African Journal of Plant Science Vol. 5(9), pp.

531-536, 6 September, 2011

Available online at http://www.academicjournals.org/ajps
ISSN 1996-0824 ©2011 Academic Journals

Full Length Research Paper

Effect of heavy metal pollutants on sunflower

Rajeev Gopal* and Neena Khurana
Department of Botany, University of Lucknow, Lucknow-226007, India.
Accepted 11 July, 2011

The aim of this present study was to assess the tolerance of pollutant elements (Co, Ni, Cd, Cr and Pb)
on visible foliar symptoms, tissue concentration and some biochemical parameters in sunflower plants
grown at 0.25 mM in soil pot culture. At equimolar concentrations (0.25 mM), Cd induced the most
severe visual toxicity effects and exhibited maximum oxidative damage as observed by accumulation of
ThioBarbituric Acid Reactive Substances (TBARS) and lower antioxidant capacity than the plants is
exposed to Co, Cd, Cr and Pb. The study suggests that the degree of oxidative damage assessed by the
manifestation of external visual toxicity effects, tissue concentration and alteration in biochemical
parameters were found to be in the order Cd>Cr>Ni>Co>Pb.

Key words: Sunflower, visible symptoms, TBARS, antioxidant, tissue concentration.

INTRODUCTION

Heavy metal contamination of soils is the major global High concentrations of these metals cause toxicity in
environmental problem. It has increased considerably in plants and have been shown to generate oxidative stress
last several years and a part is responsible for limiting the usually accompanied by an increase of reactive oxygen
crop production. Elevated concentrations of both species including the level of hydrogen peroxide (H2O2),
essential (Co and Ni) and non-essential (Pb, Cd and Cr) superoxide (O.2) and hydroxyl radicals (OH-) (Stohs and
heavy metals pose a risk to the health of vegetarian Baghi, 1995; Pietrini et al., 2003; Milone et al., 2003). Cd
wildlife and human beings (Salt et al., 1998). Among and Pb are also known to interrupt the photosynthetic
these metals Cd and Pb are considered as the most toxic electron transport chain which leads to generation of
metals in the environment (Oliveira et al., 2005; Fritioff superoxide radical and singlet oxygen (Asada and
and Greger, 2006) and are released into the environment Takahashi, 1987) and thus enhance the peroxidant status
from various sources (Landis and Yu, 1999; Sanita and of the cell by reducing the antioxidant glutathione (GSH)
Gabrielli, 1999). Even though these metals are not pool, activating calcium dependent systems affecting the
essential for plants but are readily absorbed by the most iron-mediated processes (Pinto et al., 2003). Cobalt and
root systems because of their water solubility. The growth nickel being essential micronutrients are component of
and metabolism of plants are adversely affected by the several enzymes and co-enzymes (Marschner, 2002) but
increasing levels of these metals in the soil environment excess levels of these have detrimental impact on growth
(Balestrasse et al., 2003; John et al., 2009), besides this, of plants where they disturb several physiological
Cd and Pb are known to accumulate in different plant processes (Parida et al., 2003; Gazewska and
parts and enter into the food chain. Therefore, pollution Sklodowska, 2007). However evidences indicate that Co
due to heavy metals is significant from nutritional and and Ni phytotoxicity may be attributed to oxidative
environmental point of view. metabolism of plants (Pandey et al., 2009). Chromium is
also considered as a serious environmental pollutant
(Khan, 2007). Chromium exposure has also been
described in plants to cause alterations in metabolism
*Corresponding author.E-mail: [email protected]. either by causing a direct effect on enzymes or other
metabolites. Cr is a strong oxidant with a high redox
Abbreviations: TBARS, Thiobarbituric acid reactive potential accounting for a rapid induction in generation of
substances; GSH, glutathione; ROS, reactive oxygen species; Reactive oxygen species (ROS) and its resultant toxicity
TBAR, thiobarbuteric acid reaction; TCA, trichloroacetic acid;
NBT, nitro blue tetrazolium; BDL, below detect level; EDTA,
(Shanker et al., 2005). The studies on the antioxidative
ethylenediaminetetraacetic acid; LSD, least significant efficiency under metal stresses are well documented but
difference; MDA, malondialdehyde. reports on the changes in the antioxidative efficiency due
532 Afr. J. Plant Sci.

