Plant Science 164 (2003) 645 /655
www.elsevier.com/locate/plantsci
Lead toxicity induces lipid peroxidation and alters the activities of
antioxidant enzymes in growing rice plants
Shalini Verma, R.S. Dubey *
Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India
Received 30 July 2002; received in revised form 8 January 2003; accepted 8 January 2003
Abstract
When seedlings of two rice (Oryza sativa L. ) cultivars were raised in sand cultures under 500 and 1000 mM Pb(NO3)2 in the
medium, lengths as well as weights of roots and shoots decreased with increase in Pb concentration. Pb-treated seedlings showed
elevated levels of lipid peroxides with a concomitant increase in the activities of the enzymes superoxide dismutase (SOD), guaiacol
peroxidase, ascorbate peroxidase and glutathione reductase compared to controls. Though Pb was readily absorbed by growing
seedlings, its localization was greater in roots than shoots. The level of Pb accumulation in seedlings was far higher than the supplied
one. Seedlings grown for 5 /20 days in presence of 1000 mM Pb(NO3)2 showed about 21 /177% increase in the level of thiobarbituric
acid reacting substances (TBARS) in shoots indicating enhanced lipid peroxidation compared to controls. With increase in the level
of Pb treatment in situ peroxidases showed more increase in activity than SOD. Under both controls as well as Pb treatments roots
maintained higher activity of these enzymes than shoots. About 87 /100% increase in SOD activity, 1.2 /5.6 times increase in
guaiacol peroxidase activity and 1.2 /1.9 times increase in ascorbate peroxidase activity was observed in the roots of seedlings grown
for 15 days in presence of 1000 mM Pb in the medium. Under similar treatment conditions about 128 /196% increase in glutathione
reductase activity was recorded in roots and 69 /196% increase in shoots compared to control grown seedlings. Pb treatment resulted
in a decline in catalase activity in roots whereas in shoots catalase activity increased in seedlings grown at moderately toxic Pb (500
mM) level whereas a highly toxic Pb (1000 mM) level led to a marked inhibition in enzyme activity. Two catalase isoforms were
detected in roots and three in shoots of the seedlings. A highly toxic Pb (1000 mM) level led to decrease in the intensity of two preexisting catalase isoforms in shoots. Results suggest that Pb induces oxidative stress in growing rice plants and that SOD,
peroxidases and GR could serve as important components of antioxidative defense mechanism against Pb induced oxidative injury
in rice.
# 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Catalase; Glutathione reductase; Isoenzyme; Lead; Lipid peroxides; Peroxidase; Rice; Superoxide dismutase
1. Introduction
Lead is one of the hazardous heavy metal pollutants
of the environment that originates from various sources
like mining and smelting of lead-ores, burning of coal,
effluents from storage battery industries, automobile
exhausts, metal plating and finishing operations, fertilizers, pesticides and from additives in pigments and
gasoline [1]. Its increasing levels in soil environment
inhibit germination of seeds and exert a wide range of
* Corresponding author. Tel.: /91-542-231-7190; fax: /91-542236-8174.
E-mail address: [email protected] (R.S. Dubey).
adverse effects on growth and metabolism of plants [2 /
4]. A variety of environmental stresses like soil salinity,
drought, extremes of temperature and heavy metals are
known to cause oxidative damage to plants either
directly or indirectly by triggering an increased level of
production of reactive oxygen species (ROS) [5 /10].
These ROS include superoxide radical (O2+ ), hydroxyl
radical (OH+ ) and hydrogen peroxide (H2O2) that are
produced as by products during membrane linked
electron transport activities as well as by a number of
metabolic pathways [7] and in turn cause damage to the
biomolecules such as membrane lipids, proteins, chloroplast pigments, enzymes, nucleic acids, etc. [8].
To combat the oxidative damage plants have the
antioxidant defense system comprising of enzymes
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved.
doi:10.1016/S0168-9452(03)00022-0
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S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
catalase (EC 1.11.1.6), peroxidases (EC 1.11.1.7), superoxide dismutases (SOD, EC 1.15.1.1) and the nonenzymic constituents a-tocopherol, ascorbate and reduced glutathione which remove, neutralize and scavenge the ROS [7]. The enzymes of Halliwell-Asada
pathway or ascorbate/glutathione cycle such as ascorbate peroxidase (EC 1.11.1.11), monodehydro ascorbate
reductase (MDAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase
(GR, EC 1.6.4.2) also play a significant role in scavenging H2O2 mainly in chloroplasts and in maintaining the
redox status of the cell [11].
