Role of reactive oxygen species and antioxidant enzymes in hormone
regulating programmed cell death of wheat aleurone layer
Amangeldy K. Bissenbaev*, Nazgul A. Altybaeva, Gulderay A. Kolbaeva
Department of Molecular Biology and Genetics, Faculty of Biology, Kazakh National University, 050078,
Almaty, al-Faraby 71, Kazakhstan
(* author for correspondence; Aman@kazsu.kz)
Received 13 October 2006; Accepted 14 April 2007
Hormonal regulation of reactive oxygen species and antioxidant enzyme production in wheat aleurone layer was
revealed. In the presence of gibberellic acid in wheat aleurone layer significant increase of reactive oxygen species
was observed. This effect of gibberellic acid was associated with maximal decrease in the antioxidant enzyme
activity. The present study showed that in the given model system influence of abscisic acid was directed to the
suppression of ROS production through increase in antioxidant enzyme synthesis. Strict correlation between
progression of wheat aleurone programmed cell death, ROS production and changes in activity of antioxidant
enzymes was revealed. Relying on the obtained data it was suggested that ROS and antioxidant enzymes could play
important roles in realization of hormone induced programmed cell death of wheat aleurone layer.
Key Words: Aleurone layer, plant hormone, superoxide, superoxide dismutase, ascorbate peroxidase.
Reaktif oksijen türleri ve antioksidan enzimlerin bu¤day alevron tabakas›ndaki hormon
düzenlemeli programl› hücre ölümü üzerindeki rolü
Journal of Cell and Molecular Biology 6(1): 41-48, 2007.
Haliç University, Printed in Turkey.
Alevron tabakas›ndaki reaktif oksijen türleri ve antioksidan enzim üretimi düzenlenmesi ortaya ç›kar›lm›flt›r. Alevron
tabakas›ndaki giberilik asidin varl›¤› reaktif oksijen türlerinin h›zl› bir flekilde art›fl›na sebep olmufltur. Giberilik
asidin bu etkisi antioksidan enzim miktar›ndaki maksimum derecedeki düflüfl ile iliflkilendirilmifltir. Mevcut çal›flma,
bu modelde absisik asid miktar›ndaki art›fl›n ROS üretimini bask›lad›¤›n› ve bu bask›y› antioksidan enzim
sentezlemek suretiyle yapt›¤›n› göstermifltir. Alevron tabakas›ndaki programl› hücre ölümü ve ROS üretimi ile
antioksidan enzim aktivitesi aras›ndaki s›k› iliflki tespit edilmifltir. Elde edilen bilgiler ›fl›¤›nda, ROS ve antioksidan
enzimlerinin hormon teflvikli programl› hücre ölümü üzerinde rolü oldu¤u söylenebilir.
Anahtar Sözcükler: Alevron tabakas›, bitki hormonu, süperoksit, süperoksit dismutaz, askorbat peroksidaz.
Studying the regulation mechanisms of secretory
activity and cell death of cereal aleurone layer on
different stages of plant ontogenesis is very important
in solving important problems of agriculture and
biotechnology (harvest formation and storage,
regenerative plant grows, biological and technical
42 Amangeldy K. Bissanbaev et al.
quality of cereals, and other) (Young et al., 1997).
During their growing process aleurone layer of
wheat grain synthesize and secrete number of
hydrolytical enzymes (including α- a m y l a s e ) .
Induction of synthesis of the given hydrolases depends
on the presence in tissue gibberellic acid (GA). GAdepending
hydrolases synthesis is inhibited by natural
antagonist of the GA – abscisic acid (ABA)
(Bissenbaev et al., 1992; Bethke et al., 1997). On the
subsequent ontogenesis stages wheat aleurone layer
cells are eliminated. It is suggested that aleurone cell
death is programmed, or genetically determined
(apoptosis) (Fath et al., 2000; Bissenbaev et al., 2001;
Bissenbaev et al., 2004).
It is known that reactive oxygen species such as
superoxide (O2 - ), hydrogen peroxide (H2O2) and
hydroxide radical (OH - ) play crucial role in initiation
of programmed cell death (PCD) (Jab, 1999).
