Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2011, 1 (4): 124-130
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
Phenols and their antioxidant activity in Brassica juncea seedlings
growing under HgCl2 stress
Yaksha Singh and C P Malik
School of Life Sciences, Jaipur National University, Jaipur
_____________________________________________________________________________
ABSTRACT
Lab experiments were conducted to investigate the relationship between Hg-induced
phytotoxicity and oxidative stress and the ameliorative effects of total phenols and putrescine.
Seedlings were treated with HgCl2 at the seedling stage. Seeds were prehardened with putrescine
(0.5mM). Reactive oxygen species (ROS) accumulation enhanced. Putrescine was effective in
decreasing the superoxide anion content and H2O2 content. The decrease was concentration
specific. Lipid peroxidation enhanced by % with HgCl2 which caused increased leakage.
Putrescine decreased lipid peroxidation and hence electrolyte leakage. The activities of SOD,
ascorbate peroxidase and catalase increased with treatments. Hg had a deleterious effect on
most parameters but putrescine was markedly effective in overcoming the toxic effect of Hg.
Enhancement in total phenols as a response to heavy metal stress is reported. The role of
phenolics in acting as metal chelators and scavenging molecular species of active oxygen is
proposed.
Keywords: Phenol, SOD, ascorbate peroxidase and catalase.
INTRODUCTION
Crop plants are affected by a variety of abiotic stresses like salinity, drought, low and high
temperature and heavy-metal as well as biotic stresses like pathogens. These stresses result in
significant loss of crop yield and quality. Heavy metals are important environment pollutants and
many of them are toxic even at very low concentrations. Toxic metal contamination of soil,
aqueous waste streams and groundwater poses a major environmental and human health
problem. The influence of metals on development and reproduction of plants can be firstly
quantified by determining the germination traits of seeds and growth performance of seedling. In
the presence of high concentrations of some heavy metals, most of plant species performed the
reduction of seed germination and seedling growth. Different industrial and other anthropogenic
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activities have increased the levels of heavy metals in the environment. The metals
contamination restricts the plant growth, threatening the human life.
Germination of seed is a crucial step in seedling establishment and crop productivity. Seed
priming is a controlled hydration treatment of seeds sufficient to permit pre-germinative
metabolic events to take place but insufficient to allow radical emergence and are followed by
drying back of the seeds to their original moisture content. Seed priming is a technique which
involves uptake of water by the seed followed by drying to initiate the early events of
germination up to the point of radical emergence. Its benefits include rapid, uniform and
increased germination, improved seedling vigour and growth under a broad range of
environments resulting in better establishment and alleviation of phytochrome-induced
dormancy in some crops.
The polyamines (PAs) are small aliphatic amines that are ubiquitous in all living organisms. In
plants, the most common PAs are diamine putrescine, triamine spermidine and tetramine
spermine which have been implicated in a wide range of plant growth and developmental
processes including cell division, embryogenesis, morphogenesis, fruit development, fruit
ripening, leaf senescence and response to environmental stresses [1,2,3,4]. In this study we have
used putrescine as a pre hardening agent.
MATERIALS AND METHODS
The seeds of Vasundhra were obtained from National Research Center on Rape Seed and
Mustard, (Indian Council of Agriculture Research) Bharatpur - 321303, Rajasthan. Uniformly
selected seeds were sterilized with 0.1 % HgCl2 for 1 min and subsequently repeatedly washed
under running tap water followed by distilled water. Seeds were presoaked in putrescine (1 mM)
for 8 hrs. Thereafter, these were dried and brought to original weight, Seeds were germinated in
Petri dishes lined with blotting paper and irrigated with heavy metal solution. For imposing
HgCl2 stress 1 mM concentration was used in each Petri dish. Nearly 20 seeds were sown in each
Petri dish and incubated in BOD incubator set at 25°C. Petri dishes were watered with a desired
concentration of heavy metal and left to germinate. Following parameters were studied after 15
days of sowing (DAS). Different seed, seedling, physical and biochemical characters were
analyzed. Each treatment was replicated thrice and the data represented as average values. Data
on seed germination and seedling growth shall be published elsewhere.
