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Antioxidative and Biochemical Responses in Dalbergia sissoo Roxb Seedlings

Growing Under Cobalt and Lead Stress

Article · January 2016

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Indian Journal of Forestry 39(3) 1-4, 2016

Antioxidative and Biochemical Responses in Dalbergia sissoo Roxb

Seedlings Growing Under Cobalt and Lead Stress

A.K. Tripathi, M.K. Gupta, Nemit Verma, Sohni Sinha and Amrendra Bhushan

Abstract : Dalbergia sissoo Roxb. is one of the most important timber species of India. It is used for high quality furniture,
cabinets making and has pesticidal property. It is commonly planted tree species in urban and suburban areas, roadsides and
industrial areas where the soil may be polluted with heavy metals such as Co or Pb. Lead (Pb) and Cobalt (Co) are one of the
non essential and toxic heavy metals which can cause oxidative stress in plants. The concentrations of these heavy metals in the
environment are currently increasing, due mainly to anthropogenic activities. The effect of these heavy metals on biochemical
parameters and antioxidant activity were studied in leaves of Dalbergia sissoo Roxb. grown under control laboratory conditions
in pot culture and four levels of heavy metal stress (10, 20, 30 and 40ppm Pb2+, Co2+). Six months exposure of Dalbergia sissoo
Roxb. plantlets to different concentration of Co or Pb in normal soil conditions elicited an antioxidative response, measured in
terms of ascorbic acid, proline accumulation and biochemical responses in terms of protein and free amino acids. The results
indicate that the proline, ascorbic acid contents increased with increase in Co and Pb amount in soil whereas the protein and total
chlorophyll contents showed opposite trend.

KeYwords: Lead, Cobalt, Dalbergia sissoo Roxb., Antioxidant, Seedlings.