Catalases are involved in scavenging H2O2 generated
during the photo-respiration and b-oxidation of fatty
acids [12]. Peroxidases are heme containing proteins that
utilize H2O2 in the oxidation of various organic and
inorganic substrates [13]. Peroxidases utilizing guaiacol
as electron donor in vitro are guaiacol peroxidases and
participate in developmental processes, lignification,
ethylene biosynthesis, defense, wound healing, etc. [14].
The other group of peroxidases scavenge H2O2 in cell
and utilize glutathione, Cyt c, pyridine nucleotide and
ascorbate as electron donors in vitro [14]. Guaiacol
peroxidases are glycoproteins, located in cytosol, vacuole, cell wall and in extracellular space, while the other
group is non glycosylated and localized in chloroplasts
and cytosol [14]. SODs represent a group of multimeric
metalloenzymes catalyzing the disproportionation of
superoxide free radicals, generated by univalent reduction of molecular oxygen to H2O2 and O2 in different
cellular compartments [15]. Glutathione reductase is a
member of flavoenzyme family which catalyzes the
NADPH dependent reduction of glutathione disulphide
(GSSG) to glutathione (GSH). This reaction maintains a
proper GSH/GSSG concentration ratio in cells [16].
The ROS are chemically aggressive species and the
attack of free radicals on the polyunsaturated fatty acid
components of membrane lipids initiates lipid peroxidation, an autocatalytic process that changes membrane
structure and function [7]. Measurement of the level of
thiobarbituric acid reactive substances (TBARS) in the
tissues is widely used as an index of lipid peroxidation
[17].
The heavy metals Cd, Pb, Al, Zn are known to
produce ROS and induce oxidative stress in certain
plant species [7,10,18 /20]. Our earlier studies suggested
that rice plants freely absorbed Cd and the accumulation of Cd in the tissues paralleled with enhanced lipid
peroxidation and marked elevation in the levels of the
antioxidant enzymes SOD and peroxidase [7]. As Pb is
one of the most abundant heavy metal pollutants in
both aquatic as well as terrestrial environments and rice
which is partially halophytic crop that serves as staple
food for the majority of world population, the present
study was undertaken to examine the uptake and
distribution pattern of Pb in rice seedlings, to determine
Pb-induced possible induction of the oxidative stress
and likely alterations in behaviour of the enzymes of
antioxidant defense system in rice plants.
2. Materials and methods
2.1. Plant material and stress conditions
Two rice (Oryza sativa L.) cvs. Ratna and Jaya were
used. Seeds were surface sterilized with 0.1% sodium
hypochlorite solution for 10 min and then rinsed with
double distilled water. After 24 h imbibition of seeds in
water seedlings were raised in sand cultures in plastic
pots saturated with either Hoagland nutrient solution
[21] which served as control or nutrient solutions
supplemented with Pb(NO3)2 to achieve concentrations
of 500 mM (103.6 ppm Pb2) and 1000 mM (207.2 ppm
Pb2) which served as treatment solutions. The choice
of 500 and 1000 mM Pb represents the moderate and
high concentrations mimicking polluted soils. Our data
with these two concentrations of Pb serve as signposts
for effects at other concentrations. Pots were maintained
at field saturation capacity and received control and
respective treatment solutions when needed to saturate
the sand. Pots were kept for growth of seedlings in a
biological oxygen demand (B.O.D.) cum humidity
incubator at 289/1 8C under 80% relative humidity
and 12 h photoperiod with 40/50 mmol 2 s 1 irradiance. Seedlings were uprooted at 5-day intervals up to
20 days and all experiments were performed in triplicate.
