Embed Size (px)
Citation preview
Ecotoxicology and Environmental Safety 85 (2012) 72–81
Contents lists available at SciVerse ScienceDirect
Ecotoxicology and Environmental Safety
0147-65
http://d
Abbre
hyde; T
APX, as
GR, glut
guaiacon Corr
E-m
dr.renu
pkpati@
journal homepage: www.elsevier.com/locate/ecoenv
Mitigation of adverse effects of chlorpyrifos by 24-epibrassinolideand analysis of stress markers in a rice variety Pusa Basmati-1
Isha Sharma a, Renu Bhardwaj b, Pratap Kumar Pati a,n
a Department of Biotechnology, Guru Nanak Dev University, Amritsar 143005, Punjab, Indiab Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar 143005, Punjab, India
a r t i c l e i n f o
Article history:
Received 26 April 2012
Received in revised form
6 July 2012
Accepted 6 July 2012Available online 29 August 2012
Keywords:
Chlorpyrifos
Brassinosteroids
Antioxidant enzymes
Rice
Stress
Insecticides
13/$ - see front matter & 2012 Elsevier Inc. A
x.doi.org/10.1016/j.ecoenv.2012.07.003
viations: CPF, chlorpyrifos; EBL, 24-epibrass
BARS, thiobarbituric acid reactive substance;
corbate peroxidase; CAT, catalase; DHAR, deh
athione reductase; MDHAR, monodehydroas
l peroxidase; ROS, reactive oxygen species
esponding author. Fax: þ91 183 2258272.
ail addresses: [email protected] (I. S
[email protected], renu_bhardwaj@rediffma
yahoo.com (P.K. Pati).
a b s t r a c t
The present paper first time reports the role of 24-epibrassinolide (EBL) in mitigating the adverse
effects of Chlorpyrifos (CPF), a broad spectrum organophosphate insecticide by regulating the
antioxidant defence system in an elite indica rice variety Pusa Basmati-1. It investigates the effect of
CPF (0.02%, 0.04% and 0.06%) and EBL (10–11, 10�9 and 10�7 M) treatments alone and in combination
on rice seedlings. Various growth parameters, protein, proline and malondialdehyde (MDA) content and
activities of antioxidant enzymes of seedlings were analysed. CPF showed an adverse effect on growth
and protein content of seedlings whereas it leads to an enhancement in the level of MDA and proline
content. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate
peroxidase (APX), catalase (CAT), guaiacol peroxidase (GPX) and monodehydroascorbate reductase
(MDHAR) increased after treatment with CPF. Application of different concentrations of EBL along with
CPF resulted in an overall improvement in the growth, level of protein and proline content and in the
activity of various antioxidant enzymes whereas a decline in the levels of MDA content was observed.
The work also investigated the changes at the transcript level of some key antioxidant enzymes like
Cu/Zn-SOD, Fe-SOD, Mn-SOD, APX, CAT and GR. The expression of most of the genes was enhanced in
response to CPF treatment. Application of EBL in conjunction with CPF resulted in a distinct
enhancement in the transcript accumulation of Fe-SOD and CAT showing their important role in EBL
mediated amelioration of CPF induced stress.
& 2012 Elsevier Inc. All rights reserved.
1. Introduction
To provide adequate global food supplies during the time ofrapidly rising population, increasing food demand and decliningnatural resources, is a critical concern for the present century.In order to meet these challenges, it is becoming imperativeto use modern agricultural practices. However, for achievingsustainable agriculture, the ecological considerations in modernagriculture for improvement in crop production are of paramountimportance (Swaminathan, 2006). Pesticides have been the mostrapidly growing input in agriculture to enhance the productionover the last half-century (Ruttan, 2002). These compounds have
ll rights reserved.
inolide; MDA, malondialde-
SOD, superoxide dismutase;
ydroascorbate reductase;
corbate reductase; GPX,
harma),
il.com (R. Bhardwaj),
negative effect on various physio-morphological attributes suchas visible injuries (chlorosis, necrosis, vein discoloration) andreduction in growth and biomass. They cause inhibition ofphotosystems and photosynthetic pigments thereby decreasingthe photosynthetic efficiency and alter nitrogen and/or carbonmetabolism leading to their lower availability for plant growth(Kana et al., 2004). They also hamper the development ofreproductive organs, which greatly damage fruit and seed forma-tion (Saladin and Clement, 2005).
Although, several strategies to replace pesticides have beenevolved including use of biocontrol agents (Pandya and Saraf,2010) and genetically modified crops (Channapatna, 2001) butsuch techniques are not extensively employed in agriculture.In modern agriculture, chemical pesticides are still an effectivemethod for controlling destructive biological agents and areimportant in crop production. Brassinosteroids (BRs) a class ofubiquitously present plant-specific steroid hormones that areessential for plant growth and development have received muchattention for their wide range of applications in agriculture(Gudesblat and Russinova, 2011). They bind to a small family ofleucine-rich repeat receptor kinases (BRI1) at the cell surface,thereby initiating an intracellular signal transduction cascade that
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–81 73
results in the altered expression of hundreds of genes which areinvolved in diverse functions (Clouse, 2011). Recently, the exo-genous application of BRs has been implicated for a range of bioticand abiotic stress (Vriet et al., 2012) and the BRs inducedtolerance against a variety of stresses have been linked withreactive oxygen species (ROS) scavenging mechanism (Choudharyet al., 2012).
Accumulation of ROS after abiotic stress is a common phe-nomenon which affects many cellular functions by damagingproteins, lipids, carbohydrates, DNA and ultimately results inoxidative damage (Lee and Park, 2012). The molecular responsesof plants to abiotic stress are complex processes encompassingreprogramming metabolism, maintaining the delicate balancebetween production and removal of reactive oxygen species,modulation of transcriptional activity of stress related genesand gaining a new equilibrium between growth, developmentand survival (Mazzucotelli et al., 2008). Oxidative stress elicits theROS scavenging machinery such as SOD, CAT, APX which work inconcert with each other to control the cascades of uncontrolledoxidation and protect plant cells from oxidative damage byscavenging of ROS (Sharma et al., 2012).
In spite of many studies conducted on stress ameliorationproperties of brassinosteroids, very little is known about themechanisms by which BR controls plant stress responses andregulates the expression of stress response genes (Divi et al.,2010). In the present study, for the first time a multidimensionalassessment of one of the most widely used organophosphateinsecticides, chlorpyrifos, was conducted on seedlings of an eliteindica rice variety Pusa Basmati-1 and the effect of exogenousapplication of 24-epibrassinolide (EBL) alone and in conjunctionwith CPF was also evaluated. The gene expression study reveals adistinct role of Fe-SOD and CAT in BRs induced stress response inrice against CPF stress.
2. Materials and methods
2.1. Plant material
Rice seeds (Oryza sativa L. var. Pusa Basmati-1 cv. indica) were procured from
Indian Agricultural Research Institute, New Delhi, India. Healthy seeds were
dehusked and were surface sterilised with 70% alcohol and 0.4% sodium hypo-
chlorite with tween-20 as a surfactant for 30 min followed by repeated washings
with distilled water.
2.2. Chemical preparation, treatment and growth conditions
EBL was obtained from Sigma Chemicals, USA. A stock solution of 10�4 M EBL
was prepared in ethanol. The working concentration of EBL (10–11, 10�9 and
10�7 M) was prepared by diluting the stock with distilled water. The surface
sterilised seeds were then soaked for 8 h in distilled water (control) and in
different concentrations of EBL (10–11, 10�9 and 10�7 M).
Commercial formulation of chlorpyrifos (O,O-diethyl-0-(3,5,6-trichlor-2-pyridyl)
phosphorothioate; 20% active ingredient, Hyderbad chemical Ltd., India) was diluted
with distilled water to make working concentrations of 0.02%, 0.04% and 0.06%.