to excess Co, Ni, Cr, Cd and Pb induced oxidative stress and decrease in the absorbance was recorded after every 30 s
under identical experimental conditions are very scarce. interval up to 3 min. The amount of ascorbate oxidized was
calculated using extinction coefficient of 2.8 mM cm-1. For the
The present study focuses on the relative response of
determination of dry matter, plants were collected and sampled
sunflower to stresses of heavy metals that is, Co, Ni, Cd, from each treatment at day-45, washed thoroughly to avoid loss
Cr and Pb in plants grown in soil-pot culture at an excess and injury to roots with distilled water, separated into different parts
supply of these elements causing growth retardation and and weighed for the determination of dry matter after oven drying at
inducing visible foliar symptoms. Attempt has also been 70°C. All the dried samples were digested with a mixture of
made to identify the metabolism of tolerance in sunflower HNO3:HClO4 acids (10:1) and analysed for the concentration of Co,
Ni, Cd, Cr and Pb in digested plant material by atomic absorption
to stresses of these metals. spectrophotometer. All determinations were made in duplicate and
the data have been tested statistically for significance at 5% level of
probability (Fisher Lysergic acid diethylamide method (LSD) using
MATERIALS AND METHODS SigmaStat (ver. 2.03) software.

Sunflower (Helianthus annuus L. cv ASFH 378), plants were grown

in soil contained in pots under glass house conditions at an excess RESULTS
supply of Co, Ni, Cd, Cr and Pb. The soil used in the experiment
was taken from the sub-surface horizon of normal alluvial soil from
district Barabanki, U.P. having a pH 7.1 (1:2 soil-water suspension), Visible symptoms
EC 0.3 dSm-1, CaCO3 0.57% and organic carbon 0.3%. A basal
dose of NPK (2:1:2) was missed in the processed soil and 5 Kg of Cobalt
air dried soil was filled in each polyethylene pot. The soil was
watered to field capacity and incubated in the glass house for one The effect of Co supply appeared after 5 days of metal
week before seedling. Ten seeds were sown in each pot and after
supply as interveinal chlorosis of young leaves starting
germination 4 seedlings of equal size were retained. After 20 days
of normal growth, soil in each pot was treated with 500 ml of from the base in addition to depression in growth and leaf
aqueous solution of Co, Ni, Cd (as SO4), Cr (as dichromate) and Pb area. The chlorosis later extended to apical part of lamina
(as nitrate) at the rate of 0.25 mM. For each treatment three and intensified with increasing duration. The leaf size and
replicates were taken. One set of pot was allowed to grow plants height of plants was reduced to 50% of untreated plants.
without treatment to serve as control simultaneously. The pots were
arranged in randomized block design. As visible symptoms of metal
toxicity were initiated, fresh leaf samples from each treatment were
Nickel
collected and estimated for non-protein thiol and lipid peroxidation
content and specific activity of antioxidant enzymes – peroxidase,
superoxide dismutase and ascorbate peroxidase. Lipid peroxidation Visible symptoms of Ni toxicity were initiated after 6 days
was estimated as the amount of malondialdehyde produced by as marked depression in growth followed by interveinal
thiobarbuteric acid reaction (TBAR) (Heath and Packer, 1968) with chlorosis of young leaves initiating from the base
2 ml of leaf extract in 0.1% trichloroacetic acid (TCA). extending to the middle part of lamina on continued
The leaves were homogenized (1:10 w/v) in 50 mM potassium
exposure. Subsequently the interveinal chlorotic areas
phosphate buffer (pH 7.0) containing 1.0% polyvinyl pyrrolidone.
For ascorbate peroxidase, 1mM sodium ascorbate was also added became necrotic forming necrotic patches near the veins.
to the extraction medium. After centrifugation at 20,000 g for 20 min These necrotic areas enlarged, dried and the leaves
the supernatant was used for the assay of POD, SOD and APOD. collapsed. Leaf area and height of these plants were
POD activity was measured by a modified method of Luck (1963), reduced and depression was 61 and 54%, respectively of
in a reaction mixture containing 500 µ moles phosphate buffer (pH the control. Plants collapsed after 20 days of metal
6.0), 1ml 0.5% p-phenylenediamine, 1 ml 0.01% H2O2 and 1ml
addition.
suitably diluted enzyme extract. After 5 min, reaction was stopped
by adding 2 ml 5 N H2SO4 to the reaction mixture and change in
absorbance of the supernatant was measured at 485 nm. Total
SOD activity was assayed according to the method of Beauchamp Cadmium
and Fridovich (1971) based on the ability of the enzyme to inhibit
the photochemical reduction of nitro blue tetrazolium (NBT). The 3 Cadmium in excess caused reduction in size of young
ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), leaves with a marked decrease in growth after 5 days of
13mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1mM metal addition. The lower leaves of Cd exposed plants
ethylenediaminetetraacetic acid (EDTA) and soluble enzyme
extract. Riboflavin was added in the last and the mixture was
were limped and basal part of the stem developed purple
illuminated with cool fluorescent light for 30 min. The reaction was pigmentation which extended upwards in due course.
stopped by switching the light off. Corresponding unilluminated Compared to control plants, flowering was delayed and
blanks were also taken. head size was smaller in Cd treated plants. The
The absorbance of solution was measured at 560 nm. One unit depression in height and leaf area was 77% of untreated
of SOD represented the amount of enzyme that causes inhibition of
plants.
NBT by 50%. The APOD activity was assayed following the
oxidation of ascorbate to dehydroascorbate at 290 nm by the
method of Nakano and Asada (1981).
The reaction mixture consisted of 50 mM phosphate buffer (pH Chromium
7.0), 0.5 mM ascorbate, 0.1 mM H2O2, 1 mM EDTA and enzyme
extract (10 to 20 µg protein). Addition of H2O2 started the reaction The symptoms of chromium toxicity appeared earlier that
 Gopal and Khurana 533

