКСПЕРИМЕНТАЛЬНЫЕ
СТАТЬИ
УДК 581.1
EFFECTS OF STATIC MAGNETIC FIELD PRETREATMENT WITH AND WITHOUT PEG 6000 OR NaCl EXPOSURE ON WHEAT
BIOCHEMICAL PARAMETERS1 © 2014 A. Sen, S. Alikamanoglu
Istanbul University, Faculty of Science, Department of Biology, Vezneciler, Istanbul, Turkey
Received February 10, 2013
In this study, the static magnetic field (SMF) flux of 2.9—4.7 mT was applied to mature embryo explants of Tekirdag and Selimiye wheat cultivars (Triticum aestivum L.) under tissue culture conditions at three different pretreatment times of 0, 2.2, and 19.8 s, at the rate of 1 m/s with and without 60 g/L PEG 6000 or 100 mM NaCl. Changes in different biochemical parameters were investigated. SMF pretreatment with and without 60 g/L PEG 6000 or 100 mM NaCl increased chlorophyll and carotenoid contents, FRAP values, and antioxidant enzyme (SOD, CAT, POX, and APX) activities in all experimental groups except for the activities of Mn-SOD in the cv. Selimiye root samples. Among SOD isozymes, Fe-SOD was affected by SMF stronger than other izozymes. The combinations of SMF pretreatment with and without 60 g/L PEG 6000 or 100 mM NaCl differently affected SOD isozyme activities besides its effects on other biochemical parameters.
Keywords: Triticum aestivum — antioxidant defense — pigment content — static magnetic field — NaCl — PEG 6000
DOI: 10.7868/S0015330314050145
INTRODUCTION
Since the Industrial Revolution in the 1850s, the concept of artificial magnetic field (MF) has come into our lives together with rapid technological development. Therefore, in addition to geomagnetic field, human-made MF is an inevitable environmental factor for microorganisms, plants, and animals. Because of this, since the 1930s a lot of investigations have been made to clarify how artificial MF influences on biological systems besides geomagnetic field (35—70 ^T) [1]. Many researchers have previously reported that suitable magnetic pretreatment, which depends on time, duration, and flux density, favorably affected plant growth parameters and changed biochemical parameters, such as the amount of chlorophyll (Chl) and carotenoids (CAR), and the activities of superoxide dismutase (SOD), polyphenol oxidase (PPO), cata-lase (CAT), and peroxidase (POX) [2-5]. Although
1 This text was submitted by the authors in English.
Abbreviations'. APX — ascorbate peroxidase; CAR — carotenoids; CAT — catalase; Chl — chlorophyll; FW — fresh weight; MF — magnetic field; POX - peroxidase; SMF - static MF; SOD - superoxide dismutase; TPTZ — 2,4,6-tri(2-pyridyl)-s-triazine. Corresponding author. Ayse Sen. Istanbul University, Faculty of Science, Department of Biology, "Vfezneciler, Istanbul, 34143 Turkey; e-mail. senayse@istanbul.edu.tr
MF has become a quite popular factor in recent years, the mechanisms of its action on living organisms are still unknown. Today, there are three mechanisms commonly accepted to explain cellular responses to MF in biological systems. One is "radical-pair mechanism," which consists of the modulation of singlet-triplet interconversion rates of a radical pair by weak magnetic fields. The other one is "the ion cyclotron resonance effect" and the third one is "ferrimag-netism," which is referred to the orientation of ferromagnetic particles in tissues [1].
Harsh environmental conditions, such as drought and salinity, induce both osmotic and oxidative stresses in plant apart from reducing plant growth, crop productivity and quality. Oxidative stress is defined as a serious imbalance between ROS production and antiox-idant defense, and this situation causes damage to proteins, lipids, carbohydrates, and DNA, which ultimately results in the cell death. ROS such as O2-,
H2O2, 1O2, HO2, 'OH, ROO", and RO' are highly reactive transient chemicals generally characterized by the presence of an unpaired electron. Under steady-state conditions, ROS are scavenged by various antioxidant defense systems. These systems are composed of enzymatic and non-enzymatic antioxidant defense
Carrier band
Magnetic field system [3]. L = 2.2 m — carrier band length; h = 0.06 m — distance between explants and magnets; d = 0.15 m — distance between magnets; V = 1 m/s — velocity of explants passing under magnetic field.
systems. Superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1. 11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and peroxidase (POX, EC 1. 11.1.7) are major members of the enzymatic defense system, and these enzymes are designated to minimize the
concentration of O^- and H2O2. Additionally, plant cells also include relatively high levels of non-enzymatic antioxidants, including ascorbate, glutathione, carotenoids, and a-tocopherol, which are efficient oxyradical scavengers [6, 7].
The aim of the present study was to contribute to our first report on the biochemical and biophysical basis of the potential biological effects of pretreatment with the artificial SMF alone and in combination with PEG 6000 (as a drought stress inducer) or NaCl (as a creator of salt stress conditions) on biochemical parameters (chlorophyll (a + b), a, b) and carotenoid contents, FRAP values, SOD, CAT, POX, and APX enzyme activities) in wheat tissue culture.
MATERIALS AND METHODS
Plant material. Wheat (Triticum aestivum L. cvs. Tekirdag and Selimiye) seeds were provided by Trakya Agricultural Research Institute in Edirne, Turkey.
Plant tissue culture conditions. Seed surfaces were sterilized according to the method of Ozgen et al. [8]. After that, mature embryos were obtained using sterile forceps and scalpel under aseptic conditions, and then embryos were sown on culture media, including MS [9] basal salt mixture, 0.1 mg/L 2,4-D, 20 g/L sucrose, and 10 g/L agar. Prior to sterilization of the medium, pH was adjusted to 5.8 with 1 M NaOH or HCl.