Total Phenolic Content
The colorimetric method [5] was used for the determination of total phenols. Aliquote of 0.5 ml
extract was diluted with distilled water. To this was added 0.5 ml of Folin-Ciocalteu reagent and
content shaken vigorously. After three minutes 1 ml of saturated sodium carbonate solution was
mixed and final volume was made to 10 ml with distilled water. The test tubes were kept in dark
for one hour after which absorbance was measured at 725 nm and for standard curve stock
solution of gallic acid was made by dissolving gallic acid (50 mg) in 50 ml of methanol.
Chlorophylls and carotenoid content
200 mg of fresh leaves were taken and homogenized thoroughly in 80 per cent (v/v) acetone
using a glass-in-glass homogenizer. The material was centrifuged at 3000 g for 10 min in dark
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and clear supernatant was collected in a test tube. The pellet was extracted again with 2 ml of 80
per cent acetone and recentrifuged. The two supernatants were pooled and the final volume was
adjusted to 10 ml. The absorbance was read at 647 and 664 nm respectively [6]. 80 per cent
aqueous acetone solution served as a blank. The absorbance of the solution was recorded at 480
nm respectively [7].
Malonaldehyde (MDA)
The level of lipid per oxidation was measured in term of malondialdehyde (MDA) content, a
product of lipid per oxidation by the method of Heath and Packer [8]. Plant tissue was
homogenized in 0.1 % TCA. The homogenate was centrifuged at 10,000 rpm for 10 min.
Supernatant was treated as extract. To 1 ml aliquot of supernatant, 4 ml of 0.5 % of TBA in 20 %
TCA was added. The mixture was heated at 95ºC for 30 min and cooled in ice bath. After
centrifugation at 10,000 rpm for 10 min, the absorbance of supernatant was recorded at 532 nm.
MDA content was calculated by extinction coefficient of 155 mM-1 cm-1 expressed as per gram
of fresh weight.
Hydrogen peroxide (H2O2)
It was estimated by method of Mukherji and Chaudhary [9]. 0.5 g Plant material was
homogenized in 5 ml chilled acetone (80 %) and filtered through Whatman No 1 filter paper. To
the above 5 ml of extract, added 4ml of titanium reagent followed by the addition of 5 ml of
ammonia solution. The mixture was centrifuged at 10,000 rpm and supernatant was discarded.
The residue was dissolved with 1 M H2SO4. Absorbance was recorded at 410 nm. Calculations
were made with standard curve plotted with pure H2O2. Concentration of H2O2 was determined
using standard curve plotted with known concentrations of H2O2.
Proline content
The proline content was determined by the method of Bates and Waldren [10]. Fresh material of
seedlings was homogenized in 3 % aqueous sulfosalicylic acid and the homogenate was
centrifuged to 10,000 rpm. Supernatant was estimated for proline. The reaction mixture
consisting of 2 ml supernatant, 2 ml acid ninhydrin and 2 ml of glacial acetic acid was boiled at
100°C for 1 hr. After termination of the reaction in ice bath, the reaction mixture was extracted
with 4 ml toluene and the absorbance was read at 520 nm. The amount of proline was calculated
from the standard curve plotted with known concentrations of proline.
Superoxide dismutase (SOD) (EC1.15.1.1)
The activity of SOD was assayed following the method of Dhindsa and Dhindsa [11].The fresh
plant material was homogenized in 50 mM chilled / ice cold phosphate buffer pH 7.0, 0.25 %
triton X-100 (m/v) and 1 % poly vinyl pyrrolidone (PVP and 10 % w/v glycerol) and centrifuged
centrifuged at 10,000 rpm for 10 min at 4ºC and supernatant was treated as enzyme extract. The
reaction mixture (3 ml) contained 13 mM methionine, 25 mM NBT, 0.1 mM EDTA, 50 mM
sodium bicarbonate, 50 mM phosphate buffer pH 7.8 and 0.1 ml of enzyme extract. The reaction
was started by addition of 2 mM riboflavin and exposed to 15 W fluorescent lights for 10 min.