These concentrations were chosen on the basis of 50% inhibitory concentration
(IC50), which was determined to be 0.04% CPF. After 8 h of EBL treatment, seeds
were sown in autoclaved sand moistened with different concentrations of CPF
(0.02%, 0.04% and 0.06%) in plastic boxes of dimensions 26 cm�17.5 cm. Each
treatment was replicated 3 times. Box of each concentration containing 100 seeds
were allowed to germinate under controlled conditions; 25 1C (day/night), 70–80%
RH (day/night) and 14 h photoperiod. After 12 day, samples were collected to assess
the following parameters.
2.3. Growth analysis
12 day old seedlings were removed from the boxes and were dipped in water
to remove adhering sand particles. A representative lot of 30 seedlings per
treatment were used for morphological analysis. Root and shoot length were
measured using metre scale and observations for fresh weight of seedlings were
made. Root number of the seedlings was also recorded. The seedlings were then
placed in an oven at 70 1C till a constant weight was achieved (Sharma et al., 2011)
and then were weighed to record the seedling dry mass after cooling them to
room temperature.
2.4. Chlorophyll content
Chlorophyll content estimation was done according to the procedure given by
Arnon (1949) with some modifications. Fresh leaves (100 mg) were taken from
each of the samples and were homogenised in liquid nitrogen. 1.5 ml of 80%
acetone was added to it and the reaction was incubated in dark for 1 h followed by
centrifugation at 15,000 rpm for 3 min. Absorbance was measured spectrophoto-
metrically at 645 and 663 nm against 80% acetone as blank. The chlorophyll
content was determined as follows:
Total Chl mg g FW�1� �
¼ 20:2 A645ð Þþ8:02 A663ð Þ � volume=1000� �
�weight of tissue
Chl A mg g FW�1� �
¼ 12:7 A663ð Þ22:29 A645ð Þ � volume=1000� �
�weight of tissue
Chl B mg g FW�1� �
¼ 22:9 A645ð Þ24:68 A645ð Þ � volume=1000� �
�weight of tissue:
2.5. Protein content
Fresh seedlings treated with or without EBL and CPF were harvested after
12 day and frozen in liquid nitrogen and stored at �80 1C. For protein extraction,
seedlings were homogenised in ice chilled 50 mM phosphate buffer (pH-7.8)
containing 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.5% (v/v) Triton X-100 and 10%
(w/v) PVP-40. The homogenate was centrifuged at 12,000 rpm for 20 min, super-
natant was collected and used for protein estimation and various enzyme assay.
The extraction procedure was carried out at 0–4 1C. Protein concentration for
various samples was determined by bradford assay (Bradford, 1976) using protein
estimation kit (Genei, India). Bovine serum albumin (BSA) was used as a standard
and the concentration was determined by plotting a standard curve between
known concentrations of BSA and their respective absorbance.
2.6. Proline content
Proline content in the seedlings was determined by following the method of
Bates et al. (1973). 0.5 g of fresh seedlings were homogenised in liquid nitrogen
and were extracted with 10 ml of 3% sulphosalicylic acid. The extract was then
centrifuged at 12,000 rpm for 15 min. 2 ml of supernatant is taken and to it an
equal volume of both glacial acetic acid and acid ninhydrin solutions were added.
Mixture was heated at boiling water bath for 1 h and reaction was put to an end
on ice. 4 ml of toluene was added and the absorbance of the toluene layer, when
separated from the aqueous layer, was measured spectrophotometrically at
520 nm. Amount of proline was calculated from the standard curve and expressed
as mmoles g�1 FW.
2.7. Lipid peroxidation
Extent of lipid peroxidation was estimated by measuring the malondialdehyde
(MDA) equivalents according to the method described by Hodges et al. (1999).
Seedlings (1 g) were homogenised with a mortar and pestle in 3 ml of 0.1% TCA
kept at 4 1C and then 3 ml of solution containing 0.5% TBA and 20% TCA was added.
The mixture was incubated at 95 1C for 30 min and then placed in ice to stop
the reaction. The samples were centrifuged at 10,000 rpm for 15 min and the
absorbance of the supernatant was measured at 532 nm and corrected for
non-specific absorbance at 600 nm. MDA concentration was calculated using
extinction coefficient of 155 mM–1 cm–1.
2.8. Antioxidant enzyme assay
2.8.1. Superoxide dismutase SOD (EC 1.15.1.1)
The activity of SOD was determined by monitoring the inhibition of photo-
chemical reduction of nitroblue tetrazolium (NBT), as described by Beauchamp
and Fridovich (1971). The reaction mixture (1 ml) contained 50 mM potassium
phosphate buffer (pH 7.8), 2 mM riboflavin, 75 mM NBT, 13 mM DL methionine,
100 mM EDTA and enzyme extract (50 ml). The reaction was initiated by illuminat-
ing the reaction mixture at 4000 lx- light intensity for 20 min at 25 1C and
absorbance was read at 560 nm. Identical tubes which were not illuminated
served as blank while those containing 50 ml of 50 mM phosphate buffer (pH 7.8)
in place of the enzyme extract served as positive control. One unit of activity was
Ta
ble
1E
ffe
cto
fE
BL
on
sho
ot
len
gth
,ro
ot
len
gth
an
dn
um
be
ro
fro
ots
of
12
-da
ys
old
Ory
zasa
tiv
ase
ed
lin
gs
un
de
rC
PF
stre
ss.
Da
tare
pre
sen
tsm
ea
n7
SE
(n¼
15
).D
iffe
ren
tle
tte
rs(a
,b,c
,d)
wit
hin
va
rio
us
con
cen
tra
tio
ns
of
CP
F(0
,0
.02
%,
0.0
4%
an
d0
.06
%)
are
sig
nifi
can
tly
dif
fere
nt
(Fis
he
rLS
D,
pr
0.0
5)
wh
ere
as
dif
fere
nt
lett
ers
(p,q
,r,s
)w
ith
inv
ari
ou
str
ea
tme
nts
of
EB
L(0
,1
0�
11,
10�
9a
nd
10�
7M
)a
resi
gn
ifica
ntl
yd
iffe
ren
t(F
ish
er
LSD
,pr
0.0
5)
an
dsi
gn
ify
inte
ract
ion
so
fd
iffe
ren
tco
nce
ntr
ati
on
so
fE
BL
wit
hC
PF
on
gro
wth
pa
ram
ete
rs.
CP
F(%
)S
ho
ot
len
gth
(cm
)R
oo
tle
ng
th(c
m)
Ro
ot
nu
mb
er
0M
EB
L1
0�
11
ME
BL
10�
9M
EB
L1
0�
7M
EB
L0
ME
BL
10�
11
ME
BL
10�
9M
EB
L1
0�
7M
EB
L0
ME
BL
10�
11
ME
BL
10�
9M
EB
L1
0�
7M
EB
L
01
3.7
17
0.3
8a
,p1
3.9
37
0.8
1a
,p1
4.8
97
0.1
0a
,pq
15
.677
0.1
0a
,q7
.817
0.8
7a
,p8
.497
0.4
0a
,pq
9.4
87
0.5
9a
,qr
10
.027
0.3
6a
,r5
.537
0.1
8a
,p5
.937
0.1
8a
,q6
.607
0.4
0a
,p6
.007
0.2
0a
,p
0.0
21
0.0
67
0.4
0b
,p1
0.6
77
0.6
3b
,p1
2.2
67
0.9
0a
,q1
1.9
67
0.7
3b
,qp
6.5
67
0.8
3a
,p8
.197
0.2
3a
,q8
.977
0.5
3a
,q8
.817
0.3
3b
,q4
.207
0.3
5b
,p4
.677
0.2
4b
,p5
.007
0.3
5b
,pq
5.5
37
0.2
9a
,q
0.0
46
.717
0.3
8c,p
6.9
17
0.9
5c,p
8.6
37
0.9
0b
,q9
.127
0.4
9b
,q4
.357
0.1
4b
,p6
.917
0.9
5b
,q8
.637
0.9
0a
,r9
.127
0.4
9a
,r3
.137
0.2
8c,p
3.5
37
0.4
3c,p
q4
.207
0.3
0b
,q4
.277
0.1
4b
,q
0.0
63
.207
0.2
5d
,p4
.047
0.4
2d
,pq
4.4
07
0.2
4c,p
q4
.997
0.4
5c,q
2.8
37
0.3
8b
,p3
.777
0.6
9c,p
q4
.027
0.4
9b
,pq
4.8
37
0.4
8c,q
2.1
37
0.0
7d
,p2
.337
0.2
4d
,p2
.477
0.0
7c,p
2.6
77
0.3
7c,p
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–8174
determined as amount of enzyme required to inhibit the photoreduction of NBT to
blue formazan by 50% and was expressed as SOD units mg protein�1.