Accumulation of these reactive oxygen species
may cause peroxidation of membrane lipids,
inactivation of proteins (enzymes) and oxidative DNA
damage (Sordet et al., 2004). The protective
mechanisms adapted by plants to scavenge free
radicals and peroxides include antioxidative enzymes
such as superoxide dismutase (SOD), ascorbate
peroxidases (APX), catalases (CAT) and others. These
enzymes are important components in preventing the
oxidative stress in plants. SOD catalyzes the
dismutation of superoxide radical to molecular oxygen
and H2O2 (Bawler et al., 1994). APX and CAT then
detoxify the produced H2O2 (Mittler et. al., 1998).
APX reduces H 2O2 to water, by using ascorbate as an
The aim of the present investigation was to
investigate the roles of reactive oxygen species and
antioxidant enzymes in hormone induced programmed
cell death of wheat aleurone layer.
Materials and methods
Plant material: Preparation of wheat aleurone layers
Wheat (Triticum aestivum, variety Kazakhstanskaya
4) seed aleurone layers isolated from grains of 2003
harvested. Aleurone layers were prepared from
deembryonated seeds as described previously
(Bissenbaev et al., 2004) and incubated in media
containing 10mM CaCl2 and 1μM GA and/or 5μM
DNAextraction and electrophoresis
Aleurone tissues were lysed in a 0,3 ml ice cold buffer
(10 mM Tris-HCl (pH 7.2), 10 mM EDTA, 5 mM
2–mercaptoethanol, 0.5% Triton X-100) (5 mM Tris-
HCl (pH 8.0), 20 mM EDTA, 2 % (w/v) sodium
dodecylsulphate (SDS), 1.4 M NaCl, 0.2 % (w/v) 2
–mercaptoethanol) and incubated for 30 min at 60 o C.
Extraction of DNA was carried out according to
Young and Gallie (1999) with some modifications.
The DNA was extracted with equal volumes of
chloroform and after 15 min centrifuged for 10
minutes at 5000 g. The aqueous phase was transferred
to a new tube and 2/3 volumes of ice-cold isopropanol
(-20 0 C) was added and centrifuged for 10 min at 5000
g. The supernatant was discarded, and the pellet
containing DNAwas dried and resuspended in 500 μl
of TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH
7.5). To the obtained suspension equal volume
phenol:chloroform (1:1) mixture was added. After 15
min centrifugation at 5000 g for 10 min, 1/10 volume
sodium acetate (pH 7.0) and 2/5 volume ice-cold
ethanol were added to aqueous phase. DNA was
allowed to precipitate at –20 0 C, pellet containing
DNA dried and resuspended in 50 μl of TE buffer.
DNAelectrophoresis was carried out for 2 h at 70V in
1.8 % agarose gels. DNA was visualised by UV
fluorescence after staining with ethidium bromide.
Determination of superoxide radical and hydrogen
O2 - was measured as described by Jiang and Zhang
(Jiang et al., 2001) by monitoring the nitrite formation
from hydroxylamine in the presence of O2 - , with some
modifications. One gram of frozen aleurone tissue leaf
segments was homogenized with 3 ml of 65 mM
potassium phosphate buffer (pH 7.8) and centrifuged at
5.000 g for 10 min. The incubation mixture contained
0.9 ml of 65 mM phosphate buffer (pH 7.8), 0.1 ml of
10 mM hydroxylamine hydrochloride and 1 ml of the
supernatant. After incubation at 25°C for 20 min, 17
mM sulfanilamide and 7 mM α-naphthylamine were
added to the incubation mixture. After reaction at 25°C
for 20min, same volume of ethyl ether in the was added
and centrifuged at 1500 g for 5 min. The absorbance in
the aqueous solution was read at 530 nm. A s t a n d a r d
curve with NO2 - was used to calculate the production
rate of O2 - and hydroxylamine.