2.2. Evaluation of seedling vigour and determination of
lead content
At different days of growth of seedlings length as well
as fresh weight of roots and shoots were determined
based on ten random samplings in triplicate. To
determine the amount of absorbed lead in the seedling,
fresh root/shoot samples were surface sterilized with 1 M
HCl and then with 1 mM Na2EDTA to resolve excess
surface bound Pb and then dried in oven at 70 8C for
4 /5 days. Dried samples were ground to a fine powder
in a mortar and pestle and digested with conc. H2SO4.
Digested samples were dissolved in deionized distilled
water and lead content was estimated using atomic
absorption spectrometer (AAS) fitted with PerkinElmer-337 Atomic Absorption Spectroscope in terms
of mmol g1 dry wt. of sample. Estimation was carried
out in triplicate. Standards from a stock solution of
Pb(NO3)2 dissolved in HCl were prepared in perchloric
acid.
S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
2.3. Estimation of lipid peroxides
The level of lipid peroxidation products was estimated
following the method of Heath and Packer [22]. Fresh
root/shoot samples (200 mg) were ground in 0.25%
thiobarbituric acid (TBA) in 10% TCA using mortar
and pestle. The mixture was heated at 95 8C for 30 min
and then quickly cooled in an ice bath and centrifuged at
10 000 /g for 10 min. The absorbance of the supernatant was read at 532 nm and correction for unspecific
turbidity was done by subtracting the absorbance of the
same at 600 nm. A total of 0.25% TBA in 10% TCA
served as blank. The concentration of lipid peroxides
together with the oxidatively modified proteins of plants
were quantified and expressed as total TBARS in terms
of nmol g1 fresh weight using an extinction coefficient
of 155 mM 1 cm 1. TBARS are an index of lipid
peroxidation [22].
647
specific activity is expressed as mmol of H2O2 oxidized
min 1 (mg protein)1.
2.6. Guaiacol peroxidase assay
Guaiacol peroxidase (EC 1.11.1.7) was assayed according to Egley et al. [25]. Fresh root/shoot samples
weighing 200 mg were homogenized in 5 ml of cold 50
mM Na-phosphate buffer (pH 7.0). The homogenates
were centrifuged at 22 000/g for 10 min and the
dialyzed enzyme extracts were used for the assay. Assay
mixture in a total volume of 5 ml contained 40 mM Naphosphate buffer (pH 6.1), 2 mM H2O2, 9 mM guaiacol
and 50 ml enzyme. Increase in absorbance was measured
at 420 nm (extinction coefficient of 26.6 mM 1 cm 1)
at 30 s intervals up to 2 min, using a Bausch and Lomb
Spectronic-20 spectrophotometer (USA). Enzyme specific activity is expressed as mmol of H2O2 reduced
min 1 (mg protein)1.
2.4. Superoxide dismutase assay
2.7. Ascorbate peroxidase assay
The activity of SOD was assayed according to Misra
and Fridovich [23]. About 200 mg fresh tissues were
homogenized in 5 ml of 100 mM K-phosphate buffer
(pH 7.8) containing 0.1 mM EDTA, 0.1% (v/v) Triton
X-100 and 2% (w/v) polyvinyl pyrrolidone (PVP). The
extract was filtered through muslin cloth and centrifuged at 22 000/g for 10 min at 4 8C. Supernatant was
dialyzed in cellophane membrane tubings against the
cold extraction buffer for 4 h with 3 /4 changes of the
buffer and then used for the assay. The assay mixture in
a total volume of 3 ml contained 50 mM sodium
carbonate /bicarbonate buffer (pH 9.8), 0.1 mM
EDTA, 0.6 mM epinephrine and enzyme. Epinephrine
was the last component to be added. The adrenochrome
formation in the next 4 min was recorded at 475 nm in a
UV-Vis spectrophotometer. One unit of SOD activity is
expressed as the amount of enzyme required to cause
50% inhibition of epinephrine oxidation under the
experimental conditions.
About 200 mg root/shoot samples were homogenized
in 5 ml of 50 mM K-phosphate buffer (pH 7.8)
containing 1% PVP, 1 mM ascorbic acid and 1 mM
PMSF as described by Moran et al. [26]. After
centrifugation at 22 000/g for 10 min at 4 8C, the
supernatant was dialyzed against the same extraction
buffer and it served as enzyme. Ascorbate peroxidase
was assayed according to Nakano and Asada [27].
