Antioxidant isozymes activities in potato plants (Solanum tuberosum L.) under salt stress

Abstract

To determine NaCl effects on growth and some antioxidant enzymes activity, the internode cuttings of four potato cultivars (Solanum tuberosum L.), Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive) were grown on callus inducing media amended with 0, 50, 100 and 150 mM NaCl at dark conditions. Callus growth of all cultivars was significantly reduced under salt stress. Total superoxide dismutase (SOD) activity decreased in all cultivars when calli were grown in the presence of NaCl. Peroxidase (POD), H 2 O 2 detoxifying enzyme activity increased under salt stress, but at higher NaCl levels the activity was reduced. On the other hand, SOD and POD isozyme profiles at 100 mM NaCl were different from that of the control. These differences were quantitative and were expressed more in terms of increased or decreased isozymes activities. In dark conditions, callus tissue underwent oxidative stress in the presence of NaCl. In these conditions, more than any other SOD isozymes, Mn-SOD seems to play a major role in the scavenging of superoxide radicals during NaCl stress. Therefore, the increased Mn-SOD activity in salt treated-calli could reflect sustained O 2 − production in the mitochondria. On the other hand, in spite of increased activities of Mn-SOD and POD, reactive oxygen species (ROS) had damaging effects on the plant cells due to the SOD reduced total activity. Generally, there was no difference between relatively salt sensitive and tolerant cultivars in response to NaCl stress.

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Journal of Sciences, Islamic Republic of Iran 17(3): 225-230 (2006)
University of Tehran, ISSN 1016-1104
http://jsciences.ut.ac.ir
225
Antioxidant Isozymes Activities in Potato Plants
(Solanum tuberosum L.) Under Salt Stress

H. Rahnama1,* and H. Ebrahimzadeh2

1 Agricultural Biotechnology Research Institute of Iran, Seed and Plant Improvement Campus,
Mahdasht Road, P.O. Box 31535-1897, Karaj, Islamic Republic of Iran
2 Department of Biology, Faculty of Sciences, University of Tehran, Tehran, Islamic Republic of Iran

Abstract
To determine NaCl effects on growth and some antioxidant enzymes activity,
the internode cuttings of four potato cultivars (Solanum tuberosum L.), Agria,
Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive)
were grown on callus inducing media amended with 0, 50, 100 and 150 mM NaCl
at dark conditions. Callus growth of all cultivars was significantly reduced under
salt stress. Total superoxide dismutase (SOD) activity decreased in all cultivars
when calli were grown in the presence of NaCl. Peroxidase (POD), H2O2
detoxifying enzyme activity increased under salt stress, but at higher NaCl levels
the activity was reduced. On the other hand, SOD and POD isozyme profiles at
100 mM NaCl were different from that of the control. These differences were
quantitative and were expressed more in terms of increased or decreased isozymes
activities. In dark conditions, callus tissue underwent oxidative stress in the
presence of NaCl. In these conditions, more than any other SOD isozymes, Mn-
SOD seems to play a major role in the scavenging of superoxide radicals during
NaCl stress. Therefore, the increased Mn-SOD activity in salt treated-calli could
reflect sustained O2− production in the mitochondria. On the other hand, in spite of
increased activities of Mn-SOD and POD, reactive oxygen species (ROS) had
damaging effects on the plant cells due to the SOD reduced total activity.
Generally, there was no difference between relatively salt sensitive and tolerant
cultivars in response to NaCl stress.