Introduction
Various anthropogenic activities like mining, metal smelting,
electroplating, gas exhaust, energy and fuel production, down
wash from power lines, intensive farming, power transmission,
sludge dumping, and military operations introduce heavy
metals in our surroundings (Kumar et al., 1995; Nedelkoska and
Doran, 2000). These heavy metals (HM) in excess amounts are
a global problem, threatening the health of plants, animals and
ultimately to the humans (Heckathorn et al., 2004, Zeller and
Feller, 1999). Some heavy metals, such as copper and zinc, are
needed by plants as parts of coenzymes and enzymatic prosthetic
groups, they functions as micronutrients (Antosiewicz, 1992),
whereas some heavy metals like lead and mercury have no
known biological functions (Xiong, 1998). Beside the ability
to take up essential nutrients, the other metals with unknown
metabolic function are also absorbed and accumulated by the
plants (Nikolic et al., 2008).
Plant species are the foremost absorber of toxic elements that
are discharged into the atmosphere from the burning of fossil
fuels and vehicular emission, release of industrial wastes and
use of agrochemicals. Throughout the world there are about
Tripathi, A.K., Verma, N. [*], Sinha, S., Bhushan, A.
Forest Ecology and Environment Division, Forest Research Institute,
P.O. New Forest, Dehra Dun 248 006 (Uttarakhand) India
e-mail: nemit.verma@gmail.com
Gupta, M.K.
Forest Soil & Land Reclamation Division, Forest Research Institute,
P.O. New Forest, Dehra Dun.
400 species from terrestrial plants which have been identified
as hyper-accumulators of various heavy metals (Baker et al.,
1994; Zhou et al., 2006); which can take up and accumulate
high amount of heavy metals without any damage to the cell.
Some heavy metals are essential for plant growth and
development such as Cu, Mg and Zn since they are constituents
of many enzymes and other proteins. However, the growth
inhibition and toxicity symptoms among the plant species
occurs due to increased concentrations of both essential and
nonessential heavy metals in the soil (Hall, 2002). Heavy
metals inhibit physiological processes such as respiration,
photosynthesis, cell elongation, plant-water relationship,
N-metabolism and mineral nutrition; (Zornoza et al., 2002).
Heavy metals mainly affect the various structural and
physiological functioning of the plants such as membrane
damage, structural disorganization of organelles and ultimately
reduce the growth of the plant. There are also some other effects
like which are also caused by the increased concentration of
heavy metals (Kimbrough et al., 1999; Chien and Kao, 2000;
Long et al., 2003; Zhang and Chen, 2002).
In Cholorophyll synthesis, the heavy metals lead to the direct
inhibition of an enzymatic reactions or by inducing deficiency
of an essential nutrient (van Assche and Clijsters, 1990). Heavy
metal stress leads to increase in proline content in different
plant parts by emphasizing its protective role and mark it as
an active indicator of environmental stress (Alia and Saradhi,
1991). According to some scientists proline accumulation in
plant tissues has been result from either a decrease in proline
degradation or an increase in proline biosynthesis or a decrease
2 Indian Journal of Forestry 39(3) 1-4, 2016
in protein synthesis or proline utilization or hydrolysis of
proteins (Charest and Phan, 1990).
Therefore, in order to better understand how the Dalbergia
sissoo Roxb. respond to heavy metal concentration, we have
examined the effects of two heavy metals (cobalt and lead) on
the content of total chlorophyll, free amino acid, proline and
ascorbic acid in Dalbergia sissoo Roxb. seedlings.
MATERIALS AND METHODS
Plant Material and Treatment
Species was selected on the basis of survey in different sites/
locations, which are highly affected by heavy transport and
industrial effluent. Six month old seedlings of Dalbergia
sissoo Roxb. were selected to conduct the study. Heavy metal
treatment of Pb (C2H3O2)2.3H2O and CoCl2 with four different
concentrations of 10, 20, 30 and 40 ppm were prepared in the
laboratory. Six month exposure of treatment was given to the
plants. Six replicates of each plant along with control were
treated with different concentrations. Plants were treated with
the doses of different concentrations of each heavy metal to the
plant’s root.
Determination of Chlorophyll Content
The chlorophyll contents (mg/g fresh leaf), were analysed
by following Holm, (1954) method. Leaf samples were
homogenized in 80% acetone and optical densities were
measured at 645 and 663 nm with Spectrophotometer (Spectra
scan UV 2700).
Determination of Free Amino Acid and Proline
Free amino acid was measured by the method prescribed by of
Moore and Stein (1948). Absorbance was recorded at 570 nm
using Spectrophotometer (Spectrascan UV 2700). Leucine was
used as the standard. Proline was extracted from the leaves and
estimated by the methods of Bates et al. (1973). For proline
estimation, 0.5g of plant material was homogenized in 10 ml
of 3% sulphosalicylic acid. 2ml each of glacial acetic acid
and ninhydrin were added to 2 ml of plant extract followed by
heating at 100ºC for an hour and immediate cooling in ice. 4ml
of toluene was added in the reaction mixture, vortexed for a
minute followed by separation of layers. Red colour toluene
layer was checked for absorbance at 520nm. Proline Content
was calculated from the standard proline curve.
Determination of Ascorbic Acid
Estimation of ascorbic acid was done by method given by
Roe and Kuether (1943). The plant extract was prepared in
Tris buffer (50mM, pH-10) under ice cold conditions. The
reaction was started with 0.5ml of extract, 100 mg activated
charcoal, 4ml distilled water and 0.5ml of 50% TCA. It was
mixed well then and filtered with Whatman filter paper No.1
and 0.4ml of DNPH reagent was added to 1ml of filtrate. The
reaction mixture was incubated for 3 hours at 37ºC followed
by cooling in ice bath and addition of cold H2SO4 (65%). The
absorbance was read at 520nm. The content was calculated by
using standard ascorbic acid.
Determination of Protein
The total soluble proteins in the leaves were estimated by the
method given by Lowry, (1951). 100mg of fresh leaf tissue was
homogenized in 0.1m Tris-buffer (pH 7.5) and 0.5 ml Folin
reagent was added for colour development. Absorbance was
measured at 660nm against reagent blank in PC based UV-VIS
spectrophotometer (Perkin Elmer Lambda 2S, German made).
The standard curve was prepared with Bovine Serum Albumin
(BSA). The total protein content was expressed in mg/g fresh
weight of leaves.
Determination of Total Polyphenols
Polyphenol estimation was done by the method given by
Schandari, (1970). 0.5gm of powdered material was boiled
with distilled water for 30 minutes. Volume of the supernatant
was made up to 100ml by adding distilled water. Folin Denis
reagent and sodium carbonate solution were added in known
aliquots and absorbance was read at 700nm after 30 minute.
Statistical Analysis
All experiments were carried out in six replications. Data was
presented as mean ± standard error. The data was subjected
to one-way analysis of variance (ANOVA). The data was
considered significant at p ≤ 0.05. Increases or decreases are
expressed as percentages versus control.
RESULTS
Effect of Cobalt and Lead on Chlorophyll Content
It was observed that different concentrations of each Co and
Pb used individually marginally decreased the chlorophyll
(chl a, chl b and total chlorophyll).The soil application of
10ppm cobalt and lead did not alter the content of chl a and
b significantly, the higher concentrations of 40ppm cobalt and
lead in the soil reduced the chlorophyll contents in Dalbergia
sissoo Roxb. seedlings significantly by about 50.76%and
66.07%, respectively. This decline in the total chlorophyll
contents indicated the poisoning effect of high Cobalt and Lead
concentrations to green leaves (Fig. 1). According to the results
lead has more toxicity to the chlorophyll content.
Effect of Cobalt and Lead on Proline and Ascorbic Acid
Increase in the osmoprotectant proline content was directly
proportional to the heavy metal concentration. The percentage
increases were 60 to 61% at 10ppm and 20ppm concentration
Indian Journal of Forestry 39(3) 1-4, 2016 3

