Journal of Plant Stress Physiology ● Vol 2 ● 2016 9
INTRODUCTION
Better understanding of mechanistic details of plants’
response to environmental toxicants can pave the way to
develop safe crop in future. Arsenic (As) is a wide-spread
toxic and carcinogenic metalloid. As can induce growth
inhibition, low productivity, and poor grain quality by
inducing oxidative stress in crop plants (Gunes et al., 2009;
Tripathi et al., 2012). Plant experiences oxidative imbalance
due to excess generation of reactive oxygen species (ROS)
and loss of delicate balance of ROS homeostasis (Finnegan
and Chen, 2012). Grain legumes are highly sensitive to As
toxicity and being grown in aerobic fields are generally
exposed to arsenate (As V) form of As species (Gunes
et al., 2009). At the cellular level, As V interferes with
normal enzymatic functions and disrupts plant growth and
metabolisms (Finnegan and Chen, 2012).
Integration among different defense circuits is pivotal
during As-tolerance of crop plants. Ascorbate (AsA) and
GSH are the two key players in non-enzymatic defense
components, and their redox states are more important
than their total amount in cell (Foyer and Noctor, 2011;
Noctor et al., 2012). GSH as a thiol buffer interacts
with numerous cellular components and maintains
redox homeostasis favorable to reducing environment.
The AsA peroxidase (APX), dehydroascorbate (DHA)
reductase (DHAR) and glutathione (GSH) reductase
(GR) within AsA-GSH cycle and catalases (CAT) and
GSH-s-transferase (GST) outside this cycle are the
predominant enzymatic antioxidant defense components
against ROS-induced oxidative imbalance in cell
(Foyer and Noctor, 2003; 2011; Noctor et al., 2012).
Besides GSH and GSH-dependent antioxidant defense,
methylglyoxal (MG) detoxification systems powered by
glyoxalase (Gly) I and II enzymes play significant roles in
drought and salinity stress and heavy metal detoxification
system in plants (Yadav et al., 2005; Singla-Pareek et al.,
2008; Hossain and Fujita, 2010; Hossain et al., 2010).
MG is highly toxic to plant cells and by reacting with
Exogenous thiourea modulates antioxidant defense
and glyoxalase systems in lentil genotypes under
arsenic stress
Dibyendu Talukdar*
Department of Botany, RPM College (University of Calcutta), Uttarpara, Hooghly, West Bengal, India
ABSTRACT
Arsenic (As) is a wide-spread toxic and carcinogenic metalloid, affecting crop productivity worldwide. Lentil, an
edible grain legume, is increasingly exposed to soil As contamination. However, our understandings regarding
mechanistic details and mitigation strategies against As toxicity in edible legume are extremely poor. The main
purpose of this study was to investigate the As-effects and its mitigation by thiourea (TU), a sulfhydryl bioregulator,
in lentil. Four widely grown lentil genotypes were grown in nutrient media, supplemented with 30 μM sodium
As, As + 6.5 mM TU, and As + 13 mM TU, keeping an untreated control for 10 d. As severely affected plant
dry weight by accumulating in shoots and roots. However, TU application sequestered As in crop roots and
prevented upward translocation of As. TU coordinately modulated glyoxalase (Gly) system I and II (Gly I and II)
and ascorbate (AsA)-glutathione (GSH) redox, and antioxidant defense enzymes in both leaves and roots of
four genotypes. Elevation of Gly system prevented toxic methylglyoxal overaccumulation, whereas stimulated
AsA-GSH cycle enzymes and GSH-s-transferase and catalase effectively scavenged H
2
O
2
and prevented reactive
oxygen species (ROS)-mediated onset of oxidative damage in four genotypes, as was evident from the ROS-
imaging study. Results suggested exogenous TU stimulated the Gly and antioxidant defense in fine tune against
As-induced oxidative damage in lentil genotypes.
