Biologia 67/6: 1119—1128, 2012
Section Botany
DOI: 10.2478/s11756-012-0110-1
Differential responses of hydrogen peroxide, lipid peroxidation
and antioxidant enzymes in Azolla microphylla
exposed to paraquat and nitric oxide
Anjuli Sood1*, Charu Kalra1, Sunil Pabbi2 & Prem L. Uniyal1
1
Department of Botany, University of Delhi, Delhi-110007, India; e-mail: [email protected]
Centre for Conservation and Utilisation of Blue-Green Algae, Indian Agricultural Research Institute, New Delhi-110012,
India
2
Abstract: The present investigation was carried out to decipher the interplay between paraquat (PQ) and exogenously
applied nitric oxide (NO) in Azolla microphylla. The addition of PQ (8 µM) increased the activities of superoxide dismutase
(SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) by 1.7, 2.7, 3.9 and 1.9 folds respectively
than that control in the fronds of Azolla. The amount of H2 O2 was also enhanced by 2.7 times in the PQ treated plants
than that of control. The supplementation of sodium nitroprusside (SNP) from 8–100 µM along with PQ, suppressed the
activities of antioxidative enzymes and the amount of H2 O2 compared to PQ alone. The drop in the activity of antioxidative
enzymes – SOD, GPX, CAT and APX was highest (39.9%, 48.4%, 41.6% and 41.3% respectively) on the supplementation
of 100 µM SNP with PQ treated fronds compared to PQ alone. The addition of NO scavengers along with NO donor in
PQ treated fronds neutralized the effect of exogenously supplied NO. This indicates that NO can effectively protect Azolla
against PQ toxicity by quenching reactive oxygen species. However, 200 µM of SNP reversed the protective effect of lower
concentration of NO donor against herbicide toxicity. Our study clearly suggests that (i) SNP released NO can work both
as cytoprotective and cytotoxic in concentration dependent manner and (ii) involvement of NO in protecting Azolla against
PQ toxicity.
Key words: Azolla; lipid peroxidation; nitric oxide; paraquat; reactive oxygen species
Introduction
The increased use of herbicides in agriculture to enhance yield of agronomically important crops is posing a serious threat to the environment sustainability
and growth of different non-target aquatic organisms
(Mohammad et al. 2010). Their exposure is usually not
limited to the location where they are applied. They enter aquatic ecosystems through surface runoff or heavy
rainfall, and become the most frequent organic pollutant in the aquatic ecosystems. Therefore, it is most
important to conduct extensive research to access the
toxic effects of herbicides on non-target organisms such
as Azolla.
Azolla is a small aquatic fern harbouring nitrogenfixing cyanobacteria, Anabaena azollae in its dorsal
leaf lobe cavities. Because of nitrogen-fixing potential,
the fern have long been applied to paddy fields as nitrogenous biofertilizers to reduce the cost and ill effects caused by chemical nitrogen fertilizers (Sood &
Ahluwalia 2009; Sood et al. 2011). Both Azolla and rice
share similar growth conditions and hence, Azolla becomes an integral component of rice ecosystem (Pabby
et al. 2004; Sood & Ahluwalia 2009).
Weed management is a serious challenge in the
paddy fields. Many herbicides such as bensulfuron
methyl, mefenacet, quinoclamine, simetryn, thiobencarb and paraquat etc. are being applied at different
stages of growth of crop (Kumar et al. 2008; Wu et al.
2010). The sensitivity of non-targeted plants to these
herbicides varies greatly. Aida et al (2005) reported that
rice herbicide (bensulfuron methyl) had a pronounced
effect on relative growth rate of Azolla japonica, Salvina
natans and Lemna minor with an EC50 of 5.0, 0.54, and
10 nM, respectively. Courtis et al (2011) also observed
that Azolla filiculoides and Lemna minor were most
sensitive to exposed herbicide mixture (50% atrazine,
35% isoproturon and 15% alachlor) than that of Ceratophyllum demersum, Elodea canadensis, Myriophyllum spicatum and Vallisneria spiralis.
