Thesis MS Final 9 July PDF

Heavy Metals Induced Toxicity and Levels of
Antioxidant Enzymes to Different Plants of

Hattar Industrial Estate
By
Judat Imran
CIIT/FA15-R07-003 /ATD
MS Thesis
COMSATS Institute of Information Technology
Abbottabad-Pakistan
Fall, 2017
COMSATS Institute of Information Technology
Thesis Title Heavy Metals Induced Toxicity and Levels of

Antioxidant Enzymes to Different Plants of Hattar
Industrial Estate
A Thesis Presented to
COMSATS Institute of Information Technology, Abbottabad

In partial fulfillment
of the requirement for the degree of
Master of Science
in
Environmental Sciences
By
Judat Imran
CIIT/FA15-R07-003/ATD
Fall, 2017
ii
Industrial Estate
A Graduate Thesis submitted to the Department of Environmental Sciences as
partial fulfillment of the requirements for the award of Degree of Master of
Science (MS) in Environmental Sciences.
Name Registration Number
Judat Imran CIIT/FA15-RO7-003/ATD
Supervisor
Dr. Bilal Ahmed Zaffar Amin

Assistant Professor, Department of Environmental Sciences
COMSATS Institute of Information Technology (CIIT)
Abbottabad Campus.
3 July, 2017
iii
Final Approval
This thesis titled

Industrial Estate
By
Judat Imran
Has been approved
For the COMSATS Institute of Information Technology, Abbottabad
External Examiner: _________________________________________________

Dr. Examiner Name
Department
University/Campus
Supervisor: _________________________________________________________
Dr. Bilal Ahmed Zaffar Amin, Assistant Professor
Department of Environmental Sciences,
CIIT Abbottabad
Member Supervisory Committee: _______________________________________

Dr. Qaisar Mahmood TI, Associate Professor
CIIT Abbottabad

Dr. Zulfiqar Ahmad Bhatti, Assistant Professor
CIIT Abbottabad

Dr. Muhammad Shahzad, Assistant Professor/DOO ES
CIIT Abbottabad
iv
Declaration
I, Judat Imran (CIIT/FA15-R07-003/ATD) hereby declare that I have produced the

work presented in this thesis, during the scheduled period of study. I also declare that I
have not taken any material from any source except referred to wherever due. If a
violation of HEC rules on research has occurred in this thesis, I shall be liable to
punishable action under the plagiarism rules of the HEC.
Date: 3-07-2017 Signature of the student: __________________
(Judat Imran)
(CIIT/FA15-R07-003/ATD)
v
Certificate
It is certified that Ms. Judat Imran (CIIT/FA15-R07-003/ATD) has carried out all the
work related to this thesis under my supervision at the Department of Environmental
Sciences, COMSATS Institute of Information Technology, Abbottabad and the work
fulfills the requirement for award of MS degree.
Date:____________________ Supervisor: _______________________
Dr. Bilal Ahmed Zafar Amin

Assistant Professor
Submitted through:
Prof. Dr. Arshid Pervez

Chairman/Head, Department of Environmental Sciences
CIIT, Abbottabad
vi
DEDICATION
To Almighty ALLAH and the Holy Prophet