is within 2 to 3 days of chromium addition as wilting of in roots, 149 µg Cd g-1 in stem and 137 µg Cd g-1 in
older leaves. Further growth of these plants was almost leaves as 0.72, 0.72 and 1.02 µg Cd g-1 in leaves, stem
stopped and old leaves showed induction of senescence and root of control plants, respectively.
as yellowing at leaf apex. Later the entire lamina turned
yellow along with leaf petiole and stem. Plants treated
with Cr failed to survive beyond 10 days and collapsed Chromium
due to severe Cr toxicity. A marked reduction was
observed in height (79%) and leaf area (90%) at excess The chromium concentration was also highest in roots
Cr. 610 µg Cr g-1 followed by stem 312 µg Cr g-1 and leaves
298 µg Cr g-1 while in control plants Cr was undetectable.

Lead
Lead
Sunflower plants appeared considerably tolerant to Pb
excess. A supply of excess Pb was unable to induce any In sunflower treated with excess Pb the concentration of
-1 -1
characteristic symptoms except for reduction in overall Pb was 2224 µg Pb g in roots whereas 552 µg Pb g in
-1
growth. Height of plants was reduced to 20% of control leaves and 135 µg Pb g in stem (Table 2).
whereas reduction in leaf area was 28%. Compared to
control, at excess Pb lower leaves appeared wilted and
flowering was delayed and the size of head was Enzyme activity and lipid peroxidation
decreased from that of control.
The specific activities of antioxidant enzymes POD, SOD
and APOD increased at excess each of Co, Ni, Cd and
Biomass Pb. The induction of POD activity was highest at excess
Ni and lowest in Cd treated plants. However, the increase
Total biomass of sunflower was reduced after 15 days in SOD activity was maximum at excess Pb and that of
exposure to excess supply of each Co, Ni,Cr, Cd and Pb. APOD at excess Ni (Table 1). The 3,4-
Compared to control reduction in biomass was 94, 90, Methylenedioxyamphetamine (MDA) concentration also
81, 69 and 20%, respectively in excess supply of Cr, Ni, increased at excess supply each of Ni, Co, Cd and Pb
Co, Cd and Pb (Table 1). and the increase was highest at excess Pb (Table 1).