Among different herbicides, Paraquat (1,1’-dimethyl-4,4’-bipyridinium, PQ) is the most widely used
herbicide in the world because of its greater efficiency
and low cost (Qian et al. 2009a; Moran et al. 2010). It
blocks photosynthesis by accepting electrons from photosystem I (PSI) and causes overproduction of reactive
oxygen species (ROS) which caused alteration of the
main biomolecule classes, leads to structural and func-
* Corresponding author
c 2012 Institute of Botany, Slovak Academy of Sciences
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tional changes in lipids, proteins, chlorophylls, and nucleic acids (Qian et al. 2009a; Moran et al. 2010; Sood et
al. 2011). Holst et al. (1982) observed that bipyridilium
and phenolic herbicides were detrimental to A. mexicana, causing up to 75% reduction in N2 fixation. Singh
& Singh (1988) also reported that PQ at a concentration of 0.1 ppm resulted in partial inhibition of growth
and N2 of Azolla where as, a total suppression of N2 fixation was observed at 1 ppm under field conditions. An
earlier study from our laboratory has shown that Azolla
microphylla survived only at levels up to 2–6 µM of PQ.
The visual signs of toxicity (browning and fragmentation of fronds) appeared after 24 h on exposure to 8 µM
of this herbicide (Sood et al. 2011). The level of oxidative markers (H2 O2 content and lipid peroxidation) and
activity of antioxidant enzymes- SOD, CAT, APX and
GPX was also highest at 24 h in fronds exposed 10 µM
of PQ respectively (Sood et al. 2011).
Nitric oxide (NO) is a signaling molecule which
plays important roles in a variety of physiological processes such as growth, seed germination (Liu et al.
2011), iron availability (Ramirez et al. 2011), caulogenesis and rhizogenesis (Kalra & Babbar 2012). Literature
have shown that application of the NO donor reduces
harmful effects of salinity (Chen et al. 2010), high light
levels (Xu et al. 2010), herbicides (Qian et al. 2009b;
Ferreira et al. 2010) and heavy metals on plants (Moran
et al. 2010; Siddiqui et al. 2011). However, the effect of
NO can either be protective or toxic, depending on its
concentration and on the site of action (Siddiqui et al.
2011). Qian and co-workers (2009b) have shown that
exogenous NO protects Chlorella vulgaris against toxicity of atrazine and glufosinate herbicides by increasing
the transcription of psbC psaB chlB and rbcL genes. In
soybean plants, NO decreased in the availability of substrate for antioxidant enzymes that are essential to protect soybean plants under oxidative stress (Ferreira et
al. 2010). The ability of NO to scavenge ROS and regulate the activity of antioxidant enzymes indicates the
existence of an interplay between NO and ROS. However, there is no comprehensive information available
with respect to the response of Azolla microphylla on
exposure to paraquat and NO.
A. microphylla can tolerate relatively high temperature than thatA. pinnata, an indigenous species and
temperature is one of the major constraints limiting
exploitation of Azolla in paddy fields of Northern region of India. (Pabby et al. 2002). Therefore, the aim
of this investigation was to study (i) the physiological response of PQ treated Azolla microphylla to exogenously supplied different concentrations of sodium
nitroprusside (SNP), a NO donor (ii) involvement of
NO in ameliorating PQ toxicity. To gain a comprehensive view, we have measured the accumulation of
H2 O2 content, lipid peroxidation and activity of oxidative stress related enzymes. We used another NO donor
(a combination of AsA and sodium nitrite, SN) and NO
scavengers- hemoglobin (H) and methylene blue (MB)
to strengthen the role of NO in protection against PQ
toxicity.
A. Sood et al.
Material and methods
Plant material
Azolla microphylla Kaul. was procured from the Centre for
Conservation and Utilisation of Blue Green Algae, Indian
Agricultural Research Institute, New Delhi, India. The fern
was grown vegetatively in nitrogen free chemically defined
Espinas and Watanabe medium (pH 6.5) (Watanabe & Espinas 1979) in polyhouse under natural light and controlled
temperature (25 ± 2 ◦C) for 15 days. Thereafter, the healthy
fronds were surface sterilized with 0.1% (w/v) mercuric chloride for 10 min, and washed with sterile water. Each plastic pot (4”) containing 200 mL of Espinas and Watanabe
medium was inoculated with 500 mg of Azolla. The pot containing Espinas and Watanabe medium alone were served
as control.