The content of H2O2 was measured by monitoring
the A415 of the titanium-peroxide complex following
the method described by Brenan and Frenkel (Brenan
et al., 1977). Absorbance values were calibrated to a
standard curve generated with known concentrations
Assays of antioxidant enzyme activities
Extraction of antioxidant enzymes was carried out
according to Jiang and Zhang (2001) with some
modifications. Aleurone layers were ground to a fine
powder in liquid N2, extracted in 300 µl buffer (60 mM
K2HPO4,-pH 7.8-, 0.1 mM EDTA, 20 µM E64, 20 µM
leupeptin) and the homogenate was centrifuged for 15
min at 4000 rpm at 4°C. Samples of the homogenate
were separated on 12.5 % native PAGE at 100 V.
SOD activity was assayed using the method as
described (Beauchamp et al., 1971). A f t e r
electrophoresis gels were immersed in 2.45 mM nitro
blue tetrazolium for 20 min and soaked in a solution
containing 28 mM T E M E D
(tetramethylethylenediamine), 28 µM riboflavin and
36 mM K2HPO2 (pH 7.8) for 15 min. SOD activity
was detected by illuminating the gel which causes it to
Programmed cell death in wheat aleurone 43
turn uniformly blue except at positions exhibiting
SOD activity. When maximum contrast was achieved,
rinsing the gel with H2O stopped the reaction.
APX activity was assayed using the method as
described (Mittler et al., 1993). After electrophoresis
the gel was immersed in 50 mM sodium phosphate
(pH 7.0) and 2 mM ascorbate for 30 min, changing the
solution every 10 min. The gel was soaked in 50 mM
sodium phosphate (pH 7.0), 4 mM ascorbate and 2
mM H2O2 for 20 min before briefly washing with 50
mM sodium phosphate, pH 7.0. Finally, the gel was
incubated in 50 mM sodium phosphate (pH 7.8), 28
mM TEMED and 2.45 mM nitro blue tetrazolium until
the gel turned uniformly blue except at positions
exhibiting APX activity. When maximum contrast was
achieved, rinsing the gel with H2O stopped the
Results and discussion
Study of the time course of GA’s action on the DNA
fragmentation of aleurone layer cells showed that
treatment of 1 μM GA significantly enhanced DNA
fragmentation only after incubation of aleurone layer
for 72 hour. Subsequent increase of incubation time
Figure 1. A) Effect of GAon DNAfragmentation in time dependent manner (M: marker); B) Effect of ABAon GAstimulated
DNAfragmentation in dose dependent manner (Aleurone layers were incubated for 96 hours; 1- GA(1μM); 2-GA(1 μM)+ABA
(1 μM); 3- GA (1 μM) + ABA(0,1 μM); 4- GA(1 μM) + ABA(0,01 μM)
44 Amangeldy K. Bissanbaev et al.
Figure 2. Effect of GAand ABA on the generation of superoxide radicals in wheat aleurone layer (1- 24 hours; 2- 48 hours; 3-
72 hours; Dosage of GA: 1 μM; Dosage of ABA: 5 μM)
was accompanied with significant enhancement of
DNA fragmentation. Under these conditions different
sized fragments formed characteristic
internucleosomal ladders when on agarose gel
electrophoresis (Figure 1A). Addition of 0.001-1μM
ABAto the incubation medium entirely abolished GAstimulated
fragmentation of DNAto oligonucleosomal
These data support our previous suggestions about
apoptotic characters of wheat aleurone cell death that
are initiated by GA. It seems probably that wheat
aleurone cell death is initiated in 48 hours after GA
treatment and it reaches maximal level in 96 hours
after treatment. This process accompanied by
internuclesomal DNA fragmentation that is a critical
hallmark of apoptosis (Gilchrist, 1998).
ROS are the products of cellular metabolism. In
plant cells the production of ROS has been shown in
mitochondria, chloroplasts, the plasma membrane,
peroxisomes and the apoplastic space. ROS may also
be generated by ionizing or ultraviolet radiation.
Equally, certain exogenous chemicals may redox cycle
following metabolism by the cell, with the subsequent
production of electrons that can be transferred to
molecular oxygen producing superoxide. Irrespective
of their origin, reactive oxygen species may interact
with cellular biomolecules, such as protein, lipids and
DNA, leading to modification and potentate cell death.