Reaction mixture in a total volume of 1 ml contained
50 mM K-phosphate buffer (pH 7.0), 0.2 mM ascorbic
acid, 0.2 mM EDTA, 20 mM H2O2 and enzyme. H2O2
was the last component to be added and the decrease in
absorbance was recorded at 290 nm (extinction coefficient of 2.8 mM 1 cm 1) using a UV-Vis spectrophotometer (ELICO, India) at 30 s intervals up to 7 min.
Correction was made for the low, non enzymic oxidation of ascorbic acid by H2O2. The specific activity of
enzyme is expressed as mmol ascorbate oxidized min 1
(mg protein) 1.
2.5. Catalase assay
2.8. Glutathione reductase assay
The activity of catalase was assayed according to
Beers and Sizer [24]. Fresh samples (200 mg) were
homogenized in 5 ml of 50 mM Tris /NaOH buffer (pH
8.0) containing 0.5 mM EDTA, 2% (w/v) PVP and 0.5%
(v/v) Triton X-100. The homogenate was centrifuged at
22 000 /g for 10 min at 4 8C and after dialysis supernatant was used for enzyme assay. Assay mixture in a
total volume of 1.5 ml contained 1000 ml of 100 mM
KH2PO4 buffer (pH 7.0), 400 ml of 200 mM H2O2 and
100 ml enzyme. The decomposition of H2O2 was
followed at 240 nm (extinction coefficient of 0.036
mM 1 cm 1) by decrease in absorbance. Enzyme
Glutathione reductase was assayed according to
Schaedle and Bassham [28]. Fresh root/shoot samples
weighing 200 mg were homogenized using chilled mortar
and pestle in 5 ml of 50 mM Tris /HCl buffer (pH 7.6).
The homogenate was centrifuged at 22 000 /g for 30
min at 4 8C and the supernatant after dialysis was used
for enzyme assay. The reaction mixture in a total
volume of 1 ml contained 50 mM Tris /HCl buffer
(pH 7.6), 0.15 mM NADPH, 1 mM GSSG, 3 mM
MgCl2 and 200 ml enzyme extract. The reaction was
monitored by decrease in absorbance of NADPH at 340
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S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
nm. The specific activity of enzyme is expressed as mmol
NADPH oxidized min 1 (mg protein) 1.
2.9. Isoenzyme profile of catalase
To determine the influence of Pb2 toxicity in situ on
changes in isoforms of catalase in growing rice seedlings,
rice cv . Jaya was grown for 15 days under increasing
concentrations of Pb(NO3)2 in the growth medium.
Catalase was extracted from roots and shoots and
polyacrylamide gel electrophoresis was performed in
vertical slab gel following the method of Davis at 4 8C
[29]. Tris /glycine (pH 8.3) was used as electrode buffer
and 7.5% running and 3.5% stacking gels were used.
Enzyme samples corresponding to 30 mg protein mixed
with glycerol were layered on top of the stacking gel and
electrophoretic run was completed using a current of 25
mA per slab. For detection of catalase isoforms, gels
were soaked in 5 mM K-phosphate buffer (pH 7.0) and
then transferred to a 5 mM H2O2 solution in the same
buffer. After 10-min incubation, gels were rinsed with
water and stained in a reaction mixture containing 2%
(w/v) potassium ferricyanide and 2% (w/v) ferric chloride. The isozymes appeared as colourless bands on a
deep blue background.
2.10. Protein determination
In all the enzyme preparations protein was determined by the method of Lowry et al. [30] using bovine
serum albumin (BSA, Sigma) as standard.
Fig. 1. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on the length of roots and shoots of rice cvs. Ratna and Jaya
at increasing days of growth. Values are mean9/S.D. based on ten
random samples in triplicate and bars indicate S.Ds.
roots and up to 0.8008 mmol g1 dry wt Pb absorbed in
shoots.
3. Results
3.1. Seedling vigour and uptake of lead by growing rice
seedlings
When rice seedlings were grown under increasing
concentrations of lead in the growth medium, during a
5 /20 day growth period increasing lead levels caused
decrease in length as well as fresh weights of seedlings
(Figs. 1 and 2). With 1000 mM Pb in the medium up to
40% reduction in root length and 31% reduction in
shoot length was observed in 20-day grown seedlings.