Experimental design
The concentration of PQ i.e. 8 µM was selected from our
laboratory earlier work (Sood et al. 2011). The fronds were
subjected to different concentrations of SNP (0–200 µM)
along with PQ. The SNP concentration was ranging from
zero or only PQ to the concentration where SNP response
was negative on the fronds of PQ treated Azolla. Therefore, the first experimental design consisted of (i) control (no
added PQ and SNP) (ii) PQ (8 µM + 0 µM of SNP) (iii)
PQ (8 µM) + SNP (8 µM) (iv) PQ (8 µM) + SNP (50 µM)
(v) PQ (8 µM) + SNP (100 µM) (vi) PQ (8 µM) + SNP
(200 µM), which were arranged in a randomized block design with three replicates at two time intervals (i.e. 24 and
48 h) giving a total of 36 pots. The freshly prepared SNP
used as NO donor. The fronds were harvested at interval of
24 and 48 h for analyses for various biochemical parameters.
From the previous experiment, 100 µM SNP and exposure time of 48 h was selected and applied to next experiment. In the second experiment, we also used a solution
containing 100 µM AsA and 200 µM SN as another NO
donor as reported by Hsu & Kao (2004). To ascertain the
involvement of NO, specific NO scavengers H (Zhang et al.
2009) and MB (Zhao et al. 2007; Kalra & Babbar 2012) were
used alone and along with PQ + NO donors. At the same
time, one hundred µM of sodium nitrate, sodium nitrite and
sodium ferricyanide (F) were used as additional controls. In
this experiment, the pots were again distributed in a complete randomized design with a control (without PQ and
SNP) and nine treatments, each with three replicates (total
30 pots). The treatments consisted of (i) PQ (8 µM + 0 µM
SNP) indicated by PQ (ii) 8 µM PQ + 100 µM SNP (PQ
+ SNP) (iii) PQ + SNP + H (0.1%) (iv) PQ + 100 µM
AsA+ 200 µM SN (PQ +AsA + SN) (v) PQ + AsA+ SN
+ H (0.1%) (vi) PQ + H (0.1%) (vii) PQ + 100 µM sodium
nitrate (PQ + Sod. nitrate) (viii) PQ + 100 µM SN (PQ
+ SN) and (ix) PQ + 100 µM sodium ferricyanide (PQ +
F). The fronds were harvested after 48 h for biochemical
analyses.
In the both experiments, harvested fronds of Azolla microphylla were surface dried on the filter paper and analyzed
for the changes in total protein content, H2 O2 content, lipid
peroxidation and activity of antioxidative enzymes (SOD,
CAT, APX and GPX). All experiments were repeated three
times.
Protein content
The fronds (250 mg) were grounded in 1N NaOH using pestle mortar and centrifuged at 10,000 g for 15 min. The supernatant (1 mL) was taken to determine amount of proteins
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Interplay between paraquat and nitric oxide in Azolla microphylla
employing methodology of Lowry et al. (1951) using bovine
serum albumin as standard.
H2 O2 content and lipid peroxidation assays
For H2 O2 content, fronds (100 mg) were extracted in 5 mL
of TCA (0.1%, w/v) and centrifuged at 12,000 g for 15 min.
Then, 0.5 mL of the supernatant was added to 0.5 ml of
10 mM potassium phosphate buffer (pH 7.0) and 1 mL
of 1 M potassium iodide (KI) solution. The absorbance
of the supernatant was read at 390 nm. Its content was
determined using extinction coefficient 0.28 µM−1 cm−1
and expressed as µmol g−1 FW (Velikova et al. 2000).
Lipid peroxidation was estimated by measuring malondialdehyde (MDA), a major thiobarbituric acid reactive
species (TBARS) and product of lipid peroxidation (Harris & Dodge 1972). Azolla fronds (100 mg) were homogenized in 5 mL of TCA (Trichloroacetic acid) (0.1%, w/v)
and centrifuged for 10 min and 10,000 g. To 1 mL aliquot
of supernatant was added 4 mL of 0.5% thiobarbutaric acid
(TBA) in 20% TCA. The mixture was incubated at 95 ◦C
for 30 min. The reaction was stopped by cooling over ice
and contents centrifuged at 10,000 g for 10 min. The absorbance of the supernatant was recorded at 532 nm. The
value for non-specific absorbance at 600 nm was subtracted.
