2. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
THE ROLE OF REACTIVE OXYGEN SPECIES AND CALCIUM IONS 659
tion between ROS and calcium as the signaling mes
sengers during the development of BS induced resis
tance of plants to stress inducing factors is poorly
understood.
The present work was aimed at the studying of the
role of calcium ions and enzymatic systems that per
form generation and detoxication of ROS in the devel
opment of thermal resistance of wheat coleoptile cells
treated with natural BSs.
METHODS
Pieces of wheat coleoptiles (Triticum aestivum L.)
of the breed Elegia, isolated from 4 day old etiolated
plants cultivated at 20°C, were used for the experi
ments. Samples were treated with 24 epibrassinolide
and 24 epicastasterone that belong to 7 oxalactons
and 6 oxotype of BSs, respectively, and differ by their
biological activities [18]. The preparations were syn
thesized in the laboratory of steroid chemistry of the
Institute of Bioorganic Chemistry of the National
Academy of Sciences of Belarus. Nitro blue tetrazo
lium (NBT) obtained from Sigma (United States),
NADH obtained from Oriental Yeast (Japan), EGTA,
Triton X 100 obtained from AppliChem GmbH (Ger
many), and nicotinamide obtained from Shanghai
Synnad (China) were also used in the study. Other
reagents were produced in the Ukraine and Russia and
were of high purity.
The coleoptiles were incubated in sterile 2%
sucrose solution supplemented with 1000000 units of
penicillin sodium salt (control). 24 epibrassinolide
and 24 epicastasterone were dissolved in ethanol
(5 mg/50 mL) and were introduced into the coleoptile
incubation medium in a final concentration 10 nM.
The effective concentrations of BSs that induced a
maximal increase in thermal resistance of wheat were
previously estimated [12]. The incubation medium of
the control samples was supplemented with an equal
amount of ethanol instead of BS. Imidazole (1 µM)
(the inhibitor of NADPH oxidase) [19], EGTA
(50 µM) (the chelator of extracellular calcium), nicoti
namide (1 mM) (the antagonist of cyclic adenosine 5'
diphosphate ribose (cADPR) synthesis) [20], or a com
bination of these compositions with BS were used as
effectors. The effectors were introduced into the incu
bation medium of coleoptiles 3 h prior the introduction
of BS.
After 1 day of coleoptile incubation in the solutions
of the studied compositions, a part of the samples
underwent lethal heating in a water ultrathermostate
in sterile distilled water for 10 min at 43 ± 0.1°C. After
that, the coleoptiles were put into Petri dishes contain
ing a 2% sterile solution of sucrose and penicillin.
Damage of coleoptiles was assessed after 2 days by the
loss of turgor and the appearance of a specific shade
caused by tissue infiltration.
The generation of superoxide anion radicals of the
intact coleoptiles was assessed by the reduction of
NBT as described in [21]. To confirm the specificity of
generation, superoxide dismutase (SOD)
(50 units/mL), which suppressed generation of super
oxide anion radical, was used. The amount of gener
ated was estimated by the level of reduced NBT.
Superoxide producing activity was assessed by the
change of optical density (А530) of the reaction mixture
at 530 nM for 1 h incubation of one coleoptile sample
(7 mM). The optical density of the control sample was
taken as 100%.
To estimate the activities of SOD (EC 1.15.1.1) and
catalase (EC 1.11.16), plant material was homogenized
in 0.15 M K,Na phosphate buffer, pH 7.6, and supple
mented with EDTA (0.1 mM), dithiothreitol (1 mM),
phenylmethylsulfonyl fluoride (0.5 mM) and Triton
X 100 (final concentration 0.1%) at 2–4°C [22]. The
samples were centrifuged at 8000 g for 10 min at 4°C,
and the obtained supernatant was used for analysis.
Estimation of SOD activity was based on ability of the
enzyme to compete with NBT for superoxide anions,
which were formed during aerobic interaction between
NADH and phenazine methosulfate [23]. The activity
of catalase was assessed by the amount of hydrogen
peroxide digested for a time unit at a pH of 7.2 [24].