of cobalt respectively, whereas a sudden decrease was seen
at 30ppm concentration of cobalt which was followed at
40ppm concentration of cobalt that was about 1-2% decrease
respectively, In case of lead the proline content showed a entirely
different trend of decreasing concentration (Fig. 2). The percent
decrease was about 7% at 10 ppm, the decrease in proline
concentration was increased with increasing concentration of
lead. At 40ppm lead the percent decrease was about 41%.
significantly decreased in the leaf tissues with increasing
cobalt and lead supply. The decrease in protein content
was found high at 40ppm cobalt treatment that was about
24.97% which was about three times more than the decrease
at 10ppm cobalt concentration (Fig. 4). This effect, however,
was also not significantly found in soil contaminated with lead
where e less reduction in protein concentration occurred. The
free amino acid content showed slight decrease in plants treated
with at different concentration of cobalt and lead. At 10ppm, the
decrement in free amino acids content were seen, 36.12% and
11.20%,under cobalt and lead treatment respectively but with the
increases in concentration the plants under different treatment
show variability in decrease, i.e. 86.27% under 40ppm cobalt
and at 65% under 40ppm lead respectively (Fig. 5).

Fig. 1. Total Chlorophyll Content in Dalbergia sissoo Roxb.

under lead and cobalt stress.

Fig. 3. Ascorbic Acid in Dalbergia sissoo Roxb.

under lead and cobalt stress.

Fig. 2. Proline Content in Dalbergia sissoo Roxb.

under lead and cobalt stress.

Ascorbic acid is the most abundant antioxidant in plants and

has important roles in plant growth and development. Ascorbic
acid content increased significantly in plants treated with high
concentration of cobalt and lead, the increase in ascorbic acid Fig. 4. Protein in Dalbergia sissoo Roxb.
under lead and cobalt stress.
content was more in lead treatments. The increase in ascorbic
acid contents was 39% in 10ppm and 60.13% at 20ppm
respectively in cobalt treatment and the percent increase at
10ppm of lead concentration was quite high i.e. about 125%.
The plants grown under high lead treatment followed the same
trend. At 40ppm concentration of lead the amount of ascorbic
acid contents were found more than triple (Fig. 3).