The amount of TBARS was calculated using extinction coefficient of 155 mM−1 cm−1 and expressed as µmol g−1 FW.
Enzyme assays
For estimation of activity of antioxidant enzymes, 250 mg
of Azolla fronds were homogenized in ice chilled 50 mM of
Na-phosphate buffer (pH 7.8) containing 2% polyvinylpyrrolidine (PVP) in ice cold pestle mortar. The homogenate was
centrifuged at 15, 000 g for 30 min at 4 ◦C. The supernatant
was used for the estimation of activities of all the enzymes
except APX. SOD (EC 1.15.1.1) activity was measured in
terms of its capacity to inhibit photochemical reduction of
nitroblue tetrazolium (NBT) (Giannopolitis & Ries 1977).
Reaction mixture (4 mL) contained 63 µM NBT, 13 mM
L-methionine, 0.1 mM EDTA, 13 µM riboflavin, 0.05 M
sodium carbonate and 0.5 mL enzyme extract. Tubes were
kept under 15 W fluorescent lamps for 15 min at 25 ± 2 ◦C.
Blank A containing the same reaction mixture, was placed
in the dark. Blank B containing the same reaction mixture
except for the enzyme extract was placed in light along with
the sample. The reaction was terminated by turning off the
light. Absorbance of each sample along with blank B was
read against blank A at 560 nm and the difference in percent
color reduction between blank B and the sample calculated.
Fifty percent color reduction was considered as one unit of
enzyme activity and expressed as U min−1 mg−1 protein.
CAT (EC 1.11.1.6) activity was measured as decline in absorbance at 240 nm due to the disappearance of H2 O2 (Cakmak & Marschner 1992). The reaction mixture (2 mL) contained 25 mM phosphate buffer (pH 7.0), 10 mM H2 O2 , and
0.2 mL of enzyme extract. Enzyme activity was calculated
using an extinction coefficient of 39.4 mM−1 cm−1 . One unit
of specific enzyme activity determined the amount necessary
to decompose 1 µmol of H2 O2 min−1 mg−1 protein at 25 ◦C.
GPX (EC 1.11.1.7) activity was calculated in terms of oxidation of guaiacol by measuring increase in absorbance at
470 nm (Egley et al. 1983). The reaction mixture (2 mL)
contained 25 mM phosphate buffer (pH 7.0), 0.05% guaiacol (w/v), 1.0 mM H2 O2 and 0.1 mM EDTA and 0.2 mL
of enzyme extract. The enzyme activity was determined using an extinction coefficient of 26.6 mM−1 cm−1 . One unit
of specific activity for GPX was expressed as amount of
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enzyme which resulted in the formation of 1µM tetraguaiacol min−1 mg−1 protein at 25 ◦C.
APX (EC 1.11.1.11) was determined spectrophotometrically according to the method of Zhou et al. (2005). Azolla
fronds (0.3 g) were homogenized in a pre chilled pestle mortar in 3 mL of 50 mM cool phosphate buffer (pH 7.0) containing 1 mM EDTA. The homogenate was centrifuged at
13,000g for 20 min at 4 ◦C. The supernatant was used to estimate the activity of APX. The 3 mL of reaction mixture
contained 50 mM phosphate buffer (pH 7.0), 0.5 mM AsA,
0.1 mM H2 O2 and 0.1 mL of enzyme extract. APX activity
was calculated by recording the decrease in absorbance of
AsA (extinction coefficient 2.8 mM−1 cm−1 ) with in 1 min
at 290 nm. One unit of specific activity for APX was defined
as amount of enzyme which caused degradation of 1 µM of
AsA min−1 mg−1 protein at 25 ◦C.
Statistical analysis
The data were recorded in triplicate for the various parameters and subjected to one-way ANOVA in accordance with
experimental design (complete randomized block design).
The values are expressed as mean ± standard deviation
(SD) and comparison between the mean values was made by
Post Hoc Tukey HSD (Honestly Significant Different) test
at p ≤ 0.05 using SPSS statistical software version 10.0.
