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Journal of Thermal Biology 30 (2005) 524–531

www.elsevier.com/locate/jtherbio

Differential induction of enzymes and antioxidants of theantioxidative defense system in Anabaena doliolum exposed to

heat stress

Yogesh Mishra, Poonam Bhargava, Lal Chand Rai�

Laboratory of Algal Biology, Center of Advanced Study in Botany, Banaras Hindu University,Varanasi-221005, India

Received 1 April 2005; accepted 15 June 2005

Abstract

Anabaena doliolum subjected to 43, 48, 53 and 58 1C temperature for 1, 2, 3 and 4 h, showed temperature and time-

dependent increase in H2O2 production and MDA contents. All the measured enzymes of the antioxidative defense

system (SOD, CAT, APX and GR) showed increase in their activities at 43 1C after 1 h of treatment, but at higher

temperature their activity declined. The content of antioxidants (ASC, GSH, and a-TOC) increased significantly with

rise in temperature as well as duration of treatment. This study clearly demonstrates that when enzymatic defense

system becomes inactive, the antioxidants (GSH, and a-TOC) are induced to protect the cyanobacterium from heat

stress. One of the major roles of these antioxidants appears to be the protection of PSII as reflected by an effect on O2

evolution up to 53 1C.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: A. Doliolum; Antioxidants; Heat stress; O2 evolution

1. Introduction

Human activities are changing the composition as well

as behavior of the atmosphere at an unprecedented rate.

One of the most common outcomes of these activities is

a continuous increase in certain heat trapping gases

(CO2, CH4, NO2, and chlorofluorocarbons (CFC)),

which act like a blanket over the surface of earth. These

gases have capacity to return high-energy infrared

radiation to earth surface, thereby enhancing the

temperature, a phenomenon popularly known as ‘Glo-

bal Warming’.

e front matter r 2005 Elsevier Ltd. All rights reserve

erbio.2005.06.005

ing author. Tel.: +91542 2307146;

68174.

ess: lcrai@bhu.ac.in (L. Chand Rai).

Under optimum temperature conditions, plants main-

tain a balance between production and scavenging of

active oxygen species (Bowler et al., 1992). However,

when plants are subjected to heat stress, rate of

production of active oxygen species (superoxide radicals,

hydrogen peroxide, hydroxyl radical and singlet oxygen)

out-competes their scavenging (Foyer et al., 1994),

thereby creating oxidative stress. In order to encounter

the oxidative damage, plants and cyanobacteria are

known to stimulate the antioxidative defense system

(Srivastava et al., 2005), which consists of enzymatic as

well as non-enzymatic counterparts. Superoxide dismu-

tase (SOD), catalase (CAT), glutathione reductase (GR),

and ascorbote peroxidase (APX) are the key enzymes

whereas the non-enzymatic counterpart includes ascor-

bic acid (ASC), reduced glutathione (GSH), a- toco-

pherol (a-TOC), and carotenoid (CAR). Among the

d.

ARTICLE IN PRESSY. Mishra et al. / Journal of Thermal Biology 30 (2005) 524–531 525

enzymes SOD is involved in superoxide (O2.�) and

CAT, APX and GR in H2O2 scavenging (Asada, 1992;

Bowler et al., 1992). Further, among the antioxidants

GSH and ASC not only act as substrate for GR and

APX respectively but are directly involved in ROS

scavenging. Similarly a- TOC is known to protect

thylakoid membrane against lipid peroxidation (Havaux

et al., 2003).

Information available on antioxidative defense system

of plants under heat stress suggest a decreased activity of

SOD and CAT in Agrostis palustris (Liu and Huang,

2000). Contrary to this APX activity has been shown to

increase in mustard (Dat et al., 1998b), but decrease in

two cool-season grasses (Jiang and Huang, 2001) after a

long-term exposure to high temperature.

In case of antioxidants GSH accumulation in maize,

(Nieto-sotelo and Ho, 1986) and tomato (Rivero et al.,

2004) has been reported. Further, the a-TOC accumula-

tion has also been reported in Euglena gracilis (Ruggeri

et al., 1985) under heat stress. Rady et al. (1994) have

reported a decrease in activities of key enzymes, of the

antioxidative defense system (SOD, CAT, GPX), thus

resulting in increased lipid peroxidation in Synechocystis

PCC 6803 subjected to heat stress.