KEY WORDS: Antioxidant defense, arsenate, glyoxalase system, lentil, thiourea
Original Article
Journal of Plant Stress Physiology 2016, 2: 9-21
http://sciencefl ora.org/journals/jpsp/
doi:10.19071/jpsp.2016.v2.3041
Received: Received: 12.04.2016
Accepted: Accepted: 05.05.2016
Published: Published: 06.05.2016
*Address for *Address for
Correspondence:Correspondence:
Dibyendu Talukdar,
Department of Botany,
RPM College (University
of Calcutta), Uttarpara,
Hooghly, West Bengal, India.
Tel: +9103326630191.
Fax: + 9103326634155.
E-mail: dibyendutalukdar9@
gmail.com
Talukdar: Thiourea-induced arsenate tolerance in lentil genotypes
10 Journal of Plant Stress Physiology ● Vol 2 ● 2016
proteins, lipids, carbohydrates, and DNA; they can lead
to cell death in the absence of any effective protection.
In plants, MG is detoxified mainly via the Gly system
that is comprised two enzymes: While Gly I converts
MG to S-D-lactoylglutathione (SLG) by utilizing GSH,
Gly II converts SLG to D-lactic acid, and during this
reaction GSH is regenerated. Overexpressions of the
Gly pathway in transgenic tobacco and rice plants
experiencing environmental stresses can prevent ROS
and MG accumulation by maintaining GSH redox
homeostasis and Gly activity levels (Yadav et al., 2005;
Singla-Pareek et al., 2008).
Priming of existing defense mechanisms without any
genome modification has been found effective during
crops’ tolerance to stresses (Sahu and Singh 1995;
Srivastava et al., 2011). Use of sulfhydryl bio-regulator
like thiourea (TU), an ROS-scavenger, has been found
highly effective in ameliorating salt, ultraviolet, and
heat stress in cereals and oilseeds (Srivastava et al.,
2011; Akladious, 2014) and in regulating source-to-
sink relationship in Indian mustard through alterations
in antioxidant defense (Pandey et al., 2013). Among
edible legumes, exogenous TU was found to be primarily
effective against As-induced oxidative stress in mung bean
(Talukdar, 2014), but no such information is available in
lentil crop which is extensively grown as grain legume
in Indian sub-continent (IIPR, 2011). Primary reports
indicate that As exposure can inhibit plant growth and
seed yield in lentil genotypes (Talukdar, 2013b; Talukdar
and Talukdar, 2014). However, nothing is known regarding
mechanistic details of Gly systems and its interaction with
GSH-dependent antioxidant defense in any of the grain
legume crops under As stress.
Present work was, therefore, designed to: (i) Unravel
the response of antioxidant defense and (ii) reveal the
modulation of Gly systems under As alone and As +
TU applications. The study for the first time indicates
coordinated responses between primary antioxidant
defense components and MG-detoxification systems
during As exposure and their modulations during TU-
mediated amelioration of As-stress in lentil genotypes.
Apart from physiological and biochemical studies, this
fact has been confirmed by ROS imaging analysis in this
study.
MATERIALS AND METHODS
Plant Materials and Treatment Protocols
Fresh and healthy seeds of four lentil genotypes (Lens
culinaris Medik. cv. L 9, Pusa 4, K 75, and PL 234) were
surface-sterilized with NaOCl (0.1%, w/v), washed under
running tap water followed by distilled water, and were
allowed to germinate in the dark in three separate sets
on moistened filter paper at 25°C. Germinated seedlings
were immediately placed in polythene pots (10 plants/
pots) containing 300 ml of Hoagland’s No. 2 nutrient
media following earlier protocol (Talukdar, 2013a),
and were allowed to grow for 10 days. The plants were,
then, subjected to the following treatment protocols
as: (a) Untreated control, (b) 30 μM sodium As (As,
MW 312.01 g/mol; technical grade, purity 98.5%,
Sigma-Aldrich), (c) 30 μM As + 6.5 mM TU, and (d)
30 μM As + 13 mM TU. Each treatment was replicated
four times. TU (Sigma-Aldrich, Bengaluru, India), a
sulfhydryl bio-regulator, was used to pre-soak the seeds
in the last two protocols. Pilot experiments were carried
out to determine the effective concentrations of TU and
As. Control and treated plants were allowed to grow for
another 10 days. Nutrient solution was refreshed in every
alternate day to prevent depletion of nutrients, TU as well
as As in the course of the plant’s exposure to them. The
experiment was carried out in a completely randomized
block design in a controlled growth chamber under a
14 h photoperiod, 26/16 (±2°C), relative humidity of
72 ± 2% and a photosynthesis photon flux density of
180 μmol m
−2
s
−1
. Plants were harvested after a stipulated
period. Plant parts were separated and thoroughly washed
and oven dried at 72°C for 48 h to measure dry weights
of shoots and roots.