Results
Effect of different concentrations of SNP
As shown in Fig. 1, PQ significantly (p ≤ 0.05) decreased protein content in fronds of Azolla by 37.0%
and 50.6% of control after 24 and 48 h respectively. Exogenous application of different concentrations of SNP
(8–200 µM) did not significantly (p ≤ 0.05) change protein content in fronds of Azolla compared to PQ treated
plants after 24 h. While at 48 h, a gradual increase in
protein content was observed with application of SNP
from 8–100 µM than that of PQ alone. It was maximum
(1.7 fold) in PQ treated fronds on application of 100 µM
of SNP. However, supplementation of 200 µM SNP in
PQ treated fronds, a drop in the protein content was
observed. Since, the application of 8–200 SNP µM did
not also show marked effect on the content of H2 O2 ,
lipid peroxidation and the activity of antioxidant enzymes – SOD, CAT, APX and GPX after 24 h in PQ
treated fronds (Data not shown), only 48h data is presented for next parameters.
Compared to control, PQ application significantly
(p ≤ 0.05) increased (2.2 folds) H2 O2 accumulation in
fronds after 48 h of treatment (Fig. 2a). The addition of
exogenous SNP from 8– 100 µM along with PQ dropped
H2 O2 content, which was minimum (6.72 µmol g−1
FW) at 100 µM SNP. With further increase in concentration of SNP from 100 to 200 µM, a significant (p
≤ 0.05) increase in H2 O2 content was noticed.
Lipid peroxidation was estimated by TBARS concentration. Similar to H2 O2 change, PQ significantly
(p ≤ 0.05) increased TBARS concentration in fronds of
Azolla compared to control after 48 h (Fig. 2b). Supplementation of SNP at equimolar concentration to PQ
decreased TBARS content by 16.6% compared to PQ
treated Azolla. At 50 and 100 µM of SNP, it decreased
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A. Sood et al.
Fig. 1. Effect of different concentrations of exogenous supplemented SNP (NO donor) along with PQ on the total protein content
(µg mL−1 ) at different time intervals.
Fig. 2. Effect of different concentrations of exogenous supplemented SNP (NO donor) along with PQ on the (a) H2 O2 content (µmol g−1
FW) (b) TBARS content (µmol g−1 FW) at 48 h.
further by 25% and 32.1% against PQ treated fronds.
However, addition of 200 µM SNP along with PQ, increased TBARS content compared to 100 µM SNP+
PQ.
The effect of different concentrations of SNP on
antioxidant enzymes of PQ treated Azolla is shown in
Figs 3a–d. All enzymes (CAT, SOD, GPX and APX)
showed stimulation of activities in PQ treated fronds of
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Interplay between paraquat and nitric oxide in Azolla microphylla
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Fig. 3. Effect of different concentrations of exogenous supplemented SNP (NO donor) along with PQ on the (a) CAT activity (µmol of
H2 O2 min−1 mg−1 protein) (b) SOD activity (U mg proteins−1 ) (c) GPX activity (µmol of guaiacol min−1 mg−1 protein) (d) APX
activity (µmol of AsA min−1 mg−1 protein) at 48 h.
Azolla. The exogenous supplementation of 8 µM SNP
along with PQ (8 µM) remarkably reduced activities
of CAT, SOD, GPX and APX by 6.71%, 8.66%, 5.98%
and 6.44% respectively compared to PQ alone or PQ
with no SNP. The supplementation of 50 and 100 µM
SNP along with PQ, further suppressed the activities
of these antioxidative enzymes. However, addition of
200 µM of SNP with PQ, an increase in the activities
of CAT, SOD, GPX and APX was observed than that
of SNP (100 µM) + PQ. The increase was significant
(p ≤ 0.05) only in SOD and APX enzyme activities.
The results showed that 100 µM SNP was most
effective in neutralizing PQ toxicity. However, 200 µM
SNP was noticed to reverse this neutralizing effect of
lower concentration of NO donor in PQ treated fronds
of Azolla. Therefore, in the following experiments, we
applied 100 µM SNP as NO donor.
