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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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