In view of the fact that heat shock suppresses the

translation of many proteins except HSPs (Vierling,

1991), it was presumed that heat stress may inactivate

the enzymes of the defense system thereby providing

opportunity to antioxidants (ASC, GSH and a-TOC) in

offering protection against heat stress. Further, in view

of the report that g ECS (g-glutamylcystein synthetase),

is an important enzyme for the synthesis of reduced

glutathione (GSH) is HSP 70 (Kondo et al., 1993) and

induction of expression of genes gsh1 and gsh2, under

heat stress, in Saccharomyces cerevisiae, (Sugiyama et

al., 2000) it was hypothesized that GSH may play role in

protecting Anabaena doliolum from heat stress.

To test this hypothesis, impact of temperature on A.

doliolum was studied in term of lipid peroxidation,

oxygen evolution, H2O2 content, and enzymes and

antioxidants of the antioxidative defense system of the

above cyanobacterium.

2. Materials and methods

2.1. Organism and growth conditions

The test cyanobacterium A. doliolum was grown

axenically in a modified Allen and Arnon (1955)

medium buffered with Tris/HCl at 2472 1C under

72mmol photonm�2 s�1 PAR (photosynthetically active

radiation) irradiance with a photoperiod of 14:10 h

(light: dark) at pH 7.5. The cultures were shaken by

hand two to four times daily. All the experiments were

conducted in triplicate and repeated at least twice to

confirm the reproducibility of the results.

2.2. Heat shock treatments

The exponentially growing cells of A. doliolum (OD

0.5) was shifted to temperature-controlled incubator for

heat treatments under continuous radiance of 72 mmol -

photon m�2 s�1 PAR (photosynthetically active radia-

tion) provided by fluorescent lamps throughout the heat

treatments. The doses selected for temperature treat-

ment were LC25, LC50, LC75 and lethal, which were 43,

48, 53 and 58 1C, respectively. These were obtained by

colony count method of Rai and Raizada (1985).

2.3. O2 evolution

Photosynthetic O2 evolution was measured with

polarographic oxygen electrode enclosed in 10ml air

tight reaction vessel and connected to an oxygen

analyzer (Digital Oxygen system Model-10 Rank

brother, UK).

2.4. Lipid peroxidation

Oxidative damage of lipid was measured in terms of

the total content of 2 thio barbituric acid reactive

substances (TBA) and expressed as equivalent of MDA

(malonildialdehyde) with minor modifications (Cakmak

and Horst, 1991). These reactive substances were

extracted in 3ml of 0.1% (w/v) trichloroacetic acid

(TCA) at 4 1C following centrifugation at 13 000� g for

2min. An aliquot of 0.5ml from the supernatant was

added to 1.5ml TBA (0.5% in 20% TCA). Samples were

incubated at 90 oC for 20min and the reaction was

stopped under ice bath. Centrifugation at 1000� g for

5min was performed and absorbance of the supernatant

was measured at 532 nm and corrected for non-specific

turbidity by subtracting the absorbance at 600 nm. The

concentration of MDA was calculated at its extinction

coefficient (155mM�1 cm).

2.5. Peroxide assay

The total peroxide was measured according to

Sagisaka (1976). The cell pellets suspended in cell lysis

buffer were subjected to sonication. Five percent of

TCA was added and the resulting suspension was

centrifuged, 1.6ml of the resulting supernatant was

mixed with 0.4ml 50% TCA, 0.4ml 10mM ferrous

ammonium sulfate and 0.2ml 2.5M potassium thiocya-

nate. This was then centrifuged and the absorbance of

the supernatant was measured at 480 nm. A standard

curve was used for measuring the concentration of the

peroxide.

