BIOCHEMICAL STUDIES ON ALLEVIATION OF CADMIUM INDUCED TOXICITY IN PEA (Pisum ...

231 Pages·2013·4.18 MB·English

BIOCHEMICAL STUDIES ON ALLEVIATION OF
CADMIUM INDUCED TOXICITY IN PEA (Pisum sativum
L.) AND PALAK (Beta vulgaris var. orientalis)

Dissertation

Submitted to the Punjab Agricultural University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
in
BIOCHEMISTRY
(Minor Subject: Biotechnology)

By
Prabhjot Kaur Saini
(L-2008-BS-24-D)

Department of Biochemistry
College of Basic Sciences and Humanities
© PUNJAB AGRICULTURAL UNIVERSITY
LUDHIANA-141 004

2013
CERTIFICATE I

This is to certify that the dissertation entitled, “Biochemical studies on alleviation of cadmium
induced toxicity in pea (Pisum sativum L.) and palak (Beta vulgaris var. orientalis)”, submitted for the
degree of Ph.D in the subject of Biochemistry (Minor subject: Biotechnology) of the Punjab Agricultural
University, Ludhiana, is a bonafide research work carried out by Prabhjot Kaur Saini (L-2008-BS-24-D)
under my supervision and that no part of this dissertation has been submitted for any other degree.
The assistance and help received during the course of investigation have been fully acknowledged.

______________________
(Dr. A. K. Gupta)
Major Advisor
Professor and Head
Department of Biochemistry
Punjab Agricultural University
Ludhiana - 141004
CERTIFICATE II

This is to certify that the dissertation entitled, “Biochemical studies on alleviation of cadmium
induced toxicity in pea (Pisum sativum L.) and palak (Beta vulgaris var. orientalis)”, submitted by
Prabhjot Kaur Saini (L-2008-BS-24-D) to the Punjab Agricultural University, Ludhiana, in partial
fulfillment of the requirements for the degree of Ph.D, in the subject of Biochemistry (Minor subject:
Biotechnology) has been approved by the student’s Advisory Committee along with Head of the
Department after an oral examination on the same.

_________________________ _______________________
(Dr. A. K. Gupta) (Dr. Balwant Singh)
Major Advisor External Examiner
Professor (Retired)
Department of Molecular Biology and
Biochemistry
Guru Nanak Dev University
Amritsar-143005

_________________________
(Dr. (Mrs.) Bavita Asthir)
Senior Biochemist and Head of the Department

_________________________
(Dr. Gursharan Singh)
Dean Post-Graduate Studies
Acknowledgements
Foremost, I bow before THE ALMIGHTY GOD for vesting freedom to all my wishes and standing by
me at every step.
A precious debt as that of learning is the only debt that is difficult to repay except perhaps through
gratitude. I feel privileged to express my deep sense of gratitude to my esteemed Major Advisor – Dr. Anil
Kumar Gupta, Professor of Biochemistry and Head, Department of Biochemistry, Punjab Agricultural
University, Ludhiana for his unceasing encouragement, impeccable and benevolent guidance during the
pursuance of this study. I shall remain forever indebted to him for training my mind to think
systematically and logically.
It is my profound privilege to express my deep sense of gratitude to Dr. (Mrs.) Narinder Kaur,
Emeritus Scientist, CSIR for her moral encouragement and valuable suggestions rendered during the
course of my study.
I owe my sincere thanks to Dr. B. S. Sohal, Professor of Biochemistry, Controller of Examinations and
Dean PGS Nominee, Department of Biochemistry and Late Dr. J. S. Sital, Professor of Biochemistry,
Department of Biochemistry for their encouraging attitude and valuable suggestions during the period of
my investigation. I record my cordial thanks to Dr. S. S. Gosal, Professor of Biotechnology and Director of
Research, PAU, Ludhiana for his apt suggestions and cooperation during this investigation.
I am grateful to Dr. (Mrs.) Satvir Kaur, Assistant Biochemist, Department of Biochemistry, Dr. (Mrs.)
Anita Garg, Chemist, Department of Chemistry and Dr. M. L. Sharma, Professor of Chemistry (Retired),
Department of Chemistry for their help to elucidate and eliminate my problems with unprecedented
enthusiasm, perceptive encouragement and deep affection to make this study a success.
Words seem inadequate to express deep sense of regards towards my grandparents, parents, sister
and brother whose invaluable blessings and constant encouragement brought me upto this stage of my
career.
A word of appreciation will not compensate for the ever-willing help and cooperation provided to
me by Dr. (Mrs.) Kamaljit Kaur, Dr. (Mrs.) Rachana, Dr. (Mrs.) Harinderjeet, Dr. Vishal, Dr. (Mrs.) Smriti,
Dr. Sondeep, Dr. Harpreet, Dr. Arvind, Imran bhaiya, Yadhu, Sarabjit, Rimaljeet, Patil and Vivek.
Deep and inexpressible thanks to my dear friends Meenakshi, Navdeep, Shashi, Shweta, Parminder,
Abhay, Jaspreet and Balrinder whose special affection and ever-willing support always inspired me. I am
very thankful to my classmates Reeti and Gurpreet for their sincere and in time help.
Financial assistance in the form of Bharti Field Fresh Fellowship, Ladhowal, Ludhiana is gratefully
acknowledged.
I owe my sincere thanks to Mr. Saroop Singh, Mr. Amarjeet, Mr. Ramesh and Mr. Rajinder Kumar
for their generous help in the laboratory.