The Photo – reduction of NBT resulted in the formation of purple formazon. The absorbance was
read at 560 nm and the total SOD activity of the samples was assayed by measuring its ability to
inhibit the photochemical reduction of nitro-blue-tetrazolium (NBT). 1 unit of SOD activity was
defined as the amount of enzyme, which causes 505 inhibition of the photochemical reduction of
NBT.
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Yaksha Singh et al
J. Microbiol. Biotech. Res., 2011, 1 (4):124-130
______________________________________________________________________________
Catalase (CAT) (EC 1.11.1.6)
Catalase activity was determined by the method of Aebi [12]. The reaction mixture in final
volume of 3 ml comprised 0.1 M phosphate buffer (pH 7.0). The reaction mixture was initiated
by adding H2O2 and enzyme activity was determined following degradation of at H2O2 at 240 nm
for 2 min. The catalase activity was measured using the extinction coefficient 0.0394.
Ascorbate peroxidase (APX) (EC 1.11.1.11)
APX activity was determined by following oxidation of ascorbate as a decrease in absorbance at
290 nm [13]. Ascorbate (2 mM) was added to extraction medium to the inactivation of enzyme.
Plant material was homogenized in ice cold. 50 mM phosphate buffer pH 7.0, 0.1 mM EDTA,
0.5 mM ascorbic acid, and enzyme extract. The change in A290 was recorded at 30 seconds
intervals after addition of H2O2. The rate constant was calculated using the extinction coefficient
of 2.8 mM-1cm-1.
Reactive oxygen species (ROS)
ROS production was measured as described by Able et al., [14] by monitoring the reduction of 2,
3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide-inner salt (XTT) in
presence of ROS, with some modifications. Leaves (250 mg) were homogenized with 1.5 ml of
50 mM K-phosphate buffer (pH 7.8) and centrifuged at 5000 rpm for 10 min. The reaction
mixture (1 ml) contained 50 mM K-phosphate buffer (pH7.8), 500 µl of 0.5 mm XTT. The
reaction of XTT was determined at 470 nm for 3 min. Corrections were made for the absorbance
of chlorophyll. ROS production was calculated by using extinction coefficient of 2.163104M-1
cm-1.
RESULTS AND DISCUSSION
Figure 1. Effect of HgCl2 on total phenols (left) and ROS accumulation ( right) in leaves of B.juncea after 15
days of treatment
Total Phenols
12
µM g-1 FW
µ g-1 FW
10
8
6
4
2
0
Control
1 mM Hg
Put
Treatments
1 mM Hg +
Put
ROS
450
400
350
300
250
200
150
100
50
0
Control
1 mM Hg
Put
Treatments
1 mM Hg +
Put
Figure 1 shows increase level of total phenols with mercury and putrescine. When both were
used the phenolic contents enhanced. Enhanced ROS accumulation was observed with Hg. Hg +
putrescine were effective in decreasing the level of ROS.
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Table 1 Effect of HgCl2, putrescine (1mM) on different traits in leaves of Brassica juncea after 15 days of
treatment
Treatments
Level of lipid peroxidation (MDA µM g-1 FW)
H2O2 (µM g-1 FW)
Total chlorophyll (mg g-1 FW)
Carotenoids (mg g-1 FW)
Proline (µM g-1 FW)
SOD (IU)
CAT (µM min-1 g-1 FW)
APX (µM min-1 g-1 FW)
Control
1.85
375.00
0.300
0.126
1.76
0.40
32.84
9.24
1 mM Hg
2.01
551.28
0.230
0.106
4.36
0.80
49.61
10.00
Put
1.79
221.15
0.336
0.145
2.16
0.66
43.71
11.72
1 mM Hg + Put
1.93
416.66
0.261
0.141
5.10
1.06
58.39
16.55
Antioxidant enzymes SOD, CAT and APX
SOD activity enhanced two fold with Hg treatment and putrescine + Hg further enhanced the
activity several fold (Table 1). Catalase activity showed similar trend.