ARTICLE IN PRESSY. Mishra et al. / Journal of Thermal Biology 30 (2005) 524–531526

2.6. Enzyme assay

Pellets collected from exponentially growing cultures

of A. doliolum were suspended in cell lysis buffer (pH 7)

and subjected to sonication in ice-cold condition. The

cell lysis buffer contained 1mM EDTA and 1% poly

vinyl pyrrolidone (PVP) with the addition of 1mM ASC

in case of APX assay. The sonicated sample was

centrifuged at 15 000� g for 30min at 4 1C and the

resulting supernatant was used for the assay of the

enzymes. Total SOD activity was assayed by monitoring

the inhibition of reduction of nitro blue tetrazolium

(NBT) according to the method of Giannopolitis and

Ries (1977). A 3ml reaction mixture contained

50mM potassium phosphate buffer (pH 7.8), 13mM

methionine, 75mM NBT, 2mM riboflavin, 0.1mM

EDTA, and 100 ml of enzyme extract. The reaction

mixture was illuminated for 20min at a light intensity of

5000 mmol photon m�2 s�1. One unit of SOD activity

was defined as the amount of enzyme required to cause

50% inhibition of NBT reduction monitored at 560 nm.

CAT activity was determined by measuring the con-

sumption of H2O2 (extinction coefficient

39.4mM�1 cm�1) at 240 nm for 3min (Aebi, 1984).

The reaction mixture contained 50mM potassium

phosphate buffer (pH 7), 10mM H2O2, and 200ml ofthe enzyme extract in a 3ml volume. APX activity was

determined by measuring the decrease in absorbance at

290 nm (A290) (extinction coefficient 2.8mM�1 cm�1) for

1min in 1ml reaction mixture containing 50mM

potassium phosphate buffer (pH 7), 0.5mM ASC,

0.1mM H2O2, and 200 ml of enzyme extract. The

reaction was started by adding enzyme extract.

Corrections were made for low, non-enzymatic

oxidation of H2O2 (Nakano and Asada, 1981). GR

activity was determined by measuring the oxidation

of NADPH at 340 nm (extinction coefficient

6.2mM�1 cm�1) for 3min in 1ml of assay mixture

containing 50mM potassium phosphate buffer

(pH 7.8), 2mM Na2EDTA, 0.15mM NADPH,

0.5mM GSSG (glutathione oxidized) and 200 ml of

enzyme extract. The reaction was initiated by adding

NADPH. Corrections were made for the background

absorbance at 340 nm without NADPH (Schaedle and

Bassham, 1977).

2.7. Assay of glutathione

Glutathione was estimated by 5,50 dithiobis-(2-nitro-

benzoic acid) (DTNB)- glutathione reductase coupled

assay as described in Anderson (1985). Cells were

harvested and resuspended in 5% sulphosalicylic acid

and vortexed vigorously for 5min. The extract was then

centrifuged and the supernatant was assayed to deter-

mine the glutathione content.

2.8. Assay of ascorbate

Ascorbate was measured as per the method of Keller

and Schawger (1977). A. doliolum pellet was sonicated in

ice- cold extracting buffer containing 0.25M oxalic acid

and 1mM EDTA. This was centrifuged at 6000� g for

15min. The supernatant was mixed with 5ml of

20mgml�1 DCPIP. The absorbance was measured at

520 nm. Ascorbic acid content was calculated with the

help of a standard curve.

2.9. Assay of a-TOC

a-TOC was extracted as per Munne-Bosch et al.

(1999). The pellet was sonicated in 5ml ice-cold

methanol containing 1% ASA. a-TOC was extracted

in 4ml hexane by vigorous mixing for 2min. After

centrifuging the samples at 1500� g for 20min, the

upper hexane layer was carefully removed and evapo-

rated to dryness under vacuum. The dried hexane

extract was dissolved in 2ml methanol and injected in

HPLC in a 10mM column (300� 3.9mm, C-18 column,

Waters Chromatography Division, CAT No.27324,

USA) and detected at 295 nm. Pure7a-TOC was used

as a standard.

2.10. Statistical analysis

Results were statistically analyzed using a two-way

ANOVA, followed by Duncan’s new multiple

range tests and correlation coefficients (r). The

numbers of independent variables for each experiment

were three.