Prabhjot Kaur Saini
Title of Dissertation : Biochemical studies on alleviation of cadmium induced
toxicity in pea (Pisum sativum L.) and palak (Beta
vulgaris var. orientalis)

Name of the Student : Prabhjot Kaur Saini
Admission Number : L-2008-BS-24-D
Major Subject : Biochemistry

Minor Subject : Biotechnology

Name and designation : Dr. Anil Kumar Gupta
of Major Advisor Professor and Head
Degree to be awarded : Ph.D

Year of award of Degree : 2013

Total pages in Dissertation : 219 + Vita

Name of the University : Punjab Agricultural University, Ludhiana-141004

ABSTRACT

The present study was carried out on pea (Pisum sativum L.) seedlings and palak (Beta vulgaris var.
orientalis) plants to determine the effect of exogenous cadmium on their growth and antioxidative
defence system, and to explore the role of different amendments like calcium chloride, potassium
chloride, calcium hydrogen phosphate, calcium sulphate and sodium nitroprusside in ameliorating the
effects of cadmium toxicity. P. sativum seedlings grown on agar supplemented with different amending
agents like calcium chloride, potassium chloride and sodium nitroprusside showed higher activities of
H O scavenging enzymes. P. sativum seedlings also showed lower contents of H O and
2 2 2 2
malondialdehyde in these cases suggesting a role for the amendments in increasing seedling tolerance
towards cadmium. Out of the three amendments calcium chloride was most effective in lowering
cadmium content. In B. vulgaris plants grown in pots, addition of cadmium (0-100 mg Cd kg-1 soil) led to
increase in the contents of hydrogen peroxide and malondialdehyde and activities of superoxide
dismutase, catalase and glutathione reductase in leaves and roots. On addition of calcium chloride,
potassium chloride, calcium hydrogen phosphate and calcium sulphate to the soil, a reduction in the
activities of antioxidative enzymes was observed at higher doses of amendments along with reduction in
hydrogen peroxide, malondialdehyde and cadmium content. Also improved growth was observed in
these cases. In B. vulgaris, leaves are the edible parts and results in lowering cadmium with CaSO were
4
almost as good as that of KCl while in roots CaHPO was found to be more effective.
4

Keywords: Pisum sativum L., Beta vulgaris, antioxidative enzymes, cadmium, hydrogen peroxide

_______________________ ____________________
Signature of Major Advisor Signature of the Student
Koj pRbMD dw isrlyK : mtr (pweIsm sYtIvm AYl.) Aqy pwlk (bItw vlgYirs
vr. aru IAYntYils) au~qy kYfmIAm v`loN pYdw kIqy gey
zihrIlypn nUM Gtwaux leI jIv-rswiexk KojW[

ividAwrQI dw nwm : pRBjoq kOr sYxI
Aqy dwKlw nUM AYl-2008-bI AYs-24-fI

mu`K ivSw : jIv-rswiex ivigAwn

inmn ivSw : jIv-qknolojI

mu~K slwhkwr dw nwm : fw. Ainl kumwr gupqw
Aqy Ahudw pRoPYsr Aqy mu~KI

hwsl hox vwlI ifgrI : pI.AY~c.fI.