Effect of Cobalt and Lead on Protein and Total Free Amino

Acids
Abiotic stress may inhibit a synthesis of some proteins and
promote others (Ericson and Alfinito, 1984) with a general trend Fig. 5. Total Free Amino acids in Dalbergia sissoo Roxb.
of decline in the overall content. The concentration of protein under lead and cobalt stress.
4 Indian Journal of Forestry 39(3) 1-4, 2016
Effect of Cobalt and Lead on Polyphenols
Under the model conditions of pot trial the polyphenols
accumulation in Dalbergia sissoo Roxb. leaves depending on
the extent of soil contamination by cobalt and lead that was
applied to the soil in the form of solutions of its soluble salts
was observed. The plants under control condition were having
1.355±0.078 mg/kg polyphenols in normal soil whereas the
plants under cobalt stress showed a high amount of polyphenols
i.e. 3.163±0.017 mg/kg. The amount of polyphenols rises
with the increase in concentration of cobalt. The lower dose
(10ppm) of lead has not significantly affected the polyphenol
concentration but as the concentration increased to 20ppm
the polyphenol concentration increased to 2.924±0.003mg/kg
(Fig. 6). The plants grown under 40ppm lead concentration
followed the same trend.
Fig. 6. Polyphenols in Dalbergia sissoo Roxb.
under lead and cobalt stress.
DISCUSSION
Superoxide radical (O2-), hydrogen peroxide (H2O2), hydroxyl
radical (HO·), and singlet oxygen (1O2), collectively termed
Reactive oxygen species (ROS) generation in higher plants is
induced by heavy metals which cause oxidative stress (Devi
and Prasad, 1998). All types of amino acids, nucleic acids,
proteins, lipids are attacked by ROS rapidly (Mehta et al., 1992;
Luna et al., 1994), leading to irreparable metabolic dysfunction
and cell death. Therefore, the induction of antioxidants is an
important protective mechanism to minimize oxidative damage
in polluted environments.
Effect of heavy metals on chlorophyll contents depends upon the
concentration. The decrease in chlorophyll content of different
plants is widely studied by many scientists (Yang et al. 1999a, Sun
and Wang, 1985, Ahmed et al., 2007). In the higher plants several
reports shows that the heavy metals inhibits the chlorophyll
biosynthesis (Prasad and Prasad, 1987). As per previous studies,
the chlorophyll proteins, which took protons for photosynthesis
in PS II, were decomposed and decreased under Cd stress.
The previous studies indicated that the structural changes in
chloroplast are caused by heavy metal stress and will further
leads to the damage of membrane system (Peng and Wang,
1991). Different heavy metals have different effects on protein
contents of plants. The role of lead on biochemical process is
not clear but it is reported in the previous studies that increased
lead concentration has decreased the protein content in the
plants. (Chatterjee et al., 2004; Mishra et al., 2006; Garcia et
al., 2006; Piotrowska et al., 2009). Increase in dissolubility
of protein under cadmium stress was reported by Hong et
al. (1991) and Li et al. (1992), which might be a detoxifying
mechanism. Whereas, the results of Qin et al., (1994) showed
that the increased concentration of Cd higher than 0.1mg/L has
decreased the dissolubility of proteins in B. chinensis seedlings.
Chen et al. (1999) have noticed the degradation in polypeptide
composition of B. schreberi under Hg and Cd treatment, and the
effect of Hg was more significant than that of Cd.
Our results shows, the decrease in protein contents and amino
acid content with increase in heavy metal concentration in
Dalbergia sissoo Roxb. which can be a detoxifying mechanism
under oxidative stress similar results, were reported by Jaleel et
al., 2009. However, certain amino acids, like proline, increase
under lead stress (Qureshi et al., 2007). Proline, commonly
known amino acid gets accumulated in wide variety of
organisms ranging from bacteria to higher plants on exposure
to various abiotic stress (Saradhi et al., 1993, Ahmad et al.,
2006 and Tang, 1984). Such amino acid plays a major role in
the tolerance of the plant to lead. Proline also acts as a major
reservoir of energy and nitrogen, which can be used in resuming
the growth after the stress removal. In Dalbergia sissoo Roxb.,
Proline contents increased with increase in concentration of
Cobalt and lead.
Plant responses to heavy metal stress are multigenic,
involving both osmotic and ionic homeostasis, as well as cell
detoxification. The efficiency of the latter depends upon the
antioxidant defence mechanisms (Zhang et al., 2007).
Ascorbic acid, natural antioxidant in plant has been shown
to play an important role in tolerance (Chen et al., 1990). Pb
and Cd were reported to cause an increase in ascorbic acid
in Brassica juncea. Tripathi and Tripathi, (1998) for Albizia
lebbeck reported that the increase of ascorbic acid content in
leaves with increasing concentration of each metal shows plant
susceptibility to heavy metals, similar results were observed in
present investigation i.e. the plants exhibited an increase in the
production of ascorbic acid in different treatments due to the
heavy metal stress which revealed the defence mechanism of
the plants. As reported by Halliwell and Gutteridge, (1989),
powerful reductant activity is the major reason for increase
in ascorbic acid concentration which further maintains the
stability of cell membranes during the heavy metal stress and
discards the cytotoxic free radicals.
Indian Journal of Forestry 39(3) 1-4, 2016 5
Polyphenol is found to be present in the plants as defense
compound. The phenolic compounds scavenge the ROS and
further prevent the oxidative damage by interfering with the
radical chain reactions during lipid per oxidation. The increase
in phenolic contents under lead stress is also reported earlier
(Hamid et al., 2010). High level of polyphenol may be due
to severe foliar injuries like chlorosis and necrosis caused by
metal toxicity on plants.
It can be inferred from present investigation that short term
exposure of heavy metal lead and cobalt induced acclimation of
the antioxidation defence system in Dalbergia sisoo seedlings
and It have potentiality to resist heavy metal stress up to some
extent, the high concentration of heavy metal Co and Pb could
be lethal to plants.
ACKNOWLEDGEMENT
Authors are thankful to the Director Forest Research Institute
and Indian Council for Forestry Research and Education
(ICFRE) for providing financial support for this research.
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