However, ROS can be produced specifically as part of
programmed cell death (Jab, 1999).
The initial experimental evidence that ROS also
acted as a signal in plant PCD was obtained incell
suspensions by demonstrating that H2O2-induced cell
deathcould be blocked by cycloheximide and protease
inhibitors (Levineet al., 1994). It has been shown that
two mutants of A r a b i d o p s i s , l e s i o n - s t i m u l a t i n g
d i s e a s e 1 (l s d 1) and radical-induced cell death1
(rcd1), elevate ROS levels are necessary and sufficient
to induce spreading of cell death ( Jabs et al., 1996;
Overmyer et al., 2000; Mateo et al., 2004). It was
demonstrated that the conditional f l u o re s c e n t (f l u)
mutant of Arabidopsis that generates singlet oxygen
upona dark-to-light shift initiate a cell death response
immediatelyafter the release of singlet oxygen (Camp
et al., 2003).
These data suggests that ROS generation is a
necessary link to initiate a response cascade that
results in cell death. Among the questions that remain
to be resolved about ROS in plants, especially in the
context of cell death, are identities of the ROS species
that participate in PCD.
Therefore in our following experiments the time
course of GA’s action on the accumulation of
superoxide and peroxide radicals in the wheat
aleurone layers were investigated. It was revealed that
presence of 1 μM GAin incubation medium for 24 and
48 hours significantly increased superoxide
production, when compared with the control (Figure
At 72 h, the levels of superoxide decreased
significantly, and returned almost to the level of
control. Addition of 5μM ABA to the incubation
medium entirely abolished GA-stimulated
accumulation of superoxide in the wheat aleurone
layer within the all time of GA treatment. T h e
presence of 5 μM ABA alone significantly decreased
the level superoxide compared with control and with
the effect of GA within the all time of incubation.
The study of hormone dependent accumulation of
H2O2 in aleurone layer showed that presence 1 μM
GA in incubation medium resulted in continuous
increase in the level of H2O2 within all time of
Programmed cell death in wheat aleurone 45
Figure 3. Effect of GAand ABAon the generation of hydrogen peroxide in wheat aleurone layer (1-24 hours; 2- 48 hours; 3- 72
hours; Dosage of GA:1 μM, Dosage of ABA: 5 μM)
incubation (Figure 3). Addition of 5μM ABA to the
incubation medium entirely blocked GA-stimulated
accumulation of H2O2 in the wheat aleurone layer
within the all time of GA treatment.
Thus, these data indicate that in the presence of
GA in aleurone layer the processes of superoxide and
peroxide production were really increased. Also most
significant superoxide anion accumulation was
observed in 48 hours after GA treatment. Whereas
GA-depended H2O2 content in the aleurone layer
reached its maximal point in 72 hours of incubation.
It is known that ROS are products of cell
metabolism and their intracellular level is regulated by
number of antioxidant enzymes (superoxide
dismutase, aspartate peroxidase, catalase and others).
As mentioned above, SOD catalyzes the
dismutation of superoxide radical to molecular oxygen
and more less toxic H2O2. More than 80% of SOD
activity is identified in cytosol, and other 20% - in
organelles, mainly in mitochondria (Bawler et al.,
1994). APX and CAT then detoxify the produced
This might suggest that observed differences in
production of superoxide and H2O2 to GA response is
46 Amangeldy K. Bissanbaev et al.
Figure 4. Effect of GA and ABA on the isoenzymes of superoxide dismutase of wheat aleurone layer (Dosage of GA: 1 μM,
Dosage of ABA: 5 μM; Mn: mitochondrial SOD, Fe: Plastid SOD; CZ: cytosolic SOD)
supplied by dismutation of superoxide-anion into
H2O2through activation of superoxide dismutase
reaction. A noticeable fact that is increases of cellular
concentration of H2O2, which was produced as a result
of superoxide dismutase reaction, can be dangerous to
the cell itself because of hydroxide radical production
p r o b a b i l i t y. That is why H2O2must be constantly
inactivated in the reaction, which is catalyzed by
ascorbate peroxidase and/or catalase (Shigeoka et al.,
Therefore in subsequent experiments we have
investigated the effects of GA and A B A on the
activation pattern of wheat aleurone layer antioxidant
enzymes, especially SOD and APX.
