Effect of Gamma Radiation on Growth Factors,Biochemical Parameters, and Accumulationof Trace Elements in Soybean Plants (Glycine max L. Merrill)

Abstract

The toxicity of iron, copper, and zinc was studied in soybean seeds of the NE 3297 variety irradiated at different dosages of gamma rays. After cultivating in plastic
boxes for 14 days, the average plant heights, fresh weight, and chlorophyll content
decreased in inverse proportion to radiation dose. As the radiation dose increased, the
concentrations of iron, copper, zinc, soluble protein, and malondialdehyde increased, but the activities of superoxide dismutase and peroxidase enzymes activities were significantly decreased. The activities and the number of the superoxide dismutase isoenzymes also varied depending on the irradiation dosages.

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Effect of Gamma Radiation on Growth Factors,
Biochemical Parameters, and Accumulation
of Trace Elements in Soybean Plants
(Glycine max L. Merrill)
Sema Alikamanoglu & Orkun Yaycili & Ayse Sen
Received: 11 March 2010 /Accepted: 19 April 2010 /
Published online: 7 May 2010
# Springer Science+Business Media, LLC 2010
Abstract The toxicity of iron, copper, and zinc was studied in soybean seeds of the NE
3297 variety irradiated at different dosages of gamma rays. After cultivating in plastic
boxes for 14 days, the average plant heights, fresh weight, and chlorophyll content
decreased in inverse proportion to radiation dose. As the radiation dose increased, the
concentrations of iron, copper, zinc, soluble protein, and malondialdehyde increased, but
the activities of superoxide dismutase and peroxidase enzymes activities were significantly
decreased. The activities and the number of the superoxide dismutase isoenzymes also
varied depending on the irradiation dosages.
Keywords Antioxidantenzymes.Gammaradiation.Soybean.Traceelements
Abbreviations
EDTA
IAA
Cu/Zn-SOD
Fe-SOD
MDA
Mn-SOD
NBT
PAGE
POX
ROS
SE
SOD
TEMED
Ethylenediaminetetraacetic acid
Indole-3-acetic acid
Cu/Zn-superoxide dismutase
Fe-superoxide dismutase
Malondialdehyde
Mn-superoxide dismutase
Nitroblue tetrazolium chloride
Polyacrylamide gel electrophoresis
Peroxidase
Reactive oxygen species
Standard error
Superoxide dismutase
Tetramethylethylene diamine
Biol Trace Elem Res (2011) 141:283–293
DOI 10.1007/s12011-010-8709-y
S. Alikamanoglu (*):O. Yaycili:A. Sen
Department of Biology, Faculty of Science, Istanbul University, Vezneciler, 34459 Istanbul, Turkey
e-mail: semakaman@istanbul.edu.tr

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Introduction
Major and trace elements play an important role on plant, human, and animal life
participating in various metabolic processes such as enzyme catalysis, in the synthesis of
proteins and cell growth [1]. Iron, copper, and zinc are important metallic elements that are
essential for metabolic processes in plants [2].
Iron participates in enzymes like Fe-superoxide dismutase (Fe-SOD), electron transport
systems such as cytochrome, in the synthesis of chlorophyll and in sustaining structure and
functions of chloroplasts [3]. Copper is a cofactor of various enzymes such as plastocyanin,
Cu/Zn-SOD, and cytochrome c oxidase, also playing a role in electron transport [4]. Zinc
also participates in many enzymes and plays a definite role on protein and indole-3-acetic
acid synthesis, and in carbohydrate, lipid, and nucleic acid metabolism [5].
If the concentration of trace elements exceeds the required level, they may have negative
effects on living systems. In plants, determining the concentrations of such essential trace
elements is important in assessing their effects. Excessively high concentrations of Fe, Cu,
and Zn in plant tissues and organs can trigger events leading to their death. For example, it
has been reported that excessive uptake and accumulation of these elements slow down
plant growth and causes significant DNA damage [6]. Other studies focus on the negative
effects of accumulation of these metals on transpiration, photosynthesis, enzyme activity,
protein synthesis, and germination [7, 8].