Determination of Chlorophyll (Chl) and Total
Carotenoids
Leaf Chl and carotenoid contents were determined
(Lichtenthaler, 1987). Leaf tissue (50 mg) was homogenized
in 10 ml chilled acetone (80%). The homogenate was
centrifuged at 4000 g for 12 min. The absorbance of the
supernatant was recorded at 663, 647, and 470 nm for Chl A,
Chl B, and carotenoids, respectively. The contents were
expressed as mg Chl or carotenoids g
−1
fresh weight (FW).
Measurement of As Content
As concentration in dried shoot and root samples was
measured by digestion methods (HNO
3
-HClO
4
mixture at
3:1, v/v) using flow injection-hydride generation atomic
absorption spectrophotometer (Perkin-Elmer, FIA-HAAS
Analyst 400) and keeping Standard Reference Materials
of tomato leaves (item number 1573a, from National
Institute of Standards and Technology, USA) as part of
the quality assurance/quality control protocol (Talukdar,
2013a). The translocation factor (TF) is the ratio of the
level of As in shoots on roots.
Talukdar: Thiourea-induced arsenate tolerance in lentil genotypes
Journal of Plant Stress Physiology ● Vol 2 ● 2016 11
Determination of GSH, Ascorbate Content, and Assay
of Antioxidant Defense Enzymes
Reduced and oxidized form of AsA and GSH were
determined according to Law et al. (1983) and Griffith
(1980), respectively. For enzyme assay, plant tissue of
250 mg was ground in liquid nitrogen and homogenized
in 1 ml of 50 mM K-phosphate buffer (pH 7.8) containing
1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM
dithiothreitol (DTT), and 2% (w/v) polyvinyl pyrrolidone
using a chilled mortar and pestle kept in an ice bath.
The homogenate was centrifuged at 15,000 g at 4°C for
20 min. Clear supernatant was used for enzyme assays.
For measuring APX activity, the tissue was separately
grounded in homogenizing medium containing 2.0 mM
AsA in addition to the other ingredients. All assays were
done at 0-4°C. Soluble protein content was determined
using BSA as a standard (Bradford, 1976).
Superoxide dismutase (SOD) (EC 1.15.1.1) activity was
measured by nitro blue tetrazolium (NBT) photochemical
assay (Beyer and Fridovich, 1987). One unit of SOD was
equal to that amount causing a 50% decrease of SOD-
inhibited NBT reduction. APX (EC 1.11.1.11) activity
(μmol AsA oxidized/min/mg protein) was assayed
following the protocol of Nakano and Asada (1981).
Three ml of the reaction mixture contained 50 mM
potassium phosphate buffer (pH 7.0), 0.1 mM EDTA,
0.5 mM AsA, 0.1 mM H
2
O
2
, and 0.1 ml enzyme extract.
The H
2
O
2
-dependent oxidation of AsA was followed by a
decrease in the absorbance at 290 nm (ε = 2.8/mM/cm).