As it is seen in Figure 4, in the presence of GA
activity of SOD isoenzymes of aleurone layer
decreased in correlation with incubation time.
Insignificant suppressing effect of GAwas observed in
48 hour after incubation. After 72 hours of incubation
electrophoretic SOD activity almost disappeared and
was presented only by one electronegative protein
zone with SOD activity. Analysis of time-dependent
ABA effect demonstrated that ABA strengthens the
activity of all intracellular SOD forms independently
from duration of incubation time. ABAintroduction in
5 μM dosage into aleurone layers incubating with GA
significantly blocked inhibitory effect of GA o n
activity of intracellular SOD forms.
As it can be seen in Figure 5, in the GA presence
activity of aleurone layer APX isoenzymes were
decreased in correlation with increase of incubation
time. Incubation of isolated aleurone layer with GA(1
μM) for 48 hours significantly inhibited the activity of
APX isoenzymes. After 72 hours of incubation with
GA activity of APX isoenzymes was not observed on
electropherogram. Analysis of time-dependent ABA
e ffect demonstrated that A B A strengthened the
activity of all intracellular types of APX. A B A
introduction in 5 μM dosage into culture of aleurone
layers, incubating with GA, significantly blocked the
inhibitory effect of GA on activity of intracellular
APX forms of wheat seed aleuron layers.
Obtained data indicate the important role of
hormones in regulation of antioxidant enzymes of
wheat seed aleurone layer. ABAstimulates the activity
of antioxidant enzymes and, as a consequence,
suppresses production of ROSin wheat aleurone layer.
In contrast, influence of GAin the given model system
directed to suppress the activity of antioxidant
enzymes. Also the maximal effect of GA on APX and
SOD activity was observed in three days after
incubation, which indicates the direct correlation
between decrease antioxidant enzymes activity,
enhancement of ROS production and progression of
aleurone cells PCD, which identified by
oligonucleosomal degradation of DNA.
Presented results may indicate the key role of ROS
and antioxidant enzymes in hormone induced PCD of
wheat aleurone layer. GAstrengthens apoptosis, which
is followed by oligonucleosomal DNA fragmentation
– the critical apoptosis hallmark. Probably, before
PCD intracellular concentration of ROS is increased
due to GA-dependent inhibition of enzymes of ROS
metabolism. Decrease of ROS metabolizing enzyme’s
activity in GA treated cells significantly strengthens
the process of accumulation of ROS and PCD of
aleurone layer. In contrast, during the incubation of
aleurone layers in the presence of ABA they do not
undergo PCD (Bissenbaev et al., 2001) and they have
high level of ROS metabolizing enzymes activity in
comparison with cells, which were incubated only in
the presence of GA.
This research was supported by grant no. 3-2-3.7-
2(78) from the Biological Research Center of Ministry
of Education and Science, Kazakhstan.
Programmed cell death in wheat aleurone 47
Figure 5. Effect of GA and ABA on the isoenzymes of ascorbate peroxidase of wheat aleurone layer (Dosage of GA: 1 μM,
Dosage of ABA: 5 μM)
Bawler C, Camp VW, Montagu VM., Inze D. Superoxide
dismutase in plants. Critical Reviews in Plant Science.
13: 199-218, 1994.
Beauchamp C, Fridovich I. Superoxide dismutase: improved
assay and an assay applicable to acrylamide gels.
Analytical Biochemistry. 44: 276-287, 1971.
Bethke PC, Schuurink R and Jones RL. Hormonal signaling
in cereal aleurone. J. Exp. Bot. 48: 13337-13356, 1997.
Bissenbaev AK, Tairov MM and Bersimbaev RI. Role of
protein synthesis and Ca2+ in the effect of gibberellic
acid on amylase secretion from isolated aleurone layer
of wheat seed. Russian J. of Biochem. 57 (12): 1834-
Bissenbaev AK, Keniev AM and Bersimbaev RI. Effect of
gibberellic abscisic acid on apoptosis of wheat aleurone
cells. Bulletin of Kazakh National University. 1 (13): 52-
Bissenbaev AK., Keniev AM, Bersimbaev RI. Endogenous
deoxyribonucleases involved in nuclear DNA
degradation of wheat aleurone cells. Journal of Cell and
Molecular Biology. 3: 77-81, 2004.