As an alternative to conventional breeding, mutation breeding is often used to improve
current varieties and to generate new varieties. In many studies, application of physical and
chemical mutagens in combination using in vivo and in vitro techniques generates new
varieties faster than with conventional breeding.
Gamma radiation is also used to increase genetic variation in plants and to render them
more productive and resistant. Many plant varieties with commercial and agricultural
importance have been developed using gamma radiation [9–12]. Assessing the negative
effects of irradiation after exposure, as well as determining exposure conditions, radiation
dose–response curve, and mutation dosages, is of critical importance. Among other
parameters, the level of trace elements may change due to radiation.
Oneoftheeffectsofgammaradiationistoincreasetheproductionofreactiveoxygenspecies
(ROS). Plants have developed enzymaticantioxidant mechanisms like SOD(E.C. 1.15.1.1)and
peroxidase (POX, E.C. 1.11.1.7) to protect themselves against the harmful effects of ROS.
TherearethreetypesofSOD,dependingontheirmetalcofactorsandcellularlocalizations.Mn-
SOD exists in mitochondria, Fe-SOD is found in chloroplasts, and Cu/Zn-SOD occurs in
chloroplasts and cytosol. POXs, on the other hand, are enzymes that catalyze hydrogen
peroxide-dependent oxidation of a widerange ofsubstrates, mainly phenolderivatives [13, 14].
The present study aims to determine the GR50dosage for an irradiation mutagenesis study on
the soybean variety NE 3297 and to determine the changes in Fe, Cu, and Zn accumulation after
the irradiation while measuring the amounts of chlorophyll, soluble protein, and malondialde-
hyde (MDA), as well as the activities of SOD and POX and their effect on plant growth.
Materials and Methods
Plant Materials and Irradiation Technique
The soybean variety NE 3297 was obtained from the Black Sea Agriculture Research
Institute in Samsun, Turkey. The water content of the soybean seeds was 9.5%. Soybean
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seeds were irradiated with a Cs-137 gamma-ray source at the IBL 437C Irradiation Facility
of the Our Leukemia Children Foundation. The dosage rate per minute is 10 Gy with
dosages of 0 (as controls), 100, 200, 300, 400, and 500 Gy. In the same day, intact and
irradiated soybean seeds were sown into 30×38×8 cm plastic boxes. The boxes were then
filled with standard experimental soil. Soybean seeds were grown up in a climate-controlled
chamber, with 16/8 h light/dark day periods at a temperature of 26°C. All the experiments
were conducted in triplicate using 30 seeds for each experiment.
Growth Measurements
Following harvesting at day 14, the fresh weights of the plants were measured, and the
GR50values were determined. The GR50values are defined as the dosage that causes a
decrease in the normal seedling height by 50%; its value was determined for each plant by
measuring the seedling height.
Chlorophyll and Protein Content Determination
Fourteen-day-old plant leaves were extracted in 80% ice-cold acetone, and the absorbance
of the extracts was measured at 663 and 645 nm in a Shimadzu UV-1601 spectrophotom-
eter (Japan). Chlorophyll-a, chlorophyll-b, and total chlorophyll quantities were calculated
according to Arnon's method [15]. Soluble proteins were determined according to the
Bradford method [16].
MDA Content Determination
The MDA content in the soy germinates was determined according to Buege and Aust [17].
The MDA concentrations were calculated using an extinction coefficient of 156 mM−1cm−1
reported by Zhanyuan and Bramlage [18].
Trace Elements Determination
The 14-day-old plant samples were dried at 65°C for 48 h. Then, 1 g of sample was placed
in a 250-ml digestion tube to which 10 ml of concentrated HNO3was added. The sample
was heated for 45 min at 90°C and then at 150°C for at least 8 h until a clear solution was
obtained. The solution was filtered and diluted to 10 ml with deionized water. These
solutions were then analyzed for Fe, Cu, and Zn using an atomic absorption
spectrophotometer (Shimadzu AA 680, air-C2H2, background correction mode). All
measurements were done in triplicate. The blank solutions were prepared under the same
conditions to serve as controls for possible contaminations during the preparation
procedures [19].