Tissue concentration DISCUSSION

Cobalt Plants response to various types of stresses depends on

its genetic make up therefore it behaves differently
Compared to 11.2, 10.2 and 15.9 µg Co g-1 in leaves, according to the tolerance mechanism it inhabits.
stem and roots of untreated plants, the concentration of Sunflower appears to be quite resistant to various
Co in respective plant parts was 241, 191 and 964 µg Co pathogens because of its capacity to synthesize and
g-1 in Co excess, respectively. At 0.25 mM Co, the accumulate phenolic compounds. Growth and
concentration of Co was maximum in roots followed by metabolism of sunflower were affected variably by the
leaves and stem. stress of heavy metals such as Co, Ni, Cd, Cr and Pb.
The excess of different metals under study resulted in
stunted growth, reduced biomass accumulation and
Nickel produced characteristic visible effects similar to those
described by other workers in different plant species
In leaves, stem and roots of Ni treated plants, the (Tewari et al., 2002; Zhou and Qiu, 2005; Gajewaska and
concentration of Ni was as high as 440, 660 and 1276 µg Sklodowska, 2007). These observations are
Ni g-1 as compared to 28, 8 and 15 µg Ni g-1 in respective substantiated by a significant enhancement in the level of
parts of the control plants. The accumulation of Ni was the metal in the plant tissue. The reduction in biomass
about three times more in roots than leaves at 0.25 mM was maximum in Cr excess followed by Ni, Co, Cd and
Ni. Pb as compared to control plants. This may represent the
cumulative effect of damaged or inhibited physiological
function under stress condition. The observed decrease
Cadmium in dry matter production as a result of metal stress is in
agreement with that reported earlier in plants other than
At 0.25 mM Cd, the distribution of Cd was 321 µg Cd g-1 sunflower (Ryser and Sauder, 2006; Pandey and Pathak,
534 Afr. J. Plant Sci.

Table 1. Effect of pollutant elements on various physical and biochemical parameters of sunflower in soil-pot culture.

Control Cr 0.25 mM Co Ni Cd Pb
Height (cm)
37.5 7.75* 19.5* 17.5* 8.75* 30

Leaf area (cm2)

62.6 6.50 31.5* 24.2* 14.2* 44.8
-1
Biomass (g dry matter plant )
3.27 0.192 1.208* 1.322* 1.002* 2.385*
-1
Peroxidase (Change in O.D. mg protein)
0.013 0.142* 0.188* 0.787 0.112* 0.152*

Superoxide dismutase (EU)

1.71 2.44* 2.82 3.10 2.39* 2.50*
-1
Ascorbate peroxidase (µ mol ascorbate mg protein oxidized)
0.243 0.338* 0.561* 0.867 0.284* 0.614*

Lipid peroxidation (n moles MDA 100 mg-1 fresh weight)

11.2 12.1* 13.3* 13.8 14.2* 11.4*
The values carrying superscript star are significantly different from those of respective controls.

Table 2. Effect of pollutant elements on tissue concentration in different plant parts of sunflower in soil-pot culture (±SE).

Plant part
Treatment
Leaves Stem Root
µg Co g-1
Control 11.2±0.23 10.2±0.46 15.9±1.22
0.25 mM Co 241±8.15 191±6.24 964±45.3

µg Ni g-1
Control 28.0±1.22 8.30±0.35 15.8±0.65
0.25 mM Ni 440±33.2 660±42.3 1276±55.3

µg Cd g-1
Control 0.72±0.09 0.72±0.08 1.02±0.02
0.25 mM Cd 137±4.56 149±4.33 321±7.34

µg Cr g-1
Control BDL BDL BDL
0.25 mM Cr 298±7.78 312±8.43 610±21.2

µg Pb g-1
Control BDL BDL 9.72±0.56
0.25 mM Pb 552±21.4 135±3.58 2224±52.6
BDL = below detect level.

2006). Heavy metal similarly to other stress factors have peroxidase and ascorbate peroxidase, that scavenge
been reported to induce over production of ROS in plant reactive oxygen species (Gratao et al., 2005) and avoid
tissues to protect the plant from damages caused by oxidative damage to plants.
ROS, plants possess low molecular weight antioxidants In the present study Co, Ni, Cd and Pb toxicity in
(ascorbic acid, reduced glutathione, carotenoids) and sunflower led to significant alterations in the activity of
antioxidant enzymes such as superoxide dismutase, antioxidant defense mechanism. The activity of superoxide
 Gopal and Khurana 535

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