Brenan T, Frenkel C. Involvement of hydrogen peroxide in
the regulation of senescence in pear. Plant. Physiol . 59:
48 Amangeldy K. Bissanbaev et al.
Camp W, Capiau K., Van Montagu M, Siooten L.
Enhancement of oxidative stress tolerance in transgenic
tobacco plants overproducing Fe-superoxide dismutase
in chloroplasts. Plant Physiol. 112: 1703-1714, 2003.
Fath A, Bethke PC, Lonsdale J., Meza-Romero R, Jones R.
Programmed cell death in cereal aleurone. Plant
Molecular Biology. 44: 255-256, 2000.
Gilchrist PC Programmed cell death in plant disease: The
purpose and promise of cellular suicide. Ann. Rev.
Phytopathol., 21: 345-358. 1998.
Jabs T, Dietrich RA, Dangl JL. Initiation of runaway cell
death in an Arabidopsis mutant by extracellular
superoxide. Science. 273: 1853–1856, 1996.
Jab TI. Reactive oxygen intermediates as mediators of
programmed cell death in plant and animals. Biochem.
Pharm. 57: 231-245, 1999.
Jiang M, Zhang J. Effect of abscisic acid on active oxygen
species, antioxidative defense system and oxidative
damage in leaves of maize seedlings. Plant Cell Physiol.
42(11): 1265-1273, 2001.
Levine A, Tenhaken R, Dixon R, Lamb C. H2O2 from the
oxidative burst orchestrates the plant hypersensitive
disease resistance response. Cell. 79: 583–593, 1994.
Mateo A, Muhlenbock P, Rusterucci C, Chi-Chen Chang C,
Miszalski Z, Karpinska B, Parker JE, Mullineaux PM,
Karpinski S Lesion stimulating disease 1 is required for
acclimation to conditions that promote excess excitation
energy. Plant Physiol., 136: 2818–2830, 2004.
Mittler R, Xuqiao F, Cohen M. Post-transcriptional
suppression of cytosolic ascorbate peroxidase
expression during pathogen-induced programmed cell
death in tobacco. The Plant Cell. 10: 461–473, 1998.
Mittler R, Zilinkas BA. Detection of ascorbate peroxidase
activity in native gels by inhibition of the ascorbate –
dependent reduction of nitroblue tetrazolium. Analytical
Biochemistry. 212: 540-546, 1993.
Overmyer K, Tuominen H, Kettunen R, Betz C,
Langebartels C, Sandermann H Jr, Kangasjarvi J.
Ozone-sensitive A r a b i d o p s i s rcd1 mutant reveals
opposite roles for ethylene and jasmonate signaling
pathways in regulating superoxide-dependent cell death.
Plant Cell . 12: 1849–1862, 2000.
Shigeoka Sh., Ishikawa T, Tamoi M, Miyagawa Y, Takeda T,
Yabuta Y, Yoshimura K. Regulation and function of
ascorbate peroxidase isoenzymes. J. Exp.Botany, 53
(372): 1305-1319, 2002.
Sordet O, Khan QA, Plo I, Pourquier P, Urasaki, Y, Yoshida
A, Antony S, Kohlhagen G, Solary E, Saparbaev M,
Laval J, and Pommier Y. Apoptotic topoisomerase I-
D N A complexes induced by staurosporine-mediated
oxygen radicals. J. Biol. Chem. 279: 50499-50504,
Young TE, Gallie DR, and DeMason DA. Ethylene-
Mediated Programmed Cell Death during Maize
Endosperm Development of Wild-Type and shrunken2
Genotypes. Plant Physiol . 115: 737-751, 1997
Young TE, Gallie DR. Analysis of programmed cell death in
wheat endosperm reveals differences in endosperm
development between cereals. Plant Mol Biol. 39: 915-