2.8.2. Ascorbate peroxidase APX (EC 1.11.1.11)
Ascorbate peroxidase activity was determined at 290 nm by following the rate
of oxidation of ascorbate (e of ascorbate¼2.8 mM–1 cm–1) observed spectro-
photometrically at 25 1C (Nakano and Asada, 1981). The reaction mixture (1 ml)
consisted of 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM
EDTA, 0.5 mM ascorbate and 0.1 mM H2O2. The reaction was initiated by addition
of 10 ml of the enzyme extract in a quartz cuvette. One unit of enzyme activity was
calculated as the amount of enzymes required to oxidise 1 mmole of ascorbate
mg protein�1 min�1.
2.8.3. Catalase CAT (EC 1.11.1.6)
The specific activity of catalase was measured by following the method of Aebi
(1984). The reaction mixture (1 ml) contained 20 ml of enzyme extract, 10 mM
H2O2 in 50 mM phosphate buffer (pH-7). CAT activity was estimated at 25 1C by
following the decrease in absorbance of H2O2 at 240 nm and was expressed as
mmole of H2O2 decomposed mg protein�1 min�1 (e¼39.4 mM�1 cm�1).
2.8.4. Guaiacol peroxidase GPX (EC 1.11.1.7)
Guaiacol peroxidase activity was determined by following the increase in
absorbance due to oxidation of guaiacol at 25 1C (FernaaNdez-Garciaa et al., 2004).
The total reaction volume of 1 ml comprised of 50 mM phosphate buffer (pH-7),
9 mM guaiacol, 10 mM H2O2 and 33 ml of enzyme extract. Enzyme activity was
expressed as the amount of enzyme required to produce 1 mmol of GDHP mg
protein�1 min�1 (e¼26.6 mM�1 cm�1).
2.8.5. Glutathione reductase GR (EC 1.6.4.2)
Glutathione reductase activity was assayed at 25 1C as per the method of
Jahnke et al. (1991) by tracking the decrease in absorbance at 340 nm due to the
oxidation of NADPH. The reaction in a 1 ml mixture containing 50 mM phosphate
buffer (pH 7.8), 1 mM EDTA, 1 mM GSSG and 25 ml of enzyme sample was initiated
by addition of 0.1 mM NADPH (e of NADPH¼6.22 mM�1 cm�1). Enzyme activity
was expressed as mmol of NADPH oxidised mg protein�1 min�1.
2.8.6. Dehydroascorbate reductase DHAR (EC 1.8.5.1)
Specific activity of DHAR was determined according to the method of Nakano
and Asada (1981) by measuring the increase in absorbance at 265 nm
(e¼14 m�1 cm�1) at 25 1C due to formation of ascorbate from dehydroascorbate
using the reducing power provided by GSH. The reaction mixture contained
50 mM phosphate buffer (pH¼7), 0.1 mM EDTA, 0.5 mM dehydroascorbate,
2.5 mM GSH and 25 ml enzyme extract in a total reaction volume of 1 ml. One
unit of enzyme activity was calculated as the amount of enzymes required to
produce 1 mmol of ascorbate mg protein�1 min�1.
2.8.7. Monodehydroascorbate reductase MDHAR (EC 1.6.5.4)
Method proposed by Hossain and Asada (1985) was used to determine the
enzyme activity of MDHAR. The method was based on measuring the decrease in
absorbance due to consumption of NADPH at 340 nm (e¼6.2 mM�1 cm�1) at
25 1C. The reaction mixture contained 50 mM Tris–HCL Buffer (pH-7.6), 0.15 units
ascorbate oxidase enzymes, 2.5 mM ascorbic acid and 0.2 mM NADPH/NADH
making a total volume of 1 ml. One unit of enzyme activity is described as the
amount of enzyme required to oxidise 1 mmol of NADPH mg protein�1 min�1.
2.9. Gene expression of antioxidant enzymes
Total RNA was extracted from 12-day old rice seedlings using Trizol reagent
(Invitrogen; www.invitrogen.com) according to the manufacturer’s instructions.
Residual DNA was removed from the total RNA by treating it with DNase I and
subsequent purification. RNA quality and quantity were assessed by RNA agarose
gel electrophoresis and spectrophotometric detection at 260 nm, respectively.
A total of 3 mg of RNA was used as template for reverse transcription using the
oligo (dT)18 primer and Super Script First Strand Synthesis System for RT-PCR
(Invitrogen) following the manufacturer’s recommendation. Gene expression was
studied using semi-quantitative reverse transcriptase polymerase chain reaction
in a 50 ml reaction volume that included 1 ul of cDNA template. The PCR
parameters were; predenaturation at 94 1C for 4 min, followed by 35 cycles of
94 1C for 1 min, 55 1C for 1 min, 72 1C for 1 min, with a final extension step of 72 1C
for 7 min. Gene-specific primers for various enzymes are given in Table 4. Rice
elongation factor, EF1-a was amplified as the internal control. All PCRs were
repeated using at least three independent samples and product intensities were
confirmed by separation on 1.2% agarose gel containing ethidium bromide. Images
were captured by Alpha Innotech, AlphaImager digital imaging system and the
relative amount of transcripts in a single-PCR reaction were determined using
Apha 2000TM Imager Analyser and Image Quant QL software, using the integrated
density value (IDV) for each band.
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–81 75
2.10. Statistical analysis
All data obtained were subjected to two-way analysis of variance (ANOVA) for
studying the interaction of CPF with EBL and expressed as mean7SE of three
replicates. The Fisher’s LSD test was applied for multiple comparisons using
Sigmastat version 3.5 and significance of difference between CPF and EBL
treatment was set at pr0.05.