A small enhancement in APX activity was noticed with Hg and putrescine. Hg and putrescine
further enhanced the APX activity (Table 1).
Chlorophyll and carotenoid content
There was a significant difference in amount of chlorophylls between the treated and non treated
samples. The content of total chlorophyll decreased with the increasing concentration of Hg on
the 10th and 15th day. In our investigations, it was observed that in Brassica, mercury showed
decreasing effect on Hill activity. Hg treatments also caused decline in carotenoid content but
putrescine exhibited promontory effect.
MDA
Hg stress adversely affected the membrane integrity. The MDA content indicated the degree of
lipid peroxidation. Enhanced levels of lipid peroxidation resulted in membrane injury. Putrescine
effectively alleviated the toxic effect of Hg (table 1).
Proline
The proline content increased markedly in hardened seedlings over the NHS. In fact proline
contents in the 15th days old seedlings were invariably more in the NHS compared with non
hardened control. As the concentration of metal increased the proline content also enhanced. It
seems that during heavy metal stress, accumulation of nitrogen compounds like proline as well
as proteins increase. It is pertinent to state that with NHS the situation was altered with heavy
metal stress and proline contents increased markedly.
Brassica juncea (Brassicaceae), Indian mustard, is a very important oil crop which is a fast
growing plant and produces a high biomass even in heavy metal polluted soils. Thus, this plant
could be a potential candidate for phytofiltration or phytostabilzation of heavy metal
contaminated waste water.
Heavy metals in soils are an increasing concern of environmental pollution. These are
continuously being added to agricultural land with sludge, fertilizers, lime and manure. It causes
deleterious effects to plants in two ways: by inactivating several enzymes by binding with SH128
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Yaksha Singh et al
J. Microbiol. Biotech. Res., 2011, 1 (4):124-130
______________________________________________________________________________
groups, and second by intensifying the processes of production of reactive oxygen species (ROS)
leading to oxidative stress. There are several anti oxidative systems operating within the plants to
combat such toxic effects. These include antioxidants (non-enzymatic and enzymatic).
Additionally, exogenous applications of putrescine also decrease oxidative stress.
Polyamines have been reportedly concerned with responses to several stresses e.g. salt stress,
water stress and heavy metal stress. For details one may refer to the review by Sairam and Tyagi
[15] Polyamines are shown to function as metal chelators, decrease lipid peroxidation and hence
delay senescence, scavenge ROS, etc. [16]. Several stress factors e.g. potassium deficiency,
osmotic stress, low pH are shown to stimulate the accumulation of polyamines especially
putrescine in plants.
In the present investigation we have employed putrescine as an antioxidant to ameliorate the
effect of Hg toxicity by scavenging ROS. The precise role of exogenously added putrescine on
plant resistance to Hg toxicity has not been described so far. Hence, the present investigation was
undertaken to demonstrate its effect on Brassica juncea var. vasundhra.
Generally when the level of antioxidants processes and detoxification mechanisms are lesser than
the level of ROS accumulation, damages to the plant occurs. Most plants have evolved complex
protecting systems against ROS. These comprise non enzymatic and enzymatic antioxidants.
Such systems act as defensive systems against oxidation of biomolecules and block the process
of oxidative chain reactions [18].
The present investigation provides evidence of induction of phenolic metabolism as a response to
metal stress. Phenolic compounds are shown to have strong antioxidant activity in plants
growing under heavy metal stress [17]. It has been suggested that their antioxidant act resides
chiefly in their chemical structure. Phenols are oxidized by peroxidase and contribute in
scavenging H2O2.
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