Effect of cytokinins on the activity of superoxide dismutase in nitrogen deficient wheat

Abstract

Reactive oxygen species (ROS), such as O2·¯, are formed by electron transfer to a molecule with stable electron configuration, in electron transport chains in the cell. ROS are very reactive molecules which are formed at higher rates under stress, such as drought, high insolation, heath, inadequate mineral nutrition, and such conditions lead to impairment of various physiological and biochemical processes in the cell. To reduce production of ROS, and their detrimental effect, plants developed various enzymatic and non-enzymatic protective mechanisms. Superoxide dismutase (SOD) is one of the most important antioxydative enzymes, which removes superoxid anion radical (O2·¯), whose rate of production is the highest under unfavorable environmental conditions. Plant tissues that exhibit delayed senescence often have higher cytokinin content, which is accompanied by reduced amount of ROS. The focus of this paper is to examine whether foliar application of cytokinins to young wheat plants insufficiently supplied with nitrogen affects the activity of SOD and amount of O2·¯. Application of trans-zeatine (CK) reduced the activity of SOD, but this reduction was not accompanied by an increase in the amount of O2·¯. Application of benzyl adenine (BA) also reduced the activity of SOD, with concomitant increase in the amount of O2·¯ in wheat leaves.

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Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci, Matica Srpska Novi Sad,
¥ 114, 59—68, 2008
UDC 633.11:546.17
G o r a n Z. S t o p a r i ã1
I v a n a V. M a k s i m o v i ã2
1Municipality of Subotica, Municipal Council for the Environment and Health,
Trg Slobode 1, 24000 Subotica, Serbia
2Faculty of Agriculture, Trg D. Obradoviãa 8,
Novi Sad and Institute of Field and Vegetable Crops,
M. Gorkog 30, Novi Sad, corresponding author (E-mail: ivanam@ib.ns.ac.yu)
EFFECT OF CYTOKININS ON THE ACTIVITY
OF SUPEROXIDE DISMUTASE IN NITROGEN
DEFICIENT WHEAT
ABSTRACT: Reactive oxygen species (ROS), such as O2·¯, are formed by electron
transfer to a molecule with stable electron configuration, in electron transport chains in the
cell. ROS are very reactive molecules which are formed at higher rates under stress, such as
drought, high insolation, heath, inadequate mineral nutrition, and such conditions lead to
impairment of various physiological and biochemical processes in the cell. To reduce pro-
duction of ROS, and their detrimental effect, plants developed various enzymatic and
non-enzymatic protective mechanisms. Superoxide dismutase (SOD) is one of the most im-
portant antioxydative enzymes, which removes superoxid anion radical (O2·¯), whose rate of
production is the highest under unfavorable environmental conditions. Plant tissues that
exhibit delayed senescence often have higher cytokinin content, which is accompanied by
reduced amount of ROS. The focus of this paper is to examine whether foliar application of
cytokinins to young wheat plants insufficiently supplied with nitrogen affects the activity of
SOD and amount of O2·¯. Application of trans-zeatine (CK) reduced the activity of SOD,
but this reduction was not accompanied by an increase in the amount of O2·¯. Application
of benzyl adenine (BA) also reduced the activity of SOD, with concomitant increase in the
amount of O2·¯ in wheat leaves.
KEY WORDS: wheat, nitrogen supply, cytokinin, trans-zeatine, benzyl adenine, su-
peroksid-dismutase, superoxid anion radical
INTRODUCTION
Reactive oxygen species (ROS) are extremely reactive molecules because
they have unpaired electron in their structure. ROS formation takes place in all
parts of the cell, in which electron transport takes place (E l s t n e r, 1991).
Superoxide anion radical (O2·¯), one of the ROS, is formed by electron transfer
to molecular oxygen (O2) (M e h l e r, 1951): • (e) + O2? O2·¯. This reaction
takes place during the electron transport processes in a number of cell com-
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partments. The highest amount of O2·¯ in plant cells are formed in chloro-
plasts, mitochondria and peroxisomes (F r i d o v i c h, 1986).