ifgrI id`qy jwx dw swl : 2013

Koj-pRbMD iv`c ku`l pMny : 219+vItw

XUnIvristI dw nwm : pMjwb KyqIbwVI XUnIvristI, luiDAwxw-141004

swr-q`q

ieh Koj mtr (pweIsm sYtIvm AYl.) dy nvyN pOidAW Aqy pwlk (bItw vlgYrIs vr. aurIAYntYils) dy
pOidAW au~qy kIqI geI[ ies Koj dw mMqv bwhrlI id`qI geI kYfmIAm dw aupj Aqy AYNtIaku sIfyitv bcwv
pRxwlI qy Asr dw pqw lgwauxw Aqy v`K-v`K sMSoDnW ijvyN ik kYlSIAm klorweIf, potYSIAm klro weIf,
kYlSIAm hweIfRjo n POsPyt, kYlSIAm slPyt Aqy sofIAm nweItroprusweIf dw kYfmIAm dy ivSYlypn iv`c
suDwr krn dIAW BUimkwvW dw mulWkx krnw[ pI. sYtIvm dy nvyN pOdy ijnHW nUM AYgr Aqy hor v`K-v`K pdwrQW
ijvyN ik kYlSIAm klorweIf, potYSIAm klro weIf Aqy sofIAm nweItroprusweIf nwl augwieAw igAw, auhnW
iv`c hweIfRojn praku sweIf nUM Kqm krn vwly eynzweImz dIAW srgrmIAW vDyry pweIAW geIAW[iehnW
nvyN pOidAW iv`c hweIfRjo n prauksweIf Aqy mYlnfweIAYlfIhweIf dIAW mwqrwvW vI G`t pweIAW geIAW,
ijs qoN ieh pqw lgdw hY ik ieh sMSoDnW nvyN pOidAW dI kYfmIAm dI ivSYlqw nUM sihn dI SkqI
vDwauNdIAW hn[iqMn sMSoDnW iv`coN kYlSIAm klorweIf dw kYfmIAm dI mwqrw Gtwaux au~qy sB qoN izAwdw
Asr ivKweI id`qw[gmilAW iv`c augwey gey bI. vlgYirs dy pOidAW dy p`iqAW Aqy jVHW iv`c kYfIAm dI
mwqrw (0-100 imlIgRwm/iklogRwm im`tI) dyx kwrn hweIfRojn praku sweIf Aqy mYlnfweIAYlfIhweIf Aq y
suprauksweIf ifsimaUtyz, kYtwlzy Aqy gltu wQweyaun irfktyz dIAW srgrmIAW iv`c vwDw pwieAw igAw[
kYlSIAm klorweIf, potYSIAm klorweIf, kYlSIAm hweIfRojn POsPyt Aqy kYlSIAm slPyt dI izAwdw
mwqrw im`tI iv`c dyx kwrn, AYNtIauksIfyitv eynzweImz dIAW srgrmIAW G`t ivKweI id`qIAW Aqy nwl dy
nwl hweIfRjo n prauksweIf, mYlnfweIAYlfIhweIf Aq y kYfmIAm dI mwqrw iv`c kmI pweI geI[ iehnW
pOidAW dy ivkws iv`c vI vDyry suDwr pwieAw igAw[ bI. vlgYirs iv`c p`qy Kwx Xog ih`sw hn Aqy kYfmIAm
dI mwqrw G`t krn iv`c kYlSIAm slPyt Aqy potYSIAm klorweIf donW dw brwbr dw Asr pwieAw igAw
pr jVHW iv`c kYlSIAm hweIfRojn PosPyt vDyry Asrdwr pwieAw igAw[

pRmu`K Sbd : pweIsm sYtIvm AYl., bItw vlgYirs, AYNtIaku sIfyitv eynzweImz, kYfmIAm, hweIfRojn
prauksweIf[

m~uK slwhkwr dy hsqwKr ividAwrQI dy hsqwKr
CONTENTS

Chapter Topic Page

I. INTRODUCTION 1-3
II. REVIEW OF LITERATURE 4-44
III. MATERIALS AND METHODS 45-56
IV. RESULTS AND DISCUSSION 57-188
V. SUMMARY 189-191
REFERENCES 192-219
VITA
____________________________________________________________________________________
LIST OF ABBREVIATIONS

BSA : Bovine serum albumin
cm : Centimeter
FW : Fresh weight
EDTA : Ethylene diamine tetra acetic acid
g : Gram
GSH : Reduced glutathione
GSSG : Oxidised glutathione
kg : Kilogram
µg : Microgram(s)
µl : Microlitre(s)
µM : Micromolar
µmol : Micromole(s)
mg : Milligram(s)
ml : Millilitre(s)
mM : Millimolar
min : Minute
NADPH : Reduced nicotinamide adenine dinucleotide phosphate
nmole : Nanomole(s)
nm : Nanometer
PAGE : Polyacrylamide gel electrophoresis
pH : Potentia hydrogenii
PVP : Polyvinylpyrrolidone
ROS : Reactive oxygen species
SD : Standard deviation
TBA : Thiobarbituric acid
TCA : Trichloroacetic acid
v/v : volume/volume
w/v : weight/volume
CHAPTER- I
INTRODUCTION