3. Results
3.1. Analysis of growth parameters
Adverse effects of CPF on growth parameters and the signifi-cance of application of EBL alone and its co-treatment with CPF ispresented in Table 1. A continuous decline in shoot length (cm)was observed with the application of increasing concentration ofCPF (0.02, 0.04, and 0.06%). At the highest concentration of CPF(0.06%), shoot length decreased to 76% (3.270.25) as comparedto control seedlings (13.770.38). Application of different concen-trations of EBL (10�11, 10�9, 10�7 M) to samples growing indistilled water led to a significant enhancement in the shootlength. The binary combination of EBL and CPF was effective inenhancing the shoot length of seedlings. Among CPF stressedseedlings, the maximum increase in shoot length (9.1270.49)was recorded for the combination of 10�7 M EBLþ0.04% CPFas compared to the seedlings growing in 0.04% CPF alone(6.7170.38). Similar decline in the root length (cm) of theseedlings was observed on application of different concentrationsof CPF. Application of 0.04% CPF led to a sharp decline in rootlength while maximum decline in the value was observed at0.06% CPF (2.8370.38) as compared to control seedlings(7.8170.87). On supplementation of seeds with different con-centrations of EBL, a significant improvement in root length wasnoticed. The highest value of root length (10.0270.36) wasobserved for seeds supplemented with 10�7 M EBL and thengrown in distilled water (DW) as compared to control seedlings.Seeds pre-soaked with 10�7 M EBL and grown in 0.06% CPFdemonstrated the maximum increase of 70% (4.8370.48) in rootlength over 0.06% CPF alone (2.8370.38). The root number of riceroots also showed a continuous decrease with the increase in theconcentration of CPF. Significant enhancement in the root numberwas observed on supplementation of higher concentrations of EBLto both stressed and control samples. Maximum enhancement(31%) in the root number was observed for samples treated with10�7 M EBLþ0.04% CPF (4.2770.14) as compared to 0.04% CPF(3.1370.28) alone. Values for fresh and dry weight (g/seedling)were also observed to fall significantly on the application of CPF(Table 2). Maximum decline in fresh weight (0.020370.002)was observed in seedlings growing in 0.06% CPF as comparedto control seedlings (0.080770.002). Application of differentconcentrations of EBL to seeds growing in water and pesticidesolution resulted in seedlings with enhanced fresh weight.Maximum value for fresh weight (0.0970.001) was observed in
Table 2Effect of EBL on fresh weight and dry weight of 12-days old Oryza sativa seedlings und
various concentrations of CPF (0, 0.02%, 0.04% and 0.06%) are significantly different (Fish
(0, 10�11, 10�9 and 10�7 M) are significantly different (Fisher LSD, pr0.05) and signi
CPF (%) Fresh weight (g/seedling)
0 M EBL 10�11 M EBL 10�9 M EBL 10�7 M EBL
0 0.0870.002 a,p 0.08670.002 a,q 0.09070.001 a,q 0.08770.001 a,q
0.02 0.06170.002 b,p 0.06370.002 b,p 0.07370.003 b,q 0.07570.001 b,q
0.04 0.04270.001 c,p 0.04570.002 c,pq 0.04970.002 c,qr 0.05170.001 c,r
0.06 0.02070.002 d,p 0.02570.001 d,pq 0.02770.003 d,q 0.03370.004 d,r
samples treated with 10�9 M EBLþDW as compared to DW alone.Also, in seedlings treated with CPF, the combination of 10�7 MEBLþ0.06% CPF resulted in a maximum rise of 39% in fresh weight(0.03370.004) as compared to seedlings in 0.06% CPF alone.Similarly, a declining trend in dry weight of seedlings wasalso observed under the effect of different concentrations ofCPF. Supplementation of EBL to seeds resulted in significantenhancement in dry weight irrespective of the concentration ofCPF applied. Maximum enhancement in dry weight (0.01870.0012) was observed for samples treated with 10�7 M EBLþ0.06% CPF as compared to samples growing in 0.06% CPF alone(0.009370.0007).
3.2. Chlorophyll content
A significant decline in content of chlorophyll a, b and totalchlorophyll (mg g�1 FW) was observed in rice seedlings treatedwith increasing concentration of pesticide (Table 3). However, EBLhad significant bearing on the level of chlorophyll pigment of riceseedlings treated with pesticide and water. Seeds treated withEBL and grown in distilled water had enhanced level of pigmentas compared to control samples. Moreover, EBL concentration of10�7 M led to the maximum enhancement in the level ofchlorophyll content of pesticide treated samples. A combinationof 0.06% CPFþ10�7 M EBL led to the maximum enhancement(90%) in the level of chlorophyll b pigment (0.5870.02) ascompared to seedlings treated with 0.06% CPF (0.370.02) alone.The same combination led to 43% and 52% increase in chlorophylla and total chlorophyll content as compared to seedlings treatedwith 0.06% CPF alone.
3.3. Protein, free proline and MDA content
Seedlings growing in different concentrations of CPF mani-fested a decline in protein content (mg g FW�1) (Fig. 1). A sharpdecline in protein content was observed at 0.04% CPF concentra-tion however, maximum fall in the value (15.0471.16) wasrecorded at 0.06% CPF as against the control value (24.271.99).Sample pre-soaked with EBL led to a significant enhancement inthe protein content both under control and stress conditions.A maximum rise of 71% (25.7771.82) in the level of protein wasobserved for samples pre-soaked with 10�7 M EBL and growing in0.06% CPF as compared to 0.06% CPF alone. The concentration of10�7 M EBL proved to be the most efficient in enhancing theprotein content of stressed as well as control seedlings.
Free proline content (mmoles g FW�1) of seedlings enhancedsignificantly on treatment with increasing concentrations of CPF(Fig. 2). Maximum value of proline content (0.870.04) wasobserved for seedlings treated with 0.06% CPF as compared tocontrol seedlings (0.470.02). Supplementation of seeds withdifferent concentrations of EBL proved to be very efficient inaugmenting the proline content. Treatment of seeds growing in
er CPF stress. Data represents mean7SE (n¼15). Different letters (a,b,c,d) within
er LSD, pr0.05) whereas different letters (p,q,r,s) within various treatments of EBL
fy interactions of different concentrations of EBL with CPF on growth parameters.
Dry weight (g/seedling)
0 M EBL 10�11 M EBL 10�9 M EBL 10�7 M EBL
0.03970.0023 a,p 0.04470.0020 a,p 0.05070.0044 a,q 0.04770.0047 a,rq
0.02970.0023 b,p 0.03370.0015 b,p 0.03970.0018 b,q 0.04170.0010 b,q
0.01970.0015 c,p 0.02570.0007 c,pq 0.02570.0012 c,q 0.02870.0013 c,q
0.009370.0007 d,p 0.01170.0010 d,p 0.01570.0007 d,pq 0.01870.0012 d,q
Ta
ble
3E
ffe
cto
fE
BL
on
chlo
rop
hy
lla
,ch
loro
ph
yll
ba
nd
tota
lch
loro
ph
yll
of
12
-da
ys
old
Ory
zasa
tiv
ase
ed
lin
gs
un
de
rC
PF
stre
ss.D
ata
rep
rese
nts
me
an7
SE
(n¼
3).
Dif
fere
nt
lett
ers
(a,b
,c,d
)w
ith
inv
ari
ou
sco
nce
ntr
ati
on
so
fC
PF
(0,0
.02
%,
0.0
4%
an
d0
.06
%)
are
sig
nifi
can
tly
dif
fere
nt
(Fis
he
rLS
D,
pr
0.0
5)
wh
ere
as
dif
fere
nt
lett
ers
(p,q
,r,s
)w
ith
inv
ari
ou
str
ea
tme
nts
of
EB
L(0
,1
0�
11,
10�
9a
nd
10�
7M
)a
resi
gn
ifica
ntl
yd
iffe
ren
t(F
ish
er
LSD
,pr
0.0
5)
an
dsi
gn
ify
inte
ract
ion
so
fd
iffe
ren
tco
nce
ntr
ati
on
so
fE
BL
wit
hC
PF
on
pig
me
nt
con
ten
t.
CP
F(%
)C
hlo
rop
hy
lla
(mg
gF
W�
1)
Ch
loro
ph
yll
b(m
gg
FW�
1)
To
tal
chlo
rop
hy
ll(m
gg
FW�
1)
0M
EB
L1
0�
11
ME
BL
10�
9M
EB
L1
0�
7M
EB
L0
ME
BL
10�
11
ME
BL
10�
9M
EB
L1
0�
7M
EB
L0
ME
BL
10�
11
ME
BL
10�
9M
EB
L1
0�
7M
EB
L
01
.637
0.0
4a
,p1
.897
0.1
1a
,q1
.717
0.0
7a
,p2
.117
0.0
3a
,r1
.187
0.0
1a
,p1
.147
0.0
3a
,p1
.397
0.0
2a
,q1
.297
0.0
1a
,r2
.817
0.1
2a
,p5
.937
0.1
8a
,q3
.107
0.0
6a
,p3
.397
0.0
5a
,q
0.0
21
.577
0.0
2a
b,p
1.6
97
0.0
1b
,q1
.797
0.0
8a
,r1
.747
0.0
6b
,rq
1.1
17
0.0
1a
,p1
.027
0.0
2b
,q0
.977
0.0
2b
,q1
.187
0.0
4a
,p2
.687
0.1
0a
,p4
.677
0.2
4b
,p2
.767
0.0
6a
,p2
.927
0.0
6a
,q
0.0
41
.517
0.0
4b
,p1
.737
0.0
1b
,q1
.647
0.0
5a
,p1
.697
0.0
5b
,pq
0.5
17
0.0
3b
,p0
.557
0.0
2c,p
0.6
07
0.0
2c,q
0.5
87
0.0
3b
,q2
.027
0.0
6b
,p3
.537
0.4
3c,p
q2
.247
0.0
7b
,q2
.277
0.0
3b
,q
0.0
61
.237
0.0
1c,p
1.5
67
0.0
3c,q
1.7
37
0.0
1a
,q1
.777
0.0
4b
,q0
.307
0.0
2c,p
0.4
17
0.0
5d
,q0
.537
0.0
2c,r
0.5
87
0.0
2b
,r1
.547
0.0
5c,p
1.9
77
0.0
5b
,q2
.267
0.0
4b
,r2
.347
0.0
3b
,s Table 4List of primers used for Rt-pcr.