The protein level was measured by the Bradford
method, with bovine serum albumin used as a stan
dard [25].
All experiments were carried out independently 3–
4 times in threefold biological repeats. The confidence
of the differences between the variants was assessed by
the Student t test (p ≤ 0.05).
RESULTS AND DISCUSSION
Treatment of wheat coleoptiles with 24 epibrassin
olide and 24 epicastasterone led to an increase in their
viability after heating to the point of damage (table).
The effects of 24 epibrassinolide were a little bit stron
ger in comparison with 24 epicastasterone. However,
the differences between the effects of the two BSs were
not found to be significant at p ≤ 0.05.
Treatment with either 24 epibrassinolide or 24 epi
castasterone similarly increased generation of the
superoxide anion radical in wheat coleoptiles (Fig. 1).
The maximal effect was observed 2–5 h after treat
ment with BSs. However, the production of
decreased in 24 h in both experiments, with either
24 epibrassinolide or 24 epicastasterone treatment.
Therefore, in further experiments, generation of the
superoxide anion radical was measured in 2 h and 5 h
after treatment with BS.
The inhibitor of NADPH oxidase imidazole
decreased the formation of in wheat coleoptile
(Table) and blocked the activation of superoxide pro
duction by both BSs. Therefore, it may be suggested
that activation of ROS generation in wheat coleop
tiles induced by BSs is caused by the activation of
NADPH oxidase.
О2
−i
О2
−i
О2
−i
О2
−i
3. 660
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
KOLUPAEV et al.
This enzyme is known to be calcium dependent.
The binding of calcium to the catalytic membrane
bound subunit of NADPH oxidase plays an excep
tionally important role in the regulation of enzyme
activity [14].
Therefore, the effect of the extracellular calcium
chelator EGTA on ROS generation in wheat coleop
tiles in the presence or absence of exogenous BS has
been studied. Treatment with EGTA did not produce
any considerable effect on the generation of the super
oxide anion radical by wheat coleoptiles (table). How
ever, the chelator of calcium significantly leveled the
increase in production induced by BS.
The increase in the extracellular concentration of
calcium may also occur because of its outflow from
intracellular compartments, particularly vacuoles.
This process may be regulated by cADPR [26]. The
О2
−i
inhibitor of cADPR synthesis, nicotinamide (the
inhibitor of ADP ribosyl cyclase), was used in order to
study the role of cADPR in the regulation of the
BS induced formation of ROS in wheat coleoptiles
[20]. Treatment with nicotinamide resulted in a slight
increase in generation of the superoxide anion radical
in coleoptiles (nonsignificant at p ≤ 0.05). However,
treatment with nicotinamide partially removed the
increase in generation in coleoptiles induced by
BS. Therefore, it may be suggested that the BS acti
vated ROS generation system depends on the avail
ability of both exogenous and endogenous calcium
once it is inhibited by both calcium chelator EGTA
and nicotinamide. The observed effects confirmed the
suggestion that different treatments, which induce
changes in the cytosolic concentration of calcium,
may affect the activity of NADPH oxidase [27].
This raises the question of whether there is a
dependence of the development of thermal resistance
of wheat coleoptiles on the calcium dependent activa
tion of ROS generation induced by BS. Although the
inhibitor of NADPH oxidase imidazole insignifi
cantly affected the thermal resistance of coleoptiles, it
leveled the positive effects of 24 epibrassinolide and
24 epicastasterone on the viability of coleoptiles,
which underwent heating to the point of damage
(table). Treatment with the compositions, such as
EGTA and nicotinamide, which limit the influx of
calcium into the cytosol and level the activation of
ROS generation induced by BSs, also prevented the
development of thermal resistance of coleoptiles
induced by these phytohormones (Table). This allows
us to suggest that ROS and calcium ions work as mes
sengers during the realization of the stress protective
effect of BSs.