Superoxide anion radical (O2·¯) is very reactive molecule which acts as
progenitor of destructive chain reactions that finally result in the damaging of
the molecules close to the site of O2·¯ production (B e r l e t t and S t a d t m a n,
1997). Therefore, keeping the low cellular level of O2·¯ is of crucial impor-
tance for cell metabolism. To do that, plant cells developed a number of effi-
cient biochemical mechanisms during the evolution. The most efficient mecha-
nism for removal of O2·¯ from the cell is “ascorbate-glutathione" cycle, which
functions in chloroplasts, mitochondria and peroxisomes, where the highest
amount of O2·¯ is formed (A s a d a, 1999).
The first reaction in the “ascorbate-glutathione" cycle is transformation of
two molecules of superoxide anion radical (O2·¯), with two protons (H+), into
one molecule of hydrogen peroxide (H2O2) and one molecule of oxygen (O2).
The enzyme that catalyses this reaction is superoxide dismutase (SOD):
O2·¯ + O2·¯ + 2H+
SOD
? ???
H2O2+ O2
The importance of SOD in the removal of O2·¯ is thoroughly proven
( P e r l et al., 1993; Y u et al., 1999). Plants with increased synthesis of SOD
in their chloroplasts had higher tolerance to stress-inducing factors such as
drought, low or high temperatures, imbalanced mineral nutrition, etc. Under
stress, in plants with higher SOD activity the growth was less reduced than in
plants in which SOD activity did not increase upon the induction of stress
( Š t a j n e r et al., 2004). Therefore, the activity of SOD may be used as a pa-
rameter to evaluate the level of plant tolerance to particular stress-inducing
factor.
Cytokinins can also participate in removal of ROS from the cell. After
L e s h e m et al. (1979) the molecular structure of cytokinins allows them to
react directly with superoxide anion, but also with the other ROS, thus remo-
ving them from the cell metabolism.
As the possible direct antioxidant activity effect of cytokinins can also
be considered their effect on the activity of antioxidant enzymes, including
SOD. While studying the effects of cytokinins on senescence, several authors
(G r o s s m a n and L e s h e m, 1978; L i u et al., 1996) found that cytoki-
nins, such as zeatin and benzyl adenine, can increase the activity of some anti-
oxidative enzymes (SOD, catalase), and therefore delay senescence of the plant
tissue. Moreover, L e s h e m et al. (1981) showed that cytokinins inhibit the
activity of xanthine oxidaze, an enzyme that is one of the generators of ROS
in the cell. The senescence was delayed also by reduction of lipoxygenase
activity induced by cytokinins, which contributed to preserve the integrity of
cell membranes.
Although it is known that cytokinins have the potential to reduce oxi-
dative stress in plants, this was not tested under nitrogen starvation in wheat.
Therefore, the aim of this study was to analyze the effect of foliar application
of cytokinins (zeatine and benzyl adenine) on the activity of SOD and amount
of O2·¯ in the leaves of young nitrogen-deficient wheat plants.
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MATERIALS AND METHODS
The effect of cytokinins on the activity of SOD and amount of O2·¯ was
analyzed in bread wheat (Triticum vulgare), cultivar Renesansa, differently
supplied with nitrogen. Young wheat plants were grown in water cultures and
foliary treated with solutions of trans-zeatine (CK) and benzyl adenine (BA),
under semi-controlled conditions.
Plant growth
Germination was done in an incubator, in the dark, at 26°C. Seedlings
were divided into three groups at planting. One was supplied with the com-
plete ½ strength Hoagland nutrient solution (H o a g l a n d and A r n o n, 1950)
(control, N1, nitrogen supplied as [2.5 mM KNO3and 2.5 mM Ca(NO3)2]),
another onto the same solution but with the N concentration reduced to ½
(N1/2, nitrogen supplied as [1.25 mM KNO3and 1.25 mM Ca(NO3)2]), and for
the third group N concentration was reduced to ¼ of the full dose (N1/4,
[0.625 mM KNO3and 0.625 mM Ca(NO3)2]). Nutrient solutions were aerated
each day and replaced every 3—4 days. Fifteen days after planting, each group
was divided into 3 sub-groups of 6 pots and 8 plants per pot each, and treated
with solutions of trans-zeatine (CK) and benzyl adenine (BA).