3. Results

3.1. Peroxide and MDA content

Fig. 1 summarizes the extent of oxidative damage

measured in terms of H2O2 and MDA content in A.

doliolum subjected to different temperatures (43, 48, 53

and 58 1C) for time intervals of 1, 2, 3 and 4 h. H2O2

content showed an increase of 2.3, 2.8, 2.9 and 4.2-fold

as compared to control after 1 h treatment. The time

course data showed a similar trend, with maximum

increase after 4 h treatment at 58 1C, this being 4.9- fold

as compared to control value (Fig. 1). In tune with

H2O2, the MDA content registered increase of 1.2, 1.4,

2.2 and 4.0-fold at 43, 48, 53 and 58 1C respectively as

compared to control after 1 h treatment. Further, with

increase in time from 1 to 4 h an increase of 1.1–1.3,

1.4–2.2, 2.2–3.4 and 4.0–8.7fold was observed at 43, 48,

53 and 58 1C, respectively (Fig. 1).

ARTICLE IN PRESSµm

ol m

g-1

prot

ein

0.0

0.2

0.4

0.6

0.8

1.0 1 h2 h3 h4 h

nmol

MD

A m

g-1

prot

ein

0.0

0.1

0.2

0.3

0.4

0.5

43°C 48°C 53°C 58°CControl

(A)

(B)

Fig. 1. Effect of different temperature on (A) peroxide content

and (B) MDA content over a time period of 4 h in A. doliolum.

Y. Mishra et al. / Journal of Thermal Biology 30 (2005) 524–531 527

3.2. Enzyme activities

Of the different enzymes involved in the antioxidative

defense system SOD activity showed increase of

2.1%, 5.3%, 5.8% and 11.7% after 1–4 h treatments at

43 1C as compared to control. However, on further

increase of temperature from 48 to 58 1C the SOD

activity showed a continuous decline, and maximum

inhibition of 89% after 4 h treatment at 58 1C was

found. Likewise, CAT activity depicted a 4-fold

increase after 1 h treatment at 43 1C followed by a

continuous decline in its activity both with increase

in temperature as well as time. A 3 and 4 h treatment

at 53 1C and 58 1C produced a decrease in its

activity by 4.8%, 14%, 13% and 24%, respectively as

compared to control. Further, Fig. (2) showed an

increase in APX activity by 6.0–6.5 fold at 43 1C

followed by a continuous decline in its activity. GR

activity showed an increase of 49%, 27%, 12% and 2%

after 1 and 2 h of treatment at 43 and 48 1C, respectively.

Thereafter, a continuous decline in its activity was

observed with a maximum decline of 83% at 58 1C after

4 h (Fig. 2).

3.3. Antioxidant contents

Unlike the enzymes, antioxidants showed continuous

increase with increase in temperature as well as duration

of treatment. An increase of 1.55, 2.72, 2.80 and 2.85

fold in ASC content was observed after 1 h treatment at

43, 48, 53 and 58 1C, respectively. Further, 1.48, 1.85,

1.96 and 2.7 fold increase in GSH content was observed

at 43, 48, 53 and 58 1C, respectively, after 1 h treatment.

Like GSH, ASC and a-tocopherol contents showed a

rise of 4.3, 4.4, 4.5 and 6.0, fold respectively at 43, 48, 53

and 58 1C after 4 h. Both GSH and a-TOC showed time

dependent increase in their contents for each tempera-

ture (Table 1a–c) examined.

3.4. Oxygen evolution

The O2 evolution was measured for each temperature

and time of treatment. At 43 1C, O2 evolution showed an

increase of 1.25-fold after 4 h of treatment over the

control (Table 1d). At 53 1C and 2 h treatment the extent

of O2 evolution was approximately equal to that of

control.

4. Discussion

The results of this study showed a linear relationship

between temperature and oxidative stress measured in

terms of H2O2 and MDA content. Both these para-

meters showed a significant increase with increase in

temperature and duration of treatment (Fig. 1). Increase

in H2O2 could be due to the decline in activities of

enzymes APX and CAT, which are scavengers of H2O2.

Increase in H2O2 level in response to heat stress has also

been reported in mustard (Dat et al., 1998a), and tomato

(Rivero et al., 2004). However, increased lipid peroxida-

tion could be due to increased ROS. Once initiated by

ROS, lipid peroxidation proceeds via chain reaction.

Further, polyunsaturated fatty acids (PUFA), present in

the thylakoid membranes of cyanobacteria are suscep-

tible to oxidative damage (He and Hader, 2002) and

heat stress is known to increase its content (Mikami and

Murata, 2003). Increase in lipid peroxidation under heat

stress is in accordance with reports of Gong et al. (1997)

in maize and in A. palustris by Liu and Huang (2000).