APX activity was expressed as nmol AsA oxidized/min/
mg protein. For DHAR (EC 1.8.5.1) activity assay, the
reaction mixture contained 50 mM potassium phosphate
buffer (pH 7.0), 0.2 mM DHA, 2.5 mM GSH, and 0.1 mM
EDTA in a final volume of 1 ml. Reaction was started by
addition of suitable aliquots of enzyme extract. The increase
in absorbance was recorded at 30 s intervals for 3 min
at 265 nm. Enzyme activity was expressed as μmol AsA
formed/min/mg protein (Nakano and Asada, 1981). For
GR (EC 1.6.4.2) assay, enzyme activity was determined by
monitoring the GSH-dependent oxidation of nicotinamide
adenine dinucleotide phosphate (NADPH) (Carlberg
and Mannervik, 1985). In a cuvette, 0.75 ml 0.2 M
potassium phosphate buffer (pH 7.0) containing 75 μl
NADPH (2 mM), 2 mM EDTA, and 75 μl oxidized GSH
(20 mM) were mixed. Reaction was initiated by adding
0.1 ml enzyme extract to the cuvette and the decrease in
absorbance at 340 nm was monitored for 2 min. GR specific
activity was expressed as nmol NADPH oxidized/min/mg
protein. CAT (EC 1.11.1.6) extraction was performed in a
50 mM Tris-HCl buffer. Activity was assayed by measuring
the reduction of H
2
O
2
at 240 nm (ε = 39.4/mM/cm) and
25°C as detailed earlier (Talukdar, 2013a; 2013b). For
estimation of GSTs (EC 2.5.1.18) specific activity, 1 g of
plant samples was extracted in 5 ml medium containing
50 mM phosphate buffer, pH 7.5, 1 mM DTT, and 1 mM
EDTA. The reaction mixture contained 50 mM phosphate
buffer, pH 7.5, 1 mM 1-chloro-2,4-dinitrobenzene, and
the elute equivalent to 100 μg of protein. The reaction was
initiated with the addition of 1 mM GSH, and formation of
S-(2,4-dinitrophenyl) GSH (DNP-GS) was monitored as
an increase in absorbance at 334 nm to calculate the GST
specific activity (Li et al., 1995).
Gly I (EC: 4.4.1.5) assay was carried out according to
Hossain and Fujita (2010). Briefly, the assay mixture
contained 100 mM K-phosphate buffer (pH 7.0), 15 mM
magnesium sulfate, 1.7 mM GSH, and 3.5 mM MG in a
final volume of 0.7 ml. The reaction was started by the
addition of MG, and the activity was calculated at 240 nm
for 1 min (ε = 3.37/mM/cm).
Gly II (EC: 3.1.2.6) activity was determined following
Principato et al. (1987) by monitoring the formation of
GSH at 412 nm for 1 min. The reaction mixture contained
100 mM Tris-HCl buffer (pH 7.2), 0.2 mM DTNB,
and 1 mM SLG in a final volume of 1 ml. The reaction
was started by the addition of SLG, and the activity was
calculated (ε = 13.6/mM/cm).
Determination of MG Level
Plant tissues were homogenized in 5% perchloric acid and
centrifuged at 4°C for 10 min at 11,000 g. The supernatant
was decolorized by adding charcoal; then, centrifuged
at 11,000 g for 12 min. The supernatant neutralized
by a saturated solution of potassium carbonate at room
temperature was used for MG estimation by adding sodium
dihydrogen phosphate and 20 μl of freshly prepared 0.5 M
N-acetyl-L-cysteine to a final volume of 1 ml. Formation
of the product N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)
cysteine was recorded after 10 min at 288 nm (Wild et al.,
2012). The MG content was calculated by the standard
curve and expressed as μmol/g/FW.
Determination of H
2
O
2
Content, Membrane Lipid
Peroxidation, and Electrolyte Leakage (EL)
H
2
O
2
was estimated following Wang et al. (2007) from
the absorbance at 410 nm using a standard curve. Lipid
peroxidation rates were determined by measuring the
malondialdehyde (MDA) equivalents according to Hodges
et al. (1999). EL% was measured according to Dionisio-
Sese and Tobita (1998).
