Role of reactive oxygen species and antioxidant enzymes in ...

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Role of reactive oxygen species and antioxidant enzymes in ...

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

Abstract

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

Özet

Introduction

Journal of Cell and Molecular Biology 6(1): 41-48, 2007.

Haliç University, Printed in Turkey.

http://jcmb.halic.edu.tr

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

41


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

electron donor.

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

ABA.

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

peroxide

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

of H2O2.

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

reaction.

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

fragments.

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

2).

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

H2O2.

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.,

2002).

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.

Conclusions

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.

Acknowledgements

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.

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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)

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