S. no. Name of the gene Primer sequence
1. EF1-a F: 50-GTACAAGATCGGTGGTATT-30
R: 50-GGGTACTCAGAGAAGGTCT-30
2. Cu/Zn-SOD F: 50-CCTCAAGCCTGGTCTCCAT-30
R: 50-CAGCCTTGAAGTCCGATGAT-30
3. Fe-SOD F: 50-CTTGATGCCCTGGAACCTTA-30
R: 50-GCCAGACCCCAAAAGTGATA-30
4. Mn-SOD F: 50-GCCATTGATGAGGATTTTGG-30
R: 50-CAAGCAGTCGCATTTTCGTA-30
5. CAT F: 50-GTTCGGTTCTCCACAGTCGT-30
R: 50-CCCTCCATGTGCCTGTAGTT-30
6. APX F: 50-CCAAGGGTTCTGACCACCTA-30
R: 50-CAGTTCGGAGAGCTTGAGGT-30
7. GR F: 50-AACAGCCGATGGCATAAAAG-30
R: 50-CAACCACCAGTTTCATGACG-30
0
5
10
15
20
25
30
35
Pro
tein
con
tent
(mg
gFW
-1)
EBL (M)10-9 10-7
a,p
b,p
a,p
b,pc,p
b,q
a,q
b,q b,pqab,qa,r
a,qr
b,q
a,rb,pr b,q
0 10-11
0% CPF 0.02% CPF 0.04% CPF 0.06% CPF
Fig. 1. Effect of EBL on protein content of 12-days old Oryza sativa seedlings under
CPF stress. Bar represents mean7SE (n¼3). Different letters (a,b,c,d) within
various concentrations of CPF (0, 0.02%, 0.04% and 0.06%) are significantly different
(Fisher LSD, pr0.05) whereas different letters (p,q,r,s) within various treatments
of EBL (0, 10�11, 10�9 and 10�7 M) are significantly different (Fisher LSD, pr0.05)
and signify interactions of different concentrations EBL with CPF on protein
content.
0
0.2
0.4
0.6
0.8
1
1.2
0
Pro
line
cont
ent (
µmol
es g
FW-1
)
EBL (M)10-11 10-9 10-7
a,p
c,p
b,p a,q
c,p b,p
b,r
a,p
b,pb,qd,p
b,qc,q
a,q
b,r b,r
0% CPF 0.02% CPF 0.04% CPF 0.06% CPF
Fig. 2. Effect of EBL on proline content of 12-days old Oryza sativa seedlings under
CPF stress. Bar represents mean7SE (n¼3). Different letters (a,b,c,d) within
various concentrations of CPF (0, 0.02%, 0.04% and 0.06%) are significantly different
(Fisher LSD, pr0.05) whereas different letters (p,q,r,s) within various treatments
of EBL (0, 10�11, 10�9 and 10�7 M) are significantly different (Fisher LSD, pr0.05)
and signify interactions of different concentrations EBL with CPF on proline
content.
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–8176
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–81 77
distilled water with 10�7 M and 10�9 M EBL led to the significantenhancement in proline content. For seeds growing in sandmoistened with different concentrations of CPF, EBL treatmentresulted in a boost in proline content irrespective of the concen-tration applied. Samples treated with the binary combination of10�9 M EBLþ0.02% CPF had the highest value of proline content(1.0570.03) which was 90% more than the samples growing in0.02% CPF (0.5570.02) alone.
A sharp increase in malondialdehyde content (mmoles g�1 FW)was seen in seedlings treated with CPF (Fig. 3). At the highestconcentration of CPF (0.06%), MDA content was observed to be6.4170.33 which was twice the amount in control seedlings(3.1170.012). With the application of different concentrationsof EBL, MDA content reduced significantly for samples treatedwith CPF whereas a non-significant decline was observed incase of control samples. Maximum fall of 122% in MDA contentwas observed for samples treated with 10�7 M EBLþ0.06% CPF(2.8870.23) as compared to those growing in 0.06% CPF alone(6.4170.33).
3.4. Antioxidant enzyme analysis
Application of CPF elicited the activity of most of the enzymeswhich was further enhanced by the pre-treatment of seeds withdifferent concentrations of EBL (Fig. 4). Specific activity of SOD(units mg protein�1) was augmented significantly on the applica-tion of various concentrations of CPF (Fig. 4a). At 0.04% CPF,activity of SOD was raised to 0.03870.002 as compared to controlseedlings (0.02470.002). On supplementation of seeds withvarious concentrations of EBL, significant enhancement in theactivity of SOD was observed both under control and CPF treat-ment. Binary combination of 10�7 M EBL with various concentra-tions of CPF proved to be most efficient in promoting the activityof SOD. A maximum of 23% increase in SOD activity (0.04770.002) was observed for samples growing in the combination of10�7 M EBLþ0.04% as compared to those growing in 0.04% CPF(0.03870.0002) alone.
Specific activity of APX (mmol min�1 mg protein�1) wasobserved to enhance at 0.04% CPF (42.1371.7) as compared tocontrol seedlings (33.672.1) (Fig. 4b). Application of different
0
1
2
3
4
5
6
7
8
MD
A (µ
mol
es g
FW-1
)
EBL (M)10-9 10-7
b,p
a,p
d,p
c,rc,p
a,r
c,q
a,p
b,qb,q
a,qra,p b,s
a,pa,q
b,r
10-110
0% CPF 0.02% CPF 0.04% CPF 0.06% CPF
Fig. 3. Effect of EBL on MDA content of 12-days old Oryza sativa seedlings under
CPF stress. Bar represents mean7SE (n¼3). Different letters (a,b,c,d) within
various concentrations of CPF (0, 0.02%, 0.04% and 0.06%) are significantly different
(Fisher LSD, pr0.05) whereas different letters (p,q,r,s) within various treatments
of EBL (0, 10�11, 10�9 and 10�7 M) are significantly different (Fisher LSD, pr0.05)
and signify interactions of different concentrations EBL with CPF on MDA content.
concentrations of EBL to seeds resulted in significant enhance-ment in the activity of APX under control condition. Also,pre-treatment of seeds with 10�9 M EBL and 10�7M EBL followedby application of different concentrations of CPF resulted insignificantly enhanced APX activity. A maximum rise of 30%in APX activity (45.0373.8) was recorded for samples treatedwith 10�7 M EBL70.02% CPF as compared to 0.02% CPF(34.3871.8) alone.
Application of CPF to the seedlings led to the alteration in theactivity of CAT (mmol min�1 mg protein�1) (Fig. 4c). Significantlyenhanced CAT activity (1.5870.08) was observed in seedlingstreated with 0.04% CPF as compared to control seedlings(1.2570.07). Supplementation of seeds with different concentra-tions of EBL led to the enhancement in the activity of CAT forboth control and stressed condition. Among control samples,maximum rise in CAT activity (2.170.06) was observed for seedstreated with 10�7 M EBL as compared to seeds growing indistilled water only. Among CPF treated samples, a maximumrise (60%) in CAT activity (1.770.04) was observed for samplestreated with 10�7 M EBLþ0.06% CPF as compared to 0.06% CPF(1.0670.13) alone. However, 10�7 M EBL proved to be the mosteffective in increasing the CAT activity irrespective of the con-centration of CPF applied.