Similarly up to 43% decline in fresh weight of roots and
up to 29% decline of shoots was noticed in the seedlings
at 20 days of growth.
When rice seedlings were raised under increasing
concentrations of lead, a continuous increase in the
content of lead was observed in seedlings with increasing
days of growth (Fig. 3). The absorbed lead was localized
to a greater extent in roots than in shoots. Seedlings
grown under 1000 mM Pb (207.2 ppm) for 20 days
showed up to 1.3065 mmol g1 dry wt of Pb absorbed in
3.2. Effect of lead on lipid peroxidation
In seedlings of both the rice cvs . Ratna and Jaya
during a growth period of 5/20 days, the level of lipid
peroxides, measured in terms of TBARS, increased with
increase in the concentration of Pb(NO3)2 in the growth
medium (Table 1). A 1000 mM Pb treatment level led to
about 21 /177% increase in TBARS level in shoots
during 5 /20 days growth period of seedlings compared
to controls.
3.3. Effect of lead on superoxide dismutase activity
The activity of SOD increased gradually during early
days of growth of seedlings with maximum at 15/20
days under both control as well as Pb treatments (Fig.
4). Pb(NO3)2 treatment in situ caused an induction in
the activity of SOD. At 15th day of growth of seedlings,
a 1000 mM Pb treatment led to about 87 /100% increase
in SOD activity in roots and about 39/51% increase in
S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
Fig. 2. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on fresh weight of roots ( */) and shoots (---) of rice cvs.
Ratna and Jaya at increasing days of growth. Values are mean9/S.D.
based on ten random samples in triplicate and bars indicate S.Ds.
649
Fig. 3. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on lead content of roots and shoots of rice cvs. Ratna and
Jaya at increasing days of growth. Values are mean9/S.D. based on
three replicates and bars indicate S.Ds.
shoots. Roots maintained higher SOD activity than
shoots under both control and Pb treatments.
Table 1
Level of total lipid peroxides in shoots of seedlings of rice cvs . Ratna
and Jaya at 5 to 20 days of growth under increasing concentrations of
Pb(NO3)2
3.4. Effect of lead on catalase activity and isoforms
Age of seedlings (days) Treatments
The activity of catalase increased during early days of
growth of seedlings with maximum at 10 /15 days and it
declined thereafter (Fig. 5). With increasing levels of Pb
treatment a concomitant decline in catalase activity was
observed in roots while in shoots a higher Pb treatment
level of 1000 mM led to marked inhibition in enzyme
activity. Seedlings growing under 1000 mM Pb in the
medium showed about 27/38% decline in catalase
activity in roots and about 24/72% decline in shoots
at 20 days of growth. Compared to roots, a greater
inhibition in catalase activity was observed in shoots
with 1000 mM in situ Pb treatment. Fig. 6 shows the
isoenzyme pattern of catalase in enzyme preparations
from roots and shoots or rice cv. Jaya at 15 days of
growth. As it is evident, in enzyme preparations from
roots, two catalase isozymic bands with Rf values 0.15
and 0.29 were observed in controls as well as Pb
treatments. However, in shoot samples, three isozyme
Total lipid peroxide
TBARS (nmol g 1 f.wt.)
Ratna
5
10
15
20
Jaya
Control
24.979/1.23 26.169/1.31
500 mM Pb2
30.399/1.51 51.619/2.79
1000 mM Pb2 32.589/1.59 72.589/3.82
Control
44.529/2.29 47.429/2.27
500 mM Pb2
60.329/3.12 59.039/3.07
1000 mM Pb2 75.169/3.76 76.459/3.73
Control
59.689/3.28 53.399/2.86
500 mM Pb2
72.429/3.91 69.849/3.56
1000 mM Pb2 88.559/4.78 75.819/3.94
Control
91.639/4.48 100.369/4.98
500 mM Pb2 109.839/4.97 128.399/6.57
1000 mM Pb2 121.039/6.05 146.779/7.77
Data represent mean values9/S.D. based on three independent
determinations.
bands with Rf values 0.15, 0.29 and 0.42 were detected in
controls and Pb treated seedlings. The intensity of band
with Rf value 0.29 increased in shoots with increase in
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S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
Fig. 4. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on SOD activity in roots ( */) and shoots (---) of rice cvs.