All the measured enzymes, SOD, CAT, APX, and GR

showed an increase in their activity at 43 1C there after

the activity declined gradually. This could be because (1)

each enzyme has an optimum temperature for its

activity, (2) heat shock is known to suppress translation

of many proteins except HSPs (Vierling, 1991) and

enzymes of the antioxidative defense system are not HSP

and (3) the presence of OHd

radical can modify

proteins, disturbing their catalytic activities and making

them more susceptible to proteolytic attack (Casano

ARTICLE IN PRESSU

SO

D m

g-1

prot

ein

0

5

10

15

20

25

1h

2h

3h

4h

µmol

min

-1 m

g-1

prot

ein

µm

ol m

in-1

mg-

1 pr

otei

n

0

50

100

150

200

Control 43°C 48°C 53°C 58°C

µmil

min

-1 m

g-1

prot

ein

0

10

20

30

40

50

60

Control 43°C 48°C 53°C 58°C

(A)

(C) (D)

(B)

Fig. 2. Effect of different temperature on (A) SOD, (B) CAT, (C) APX and (D) GR activities over a time period of 4 h in A. doliolum.

Y. Mishra et al. / Journal of Thermal Biology 30 (2005) 524–531528

et al., 1994). Decline in SOD could be due to increased

H2O2 content (Fig. 1), which inactivates Cu/Zn as well

as Fe-SOD (Charles and Halliwell, 1980). Among the

enzymes APX emerged as the most robust and resistant

to temperature stress. This may be due to fact that H2O2

acts as systemic signal for the increased activity of APX,

but increased temperature does not provide an optimal

condition for its full activity so APX will neither be

completely inhibited nor show maximal induction.

These results support our hypothesis that under high

temperature stress enzymes of the antioxidative defense

system should show decrease in their activities.

Notwithstanding above all the tested non-enzymatic

components of the antioxidative defense system (ASC,

GSH and a-TOC) showed an increase in their content

with increasing temperature (Tables 1a–c) thus support-

ing our hypothesis that when enzyme activities are

inhibited, the non-enzymatic components of antioxida-

tive defense system could play a major role. Further, the

significant increase in GSH content supports our second

hypothesis that GSH accumulation may counteract

oxidative damage caused by heat stress in A. doliolum.

This increase could also be explained in the light of the

report of Sugiyama et al. (2000) that expression of gsh1

and gsh2 genes was enhanced during heat stress in S.

cerevisiae. Further increase in GSH level appears

justified due to its role in gECS, which is required for

the synthesis of reduced glutathione (GSH), which is

ARTICLE IN PRESS

Table 1

Effect of different temperature on ASC content, GSH content, a-tocopherol content, and O2-evolution over a time period of 4 h in A.

doliolum

1 h 2 h 3 h 4 h

(a) ASC content (mgmg�1 protein)

Control(1C) 39.0070.009a 39.0070.009a 39.0070.009a 39.0070.009a

43 60.5070.004a,b 61.2870.005a 91.5270.006b 117.6470.002b

(1.55) (1.57) (2.34) (3.0)

48 106.3770.007c 110.3470.005b 112.0370.008b,c 136.1870.009b

(2.72) (2.8) (2.87) (3.56)

53 109.3970.004a,b 110.8470.003b 123.6570.003c 139.2570.006b

(2.8) (2.84) (3.1) (3.57)

58 111.36710.002b 134.2770.004b 134.8470.001c 140.9370.002b

(2.85) (3.44) (3.45) (3.61)

(b) GSH content (nmolmg�1 protein)

0.55470.001a 0.55470.001a 0.55470.001a 0.55470.001a

43 0.82270.005b 0.83470.002b 0.9770.009b 1.0070.002b

(1.48) (1.50) (1.75) (1.80)

48 1.03070.001c 1.11070.001c 1.1270.003c 1.2270.001c

(1.85) (2.00) (2.02) (2.20)

53 1.09070.001c 1.16070.001d 1.2470.001d 1.2870.001d

(1.96) (2.09) (2.23) (2.31)