Enzyme Assays
Extractions
All assays and extractions involving enzymes were carried out at 4°C. A 100-mg sample of
the 14-day-old plant leaves was homogenized in 100 mM (pH 7.0) sodium phosphate
buffer. The homogenate was centrifuged at 10,000g for 30 min at 4°C. The supernatant was
collected and used for determination of SOD and POX activities.
Effect of Gamma Radiation on Trace Elements in Soybean Plants285

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SOD Determination
The activity of SOD (E.C.1.15.1.1) was estimated by following the decrease in
absorbance of formazan produced by superoxide and nitroblue tetrazolium in the
presence of the enzyme. For the reaction, 2 ml of the mixture in 100 mM pH 7.8
phosphate buffer containing 13 mM methionine, 25 mM nitroblue tetrazolium
chloride, 0.1 mM ethylenediaminetetraacetic acid, 50 mM sodium carbonate, and
50 μl of the centrifugation supernatants was added with 60 μM riboflavin, and the
tubes were kept under two 15-W fluorescent lamps for 15 min. A complete reaction
mixture without the enzyme, which produced the maximum color, served as control.
Switching off the light and bringing the tubes into a dark room stopped the reaction.
A non-irradiated complete reaction mixture served as the blank. Separate controls
(without the enzymes) were used for total SOD and inhibitor studies. The absorbance
was recorded at 560 nm, and 1 U of enzyme activity was considered as the amount of
enzyme that reduced the absorbance by 50% in comparison with the tubes lacking the
enzyme [20].
POX
The activity of guaiacol POX (E.C. 1.11.1.7) was measured spectrophotometrically [21].
Supernatants were treated with 2 ml of a solution containing guaiacol, hydrogen peroxide,
and pH 7.0 phosphate buffer in concentrations of 20, 20, and 100 mM, respectively. The
enzyme produced a colorful product by using H2O2 and guaiacol as substrates. The
absorbance of the product was monitored at 470 nm, and POX activity was expressed as
ΔA470g−1(fresh weight) min−1.
SOD Visualization
Electrophoresis was carried out under non-denaturing conditions using 10% polyacrylamide
gels preceding SOD activity staining. Gel electrophoresis was conducted at 4°C for 4 h with
a constant current of 120 V. Electrophoresis buffer and gels were prepared as described by
Laemmli [22].
The activity of SOD was determined as described by Rao et al. incubating the gels for
20 min in a solution containing 2.5 mM NRT in the dark, followed by incubation in 50 mM
phosphate buffer (pH 7.8) containing 28 µM riboflavin and 28 mM tetramethylethylene
diamine also in the dark for 20 min. These gels were then exposed to two 15-W fluorescent
lamps for 15 min at room temperature [23].
The SOD isoenzyme patterns were determined by incubating the gels with a solution of
5 mM H2O2to inhibit both Cu/Zn-SOD and Fe-SOD, while another gel was incubated with
5 mM KCN to inhibit only Cu/Zn-SOD. Mn-SOD activity is resistant to both treatments
[23]. Fe-SOD from Escherichia coli (Sigma product No. S5389) was used as a standard in
the gels.
Statistical Analysis
Statistical evaluations for controls and plants under different irradiation dosages were
performed by analysis of variance. For multiple comparisons of statistically significant
differences in plant heights, the Dunnett's test was applied setting p<0.05 as the limit
[24].
286Alikamanoglu et al.

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Results
Effects of Gamma Irradiation on Growth Parameters
The average plant heights of 14-day-old NE 3297 soybean variety was measured in the
controls and irradiated experimental groups. The GR50dosage was 258 Gy (Fig. 1). The
average plant heights of the controls was 31.09 cm and that of plants exposed to 100, 200,
300, 400, and 500 Gy gamma irradiation were 28.29, 23.32, 9.95, 8.08, and 5.29 cm,
respectively (Table 1).