Treatment of seedlings with CPF led to the significant enhance-ment in the specific activity of GPX (mmol min�1 mg protein�1)at 0.02% and 0.04% CPF as compared to control seedlings (Fig. 4d).Application of different concentrations of EBL to control seedsresulted in no significant change in the specific activity of GPX.However, application of 10�9 and 10�7 M EBL to samples treatedwith various concentrations of CPF led to the significant enhance-ment in the specific activity of GPX. A maximum rise of 26%(25.872.8) was observed in seedlings treated with 10�7 MEBLþ0.06% CPF as compared to seedlings treated with 0.06%CPF (20.3371.08) alone. While in case of GR, a decrease in theactivity (mmol min�1 mg protein�1) on treatment of seedlingswith 0.06% CPF was observed (Fig. 4e). However, application ofhigher concentrations of EBL (10�9 and 10�7 M EBL) to controlseeds significantly enhanced the specific activity of GR as com-pared to non EBL treated control seeds. For CPF treated seedlingsalso, 10�9 and 10�7 M EBL led to the significant enhancement inGR specific activity in combination with 0.04% and 0.02% CPFrespectively.
A significant decline in the specific activity of DHAR (mmolmin�1 mg protein�1) was observed on treatment of seedlingswith various concentrations of CPF (Fig. 4f). The specific activityof DHAR at 0.06% CPF (1.270.11) declined to more than half of itsvalue in control conditions (2.670.01). Pre-soaking of controlseeds with 10�9 and 10�7 M EBL led to a significant improvementin the DHAR activity. Treatment of samples with 10�7 MEBLþDW led to the maximum enhancement in the activity ofDHAR (3.3370.04) as compared to those growing in distilledwater alone (2.670.01). Also, for samples treated with CPF,application of different concentrations of EBL resulted in anoverall improvement in the activity of DHAR. A maximum of52% increase in specific activity was observed in samples treatedwith 10�7 M EBLþ0.04% (2.5670.27) as compared to 0.04% CPF(1.6870.19) alone.
Application of CPF resulted in significant enhancement in thespecific activity of MDHAR (mmol min�1 mg protein�1) (1.7270.107) at 0.02% CPF as compared to control seedlings (1.1170.102) (Fig. 4g). Pre-soaking of control seeds with 10�11 and10�9 M EBL significantly boosted the specific activity of MDHARas compared to control seeds without EBL treatment. Further,seeds pre-treated with different concentrations of EBL whensubjected to CPF doses, resulted in an overall enhancement inMDHAR activity. Irrespective of the concentration of CPF applied,
0
0.01
0.02
0.03
0.04
0.05
0.06
SA
of S
OD
(UA
mg
prot
ein-1
)
EBL (M)
a,p
c,p
b,p
ab,pqb,pq
ab,pa,q
a,q
b,pr
a,qra,q
a,r
b,r
a,qa,r
ab,r
10-9 10-70
10
20
30
40
50
60
a,pa,p
b,p b,pa,q
a,pq
c,pqc,p
b,rab,pq
a,r
c,pq
a,rb,rab,pq
c,q
0
0.5
1
1.5
2
2.5
b,r
a,ra,r
a,qra,q
b,qr
a,q
c,pq
b,qa,qa,q
ac,pa,p
b,p
a,p
b,r
0
0.5
1
1.5
2
2.5
3
3.5
a,pa,p
c,pb,p
a,r
c,p c,p
a,q
b,p
b,pqa,s
d,q
a,qa,pq
b,pa,q
0
0.5
1
1.5
2
2.5
3
3.5
4
b,p
a,p
bc,p
c,p
a,pq
a,qc,q
bq
a,pb,q
c,q
a,q
d,pq
d,q
c,pb,p
SA
of G
R
µmol
min
-1 m
g pr
otei
n-1)
SA
of D
HA
R(µ
mol
min
-1 m
g pr
otei
n-1)
SA
of G
PX
(µm
ol m
in-1
mg
prot
ein-1
)
SA
of C
AT
(µm
ol m
in-1
mg
prot
ein-1
)
SA
of A
PX
(µm
ol m
in-1
mg
prot
ein-1
)
0
5
10
15
20
25
30
35
a,pq
bc,pqc,pq
b,p
ab,pa,p
b,qb,q
a,qa,q
b,r
a,p
a,p
c,pb,rbc,pr
10-110EBL (M)
10-9 10-710-110
EBL (M)10-9 10-710-110
EBL (M)10-9 10-710-110
EBL (M)10-9 10-710-110
EBL (M)10-9 10-710-110
0
0.5
1
1.5
2
2.5
SA
of M
DH
AR
(µm
ol m
in-1
mg
prot
ein-1
)
a,qa,q
b,r
a,qr
b,rb,r
a,q b,p
b,qa,p
ab,qc,p
a,p
b,pa,p
a,p
EBL (M)10-9 10-710-110
0% CPF 0.02%CPF 0.04% CPF 0.06% CPF
Fig. 4. Effect of EBL on specific activity of (a) SOD (b) APX (c) CAT (d) GPX (e) GR (f) DHAR and (g) MDHAR of 12-days old Oryza sativa seedlings under CPF stress. Bar
represents mean7SE (n¼3). Different letters (a,b,c,d) within various concentrations of CPF (0, 0.02%, 0.04% and 0.06%) are significantly different (Fisher LSD, pr0.05)
whereas different letters (p,q,r,s) within various treatments of EBL (0, 10�11, 10�9 and 10�7 M) are significantly different (Fisher LSD, pr0.05) and signify interactions of
different concentrations EBL with CPF on specific activity of antioxidant enzyme.
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–8178
application of 10�7 M EBL to these seeds resulted in an increasein the specific activity of MDHAR. A maximum rise of 83% inspecific activity of MDHAR (2.0670.11) was observed in samplestreated with 10�7 M EBLþ0.06% CPF as compared to samplesgrowing in CPF (1.1270.08) alone.
3.5. Semi-quantitative RT-PCR analysis for antioxidant genes
Four samples were selected for gene expression analysis;seedlings grown in distilled water (C), 0.04% CPF, 10�7 M EBL,and those pre-treated with 10�7 M EBL and grown in 0.04%
EF1α EF1α
EF1α EF1α
EF1α EF1α
0
2
4
6
C CPF EBL CPF+EBL0123456
0
1
2
3
4
5
0
2
4
6
8
0
2
4
6
01234567
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
Rel
ativ
e ex
pres
sion
C CPF EBL CPF+EBL
C CPF EBL CPF+EBL C CPF EBL CPF+EBL
C CPF EBL CPF+EBL C CPF EBL CPF+EBL
Fig. 5. An ethidium bromide – stained agarose gel harbouring products from reverse transcriptase – PCR of 12 day old rice seedlings exposed to distilled water (control, C),
0.04% CPF (CPF), 10�7 M EBL (EBL) and 0.04% CPF and 10�7 M EBL EBL both (CPFþEBL) for various key antioxidant genes (a) Cu/Zn-SOD (b) Fe-SOD (c) Mn-SOD (d) APX
(e) CAT and (f) GR. Results were first normalised to the housekeeping gene EF1a, and then the relative expression of genes under various treatments was determined.
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–81 79
CPF (Fig. 5). We studied the expression of all the differentisoforms of SOD. Among various isoforms of SOD, transcriptslevel for Cu/Zn-SOD and Mn-SOD accumulated to a much higherlevel as compared to Fe-SOD under CPF stress. A maximum of6.2-fold increase in expression was observed for transcript levelof Mn-SOD under CPF treatment in contrast to control samples.EBL treatment alone resulted in the upregulation of all theisoforms of SOD. The co-treatment of EBL and CPF led to amanifold increase in the expression of Cu/Zn-SOD and Fe-SODas compared to their individual treatments. The expression ofCu/Zn-SOD was upregulated (1.2-fold) in samples treated with
CPF and EBL in combination, as compared to CPF treated seedlingsalone. In case of Fe-SOD, co-treatment of EBL and CPF led to a 3.4-fold enhancement in the transcript level of the gene as comparedto CPF treatment alone. The expression for Mn-SOD enhanced inall the treatments in comparison to control while no change in thelevel of expression was observed in samples subjected to both EBLand CPF treatments as compared to CPF treated samples. Simi-larly, an upregulation of APX and CAT was also observed indifferent treatments as compared to control. However, interest-ingly, the combined treatment of EBL and CPF led to a distinctincrease in transcript level of CAT (2.3-fold) as compared to the
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–8180
CPF treated samples. Like APX expression of GR also increasedunder all the treatments as compared to control. However,transcript level was not observed to change much when EBLwas given in conjunction with CPF as compared to CPF alone.