Environmental pollution by metals became extensive as mining and industrial activities
increased in the late nineteenth and early twentieth century. Heavy metals are environmental
pollutants and their toxicity is a problem of increasing significance from ecological, nutritional
and environmental point of view. Elements naturally occur in the earth’s crust in a range of
background levels that are generally below the critical load i.e. the amount of the element above
which there is a negative effect on biodiversity and ecosystem function. However, the
concentrations of elements in localized, naturally occurring metalliferous soils or in depositions
from anthropogenic activity (eg. mining, waste disposal etc.) are considerably higher.
The most important factor responsible for groundwater pollution in Punjab is urban and
industrial waste water. This water is often not treated before its release into sewerage drains. The
most common disposal of waste water is its use as crop irrigation. The application of sewage
sludge to agricultural soils, and irrigation of field crops with sewage water and untreated
industrial effluents are common practices used in Punjab especially in vicinity of large cities, as
these are considered reusable sources of essential plant nutrients and organic carbon (Aulakh et al
2009). Singh and Kansal (1985) reported that the concentration of potentially toxic elements was
higher in sewage water of industrial towns of Ludhiana, Jalandhar and Amritsar as compared with
less or non-industrial towns of Sangrur and Abohar. Studies conducted by PAU, Ludhiana have
indicated the presence of mercury, cadmium, chromium, copper and lead in vegetables being
grown alongside the length of Buddha Nullah.
Increase in international concern about the risks associated with long-term consumption
of crops with cadmium (Cd) concentrations has led the international food standards organization,
Codex Alimentarius Commission, to propose a 0.1 mg Cd kg-1 limit for cereals, pulses and
legumes (Harris and Taylor 2001). Uptake of Cd by crop plants is the main entry pathway into the
food chain causing serious problems to human health (Buchet et al 1990). Exposure to Cd can
result in damage to kidney, lung and bone tissue. Cd has been found responsible for the itai-itai
disease in Japan, osteoporosis and renal stones (Nordberg 2004, Hassan et al 2006). The
maximum tolerable intake of Cd for humans recommended by FAO/ WHO is 70 µg/day (Sarwar
et al 2010).
Heavy metal pollutants like Cd are readily taken up by plants and can interfere with many
physiological processes associated with normal growth and development (Artexe et al 2002).
1
Cadmium inhibits root and shoot growth and yield production, affects nutrient uptake and
homeostasis, and is accumulated by agriculturally important crops including pea with a
significant potential to impair animal and human health (Sanita di Toppi and Gabrielli 1999). In
pea, a number of toxic effects of Cd on metabolism have been reported, such as decreased uptake
of nutrient elements (Sandalio et al 2001), inhibition of various enzyme activities (Chugh et al
1992, Obata et al 1996), and induction of oxidative stress (Lozano-Rodriguez et al 1997,
Sandalio et al 2001) including alterations in enzymes of the antioxidant defence system (Dalurzo
et al 1997, Sandalio et al 2001, Romero-Puertas et al 2002, Skorzynska-Polit et al 2004).
Metal toxicity is ascribed to three main reasons, though these three mechanisms might
not exclusively account for their toxicity : (i) direct interaction with proteins due to their affinities
for thioyl-, histidyl- and carboxyl-groups, causing the metals to target structural, catalytic and
transport sites of the cell; (ii) stimulated generation of reactive oxygen species (ROS) that modify
the antioxidant defence and elicit oxidative stress; and (iii) displacement of essential cations from
specific binding sites, causing functions to collapse (Hossain et al 2012). Oxidative stress is a
condition in which ROS or free radicals, are generated extra- or intra-cellularly, which can exert
their toxic effects to the cells. These species may affect cell membrane properties and cause
oxidative damage to nucleic acids, lipids and proteins that may make them nonfunctional.
However in order to overcome oxidative stress, plants have developed very efficient antioxidative
defense machinery. This machinery comprises of a ROS detoxifying enzymatic system
(superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase and guaicol
peroxidase) and an antioxidative non-enzymatic system (such as vitamin A, vitamin C, vitamin E,
glutathione, carotenoids and phenolics) which work in concert to control the cascades of
uncontrolled oxidation and protect plant cells from oxidative damage by scavenging of ROS.
In order to cope with highly toxic metals, or to maintain the level of essential metals
within physiological ranges, plants have evolved complex mechanisms that serve to control the
uptake, accumulation and detoxification of metals. Plants species significantly differ in their
tolerance to and uptake of Cd and other heavy metals. Legume crops are less tolerant to Cd
toxicity than cereals and grasses and frequently encounter strong inhibition of biomass production
in the less than micro molar range of Cd (Inouhe et al 1994). To curb this environmental
deterioration arising from agricultural production, some management practices and remediation
techniques have been evaluated and recommended. Among the remediation techniques, the use of
chemical amendments to immobilize contaminants of interest has been on the rise. Several of the
plant nutrients like phosphorus, sulphur and calcium can be used to alleviate Cd toxicity by
different mechanisms like Cd sequestration in the vegetative parts to avoid its accumulation in
2