Effects of Irradiation on Chlorophyll Content
Depending on the irradiation dose, the contents of total chlorophyll, chlorophyll-a, and
chlorophyll-b decreased (Table 2). There was no statistically significant difference in the
content of chlorophyll between the control plants and those exposed to 100 Gy gamma
irradiation, but when the irradiation dosage was raised to 400 and 500 Gy, there was a
decrease of total and chlorophyll-a and chlorophyll-b. Total chlorophyll decrease was
81.36% at 400 Gy and 80.91% at 500 Gy.
Effects of Irradiation on Fe, Cu, and Zn Content
There was an increase of the concentrations of Fe, Cu, and Zn (Table 3). This increase
varied between 4.75% and 87.39% for Fe, 24.08% and 163.09% for Cu, and 9.86% and
45.14% for Zn with respect to the control plants.
Effects of Radiation on Soluble Protein and MDA Content
The soluble protein and MDA contents increased directly depending on radiation dose
(Table 4). Soluble protein increased 43.48% at 400 Gy and 69.57% at 500 Gy gamma-ray
exposures. For MDA, the increase at these radiation doses was 24.44% and 26.69%,
respectively.
0
20
40
60
80
100
Control100200300400 500
Seedling height (Control=100)
Dose (Gy)
Fig. 1 Effects of gamma radiation on seedling height of 14-day-old NE 3297 soybeans. The GR50dose is
258 Gy
Effect of Gamma Radiation on Trace Elements in Soybean Plants 287

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Effects of Radiation on Antioxidant Enzyme Activities
The SOD and POX enzyme activities increased 3.47–31.28% and 191.88–2,125%,
respectively, depending on the intensity of gamma irradiation at which the soybean seeds
were exposed (Table 5).
Effects of Radiation on SOD
Gamma radiation induced changes on isoenzyme patterns of SOD on the native
polyacrylamide gel electrophoresis (PAGE). After incubating the gels with H2O2 and
KCN, it was observed that the first protein band represented Mn-SOD, while the
isoenzymes marked as second to ninth corresponded to Cu/Zn-SOD (Fig. 2a). The Mn-
SOD was observed only at 400 and 500 Gy gamma radiation-treated experimental groups
(Fig. 2b, c). The activity of Cu/Zn-SOD 1 and Cu/Zn-SOD 2 isozyme was not detected in
the controls and in the 100 Gy gamma-irradiated group. Eight Cu/Zn-SOD isoenzymes
were detected in the 200–500-Gy exposed experimental groups. The intensity of Cu/Zn-
SOD isoenzyme bands 1, 3, 4, 6, and 8 directly increased with radiation dose, while the
intensity of the isoenzyme bands 2, 5, and 7 appeared to be less affected by gamma
radiation treatment (Fig. 2a).
Table 1 Average Plant Heights and Fresh Weights of 14-Day-Old NE 3297 Soybean Variety after Gamma
Irradiation at Different Doses
Gamma-ray dose (Gy) Average plant height (cm) Average fresh weight (g)
0
100
200
300
400
500
31.09±1.57a*
28.29±1.42a
23.32±1.07b
9.95±1.02c
8.08±1.09c
5.29±1.05d
1.597±0.23
1.474±0.31
1.377±0.21
1.165±0.20
1.096±0.21
0.909±0.19
Number of seeds per experiment=90. Different letters indicate significant differences relative to controls
*p<0.05 (Dunnett's test)
Table 2 Chlorophyll-a, Chlorophyll-b, and Total Chlorophyll Contents in 14-Day-Old NE 3297 Soybeans at
Different Gamma Radiation Dosages
Dose (Gy)Chlorophyll contents (mg g−1fw)
Chlorophyll-a Chlorophyll-bTotal chlorophyll
0
100
200
300
400
500
1.182±0.238
1.196±0.111
0.799±0.075
0.637±0.035
0.215±0.012
0.221±0.014
0.374±0.039
0.299±0.027
0.290±0.026
0.236±0.012
0.075±0.003
0.076±0.002
1.556±0.234
1.495±0.138
1.089±0.101
0.873±0.048
0.290±0.013
0.297±0.016
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Discussion
The physiological, biochemical, and molecular effects of gamma radiation were studied on
NE 3297 soybean variety, which has high economical and nutritional value. The effects of
radiation on the levels of Fe, Cu, and Zn of this plant have also been demonstrated.