Treatment with cytokinins
Plants were treated with water solution of trans-zeatine (CK) of the
following concentrations: 0 mg/dm3(deionized water-control, CK 0), 2.5 mg/
dm3(CK 2.5), 5,0 mg/dm3(CK 5) and 10 mg/dm3(CK 10). Benzyl adenine
(BA) was applied at following concentrations: 0 mg/dm3(deionized water-con-
trol, CK 0), 10 mg/dm3(BA 10), 40 mg/dm3(BA 40). Foliar treatments with
both CK and BA were done twice during the experiment by spraying 0.1 dm3
of each solution of 144 plants. The first treatment was done 7 days after plan-
ting and the second 14 days after planting. Plants were analyzed 7 days after
the second treatment.
Extraction of O2·¯ and SOD
O2·¯ and SOD were extracted from 1 g of fresh leaf tissue, in phosphate
buffer pH 7.0, as described by Q u y H a i et al. (1975).
Determination of the amount of O2
·¯and the activity of SOD
Superoxide radical (O2·¯). The amount of O2·¯ was determined by the
method of M i s r a and F r i d o v i c h (1972), based on the reaction of inhi-
bition of the auto oxidation of adrenaline.
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Superoxide dismutase (SOD). The total activity of SOD was determined
on the basis of the inhibition of transformation of adrenaline into adreno-
chrome in the presence of the air (M i s r a and F r i d o v i c h, 1972).
Statistical analysis
The results were statistically processed by the analysis of variance, and
calculation of the least statistically significant differences (LSD) between the
control and cytokinin treatment, for each level of nitrogen nutrition and for
probability of 5%, using the computer program STATISTICA 7.
In the figures, in the Results section, numerical values above the bars re-
present relative (in %) increase or decrease of the measured value of each
treatment with respect to the control (control = 0). The control plants are those
grown on complete ½ strength Hoagland solution and untreated with cyto-
kinins (N1, CK 0, BA 0).
Following the ranking of mean values by the Duncan test, the significan-
ce of differences between the means is marked by letters inside the figure bars.
Two means differ significantly (for p < 0.05) if they do not share any letters.
RESULTS
The effect of cytokinins on the activity of SOD
The activity of SOD ranged from 33.57 U/g FW (in treatments N1/2 and
N 1/4, CK 10) to 205.52 U/g FW (in the control). Such a high span between
measured values suggests that both nitrogen nutrition and treatment with cyto-
kinins affected the activity of SOD (Fig. 1).
An increase in N concentration in the nutrient solution increased the acti-
vity of SOD, whereas increase in CK concentration significantly reduced acti-
vity of SOD in young wheat leaves. Analysis of variance permits to conclude
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Fig. 1 — Activity of superoxide dismutase (SOD) in leaves of young wheat plants grown
in the presence of different N concentrations and treated with different concentrations of CK

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that there is an interaction between applied N and CK concentrations on the
activity of SOD. The effect of N concentration on the activity of SOD signifi-
cantly varied with the increase in the concentration of CK.
In the experiment with BA, the highest activity of SOD was found in the
control (N1 and BA0 — 186.05 U/g FW). The lowest activity was found in
plants supplied with the lowest N and the highest BA concentration (N1/4 and
BA40 — 26.00 U/g FW) (Fig. 2).
With the reduction of N concentration in the nutrient solution the activity
of SOD declined as well. On the contrary, an increase in the BA concentration
reduced the activity of SOD. However, analysis of variance showed that there
was no statistically significant interaction between N and BA concentration on
the activity of SOD.
The effect of cytokinins on the amount of superoxide radical (O2·¯)
In the experiment with CK, the highest amount of O2·¯ (4.35 mmol/g
FW) was found in plants supplied with ¼ of full dose of N and treated with
2.5 mg CK/dm3solution, whereas the lowest amount of O2·¯ (2.64 mmol/g
FW) was found in plants supplied with full dose of N and treated also with
2.5 mg CK/dm3solution (Fig. 3).
The concentration of N in the nutrient solution exhibited statistically signi-
ficant effect on the reduction of the amount of O2·¯ in wheat leaves. This re-
duction was the most outstanding in plants grown in the presence of full N
dose (N1) (Fig. 3). The effect of treatment with CK on the amount of O2·¯ in
wheat leaves was not statistically significant.
Treatment with BA significantly increased the production of O2·¯ in leaves.
The highest amount of O2·¯ (4.68 mmol/g FW) was found in leaves of wheat
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Fig. 2 — Activity of superoxide dismutase (SOD) in leaves of young wheat plants grown
in the presence of different N concentrations and treated with different concentrations of BA