Ratna and Jaya at increasing days of growth. Values are mean9/S.D.
based on three replicates and bars indicate S.Ds.
the level of Pb treatment. In shoots of 1000 mM Pb
treated seedlings, the band with Rf value 0.15 seemed to
disappear. Intensity of catalase isozymes was greater in
roots than in shoots.
Fig. 5. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on catalase activity in roots ( */) and shoots (---) of rice cvs.
Ratna and Jaya at increasing days of growth. Values are mean9/S.D.
based on three replicates and bars indicate S.Ds.
showed higher level of enzyme activity than shoots.
Under 1000 mM Pb treatment about 128 /196% increase
in enzyme activity was observed in roots and 69/196%
increase in shoots in 15 day grown seedlings.
3.5. Effect of lead on guaiacol peroxidase activity
3.7. Effect of lead on glutathione reductase activity
With in situ Pb an increase in guaiacol peroxidase
activity was observed in both roots as well as shoots of
the two rice cultivars (Fig. 7). Under both control as
well as Pb treatments roots maintained higher guaiacol
peroxidase activity than shoots. A 1000 mM Pb treatment led to about 1.2 /5.6 times increase in guaiacol
peroxidase activity in roots of rice seedlings at 15th day
of growth.
3.6. Effect of lead on ascorbate peroxidase activity
Similar to guaiacol peroxidase, the activity of ascorbate peroxidase showed a concomitant increase in
seedlings with increase in Pb treatment levels (Fig. 8)
and under both controls and Pb treatments roots
In control grown rice seedlings of both the cultivars
the activity behaviour of glutathione reductase was
different in roots than in shoots during a 5/20 day
growth period (Fig. 9). During early days of growth of
seedlings, up to 10 days, the activity of enzyme increased
in both roots as well as shoots, whereas during 10/20
day period a gradual decline in enzyme activity was
noticed in roots and not in shoots where enzyme showed
gradual increase in activity up to day 20. But, in both
the rice cultivars increasing levels of Pb treatment in situ
led to a marked increase in enzyme activity. At day 15 of
growth, seedlings growing in presence of 1000 mM Pb
showed about 52 /127% increase in glutathione reductase activity in roots and 66/114% increase in shoots.
S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
651
Fig. 6. Isoenzyme profile of catalase in enzyme preparations from roots and shoots of 15 days grown seedlings of rice cv. Jaya. Seedlings were raised
under increasing concentrations of Pb(NO3)2 in the growth medium. (C) Control; (500), 500 mM Pb(NO3)2; (1000), 1000 mM Pb(NO3)2. For details
see text.
Fig. 7. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on activity of guaiacol peroxidase in roots ( */) and shoots
(---) of rice cvs. Ratna and Jaya at increasing days of growth. Values
are mean9/S.D. based on three replicates and bars indicate S.Ds.
Fig. 8. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on activity of ascorbate peroxidase in roots ( */) and shoots
(---) of rice cvs. Ratna and Jaya at increasing days of growth. Values
are mean9/S.D. based on three replicates and bars indicate S.Ds.
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S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
Fig. 9. Effect of increasing concentration of Pb(NO3)2 in the growth
medium on the activity of glutathione reductase in roots ( */) and
shoots (---) of rice cvs. Ratna and Jaya at increasing days of growth.
Values are mean9/S.D. based on three replicates and bars indicate
S.Ds.
4. Discussion
Lead is one of the most abundant heavy metals
polluting the soil environment [1 /4]. It is readily
absorbed by plants mainly through the root system
and thereafter exerts its toxicity symptoms. Metal
phytotoxicity occurs when metals move from soil to
plant roots and are further transported to various sites
in the shoots. The effects of Pb phytotoxicity include
stunted growth, chlorosis, blackening of the root
systems [2], alteration in water and nutritional status
of plants [4] as well as various plant processes [2,4].