Measuring of seedling growth is one of the best parameters to study plant response
against radiation [25, 26]. Seedling heights of NE 3297 variety grew in inverse proportion
to radiation dose. In mutation breeding studies, applied irradiation dosages depend on the
species [27], and suitable dosage limits have to be determined for each individual plant
variety, considering different parameters. In mutation breeding studies, GR50 dose is
recommended as optimum dose limit, so it is necessary to determine the dose that reduces
seedling heights by 50% compared to non-irradiated controls [25]. In the present case, the
GR50was determined to be 258 Gy.
Chlorophyll-a, chlorophyll-b, and total chlorophyll were negatively affected by
radiation, especially at 400 and 500 Gy. At these radiation doses, the chlorophylls' content
decreased about 80%.
The trace element content is another parameter that needs to be considered in mutation
breeding studies to determine suitable mutagen dose. Studies showed that trace element
uptake increases in plants under different stress conditions. It was reported that Cu, Fe, Mn,
and Zn concentrations increased in soybean plants when exposed to acid rain [28].
In this study, the Fe, Cu, and Zn concentrations increased experimental depending on the
irradiation dose. The most pronounced trace element concentration change was determined
for copper in the 500-Gy group, in which the Cu concentration increased about 2.5-fold
Table 3 Concentrations of Iron, Copper, and Zinc in 14-Day-Old NE 3297 Soybeans at Different Gamma
Radiation Dosages
Dose (Gy)Trace element contents (mg kg−1)
FeCu Zn
0
100
200
300
400
500
343.5±1.2
359.8±1.4
432.1±3.7
540.0±2.0
569.4±7.8
643.7±13
27.45±0.16
34.06±0.35
35.77±0.82
50.67±1.43
71.58±0.26
72.22±0.38
512.2±10.9
562.7±5.10
586.2±2.10
615.6±6.08
698.9±6.45
743.4±10.6
Table 4 Soluble Protein and Malondialdehyde Levels in 14-Day-Old NE 3297 Soybeans at Different
Gamma Radiation Dosages
Dose (Gy)Soluble protein content (mg g−1fw) MDA (μmol g−1fw)
0
100
200
300
400
500
0.23±0.053
0.25±0.049
0.29±0.042
0.32±0.057
0.33±0.038
0.39±0.067
3.56±0.43
3.69±0.67
3.97±0.46
3.93±0.53
4.43±0.63
4.51±0.58
Effect of Gamma Radiation on Trace Elements in Soybean Plants289

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compared to controls. According to the results, the highest acceptable ratios in changes of
trace element contents are 41% for Fe, 54% for Cu, and 12% for Zn.
Differentstudiesshowedthatproteincontentschangeinplantsunderstressconditions[29]. In
this study, the soluble protein contents of NE 3297 soybean increased after gamma radiation. A
significant increase of 70% was observed in the group that was exposed to 500 Gy.