Keywords: Antioxidant enzymes; Salt stress; Solanum tuberosum


Introduction

*E-mail: hrahnama@abrii.ac.ir
Plants are exposed to a variety of abiotic stresses
such as drought and salinity that influence their growth,
development and productivity. Plants exposed to salt
stress, undergo changes in their metabolism in order to
cope with the changes taking place in their environment
[1]. One of the biochemical changes occurring when
plants are subjected to biotic or abiotic stresses is the
production of reactive oxygen species (ROS). The main
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bacteria, and yeast (Saccharomyces cerevisiae), such as
microbial cytochrome c peroxidase (EC 1.11.1.5),
bacterial catalase-peroxidase (EC 1.11.1.6), and
ascorbate peroxidase (EC 1.11.1.11). Class II plant
peroxidases are extracellular peroxidases from fungi,
including lignin peroxidase (EC1.11.1.14) and Mn+2-
independent peroxidase (EC 1.11.1.13). Class III plant
peroxidases (EC 1.11.1.7) were originally described as
peroxidases and are secreted outside of the cells or
transported into vacuoles.
The objective of the present study was to determine
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J. Sci. I. R. Iran
226
sites of ROS production in the plant cell during abiotic
stress are the organelles with highly oxidizing metabolic
activities or with sustained electron flow: chloroplasts,
mitochondria and microbodies [2]. In plant cells,
specially non-photosynthesizing cells, mitochondria is
the major source of ROS [3]. The relative importance of
mitochondria and chloroplasts in ROS production in
light is not known.
ROS are highly reactive and when the capacity of
plant for scavenging is less than ROS production they
can seriously disrupt normal metabolism through
oxidative damages of lipids, proteins and nucleic acids
[4-7]. Plants posses a number of antioxidant systems
that protect them from these potential cytotoxic effects.
Antioxidant enzymes are
components in the scavenging system of ROS [7].
Superoxide dismutase (SOD) is a major scavenger of
O2
H2O2. Catalase (CAT), ascorbate peroxidase (APX) [8]
and a variety of general peroxidases (POD) [9] catalyze
the breakdown of H2O2. Therefore, these enzymatic
systems eliminate the damaging effects of toxic oxygen
species.
Both SOD and POD exist in multiple isoforms in
plant tissues. The SOD isozymes are generally classified
according to their active site metal [10]: copper/zinc
(Cu/Zn-SOD), iron (Fe-SOD) or manganese (Mn-SOD).
Most plants have Fe-SOD and Cu/Zn-SOD in their
chloroplasts. Chloroplastic Fe-SOD is generally the
most abundant SOD in green leaves, while in
germinating seedlings and in etiolated materials the
cytoplasmic Cu/Zn-SOD and mitochonderial Mn-SOD
are prevalent. The study of SOD isozyme activities can
help in understanding salt effects on subcellular
compartments [11].
Peroxidases (EC 1.11.1.7) are widely found in
animals, plants and microbes and oxidize a vast array of
compounds (electron donors) in the presence of
hydrogen peroxide (H2O2). The plant peroxidase
superfamily is divided into three classes based on
differences in primary structure [12]. Class I plant
peroxidases include intracellular enzymes in plants,
the most important
− and its enzymatic action results in the formation of
the role of SOD and POD isozymes in salt tolerance of
potato plants in cellular level.
Materials and Methods
Plant Materials and Treatments
Four potato (Solanum tuberosum L.) cultivars, Agria,
Kennebec (relatively salt tolerant) Diamant and Ajax
(relatively salt sensitive) were used in this experiment.
The plants were maintained by subculture of nodal
cuttings on sterile medium consisting of MS [13] salts
and vitamins, 2 mg/l Ca-pantatonate, 2 mg/l GA3, 0.01
mg/l NAA, 1 mg/l STS, 30 g/l sucrose and 7 g/l agar.
The internode segments of seedlings were used for the
generation of callus in callus inducing medium. This
medium consisted of MS salts and myo-inositol and
(mgl−1): thiamine-HCl (10), nicotinic acid (0.5),
pyridoxine-HCl (0.5), glycine (2), kinetine (0.5), 2, 4-D
(5), sucrose 3% and agar 0.75%. For salinity treatment,
NaCl was added to this medium at four levels (0, 50,
100, 150 mM). Internode segments were cultured in
each petridish with 5 replicates for any treatment.
Cultures were grown in dark at 25°C. After one month
the calli were subcultured in fresh media. In the end of
second month the calli were harvested and their fresh
weights were determined and were stored at −20°C for