Our results indicated decrease in vigour (length and
weight) of rice seedlings when raised under increasing
levels of Pb(NO3)2 in sand culture experiments. We
conducted experiments with Pb(NO3)2 of increasing
concentrations up to 1000 mM and based on our
observations (data not reported here) we concluded a
Pb(NO3)2 level of 500 mM as moderately toxic and 1000
mM as highly toxic. These two toxicity levels were used
for raising seedlings for all other experiments. Decreased
seedling vigour in rice due to Pb could possibly be
attributed to the interference of Pb with the metabolic
and biochemical processes associated with normal
growth and development of the plant.
Our studies on Pb uptake indicated an increased
uptake of Pb in rice seedlings with increase in Pb
concentration in the growth medium and that the
absorbed lead is distributed in an organ specific manner
with its localization greater in roots than in shoots. It
has been shown that Pb is unevenly distributed in roots,
where different root tissues act as barriers to apoplastic
and symplastic Pb transport and hence Pb transport to
shoot gets restricted [31]. Our results indicated that in 20
day grown rice seedlings, the accumulated Pb level was
higher than the Pb supplied in the growth medium. This
suggests that rice, a partially halophytic plant, accumulates Pb against the concentration gradient. These
results corroborate with our earlier findings with the
heavy metal Cd which was also found to accumulate in
rice seedlings to a greater extent in roots than in shoots
and that the uptake of Cd was against concentration
gradient [7].
In many plant species heavy metals have been
reported to cause oxidative damage due to production
of ROS [7,10,18 /20]. To resist oxidative damage, the
antioxidant enzymes and certain metabolites present in
plants play an important role leading to adaptation and
ultimate survival of plants during periods of stress
[10,11]. The present study suggests that Pb toxicity in
situ leads to production of lipid peroxides and induces
some of the key enzymes of antioxidant defense system
in rice plants. Induction in the activities of antioxidative
enzymes is a general strategy adopted by plants to
overcome oxidative stress due to the imposition of
environmental stresses [10,32].
Lipid peroxidation is a biochemical marker for the
free radical mediated injury. Our results show an
increase in the level of lipid peroxides with increasing
concentrations of Pb, indicating that Pb induces oxidative stress in rice plants. Our results are in conformity
with the observations of Malecka and coworkers [10]
who reported Pb-induced oxidative stress in pea root
cells. Similar to our observations, enhanced lipid
peroxidations have been reported under severe water
stress [6], high temperature [9], UV-radiation [33], Cd
and Zn toxicity [7,20] in different plant species.
The enzymic components associated with defense
against ROS include SOD, catalase, peroxidase and
enzymes of ascorbate /glutathione cycle. SOD and
catalase have been identified as enzymatic protectors
against peroxidation reactions [34]. SOD is an essential
component of antioxidative defense system in plants and
it dismutates two superoxide radicals (O2+ ) to water
and O2. Our results show increased activity of SOD in
rice plants growing under toxic levels of Pb. SOD
S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
activity has been reported to increase under salinity [5],
water stress [6], g-radiation [35], UV-B radiation [33],
Cd, Pb, Al and Cu toxicity [7,10,18,36]. Increase in SOD
activity in response to stress appears to be probably due
to de-novo synthesis of the enzymic protein [37].
Transgenic plants over-expressing SOD, show increased
tolerance towards oxidative damage caused due to harsh
environmental conditions and among antioxidant enzymes the activity levels of SOD are of more relevance in
maintenance of the overall defense system of plants
subjected to oxidative stress [38].
Catalase is universally present oxidoreductase that
decomposes H2O2 to water and molecular oxygen and it
is one of the key enzymes involved in removal of toxic
peroxides [17]. A decline in catalase activity under Pb
toxicity was observed in our studies which suggests a
possible delay in removal of H2O2 and toxic peroxides
mediated by catalase and in turn an enhancement in the
free radical mediated lipid peroxidation under Pb
toxicity. Similar decline in catalase activity was reported
under salinity [5], chilling [39], drought [40] and hypoxia
[41]. However, unlike our studies in subcellular compartments of pea root cells increased catalase activity
was observed when plants were grown in nutrient
medium containing 0.5 or 1 mM Pb(NO3)2 [10]. A
reduction in catalase activity under stressful conditions
has been attributed to the inactivation of enzyme
protein due to ROS [42], decrease in enzyme synthesis
or change in assembly of enzyme subunits [43,44].