One may hypothesize that the antioxidant protec
tive systems of cells is induced by BSs. Indeed, the
О2
−i
Generation of the superoxide anion radical in wheat coleoptiles in 2 h and 5 h after the beginning of treatment with BSs
and survival of their samples after damaging heating (43°C, 10 min)
Experimental variant
Generation of the superoxide anion
radical, % from the control Survival, %
2 h 5 h
Control 100.0 ± 3.8 104.2 ± 3.6 51.3 ± 1.9
24 epibrassinolide (10 nM) 139.2 ± 4.2 126.2 ± 3.9 68.1 ± 2.9
24 epicastasterone (10 nM) 135.3 ± 4.0 133.0 ± 3.7 64.4 ± 2.0
Imidazole (1 µM)* 89.6 ± 3.3 91.3 ± 2.9 46.8 ±2.6
24 epibrassinolide (10 nM) + imidazole (1 µM) 96.7 ± 3.9 94.6 ± 3.5 52.4 ± 2.5
24 epicastasterone (10 nM) + imidazole (1 µM) 102.2 ± 4.3 93.2 ± 2.6 54.2 ± 2.3
EGTA (50 µM)* 107.2 ± 5.1 104.7 ± 3.3 47.9 ± 2.9
24 epibrassinolide (10 nM) + EGTA (50 µM) 108.4 ± 3.7 110.0 ± 3.1 51.9 ± 2.6
24 epicastasterone (10 nM) + EGTA (50 µM) 108.9 ± 4.2 105.6 ± 2.8 49.0 ± 3.2
Nicotinamide (1 mM)* 112.4 ± 3.7 107.8 ± 3.6 50.8 ± 3.1
24 epibrassinolide (10 nM) + nicotinamide (1 mM) 119.4 ± 3.9 109.4 ± 4.2 49.0 ± 2.8
24 epicastasterone (10 nM) + nicotinamide (1 mM) 109.8 ± 4.2 107.0 ± 3.3 49.3 ± 3.0
* Imidazole, EGTA, and nicotinamide were introduced to the incubation medium 3 h prior the introduction of BS.
%
140
120
100
80
0 2 4 6 24
h
1
2
3
Fig. 1 The dynamics of superoxide anion radical produc
tion (% from the value at the first time point in the control)
1, control; 2, 24 epibrassinolide (10 nM); 3, 24 epicastas
terone (10 nM).
4. APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
THE ROLE OF REACTIVE OXYGEN SPECIES AND CALCIUM IONS 661
arbitrary units/(mg protein min)
10
9
8
7
6
I II III IV V VI
(a)
1 2 3
µmol H2O2/(mg protein min)
900
800
700
600
I II III IV V VI
(b)
Fig. 2 The activities of SOD (a) and catalase (b) in wheat coleoptiles: I–III are 2, 5, and 24 h after the beginning of treatment
with BS; IV and V are 2 and 24 h after damaging treatment; VI is 24 h after transferring the coleoptiles that did not undergo dam
aging treatment to the medium without BS. 1, Control; 2, 24 epibrassinolide (10 nM); 3, 24 epicastasterone (10 nM).
incubation of wheat coleoptiles in the solutions of
24 epibrassinolide and 24 epicastasterone was fol
lowed by an increase in the activities of SOD and cat
alase (Fig. 2). The maximal activities of these enzymes
were observed 24 h after treatment with BSs. It was
shown that 24 epibrassinolide provided more signifi
cant activation of SOD in comparison with 24 epi
castasterone.
After damaging heating, the activities of antioxi
dant enzymes in coleoptiles also exceeded control val
ues. In experiments without damaging heating, the
activity of SOD in coleoptiles remained increased 24 h
after transference of the coleoptiles into a medium
without BSs, while the activity of catalase approached
the control values.