subsequent analysis.
Protein Extraction
One gram of frozen callus was homogenized in 1 ml
of an ice cold solution containing 100 mM phosphate
buffer (pH 7.4), 1 mM EDTA, 0.5% (v/v) Triton X-100,
1 mM ascorbic acid (for ascorbate peroxidase only)
[14]. The homogenate was then centrifuged for 30 min
at 18,000 g. The eluent was stored at −20°C for
subsequent analysis of SOD, POD. Protein content was
determined using Bradford method [15].
Enzyme Determination
POD activity was measured by the H2O2-dependent
oxidation of benzidine at 530 nm, in a reaction mixture
containing 2 ml of 0.2 M acetate buffer (pH 4.8), 0.2 ml
of 3% H2O2, 0.2 ml of 0.04 M benzidine and 0.1 ml of
the extracted protein [16]. SOD activity was measured
by monitoring the inhibition of nitroblue tetrazolioum
(NBT) reduction at 560 nm [17]. The reaction mixture
contained 50 mM phosphate buffer (pH 7), 0.1 mM Na-
EDTA, 75 μM riboflavin, 13 mM methionine and 10-25
μl enzyme extract. Reaction was carried out in test tubes
at 25°C under the illumination of a fluorescent lamp
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treatments at P≤0.05 derived from an analysis of
variance.
J. Sci. I. R. Iran Rahnama and Ebrahimzadeh
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227
(40-W). The reaction was allowed to run for 8 min and
stopped by switching the light off. Blanks and controls
were run in the same manner but without illumination
and enzyme, respectively. Under the experimental
condition, the initial rate of reaction, as measured by the
difference in increase of absorbance at 560 nm in the
presence and absence of extract, was proportional to the
amount of enzyme. The unit of SOD activity was
defined as the amount of enzyme that inhibits the NBT
photoreduction by 50%. SOD activity values are given
in units per mg of protein [10]. For enzymes POD, the
activity was defined as the changes in absorbance per
minute for 1 mg protein (OD/min/mg protein).
Electrophoresis and Enzyme Activity Visualization
Non-denaturating polyacrilamid gel electrophoresis
(PAGE) was carried out with 10% resolving gel at 4°C
and 65 mA [18]. POD isozymes were visualized by
incubating the gels in a solution consisting 80 ml of 0.2
M acetate buffer (pH 5), 8 ml of 3% H2O2, 4 ml of 0.04
M benzidine. POD isozymes appeared with brown
bands after 30-60 min at 4°C [19]. SOD isozymes were
visualized by incubating the gels for 30-45 min in the
dark in a solution consisting of 100 ml of 0.2 M Tris-
HCl (pH 8), 4 mg riboflavin, 4 mg Na-EDTA, 20 mg
NBT and illuminating of the gels until the bands
became apparent [20]. The three types of SOD, Mn-
SOD, Fe-SOD and Cu/Zn-SOD were identified using
specific inhibitors. Before staining, gels were incubated
at 25°C for 30 min, separately, in solution of 5 mM
H2O2, 2 mM KCN or both inhibitors. The sensitivity of
Cu/Zn-SOD to cyanide (KCN) has been used as a
diagnostic tool to distinguish Cu/Zn-SOD from Fe-SOD
and Mn-SOD that are unaffected by cyanide. Likewise,
Fe-SOD is irreversibly inactivated by H2O2, whereas
Mn-SOD is resistant to both inhibitors [10].
Statistic
Values in the text indicates mean values ± S.E, and
the least significant difference (LSD) between
Results and Discussion
Growth
The effects of increasing concentrations of NaCl on
the growth of potato calli are shown in Figure 1.
Although, Agria and Kennebec were classified as
relatively salt tolerant, their calli growth reduction was
similar to that of NaCl sensitive cultivars under salt
stress. Therefore NaCl tolerance mechanisms were not
sufficiently active in the calli compared to that in whole
plants. In whole plants, NaCl absorption mediated by
specialized root cells. Non-differentiated callus tissue
doesn’t have endoderm layer, which controls ion
absorption, and other controlling mechanisms that are
present in the whole plant. Then, there isn’t a difference
between salt sensitive and tolerant cultivars growth in
the presence of NaCl stress.
Enzyme Activity
As explained earlier, H2O2, OH and O2
produce under salt stress are potentially harmful to
plants. Fortunately, plants have the capacity to cope
with these reactive oxygen species by eliminating them
with an efficient scavenging system [2]. Superoxide
dismutase (SOD) is a major scavenger of superoxide
(O2
H2O2 and O2. In the present study, SOD activity reduced
in all cultivars under salt stress (Fig. 2A). However, the
basic levels of SOD activity in Agria and Kennebec
were higher than that in the sensitive cultivars. On the