Talukdar: Thiourea-induced arsenate tolerance in lentil genotypes
12 Journal of Plant Stress Physiology ● Vol 2 ● 2016
ROS Imaging
Detection and imaging of superoxide radicals in leaf and
root sections were carried out using the fluorescence probe
dihydroethidium (DHE) (Rodr´ıguez-Serrano et al., 2006).
Leaf/root segments of approximately 30 mm
2
were incubated
for 1 h at 25°C, in darkness, with 10 μM DHE prepared in
5 mM Tris-HCl buffer at pH 7.4, and samples were washed
twice with the same buffer for 12 min each. After washing,
sections were embedded in a mixture of 15% acrylamide-
bisacrylamide stock solution, and 100 mm thick sections,
were cut under 10 mM phosphate-buffered saline (PBS).
Sections were then soaked in glycerol: PBS (containing azide)
(1:1 v/v) and mounted in the same medium for examination
with a confocal LASER scanning microscopy (CLSM) system
(Carl Zeiss, LSM 780, Bengaluru, India) using standard
filters and collection modalities for DHE green fluorescence
(λ excitation 488 nm; λ emission 520 nm). H
2
O
2
was
detected by incubation with 25 μM 2'7'-dichloro fluorescein
diacetate (DCF-DA) (excitation 485 nm, emission 530 nm)
(Rodr´ıguez-Serrano et al., 2006). Preinfused sections with
1 mM tetramethylpiperidinyloxy (TMP), a scavenger of
superoxide radicals, and 1 mM AsA, a scavenger of H
2
O
2
served as negative controls.
Statistical Analysis
The results are the mean values ± standard errors of at
least four replicates. Multiple comparisons of means were
performed by ANOVA (SPSS Inc., version 10), and the
means were separated by Duncan’s multiple range test
with significance level at P < 0.05. Simple correlation was
carried out among different traits using Microsoft Excel
data analysis tool pack 2007.
RESULTS
Changes in Plant Height, Dry Weight, and As Uptake
Potential
Lentil genotypes exhibited significant growth inhibition as
both root and shoot length (SL) and dry weights reduced
significantly (P < 0.05) in comparison to control at
30 μM As. Stem and root length (RL) in lentil genotypes
were reduced by 2-2.5-fold (Table 1). In L 9 and Pusa 4,
while shoot dry weight (SDW) was reduced by about 2-2.7-
fold root dry weight (RDW) was decreased by about 3-3.3-
fold (Table 1). SDW in K 75 and PL 234 were declined by
nearly 1.8-fold and 3.1-fold, respectively, whereas RDW
was decreased by about 2.5-fold and 4.5-fold, respectively.
Co-application of 6.5 mM TU with As did not change SL
and RL as well as SDW and RDW significantly in L 9 and
Pusa 4. However, both dry weights increased over control
by about 1.5-fold at As + 13 mM TU in both the genotypes
(Table 1). SDW and RDW exhibited upward trend in K
75 and Pl 234 genotypes at As + 6.5 mM TU and further
increased at As + 13 mM TU (Table 1). At 30 μM, As
accumulated in marginally higher amount in roots of all
four genotypes than that of shoots (shoot/root TF = 1.0).
Application of TU at 6.5 mM and 13 mM significantly
increased As amount in roots compared to shoots (shoots/
roots TF <1.0) of all four genotypes but both the K 75 and
PL 234 accumulated significantly higher As content than L
9 and Pusa 4 in their roots in presence of TU (Figure 1a).