Treatment with the NADPH oxidase inhibitor
imidazole removed the increase in activity of the stud
ied antioxidant enzymes induced by BS (Fig. 3). How
ever, imidazole did not affect the activities of SOD and
catalase. These observations are consistent with the
data previously obtained in experiments with cucum
bers [3]. It was shown that the increase in the activity
of antioxidant enzymes induced by BS was leveled by
treatment with another inhibitor of NADPH oxidase
diphenyleneiodonium (DPI) [3].
EGTA induced an insignificant increase in SOD
activity but did not affect the activity of catalase (Fig. 3).
Nicotinamide did not have a significant effect on the
activities of either of these two enzymes. However, both
EGTA (the chelator of extracellular calcium) and nico
tinamide (cADPR synthesis antagonist that prevents
calcium outflow from intracellular compartments) sig
nificantly leveled the increase in activities of antioxi
dant enzymes in coleoptiles induced by BSs.
Thus, it may be suggested that the stress protective
effects of BSs on wheat coleoptile cells are mediated
by ROS [12] and calcium ions (Table, Fig. 3). Appar
ently, the key enzyme that provides BS induced acti
vation of ROS generation is NADPH oxidase. This
suggestion was confirmed by the elimination of the
BS induced increase in production of the superoxide
anion radical after treatment of the coleoptiles with
imidazole (table). Apparently, calcium ions partici
pate in the activation of NADPH oxidase after treat
ment with BS, because both binding of extracellular
calcium by EGTA and treatment of coleoptiles with
nicotinamide, which inhibits cADPR and subse
quently prevents calcium outflow through the intrac
ellular cADPR sensitive calcium channels, leveled the
increase in ROS generation in the presence of BSs
(table).
Activation of NADPH oxidase by calcium may be
implemented not only in a direct manner but also
through activation of a calcium dependent protein
kinase, which phosphorylates the catalytic subunit of
NADPH oxidase and thus increases its activity [29].
According to one of the models, the activity of
NADPH oxidase is regulated by both phosphoryla
tion of the catalytic subunit and its interaction with
calcium [14]. It is suggested that the phosphorylated
catalytic subunit of the enzyme, when binding calcium
in the area of EF hands of the calcium binding loop,
undergoes deeper conformational modifications in
comparison with the nonphosphorylated subunit and
is therefore activated more effectively [14].
5. 662
APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 50 No. 6 2014
KOLUPAEV et al.
Activation of NADPH oxidase and an increase in
the production of ROS are apparently required for
implementation of the stress protective effects of BSs.
Indeed, the increase in the activity of antioxidant
enzymes induced by BSs was leveled by imidazole (the
inhibitor of NADPH oxidase) (Fig. 3). Imidazole also
prevented the formation of an integrative reaction of
coleoptiles to BS treatment, i.e. the increase in their
thermal resistance (Table). These effects of BSs were
also removed by EGTA and nicotinamide, which pre
vent an increase in the concentration of calcium in
cytosol (Fig. 3, Table). This effect of calcium antago
nists may be due to both blocking of the activation of
NADPH oxidase and the formation of a ROS medi
ated signal induced by BSs and the participation of
calcium on other reactions, which do not deal with
ROS generation. It is noteworthy that the BS induced
increase in the activities of SOD and catalase was
obviously leveled by both EGTA and nicotinamide,
which inhibits cADPR and prevents calcium outflow
from intracellular compartments through the
cADPR dependent calcium channels (Fig. 3). It may
be suggested that the BS signal, which induces activa
tion of these enzymes, depends on the calcium influx
into the cytosol from both intercellular space and
intracellular compartments. However, this suggestion
needs experimental verification.
The obtained results bring us to the conclusion that
ROS and calcium ions work as signaling messengers in
the formation of BS induced thermal resistance of
wheat coleoptiles. Calcium ions may be involved in
activation of NADPH oxidase and transient activa
tion of ROS production in wheat coleoptiles. Hence,
BS induced signaling, which is triggered by Ca2+
and
ROS, provides activation of stress protective systems,
particularly the antioxidative system, and an increase
in the thermal resistance of plant cells.
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Translated by M. Bibov