4. Discussion
In the present study, application of various concentrations ofCPF led to a decrease in shoot and root length, root number, freshand dry weight of rice seedlings. These observations may be dueto the toxic effect of CPF on plants. Recently, the role of CPF forreducing the availability of important micronutrients like Mn andCu in plants has been established (Kaushik et al., 2010). However,pre-treatment of plants with different concentrations of EBL had apositive effect on various growth parameters and indicatetowards the stress ameliorative properties of BRs. Improvementin growth under BRs application could be attributed to effect ofBRs in accelerating the degradation of pesticide. It has beenreported that EBL leads to enhanced activity and expression ofpesticide metabolism and detoxification enzymes which facil-itates their degradation and reduction of their residual level inplants (Xia et al., 2009). Further, the role of brassinosteroids ongrowth promoting activities of plants due to its involvementin cell division, cell expansion, modification of cell wall prop-erties and synthesis of macromolecules are well documented(Clouse, 2011).
To get insights into the pesticides induced stress and stressameliorative properties of BRs, various biochemical parameterswere studied. One of the conspicuous effects of CPF treatmentwas decline in chlorophyll pigment level. This may be due to theCPF induced impairment of the photosynthetic apparatus of theplant (Xia et al., 2006) or the free radical induced oxidation ofchlorophyll pigment (Kato and Shimizu, 1985) or consequence ofthe activation of chlorophyllase (Reddy and Vora, 1986). However,EBL induced enhancement in the chlorophyll content may be eitherdue to the activation of genes responsible for chlorophyll biosynth-esis as BRs are linked to transcription and/or translation (Kalinichet al., 1985) or due to the their role in reducing the degradation ofchlorophyll (Honnerova et al., 2010). Treatment of CPF resulted indecline in protein content which is in contrast to the EBL whereincrement in total protein content was observed. Application ofBRs is known to elicit de novo synthesis of various proteins throughits effect on transcription and translation (Clouse and Sasse, 1998;Dhaubhadel et al., 1999). In the present experiment, enhancementin the level of proline was observed in CPF treated seedlings whichwas further augmented by EBL application. The increased level ofproline provides tolerance to plants against various stress (Hareand Cress, 1997). Proline has been implicated in protection andstabilisation of enzymes and membrane structures (Bandurska,2001) and ROS scavenging (Matysik et al., 2002). Increased prolinelevels in plants treated with EBL might have resulted due toactivation of genes that are responsible for proline biosynthesis.Besides proline, the lipid peroxidation content also acts as a stressresponsive marker. Hence, to know the extent of damage caused byperoxidation, quantification of malondialdehyde (MDA), the endproduct of lipid peroxidation in pesticides and EBL treated sampleswas conducted. CPF treatment resulted in considerable damage tothe biomembranes as it was manifested by the enhanced level ofMDA content in seedlings. Lowering of lipid peroxidation level insamples treated with EBL and subsequently with CPF indicates theprotective role of brassinosteroids towards membrane damage.
Plant cells actively produce ROS both under stress and normalgrowth conditions. The production and scavenging of ROS iskept under control by antioxidative defence system composedof antioxidant enzymes like superoxide dismutase, ascorbate
peroxidase, catalase and glutathione reductase of Asada–Halliwell pathway (Sharma et al., 2012). However, the environ-mental stress leads to the production of reactive oxygen speciesand the quenching activity of the antioxidant system is upset,causing oxidative stress (Mittler, 2002). In the present experi-ment, the activity of key antioxidant enzymes was determined toassess the effect of CPF alone and its co-treatment with EBL.CPF induced oxidative stress resulted in significantly enhancedactivity of SOD, APX, CAT, GPX and MDHAR. Application of EBL,particularly at its highest concentration, in conjunction with CPFresulted in further elevation in the activities of all enzymes. Therole of BRs as secondary messengers to elicit antioxidativedefence system in stressed plants and thereby effectively foragingthe ROS in plants under stress is known (Mazorra et al., 2002).
To get further insights into the dynamics of antioxidantenzymes under the effect of EBL and CPF, we studied theexpression of some key antioxidant genes. The expression ofdifferent isoforms of SOD, copper/zinc (Cu/Zn-SOD), manganese(Mn-SOD) and iron (Fe-SOD), which are localised in differentcellular compartments, was studied. Individual treatment ofpesticide and EBL induced the expression of all the isoforms ofSOD as compared to control. However, pesticide treatment inconjunction with EBL resulted in a sharp upregulation of Fe-SODin contrast to CPF and EBL treated samples alone. Initially,Fe-SOD was assumed to be absent in rice until the sequence ofFe-SOD was reported for the first time by Kaminaka et al. (1999).Rice Fe-SOD was characterised as distinct type compared to otherknown plant Fe-SODs. It is insensitive to hydrogen peroxide andhence its response to different types of stress is worth investigat-ing. Further, in general Fe-SOD and Mn-SOD are structurallyvery similar and believed to be evolved from a commonancestor (Stallings et al., 1984), while in case of rice a strikinglyphylogenetically distant relationship was observed (Kaminakaet al., 1999). In the present study, the differential response ofFe-SOD and Mn-SOD suggests that these two genes are comple-mentarily expressed in rice in response to different treatments.The balance between SODs and the different H2O2-scavengingenzymes in cells is considered to be crucial in determining thesteady-state level of O2
d� and H2O2. In the present experiment, itwas observed that the EBL and pesticide treatment together led toa relatively small increase in transcripts for APX and GR incomparison to CPF alone while mRNA level for CAT was stronglyinduced. There are adequate reports linking the overexpression ofantioxidant genes with enhanced stress tolerance (Prashanthet al., 2008; Gill and Tuteja, 2010). Thus, EBL led upregulation ofthese key antioxidant enzymes might be playing a crucial role inproviding enhanced tolerance to rice against pesticide inducedoxidative stress.
5. Conclusions
From these findings, it can be concluded that stress caused byCPF is ameliorated by the application of EBL, particularly at theconcentration of 10�7 M which proves to be the most effective.These results strongly suggest that BRs enhance plant tolerance topesticides by modulating the stress responsive markers. Further,it seems that EBL has provided two-way protection to riceseedlings, one by antioxidant enzymes mediated better scaven-ging of ROS and the other by enhancing proline content to protectenzymes and repair the injury after the degradation has occurred.The selective upregulation of Fe-SOD and CAT under the com-bined effect of EBL and CPF reveals that they might be playing apivotal role in BRs mediated defence against pesticide stress andmay be hinting towards a tightly regulated mechanism of BRinduced stress tolerance. These results can be interpreted as an
I. Sharma et al. / Ecotoxicology and Environmental Safety 85 (2012) 72–81 81
internal tolerant mechanism and may facilitate in developingacceptable strategies for reducing the risk of adverse effect ofpesticides in crop production. Moreover, in the era of modernagriculture where application of pesticides has become a part ofcrop production process, the use of brassinosteroids in conjunc-tion with pesticides will open new avenues for sustainableagriculture.
Acknowledgments
University Grants Commission (UGC), New Delhi, India is dulyacknowledged for funding the proposed work. We extend ourthanks to Dr A.K. Dixit, Senior scientist, Pesticide Residues, IARI,New Delhi, India and Dr. P.K. Nagar, former head, BiotechnologyDivision, IHBT, Palampur, India for helpful discussion.
References
Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126.Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in
Beta vulgaris. Plant Physiol. 24 (1), 1–15.Bandurska, H., 2001. Does proline accumulated in leaves of water deficit stressed
barley plants confine cell membrane injuries? II. Proline accumulation duringhardening and its involvement in reducing membrane injuries in leavessubjected to severe osmotic stress. Acta Physiol. Plant. 23, 483–490.