Magnetic field pretreatment. The magnetic field system, which was used at the Department of Biology in Istanbul University, was prepared by the magnetic field group of Joint Institute for Nuclear Research,
Laboratory in Dubna, Russia (JINR). To generate the magnetic fields, we used 10 magnets of 0.45 x 0.065 x 0.022 m dimensions. These magnets were mounted onto a belt system that rotated at the rate of 1 m/s. The distance between magnets and the belt system was 0.06 m (figure). When the magnetic field was applied, the Petri dishes with sterile culture media and sown mature wheat embryos were put on the belt system that rotated with a rate of 1 m/s at the 23 ± 2°C. Magnetic flux density was 2.9—4.7 mT, and three different exposure times were 0 (as a control under geomagnetic field conditions), 2.2, and 19.8 s. The characteristics of magnets were [3]:
B = h
where B is magnetic induction, T; is magnetic per-miability ofvacuum, = 4n x 10-7 T m/A; is magnetic permiability of air, = 1; H is magnetic field strength, H = 2300-3700 A/m.
Then embryos were immediately transferred into fresh medium with and without 60 g/L PEG 6000 (as a drought inducer) or 100 mM NaCl. After MF pre-treatment all in vitro cultures were maintained at 25 ± 2°C with the photoperiod of16 h in a growth chamber. At the 28th day, leaf samples from experimental groups were harvested and used for measuring biochemical parameters.
Determination of pigment contents. Samples of 0.1 g were homogenized in 80% cold acetone, and the absorbance of the homogenates was determined at 663, 645, and 470 nm in the UV/Visible spectrophotometer. Chl a, b, total Chl, and carotenoid quantities were calculated according to the modification of Lichtenthaler and Wellburn [10].
Non-enzymatic antioxidant power activity of samples (FRAP values). The ability to reduce ferric ions was measured using the method of Benzie and Strain [11]
Table 1. Pigment contents in 28-day-old Selimiye and Tekirdag cultivars pretreated with non-uniform SMF at various times with and without 60 g/L PEG 6000 or 100 mM NaCl under in vitro conditions
Cultivar Experimental group Chl a+b Chl a Chl b CAR
mg/g fr wt
Selimiye Control 0.167 ± 0.018a* 0.113 ± 0.011a 0.054 ± 0.007a 0.052 ± 0.005a
2.2 s 0.233 ± 0.022b 0.140 ± 0.015b 0.092 ± 0.004b 0.074 ± 0.007b
19.8 s 0.237 ± 0.021b 0.153 ± 0.013b 0.084 ± 0.009b 0.062 ± 0.004b
60 g/L PEG 6000 0.128 ± 0.023c 0.086 ± 0.007c 0.042 ± 0.008c 0.047 ± 0.003a
2.2 s + 60 g/L PEG 6000 0.214 ± 0.017b 0.138 ± 0.011b 0.076 ± 0.007ab 0.059 ± 0.008ab
19.8 s + 60 g/L PEG 6000 0.199 ± 0.015b 0.140 ± 0.012b 0.059 ± 0.005ac 0.063 ± 0.009b
100 mM NaCl 0.143 ± 0.017c 0.091 ± 0.009c 0.047 ± 0.007c 0.050 ± 0.004a
2.2 s +100 mM NaCl 0.209 ± 0.019b 0.129 ± 0.009ab 0.080 ± 0.005ab 0.060 ± 0.005b
19.8 s + 100 mM NaCl 0.184 ± 0.016ab 0.125 ± 0.012ab 0.059 ± 0.008ac 0.061 ± 0.003b
Tekirdag Control 0.120 ± 0.011a* 0.076 ± 0.010a 0.049 ± 0.009a 0.054 ± 0.005a
2.2 s 0.183 ± 0.021b 0.101 ± 0.016b 0.082 ± 0.007b 0.072 ± 0.008b
19.8 s 0.176 ± 0.024b 0.109 ± 0.014b 0.067 ± 0.001b 0.067 ± 0.006b
60 g/L PEG 6000 0.099 ± 0.019c 0.064 ± 0.008c 0.035 ± 0.001c 0.037 ± 0.005c
2.2 s + 60 g/L PEG 6000 0.155 ± 0.016ab 0.092 ± 0.004a 0.062 ± 0.007ab 0.056 ± 0.007a
19.8 s + 60 g/L PEG 6000 0.155 ± 0.019ab 0.101 ± 0.011a 0.054 ± 0.008a 0.055 ± 0.009a
100 mM NaCl 0.090 ± 0.017c 0.052 ± 0.007c 0.038 ± 0.009c 0.032 ± 0.007c
2.2 s + 100 mM NaCl 0.146 ± 0.013ab 0.088 ± 0.009a 0.058 ± 0.006a 0.043 ± 0.007d
19.8 s + 100 mM NaCl 0.128 ± 0.011a 0.082 ± 0.002a 0.046 ± 0.006a 0.040 ± 0.006d
* Different letters indicate significant differences (P < 0.05) among the experimental groups at each level (a, b, c, and d) of non-uniform SMF pretreatment at various times with and without 60 g/L PEG 6000 or 100 mM NaCl (Kruskal—Wallis test). The data represent means ± standard errors (n = 5).
using FeSO4 • 7H2O as a standard. 200 ^L of the extract was added to 2.8 mL of FRAP reagent (10 parts of 300 mM sodium acetate buffer, pH 3.6, 1 part of 10 mM TPTZ solution, and 1 part of 20 mM FeCl3 • 6H2O), and the abs
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