Changes in Leaf Photosynthetic Pigment
Leaf Chl A, Chl A/B ratio, and carotenoids reduced
significantly in four genotypes with different magnitudes
Table 1: Changes in RL (cm/plant), SL (cm/plant), SDW (g/plant), RDW (g/plant) in L 9, Pusa 4, K 75, and PL 234 genotypes of
lentils under untreated control, sodium arsenate (30 μM As), As+6.5 mM TU and As+13 mM TU for 10 days treatment duration
Treatments (traits) L 9 Pusa 4 K 75 PL 234
Control (RL) 28.88±1.8
bb
ʹ 20.89±1.3
cb
ʹ 33.45±2.1
aa
ʹ 36.87±1.9
aa
ʹ
As (RL) 11.56±1.1
bb
ʹ 8.41±1.0
bb
ʹ 16.72±1.3
aa
ʹ 16.76±1.3
aa
ʹ
As+6.5 mM TU (RL) 29.03±1.9
bb
ʹ 21.03±1.4
cb
ʹ 33.57±2.3
aa
ʹ 37.00±2.9
aa
ʹ
As+13 mM TU (RL) 33.03±2.6
ba
ʹ 28.08±1.6
cb
ʹ 36.51±2.8
aa
ʹ 37.88±3.2
aa
ʹ
Control (SL) 31.45±1.9
bb
ʹ 27.56±1.2
bb
ʹ 39.11±2.0
aa
ʹ 37.22±1.9
aa
ʹ
As (SL) 12.60±1.4
bb
ʹ 11.02±1.0
bb
ʹ 20.08±1.4
aa
ʹ 17.01±1.5
aa
ʹ
As+6.5 mM TU (SL) 31.99±2.0
bb
ʹ 28.11±1.2
bb
ʹ 40.12±2.0
aa
ʹ 37.76±1.9
aa
ʹ
As+13 mM TU (SL) 32.11±2.2
bb
ʹ 29.13±1.3
bb
ʹ 44.32±2.9
aa
ʹ 39.52±2.3
aa
ʹ
Control (SDW) 0.19±0.04
bb
ʹ 0.21±0.04
bb
ʹ 0.30±0.06
aa
ʹ 0.32±0.08
aa
ʹ
As (SDW) 0.10±0.02
bb
ʹ 0.08±0.01
bb
ʹ 0.17±0.03
aa
ʹ 0.19±0.02
aa
ʹ
As+6.5 mM TU (SDW) 0.20±0.04
bb
ʹ 0.22±0.04
bb
ʹ 0.31±0.07
aa
ʹ 0.34±0.09
aa
ʹ
As+13 mM TU (SDW) 0.29±0.09
bb
ʹ 0.32±0.10
bb
ʹ 0.43±0.12
aa
ʹ 0.46±0.17
aa
ʹ
Control (RDW) 0.24±0.03
bb
ʹ 0.26±0.04
bb
ʹ 0.36±0.09
aa
ʹ 0.39±0.10
aa
ʹ
As (RDW) 0.08±0.01
bb
ʹ 0.08±0.01
bb
ʹ 0.14±0.02
aa
ʹ 0.15±0.01
aa
ʹ
As+6.5 mM TU (RDW) 0.23±0.03
bb
ʹ 0.27±0.04
bb
ʹ 0.37±0.09
aa
ʹ 0.41±0.11
aa
ʹ
As+13 mM TU (RDW) 0.36±0.10
bb
ʹ 0.39±0.10
bb
ʹ 0.47±0.14
aa
ʹ 0.50±0.16
aa
ʹ
Means±SE of four replicates treatment
−1
. Means followed by different lowercase letters in rows (genotypes) and prime lowercase letters in
columns (treatments) are significantly different at P<0.05 by ANOVA followed by Duncan’s multiple range test. RL: Root length, SL: Shoot length,
SDW: Shoot dry weight, RDW: Root dry weight, TU: Thiourea, As: Arsenic
Talukdar: Thiourea-induced arsenate tolerance in lentil genotypes
Journal of Plant Stress Physiology ● Vol 2 ● 2016 13
(Figure 1b). As treatment alone reduced CHL A content
by about 2.5-3.2-fold in L 9 and Pusa 4 and 1.3-1.5-fold
in K 75 and PL 234 genotypes. CHL B content did not
change significantly, but Chl A/B ratio reduced markedly
in all genotypes. The carotenoid level was also severely
affected with nearly 2.2 (L 9) to 2.7-fold (Pusa 4) decrease
and 1.5 (K 75) to 1.9 (PL 234)-fold reduction in lentil
genotypes (Table 1). Application of As + 6.5 mM TU and
As + 13 mM TU considerably restored the photosynthetic
pigment levels in four genotypes. Chl A content, Chl A/B
ratio, and carotenoid levels even increased significantly
in Pusa 4 and PL 234 exposed to As + 13 mM TU
(Figure 1b). Chl A/B ratio was significantly correlated
with SDW in L 9 (r = 0.703, n = 12, P < 0.05), Pusa 4
(r = 0.710, n = 12, P < 0.05), K 75 (r = 0.813, n = 12,
P < 0.05), and PL 234 (r = 0.789, n = 12, P < 0.05)
genotypes (data not in table).