− radicals that
−) and its enzymatic action results in the formation of

Figure 1. Effect of NaCl treatment on callus fresh weights of
potato cultivars. Values are means±S.E of five replicates.
(AG) Agria; (DIA) Diamant; (K) Kennebec; (AJ) Ajax.


Figure 2. The effects of NaCl on antioxidant enzyme
activities in potato calli. (A) SOD activity; (B) POD activity.
Values are means±S.E of five replicates. (AG) Agria; (DIA)
Diamant; (K) Kennebec; (AJ) Ajax.
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Therefore, the analysis of the activity of individual POD
and SOD isozymes is important, because it can help to
understand how each stress may affect the different
subcellular compartments [11].
The electrophoretic profiles of POD and SOD
isozymes showed that the isozyme activities were
affected by NaCl treatment. The effects of NaCl were
expressed more on activity of constitutive enzyme
pools. In the present study we observed a differential
POD and SOD isozyme activities among the cultivars
(Fig. 3). For example, POD isozymes with relative
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228
other hand, at 50 mM NaCl, the reduced SOD activity in
Agria and Kennebec was less than that in the sensitive
cultivars. The present results are in contrast with the
results of Benavides et al. [21]. They reported that SOD
activity increased significantly in the salt sensitive colon
of potato and remained unchanged in tolerant ones when
exposed to NaCl stress. Other studies reported increased
SOD activity in salt tolerant cultivars and reduced
activity in sensitive ones [21-24].
Decomposition of O2
enzymatic, is always accompanied by production of
H2O2, therefore its removal by cells is essential in
mitigating the oxidative stress. In plant this is achieved
by a well organized functioning of catalase, ascorbate
peroxidase and peroxidases. There are variable
responses of H2O2 scavenging enzyme activities to NaCl
stress. In cotton, it has been reported that, under salt
stress, H2O2 scavenging enzyme activities are increased
in salt tolerant cultivars and reduced or remained
unchanged in non-tolerant ones [7,23,24]. These reports
explain that, the increase in the enzyme activity could
be associated with its salt tolerance character [24]. Rout
and Shaw [25] suggested the active involvement of at
least catalase and peroxidases among the H2O2
scavenging enzymes in salt tolerant plants. They
concluded that APX probably did not have any role in
salt tolerance. Benavides et al [20] reported that APX
was probably more important than CAT in the H2O2
detoxification in potato which had been exposed to
NaCl. In the present study POD activity was increased
in all cultivars under salt stress. At 50 mM NaCl
treatment, POD activity in Agria, Diamant, Kennebec
and Ajax increased 167%, 50%, 55% and 100%
respectively when compared with the 0 mM NaCl levels
treated calli (Fig. 2B). At higher NaCl levels, POD
activity was partially reduced.
−, whether enzymatic or non-
Isozyme Activity
It is known that enzymes that have multiple
intracellular distributions are present as different
isozyme forms in different cell compartments [26].
mobility (Rm): 0.38, 0.42, 0.45, 0.51 in Ajax, Rm: 0.4,
0.45, 0.51 in Kennebec and Rm: 0.38, 0.45, 0.51 in
Diamant and Rm: 0.45, 0.51 in Agria were specificity of
these cultivars, so that we could identify these cultivars
by POD isozymes pattern. In general, POD isozymes
had increased activity under salt stress (Fig. 3A).
There were 7-11 SOD isozymes in different cultivars
(Fig. 3B). Some of them were cultivar specific and
some had higher activity in a cultivar. For example,
isozymes with Rm: 0.606, 0.313 in Kennebec and Ajax,
Rm: 0.346 in Kennebec and Rm: 0.36 in Ajax were
unique. On the other hand, a very high activity in SOD
isozymes with Rm: 0.413 in Diamant or Rm: 0.533 in
Agria were specific to these cultivars. Therefore, we
recommend that these enzymes could be used for the
identifying of potato cultivars.
Contradictory results have been obtained in relation
to the effect of salt and drought on the activity and
protein levels of the various isozymes of SOD. Drought
increased the activity and amount of cytosolic SOD in
peas [27]. It has been reported that in NaCl tolerant pea
cultivars, leaf mitochondrial Mn-SOD and chloroplastic
Cu/Zn-SOD activities increased under salt stress, while
the cytosolic Cu/Zn-SOD activity remained unchanged
[22]. In the salt sensitive cultivar, chloroplastic SOD