Effect of exogenous NO donors and NO scavengers
As shown in Fig. 4, PQ application dropped protein
content in fronds of Azolla by 36.2% of control. The exogenous supply of NO donors (100 µM SNP and a combination of 100 µM AsA+ 200 µM SN) along with PQ
markedly elevated the inhibition by 32.6% and 29.1%
compared with PQ alone. The effect of NO was removed
partially by the addition of NO scavengers- H and MB.
However, these scavengers alone with PQ (PQ + H and
PQ + MB) did not able revert PQ toxicity. When compared, the two concentration of H (0.1% and 0.2%) and
MB (100 and 200 µM) did not able to show any significant (p ≤ 0.05) change in the content of proteins of
fronds incubated with NO donors + PQ. For next parameters, only 0.1% of H concentration was used. The
addition of sodium nitrate (PQ + Sod. Nitrate), sodium
nitrite (PQ + SN) and sodium ferricyanide (PQ + F) in
PQ treated fronds also did not markedly affect protein
content.
Levels of H2 O2 increased by 2.2 fold in fronds
treated with PQ compared to the control (Fig. 5a).
The application of both NO donors (PQ + SNP and
PQ + AsA+ SN) decreased H2 O2 accumulation by 55%
and 81.3% than that of PQ alone. The effect exogenous
NO was blocked by the addition of H with NO donors
in PQ treated fronds. Addition of sodium ferricyanide
and sodium nitrate or nitrite had no significant effect
on H2 O2 content under PQ toxicity.
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A. Sood et al.
Fig. 4. The effect of NO donors [SNP (100 µM) and SN+AsA (200 µM + 100 µM)] alone and along with NO scavenger, hemoglobin
(H) and methylene blue (MB) on the protein content in fronds of Azolla microphylla grown in medium without or with 8 µM PQ at
48 h.
Fig. 5. The effect of NO donors [SNP (100 µM) and SN+AsA (200 µM + 100 µM)] alone and along with NO scavenger, hemoglobin
(H) on (a) H2 O2 content (µmol g−1 FW) (b) TBARS content (µmol g−1 FW) in fronds of Azolla microphylla grown in medium
without or with 8 µM PQ at 48 h.
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Interplay between paraquat and nitric oxide in Azolla microphylla
1125
Fig. 6. The effect of NO donors [SNP (100 µM) and SN+AsA (200 µM + 100 µM)] alone and along with NO scavenger, hemoglobin
(H) on (a) CAT activity (µmol of H2 O2 min−1 mg−1 protein) (b) SOD activity (U mg proteins−1 ) (c) GPX activity (µmol of guaiacol
min−1 mg−1 protein) (d) APX activity (µmol of AsA min−1 mg−1 protein) in fronds of Azolla microphylla grown in medium without
or with 8 µM PQ at 48 h.
PQ application increased TBARS content 4.29
times in fronds of Azolla compared to control (Fig. 5b).
The application NO donors (SNP and AsA+SN) reduced TBARS content by 55.5% and 60.3% respectively. The effect of exogenous NO was removed by
the addition of H along with NO donors in PQ treated
fronds of Azolla. The application of sodium ferricyanide
(PQ + F), sodium nitrite (PQ + Sod. Nitrate) and
sodium nitrite (PQ + N) had non-significant (p ≤
0.05%) on the TBARS content.
Activities of CAT, SOD, GPX and APX showed
2.7, 1.8, 3.9 and 1.9 fold increase respectively in the
fronds treated with PQ (8 µM) in comparison to control
(Figs. 6a–d). The exogenous application of NO donors
reduced the activity of all antioxidant enzymes. The
activity of CAT, SOD, GPX and APX was dropped
by 44.0%, 32.9%, 55.9%, 40.5% with supplementation
of SNP and 48.6%, 28.8%, 54.1%, 37.3% with AsA+
SN respectively compare to PQ treated Azolla. The
addition of NO scavenger, H removed the effect of exogenously supplied NO donors in PQ treated fronds
and addition of sodium nitrate or nitrite, sodium fer-
ricyanide and hemoglobin did not able to change the
activities of antioxidative enzymes significantly (p ≤
0.05 %) (Figs 6a–d).