Bates, L.S., Waldeen, R.P., Teare, I.D., 1973. Rapid determination of free proline forwater stress studies. Plant Soil 39, 205–207.
Beauchamp, C.U., Fridovich, I., 1971. Improved assays for superoxide dismutaseand an assay applicable to polyacrylamide gels. Anal. Biochem. 44, 276–287.
Bradford, M., 1976. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem. 72, 248–254.
Channapatna, S.P., 2001. The genetically modified crop debate in the context ofagricultural evolution. Plant Physiol. 126, 8–15.
Choudhary, S.P., Kanwar, M., Bhardwaj, R., Yu, J., Tran, L.P., 2012. Chromium stressmitigation by polyamine-brassinosteroid application involves phytohormonaland physiological strategies in Raphanus sativus L. PLoS One 7 (3), e33210.
Clouse, S.D., 2011. Brassinosteroid signal transduction: from receptor kinaseactivation to transcriptional networks regulating plant development. PlantCell 23 (4), 1219–1230.
Clouse, S.D., Sasse, J.M., 1998. Brassinosteroids: essential regulators of plantgrowth and development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49,427–451.
Dhaubhadel, S., Chaundhary, S., Dobinson, K.F., Krishna, P., 1999. Treatment with24-epibrassinolide, a brassinosteroid, increases the basic thermotolerance ofBrassica napus and tomato seedlings Plant Mol. Biol. 40, 333–342.
Divi, U.K., Rahman, T., Krishna, P., 2010. Brassinosteroid-mediated stress tolerancein Arabidopsis shows interactions with abscisic acid, ethylene and salicylicacid pathways. BMC Plant Biol. 10, 151.
FernaaNdez-Garciaa, N., Carvajal, M., Olmos, E., 2004. Graft union formation intomato plants: peroxidase and catalase involvement. Ann. Bot. 93, 53–60.
Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery inabiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930.
Gudesblat, G.E., Russinova, E., 2011. Plants grow on brassinosteroids. Curr. Opin.Plant Biol. 14 (5), 530–537.
Honnerova, J., Rothova, O., Hola, D., Kocova, M., Kohout, L., Kvasnica, M., 2010. Theexogenous application of Brassinosteroids to Zea mays (L.) stressed by long-term chilling does not affect the activities of photosystems 1 or 2. J. PlantGrowth Regul. 29, 500–505.
Hare, P.D., Cress, W.A., 1997. Metabolism implications of stress-induced prolineaccumulation in plants. Plant Growth Regul. 21, 79–102.
Hodges, M.D., DeLong, J.M., Forney, C.F., Prange, R.K., 1999. Improving thethiobarbituric acid-reactive-substances assay for estimating lipid peroxidation
in plant tissues containing anthocyanin and other interfering compounds.Planta 207, 604–611.
Hossain, M.A., Asada, K., 1985. Purification of dehydroascorbate reductase fromspinach and its characterization as a thiol enzyme. Plant Cell Physiol.25, 85–92.
Jahnke, L.S., Hull, M.R., Long, S.P., 1991. Chilling stress and oxygen metabolizingenzymes in Zea mays and Zea diploperennis. Plant Cell Environ. 14, 97–104.
Kalinich, J.F., Mandava, N.B., Todhunter, J.A., 1985. Relationship of nucleic acidmetabolism to brassinolide-induced responses in beans. J. Plant Physiol. 120,207–214.
Kaminaka, H., Morita, S., Tokumoto, M., Yokoyama, H., Masumura, T., Tanaka, K.,1999. Molecular cloning and characterization of a cDNA for an iron-superoxidedismutase in rice (Oryza sativa L.). Biosci. Biotechnol. Biochem. 63, 302–308.
Kana, R., Spundova, M., Ilık, P., Lazar, D., Klem, K., Tomek, P., Naus, J., Prasil, O.,2004. Effect of herbicide clomazone on photosynthetic processes in primarybarley (Hordeum vulgare L.) leaves. Pestic. Biochem. Physiol. 78 (3), 161–170.
Kato, M., Shimizu, S., 1985. Chlorophyll metabolism in higher plants. VI. Involve-ment of peroxidase in chlorophyll degradation. Plant Cell Physiol. 26,1291–1301.
Kaushik, G., Satya, S., Naik, S.N., Tripath, B., 2010. Impact of Chlorpyrifos residueson micronutrient bioavailability in chickpea. Food Dig. 1, 57–61.
Lee, S., Park, C., 2012. Regulation of reactive oxygen species generation underdrought conditions in Arabidopsis. Plant Signal. Behav. 7 (6), 599–601.
Matysik, K.J., Alia, B., Mohanty, P., 2002. Molecular mechanism of quenching ofreactive oxygen species by proline under stress in plants. Curr. Sci. 82,525–532.
Mazzucotelli, E., Mastrangelo, A.M., Crosatti, C., Guerra, D., Stanca, M., Cattivelli, L.,2008. Abiotic stress response in plants: when post-transcriptional and post-translational regulations control transcription. Plant Sci. 174, 420–431.
Mazorra, L.M., Nunez, M., Hechavarria, M., Coll, F., Sanchez-Blanco, M.J., 2002.Influence of brassinosteroids on antioxidant enzymes activity in tomato underdifferent temperature. Biol. Plant 45, 593–596.
Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends PlantSci. 7 (9), 405–410.
Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specificperoxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880.
Pandya, U., Saraf, M., 2010. Application of fungi as a biocontrol agent and theirbiofertilizer potential in agriculture. J. Adv. Dev. Res. 1 (1), 90–99.
Prashanth, S.R., Sadhasivan, V., Parida, A., 2008. Over expression of cytosoliccopper/zinc superoxide dismutase from a mangrove plant Avicenia marina inindica rice var Pusa Basmati-1 confers abiotic stress tolerance. Transgenic Res.17 (2), 281–291.
Reddy, M.P., Vora, A.B., 1986. Changes in pigment composition, hill reactionactivity and saccharide metabolism in bajra (Pennisetum typhoides S&H) leavesunder NaCl salinity. Photosynthetica 20, 50–55.
Ruttan, V.W., 2002. Productivity growth in world agriculture: sources andconstraints. J. Eco. Perspect. 16 (4), 161–184.
Saladin, G., Clement, C., 2005. Physiological side-effects of pesticides on non-targetplants. In: Livingston, J.V. (Ed.), Agriculture and Soil Pollution: New Research.Nova Science, pp. 53–86.
Sharma, I., Pati, P.K., Bhardwaj, R., 2011. Effect of 24-epibrassinolide on oxidativestress markers induced by nickel ion in Raphanus sativus L. Acta Physiol. Plant.33 (5), 1723–1735.
Sharma, P., Jha, A.B., Dubey, R.S., Pessarakli, M., 2012. Reactive oxygen species,oxidative damage and antioxidative efensmechanism in plants understressful conditions. J. Bot., vol. 2012 (2012), Article ID 217037, 26 pp.http://dx.doi.org/10.1155/2012/217037.
Stallings, W.C., Pattridge, K.A., Strong, R.K., Ludwig, M.L., 1984. Manganese andiron superoxide dismutases are structural homologs. J. Biol. Chem. 259,10695–10699.
Swaminathan, M.S., 2006. An evergreen revolution. Crop Sci. 46, 2293–2303.Vriet, C., Russinova, E., Reuzeau, C., 2012. Boosting crop yields with plant steroids.
Plant Cell. 24, 842–857.Xia, X.J., Wang, Y., Zhou, Y., Tao, Y., Mao, W., Shi, K., Asami, T., Chen, Z., Yu, J., 2009.
Reactive oxygen species are involved in brassinosteroid-induced stress toler-ance in cucumber. Plant Physiol. 150, 801–814.
Xia, X.J., Huang, Y.Y., Wang, L., Huang, L.F., Yu, Y.L., Zhou, Y.H., Yu, J.Q., 2006.Pesticides induced depression of photosynthesis was alleviated by 24-Epi-brassinolide pre-treatment in Cucumis sativus L. Pestic. Biochem. Physiol.86, 42–48.