Response of Antioxidant Defense Components and
Gly System
Foliar and roots AsA and GSH content decreased
while DHA and GSH disulfide (GSSG) level increased
significantly over control during As exposure alone
(Table 2). Among the four genotypes, As-treatment
reduced AsA and GSH levels in both organs of L 9 and
Pusa 4 in higher extent than that in K 75 and PL 234
(Table 2). Compared to control, AsA and GSH redox
values decreased in all four genotypes, but foliar AsA
and GSH redox did not change significantly among the
genotypes. Respective redox values in roots, however,
were significantly higher in K 75 and PL 234 (Table 2). At
As + 6.5 mM TU, AsA level was remained low in L 9 and
Pusa 4 but comparable to control in K 75 and PL 234. GSH
and GSSG content were comparable to control in all four
genotypes (Table 2). Upon exposed to As + 13 mM TU,
root AsA, and GSH content significantly increased, but
DHA and GSSG levels decreased in the genotypes. Leaf
AsA and GSH did not change significantly in the four
genotypes compared to their respective controls. AsA and
GSH redox changed, accordingly (Table 2).
Antioxidant enzymatic activities also differed sharply
between presence and absence of TU. As exposure
alone reduced foliar and root APX, DHAR, GR, and
GSTs activities in all four genotypes but the treatment
enhanced SOD activity (Figure 2a-e). CAT activity did
not change significantly in K 75 and PL 234 but declined
significantly in L 9 and Pusa 4 seedlings (Figure 2f). Upon
imposition of As + 6.5 mM TU, foliar and root activities
of APX, DHAR, GR, and GSTs became as per control in
all genotypes, but CAT level was remained low. At As +
13 mM TU, activities of five enzymes were significantly
higher in roots but were comparable in leaves of all four
genotypes (Figure 2a-c, e, f). SOD activity, however, was
noticeably low at As + 6.5 mM TU, and further reduced
at As + 13 mM TU (Figure 2d).
As treatment significantly reduced Gly I and II activities
in leaves and roots of L 9 and Pusa 4 genotypes
(Figure 3a and b). Gly I activity did not change significantly,
but Gly II level decreased markedly in As-treated K 75
and PL 234 (Figure 3a and b). At As + 6.5 mM TU, Gly
I and II activities became comparable to control in leaves
of four genotypes but increased markedly in their roots.
At As + 13 mM TU, root Gly I and II activity increased
significantly in the genotypes, but the foliar activity of
both enzymes did not change significantly (P > 0.05) in all
four genotypes under As + 13 mM TU (Figure 3a and b).
Changes in Methylglyoxal, H
2
O
2
, and Oxidative Stress
Level
As treatment alone significantly elevated MG and other
oxidative stress markers such as H
2
O
2
, MDA, and EL%
in leaves and roots of four genotypes (Table 3). MG level
Figure 1: Changes in (a) Arsenic (As) content in shoots and roots and
(b) leaf chlorophyll (Chl) A, B, Chl A/B ratio, and carotenoid contents of
four lentil genotypes (L 9, Pusa 4, K 75, and PL 234) under untreated
control, 30 μM arsenate and As + thiourea (TU) treatments. Data are
means ± standard error of four replicates with different lowercase letters
over error bars represent signifi cant differences (P < 0.05) at ANOVA
followed by Duncan’s multiple range test
b
a