Discussion
ROS such as O−
2 and H2 O2 are constantly generated
as byproducts in chloroplasts, mitochondria and peroxisomes. A tight control exists over the production of
ROS by enzymatic or non-enzymatic mechanisms under
normal conditions in plants (Apel & Hirt 2004). However, different environmental stressors, such as light,
drought, extreme temperature, heavy metals, salinity,
UV radiation, and herbicide treatments drastically enhance the production of these free radicals that can
destroy the organelles, damage the membrane system
and inhibit related gene expression (Qian et al. 2009b;
Sood et al. 2011). Compounds that are able to reduce
the damaging effect of such stresses are of great importance from application view point. NO is one such
compound that can counteract oxidative damage and
displays a protective effect against various stressful conUnauthenticated
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1126
ditions (Hasanuzzaman et al. 2010). Despite of wealth
of information available about the impact of NO on various physiological processes in plants, an in-depth study
is required as per its interplay with different environmental contaminants because of its dual functionality.
The tolerance of plants to ROS requires the adaptation of many complex and multifaceted processes.
ROS-scavenging enzymes such as SOD catalyzes the
.
conversion of O2− to H2 O2 and O2 in the chloroplast,
mitochondrion, cytoplasm and peroxisome (Wang et al.
2011). Other enzymes – CAT, GPX, GSH-Px and APX
are important for regulation of intracellular H2 O2 . CAT
acts in the peroxisomes of cells, and the enzyme GPX
plays a role in the apoplast, chloroplast and cytosol
(Shigeoka et al. 2002). In the chloroplasts, cytosol, mitochondria and peroxisomes, there is an ascorbate – glutathione cycle in which APX plays an important role
in removing H2 O2 when catalyzing the oxidization of
AsA (Pang & Wang 2010). Literature showed that in
plants, PQ treatment causes a significant activation of
APX, GPX, SOD and CAT (Liu et al. 2009; Sood et al.
2011.) Earlier work from our laboratory showed that the
toxicity of PQ in A. microphylla resulted in changes in
the activity of antioxidative enzymes (SOD, CAT, GPX
and APX), which was maximum at 48 h at 8 µM PQ
(Sood et al. 2011). Therefore, the present investigation
is the extension of our earlier work where NO donors
and scavenger were employed to check the involvement
role of NO against PQ induced toxicity.
Results showed that PQ application caused significant accumulation of TBARS content (MDA content)
and the amount of H2 O2, indicating PQ induced stress
in the fronds of Azolla. It is known enhanced production of malondialdehyde during peroxidation of lipids
is used as indicator of stress damage (Ohkawa et al.
1979). Under light, the overproduction of ROS due to
.
PQ generated O2− might have resulted in damage of
lipid membranes and increased MDA levels in Azolla.
Similar changes were also observed in TBARS content
and amount of H2 O2 in Azolla exposed to different levels of PQ (Sood et al. 2011).
The application of lower levels of SNP (8–100 µM)
along with PQ reduced the levels of H2 O2, TBARS content and activity of antioxidant enzymes (SOD, CAT,
GPX, APX) as compared to PQ treated Azolla. However, higher concentration (200 µM) had the opposite effect increasing the amount of H2 O2 , lipid peroxidation and activity of antioxidant enzymes (SOD
and APX). The activity of CAT and GPX was also
increased at higher SNP, but this increase was nonsignificant. Wang et al. (2010) reported that exposure of
Hydrilla verticillata to 25–100 µM SNP (NO donor) decreased H2 O2 and MDA content and activities of CAT
and peroxidase on second day of exposure. However,
effect was opposite at 200–400 µM SNP. They concluded that 25–100 µM SNP increased the growth of