Marker Control 100Gy 200Gy 300Gy 400Gy 500Gy
Mn-SOD
Marker Control 100Gy 200Gy 300Gy 400Gy 500Gy
Mn-SOD
Marker Control 100 Gy 200 Gy 300 Gy 400 Gy 500 Gy
Mn-SOD
1
2
3
4
5
6
7
8
Cu/Zn-SOD
a
b
c
Fig. 2 Native polyacrylamide gel electrophoresis analysis of superoxide dismutase isoenzymes by activity
staining. a Gels with no treatment. b Treated with 5 mM KCN. c Treated with 5 mM H2O2
Table 5 Activities of Superoxide Dismutase and Peroxidase in 14-Day-Old NE 3297 Soybeans at Different
Gamma Radiation Dosages
Dose (Gy) SOD (U mg−1protein) POX (ΔA470g−1(fw) min−1)
0
100
200
300
400
500
65.64±12.83
67.92±14.33
75.37±18.08
78.79±21.43
82.69±20.08
86.17±22.83
1.60±0.053
4.67±0.83
5.44±0.73
18.9±2.63
32.8±5.07
35.6±5.03
290Alikamanoglu et al.

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Stress factors such as gamma radiation and heavy metal accumulation increase the level
of ROS such as superoxide radicals (O2·−), hydroxyl radicals (·OH), and hydrogen peroxide
(H2O2), as well as causing cellular damage. Transition elements such as Fe and Cu reduce
hydrogen peroxide, and hydroxyl radical is produced by the Fenton and Haber Weiss
reactions. Extremely reactive hydroxyl radicals (·OH) may cause base modifications, base
deletions, and strand breaks on DNA, as well as causing photolytic degradation by
oxidation, and damaging membrane structures by peroxidation. Damaging of the membrane
causes increased permeability and, eventually, cell death [26, 30, 31].
Aerobic organisms have developed various antioxidant mechanisms to prevent damage by
ROS. Enzymes such as SOD and POX that play role in plant antioxidant defense systems are
the most important electron scavengers. As a member of antioxidant defense system, SOD is a
metalloenzymethat catalyzesdismutation ofsuperoxide radicals thataccumulatein responseto
environmental stress factors such as gamma radiation, heavy metals, and hydrogen peroxide.
Screening of SOD isoenzymes is possible using cyanide and hydrogen peroxide, sensitive to
SOD. Cu/Zn-SOD is sensitive to both cyanide and hydrogen peroxide, while Fe-SOD is only
sensitive to hydrogen peroxide. Mn-SOD is not affected by any of them [5].
The activities and band numbers of SOD isoenzymes may vary depending on plant species
and on the applied stress factor [26, 32, 33]. In our study, the SOD activities increased by
3.47% and 31.28% depending on the dose of applied gamma radiation. In native PAGE
stained for SOD activity, we observed six isoenzyme bands in the non-irradiated and 100-Gy
groups. In the 200- and 300-Gy sets, there were eight bands, while there were nine bands in
the 400- and 500-Gy cases (Fig. 2a). The bands observed at the bottom of the gel belonged to
the Cu/Zn-SOD isoenzyme. The single band at the top portion of the lane containing the
samples from the plants that received 400 and 500 Gy gamma radiation belonged to an Mn-
SOD isoenzyme (Fig. 2b, c). We did not observe a band for the Fe-SOD isoenzyme.
TheactivityofPOXincreasedinadose-dependentfashion,from2.92-foldatthelowestdose
up to 22.3-fold at the highest irradiation level. This result is in good agreement with previous
studies reporting increased POX activity in plants under oxidative stress [30, 31, 34–36].
Malondialdehyde is a cytotoxic product of lipid peroxidation, which increases as a result
of oxidative stress. We found that the MDA content increased from 3.65% to 26.69%, again
depending on gamma radiation dose applied. This is also in good agreement with
previously published results [31, 34, 37, 38].
In sum, the results here presented show that the effects of gamma radiation of NE 3297
soybeans depend on the irradiation dose. In parallel to dose increase, plant height, average
plant fresh weight, and chlorophyll contents decreased, while soluble protein contents,
MDA contents, SOD, and POX enzyme activities, as well as trace element concentrations,
increased as a result of oxidative stress caused by radiation. These results may be used in
estimation of optimal radiation doses and in planning of mutation studies.
Acknowledgement This work was supported by the Research Fund of Istanbul University, project No.
BYP-535/24112004.
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