58 1.50070.001d 1.60070.001c 1.6470.001e 1.6770.002e

(2.70) (2.80) (2.96) (3.01)

(c) a-tocopherol content (mmolmg�1 protein)

0.27170.001a 0.27170.001a 0.27170.001a 0.27170.001a

43 0.39070.005 0.44070.002b 0.70070.009 1.15070.002b

(1.4)b (1.6) (2.5) (4.25)

48 0.47070.001c 0.48070.001b 0.92070.001c 1.19070.001c

(1.7) (1.77) (3.4) (4.4)

53 0.58070.001d 0.62070.001c 0.98070.001d 1.22070.001d

(2.1) (2.29) (3.6) (4.5)

58 0.68070.001e 0.84070.001d 1.12070.001e 1.64070.002e

(2.5) (3.1) (4.1) (6.0)

(d) O2-evolution (mmol oxygen evolved /mg protein)

3.270.002 c,b 3.270.002 b 3.270.002 b 3.270.002 c

43 3.270.001 c 3.470.002 c 3.670.001 c 4.070.002 d

(0.0) (1.06) (1.12) (1.25)

48 3.370.003 c 3.470.002 c 3.670.002 c 3.070.001 c

(1.03) (1.06) (1.12) (0.93)

53 3.170.001 b 3.270.001 b 3.070.001 b 2.670.001 b

(0.96) (0.98) (0.94) (0.80)

58 2.370.002 a 1.870.002 a 1.670.00 a 0.1270.00a

(0.70) (0.56) (0.5) (0.036)

All values are mean7SD of three replicates. Values having different letters are significantly different (po0.05). Different analysis was

done for each column (Duncan’s new multiple range Test). Values in parenthesis show fold change over control.

Y. Mishra et al. / Journal of Thermal Biology 30 (2005) 524–531 529

HSP 70 (Kondo et al., 1993). Moreover, since cell can

accumulate GSH to considerably high levels (4mM)

(Hell and Bergmann, 1990) and GSH is an important

OHdscavenger, enhancement in its content is further

justified. Kocsy et al. (2002) have reported such

accumulation in wheat. Increase in ASC content could

be due to the accumulation of H2O2, which triggers APX

induction, but increased temperature does not allow its

efficient functioning thereby leading to accumulation of

ASC. Further, ASC itself acts as antioxidant and

prevents a-TOC depletion during oxidative stress.

Notwithstanding this a 6-fold increase in a-TOC

content of A. doliolum has been observed. The synthetic

pathway of a-TOC requires HPT (homogentisate

phytyltransferase). The activity of this enzyme is known

to be increased in certain abiotic stresses, (DellaPenna

and Collakova, 2003). Thus increase in temperature may

also increase the activity of HPT. Further increased

ARTICLE IN PRESSY. Mishra et al. / Journal of Thermal Biology 30 (2005) 524–531530

pyruvate may help in the increased level of a-TOC

(Sugiyama et al., 2000). A significant rise in a-TOC is

also justified by its role in protecting lipid peroxidation

by deactivating singlet oxygen, superoxide anions, and

reducing fatty acyl peroxy radicals (Fryer, 1992).

To assess the extent of protection offered to the

organism by a-TOC, GSH and ASC, when the enzyme

activities were inhibited, effect of temperature on O2-

evolution was measured. High temperature did not show

perceptible inhibition upto 53 1C (2 h). This can be

explained in the light of protection of PSII and D1

protein by a-TOC (Trebst et al., 2002). Further, a-TOC

is also known to scavenge lipid and lipid peroxy radicals

produced as a result of oxidative damage.

5. Summary

This study demonstrates that at increasing tempera-

ture enzymes playing the role of antioxidants fail to

function efficiently but the other antioxidants (ASC,

GSH, and a-TOC) play a major role (as evident by

insignificant effect on oxygen evolution at high tem-

perature (53 1C)) with a-TOC providing protection to

PSII activity, ASC regenerating the a-TOC and GSH

maintaining the redox state of the cell.

Acknowledgements

Yogesh Mishra and Poonam Bhargava are thankful

to CSIR and UGC for the award of JRF. We are also

thankful to Dr. A. S. K. Sinha and Mr. B. Sharma of IT,

BHU for measuring the a-TOC.

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