Multiple isoforms of catalase have been reported in
higher plants, which are under control of different genes
[45]. Our results indicated two isoenzymic forms of
catalase in roots and three in shoots of rice seedlings.
Three genetically distinct catalase isoforms have been
detected in maize which appear to be synthesized in a
tissue specific and age dependent manner [45]. Inactivation of catalase isoenzymes due to high light intensity
and changes in its isozyme patterns have been observed
in response to g-radiation [35]. Decreased intensity of
two isoenzymic forms of catalase in shoots of Pbstressed seedlings is in conformity with decreased
activity of the enzyme under Pb treatments.
Our results indicate an enhancement in the activity of
guaiacol peroxidase, suggesting that this enzyme serves
as an intrinsic defense tool to resist Pb-induced oxidative
damage in rice plants. Peroxidases are widely accepted
as ‘stress enzymes’ [46]. Induction in peroxidase activity
has been documented under a variety of stressful
conditions such as water stress [40], chilling [47], salinity
[48], g-radiation [35] and under toxic levels of Al, Cu,
Cd, Zn [7,18,19]. As guaiacol peroxidases are located in
cytosol, cell wall, vacuole and in extracellular spaces,
increased peroxidase activity in Pb stressed seedlings
might be possibly due to increased release of peroxidases
localized in the cell walls. Under sublethal salinity and
metal toxicity conditions, level of peroxidase activity has
653
been used as potential biomarker to evaluate the
intensity of stress [7,48].
Ascorbate peroxidase (APX) and glutathione reductase (GR) are indispensable components of ascorbate/
glutathione pathway, required to scavenge H2O2 produced mainly in chloroplasts and other cell organelles
and to maintain the redox state of the cell [14]. APX
utilizes the reducing power of ascorbic acid to eliminate
potentially harmful H2O2. Our results indicate an
enhancement in the activity of APX in response to Pb
stress. Similar induction was reported in response to
mild water stress [6], chilling [39], drought [49], ozone
toxicity [50], Cu toxicity [51] and UV-B radiation [52].
APX along with catalase and SODs are considered as
key enzymes within the antioxidative defense mechanism, which directly determine the cellular concentration
of O2+ and H2O2 [14].
Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG) to
reduced glutathione (GSH). Owing to its redox active
thiol group, GSH is involved in the redox regulation of
the cell cycle [53] and has often been considered to play
an important role in defense of plants and other
organisms against oxidative stress [54]. Being a major
water soluble antioxidant in plant cells, GSH directly
reduces most active oxygen species, while GR uses
NADPH to reduce GSSG to GSH [55]. Various free
radicals and oxidants are able to oxidize GSH to GSSG
[55]. Higher cellular GSH levels are associated with
heavy metal tolerance in tomato cells [56] and heavy
metal exposure leads to accelerated GSH synthesis in
roots and cultured cells [57]. Our results show increased
GR activity in Pb treated rice seedlings which suggests
possible involvement of GR in regenerating GSH from
GSSG under Pb toxicity conditions to increase GSH/
GSSG ratio and the total glutathione pool [55]. Similar
to our findings, induction in GR activity has been
reported in the leaves of Cd and Zn stressed Phaseolus
plants [19]. Increase in the activity of GR has been
attributed to the de-novo synthesis of the enzyme
protein [6].
Our results suggest that Pb toxicity causes oxidative
stress in rice plants and the enzymes peroxidases, SOD
and GR appear to play a pivotal role in combating
oxidative stress in plants. As, unlike iron, Pb is not an
oxido-reducing metal, the oxidative stress induced by Pb
in growing rice seedlings appears to be an indirect effect
of Pb toxicity leading to production of ROS with a
simultaneous increase in tissue levels of SOD, peroxidase and GR.
Acknowledgements
Financial support for this work was provided by the
University Grants Commission, New Delhi (India) in
654
S. Verma, R.S. Dubey / Plant Science 164 (2003) 645 /655
form of Major Research Project. S.V. is grateful to the
Council of Scientific and Industrial Research, New
Delhi for providing a Senior Research Fellowship.
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