H. verticillata by alleviating the oxidative stress and
200–400 µM SNP might enhance the oxidative stress
in plant cell. Qain et al. (2009b) also observed similar
dual role of NO on ROS metabolism of Chlorella vul-
A. Sood et al.
garis where high SNP had negative effect, decreasing
antioxidant enzymes activity, increasing levels of ROS,
H2 O2, MDA content and the expression of photosynthesis related genes. The different concentrations of SNP
release NO in a concentration dependent manner (Ederli et al. 2009) and the action of generated NO as pro
or antioxidant further depends on the relative production of NO and O−
2 (Hasanuzzaman et al. 2010; Habib
& Ali 2011). In our study, at lower concentrations of
SNP, NO could have detoxified ROS produced by PQ
after reacting with O−
2 thereby generating the peroxynitrite ion (ONOO− ). These peroxynitrite are less toxic
than peroxides, thus limit cellular damage (Hasanuzzaman et al. 2010). These peroxynitrite might have reacted with H2 O2 to yield a nitrite anion and oxygen
(Martinez et al. 2000). This could be the mechanism of
cytoprotective action of NO in Azolla fronds. The observed drop in activity of SOD and CAT at lower SNP
concentrations also indicates the decrease in availability
of superoxide anion free radical O−
2 since SOD converts
O−
radical
to
H
O
,
which
is
later
hydrolyzed by CAT,
2 2
2
producing H2 O and O2 (Ferreira et al. 2010). Our findings were further supported by the observed decrease
in accumulated H2 O2 at these concentrations of SNP
in PQ treated Azolla. The observed drop in TBARS
content was also because of the lesser availability of free
oxygen radical for fatty acid oxidation. NO react with
lipid alcoxyl (LO. ) and lipid peroxyl (LOO. ) radicals
and stop the chain of peroxidation (Beligni & Lamattiana 1999). However at high SNP levels (200 µM), NO
concentrations could provoke a major oxidative insult,
since this molecule is itself a nitrogen reactive species
(Shi et al. 2005) and the peroxynitrite (ONOO− ), produced by the diffusion-limited reaction of NO and superoxide (O−
2 ) oxidise and nitrate DNA, causing DNA
damage. Therefore, the production of reactive nitrogen
oxide species (RNOS) e.g. N2 O3 or ONOO− in PQ
treated Azolla might be the cause of reversal of protective effect of SNP at higher concentration (200 µM).
Dubovskaya et al. (2007) reported that the exogenous
SNP and hydrogen peroxide exerted anatagonostic effects on tobacco leaves at micromolar concentration but
induced synergistic effect at millimolar concentration,
suggesting that NO performs a dual role in plants, acting as antioxidant (scavenger of reactive oxygen species)
and as a signaling messenger.
To strengthen the role of NO in reducing PQ toxicity in Azolla, another NO donor (AsA : SN) and decomposition product of SNP and structural analogue
of SNP (sodium ferricyanide) was also supplemented
with PQ. Our results showed that another NO donor
could also protect Azolla from PQ toxicity where as no
such response was noticed in other treatments. Ferreira
and co-workers (2010) reported that supplementation
of NO donor in herbicide treated soybean plants decrease the availability of ROS, an essential substrate for
the activity of antioxidant enzymes – SOD, CAT and
peroxidases (POD) hence, protect the plants from oxidative stress. Similarly, addition of SNP reduced cadmium (Cd) toxicity in hydroponically grown wheat root
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Interplay between paraquat and nitric oxide in Azolla microphylla
via decreasing lipid peroxidation, H2 O2 content and activity of scavenging enzymes (Singh et al. 2008). Our
findings were further justified, with the reversal protective effect on addition of hemoglobin, resulting in drop
of total proteins, increase in H2 O2 content, lipid peroxidation and activity of antioxidative enzymes. Qian et
al. (2009b) also observed decrease in activities of SOD,
CAT, POD and lipid peroxidation in Chlorella vulgaris
exposed to herbicide combined with NO donor (100µM
SNP) and this drop was reversed when NO scavenger
(cPTIO) was added.
Our study clearly demonstrated that the PQ increased the levels of H2 O2 and lipid peroxidation and
modulated the antioxidative responses in Azolla microphylla. The exogenous NO (up to 100 µM SNP) protects
A. microphylla from PQ induced oxidative stress, causing drop in H2 O2 content, lipid peroxidation and activity of antioxidative enzymes. At 200 µM SNP, exogenous NO protective effect was reversed in PQ treated
plant indicating that SNP released NO can work both
as cytoprotective and cytotoxic in concentration dependent manner.
Acknowledgements
The author (AS) is thankful to Department of Botany, University of Delhi, Delhi and Conservation and Utilisation of
Blue Green Algae, IARI, New Delhi for providing the necessary facilities to carry out this investigation.
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Received October 17, 2011
Accepted June 7, 2012
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