activity was not increased by salt, while the cytosolic
and mithocondrial SOD activity was even decreased. In
leaves of Vigna unguiculata, salt decreased Mn-SOD
activity [22]. It is questionable whether one can
integrate these contradictory data into a model and draw
general conclusions. The experiments exploit different
plant systems, with cultivars varying with respect to salt
tolerance, and the effects were measured in different
tissues and at various ages. All of these parameters
should be taken into account when the level and activity
of the enzymes are determined.
In the present study, SOD isoenzyme profile at 100
mM NaCl in calli showed less difference compared to
that of the control. In most cases, there was a reduced
activity in SOD isozymes in response to elevated levels
of NaCl concentration. For example, this reduction was
seen in isozymes with Rm: 0.17, 0.27, 0.29, 0.6 in
Kennebec, Rm: 0.17, 0.56 in Ajax and Rm: 0.56, 0.6 in
Agria.
There was no chloroplastic Fe-SOD and Cu/Zn-SOD,
because the calli were grown in the dark conditions
(Fig. 3C). Therefore, we concluded that the Fe-SOD
activity in potato callus is related to peroxisomes. In the
presence of H2O2 + KCN (Fig. 3D), it appeared that
there were two SOD isoforms (Rm: 0.413, 0.45) in
Diamant, two isoforms (Rm: 0.45, 0.48) in Agria, two
isoforms (Rm: 0.017, 0.45) in Kennebec and Ajax, all of
them belonging to Mn-SOD. In most cases, Mn-SOD
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Conclusion
J. Sci. I. R. Iran Rahnama and Ebrahimzadeh
Vol. 17 No. 3 Summer 2006
229
isozymes had higher activity than the others. SOD
isozymes with Rm: 0.27, 0.29 were related to Fe-SOD.
Other SOD isozyme activities with Rm: 0.56, 0.6, 0.65
which were Cu/Zn-SOD, generally reduced under salt
stress. On the other hand, generally Mn-SOD activity
increased in salt treated calli and this could reflect
sustained O2
Therefore, it is possible that an increase in enzyme
activities under salt stress is indicative of increased
production of ROS and a build up of a protective
mechanism to reduce oxidative damage triggered by
stress experienced by plants. However, because of
reduction in total SOD activity this was not observed in
potato calli.
The results obtained with callus tissue were not in
total agreement with those found in potato seedlings. In
opposite to callus tissue, in Agria and Kennebec
seedlings, SOD activity was increased under salt stress,
where the fresh weights were unchanged, but in
relatively NaCl sensitive cultivars the activity showed
reducing patterns [28]. H2O2 detoxifying enzymes in
callus tissues showed similar changes as potato
seedlings. Based on the present results, we concluded
that in potato calli, POD probably had an important role
in H2O2 detoxification. Therefore, we would suggest
that the importance of the enzymes in ROS
detoxification depends on the experimental tissue and
the cultivar. Although, Mn-SOD activity increased
under salt stress, total SOD showed reduced activity in
the present study. Therefore, in callus tissue, probably
because of inefficient activity of SOD, superoxide
radicals had more damaging effects on the growth. It is
therefore concluded that elevated H2O2 detoxifying
enzyme activities without an accompanying increase in
the ability to scavenge superoxide radicals, result in
damaging effects in plants. The different responses of
potato seedlings and calli to NaCl were not totally
unexpected. Because gene regulation in undifferentiated
callus tissue grown on phytohormone amended media is
likely to be different from gene regulation in tissue that
has undergone normal ontogeny.
− production in the mitochondria.
In conclusion, more than any other SOD isozymes,
Mn-SOD seems to play a major role in the scavenging
of superoxide radicals during NaCl stress in potato calli.
On the other hand, because of reduced total SOD
activity, ROS had damaging effects on the plant cells
and there was no difference between relatively salt
sensitive and tolerant cultivars in response to NaCl
stress.

Figure 3. Effects of 100 mM NaCl on POD (A) and SOD (B,
C, D) isozyme activities in potato calli. Inhibitor tests for SOD
isozymes in the presence of KCN (C), H2O2 + KCN (D) and in
absence of any treatment (Control; B). (AG) Agria; (DIA)
Diamant; (K) Kennebec; (AJ) Ajax; (0) 0 mM NaCl; (100)
100 mM NaCl.

Acknowledgement
We would like to acknowledge the University of
Tehran for the financial supports.
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