Muhammad (P.B.U.H)
&
My Loving Family
vii
ACKNOWLEDGEMENTS
First and foremost I offer my sincerest gratitude to my supervisor, Dr. Bilal Ahmad
Zafar Amin, who has supported me throughout my thesis with his patience and
knowledge whilst allowing me the room to work in my own way. I attribute the level of
my MS degree to his encouragement and effort and without him this thesis, too, would
not have been completed or written. One simply could not wish for a better or
friendlier supervisor.
I will love to say special thanks to Professor Dr. Arshid Pervez, the worthy Head of
Department, & Professor Dr. Qaisar Mahmood TI, for their general encouragement and
moral support.
The Department of Environmental Sciences has provided the support and equipment I
have needed to produce and complete my thesis and the HEC (Higher Education
Commission) has funded my studies.
Finally, I thank my parents for supporting me throughout all my studies at University
and for providing a home in which to complete my writing up.
Judat Imran
viii
ABSTRACT
Now a days, environmental pollution due heavy metals is major problem. Heavy
metals are toxic and non-biodegradable. Heavy metals enter into the environment as
result of both natural and anthropogenic activities. When the amount of heavy metals
exceed, it results in the production of reactive oxygen species with in plants, animals
and humans that cause damage to them. In order to overcome that plants are well
equipped with defense mechanism comprising of enzymatic antioxidants which
includes Ascorbate Peroxidase, Guaiacol Peroxidase and Catalase etc. Generally,
phytoremediation is defined as an emerging technology which applied the use of
selected plants to clean up the contaminated environment from hazardous contaminant
in order to improve the environment quality. The present work aimed at selecting the
best native phytoremediation plant of heavy metals in Hattar Industrial Estate. To do
so, four different common plants of Hattar Industrial Estate (near volta battery
industries) were collected. The amount of heavy metals and antioxidants were
analyzed. Maximum accumulation of heavy metals and increased production of
antioxidants was shown in Rumex acetosa as compared to Cannabis sativa,
Parthenium hysterophorus and Conyza canadensis. Bio accumulation coefficient and
Translocation factor of Rumex acetosa were 35 and 0.815. The increased amount of
heavy metals was found in leaves and stem of Rumex acetosa (than roots). The levels
of three different antioxidants in Rumex acetosa varied as; GPOXs found in the leaves,
stem and roots of contaminated Rumex acetosa were 5, 4.02 and 2.9 times higher as
compare to its control plant of non-contaminated area. Secondly, Rumex acetosa had
CATs in leaves, stem and roots as 1.71, 1.7 and 1.47 times (higher than control).
Thirdly, SODs found in Rumex acetosa leaves, stem and roots as compared to control
had shown 1.67, 1.9 and 1.45folds amplified. Thus, Rumex acetosa is proved to be
environment friendly, cost efficient and ecological beneficial plant to remove heavy
metals from soil.
ix
TABLE OF CONTENTS
1. Introduction…………………………………………….….…...………… 1
1.1. Heavy metals……………....………………………………………….. 3
1.2 Lead…………………………………………………………………… 6
1.2.1 Chemical properties of lead…………………………………………… 7
1.2.2 Sources of lead…………………………………….………………….. 7
1.2.3 Lead uptake…………………………………………………………… 7
1.2.4 Lead transport………………………………………………………… 8
1.2.5 Lead toxicity………………………………………………………….. 9
1.3 Cadmium………………………………………………...…………… 10
1.3.1 Chemical properties of cadmium…………………………………….. 11
1.3.2 Sources of cadmium…………………………………………..……… 11
1.3.3 Cadmium uptake and transportation………………………………..... 11
1.3.4 Toxicity of cadmium……………………………………………….… 13
1.3.5 Cytogenetic effect of cadmium……………………………….……… 14
1.3.6 Differential cadmium tolerance in plants……………………………... 14
1.3.7 Phytotoxicity of Cd and their interactions with nutrients…………….. 16
1.4 Zinc……………………………………………………………….….. 16
1.4.1 Sources of zinc……………………………………..………………… 16
1.4.2 Uptake, translocation and functions of zinc in plants……………..…. 17
1.4.3 Zinc toxicity………………………………………………………….. 21
1.5 Antioxidants…………………………………………..…………….. 21
1.5.1 Reactive oxygen species, sites of production and their effects…..... 23
1.5.2 Types of ROS………………………………………………………... 26
1.5.3 Antioxidative Defense System in Plants…………………………...... 28
1.5.4 Non enzymatic components of antioxidative defense system…….…. 29
1.5.5 Enzymatic components………………………………………...……. 29
x
1.5.6 Superoxide Dismutase (SOD)…………………………………………...……. 30
1.5.7 Catalase (CAT)……………………………………………….……….………. 31
1.5.8 Guaiacol Peroxidase (GPX)……………………………………….…….……. 32
1.5.9 Enzymes of Ascorbate-Glutathione cycle………………………….…...…….. 32
1.5.10 Ascorbate Peroxidase (APX)………………………………….………….…....33
1.5.11 Monodehydroascorbate Reductase (MDHAR)……………………….………. 34
1.5.12 Dehydroascorbate Reductase (DHAR)……………………………............….. 35
1.5.13 Glutathione Reductase (GR)……………………………………………..…… 35
1.6 Phytoremediation…........................................................................................... 37
1.7 Statement of the Problem…………………………………………….……….. 39
1.8 Purpose of the Study………………………………………………….….…… 39
1.9 Objectives………………………………….………………………………..… 39
2 Materials & Methods………………………………………………….………….. 40
2.1 Sample collection …………………...………………………….……… 41
2.2 Sample storage…………………………………………………….…...….42
2.3 Sample preparation………………………………………………..……… 42
2.4 Heavy metals analysis…………………………………………….....…… 42
2.5 Extraction and antioxidant assays……………………………..…………. 43
3 Results………………………………………………….……………………...….45
3.1 Analysis of lead in contaminated plants…………………………………….…..... 46
3.2 Analysis of zinc in contaminated plants…………………………………..……… 49
3.3 Analysis of cadmium in contaminated plants……………………...…………….. 52
3.4 Bioaccumulation Coefficient (BAC) and translocation factor (TF)........................ 54
3.5 Antioxidants assay………………………………………………………………... 55
3.5.1 Analysis of GPOXs activity………………………………………….……....… 55
xi
3.5.2 Analysis of CATs activity……………………………………………………… 57
3.5.3 Analysis of SODs activity …………………………………….……………….. 59
4 Discussion …………………………………………………………….………...... 62
5 References………………………………………………………………….……….71
xii
LIST OF FIGURES
Fig 1: Sites of production of reactive oxygen species (ROS) in plants……………….24
Fig 2: Reactive oxygen species (ROS) as second messengers in several plant hormone
responses, including stomatal closure etc……………………………………….…….25
Fig 3: Reactive oxygen species (ROS) induced oxidative damage to lipids, proteins,
and DNA………………………………………………………………………………26
Fig 4: Schematic presentation of generation of reactive oxygen species in
plants……………………………………………………………………………….….28
Fig 5: Site view and sample collection………..………………………………………41
Fig 6: Different steps involved in heavy metals analysis…………………..…………42

Fig 7: Antioxidants extraction and assays…………………………………………….44
Fig 8 a-d: Concentration of Lead with in different plant organs of contaminated
plants……………………………………………………………………………..……47
Fig 9 a-d: Concentration of lead with in different plant organs of control
plants…………………………………………………………………………………..48
Fig 10 a-d: Concentration of zinc within plant organs of different plants…................50
Fig 11 a-d: Concentration of Zn within plant organs of different plants…………...…51
Fig 12 a-d: Concentration of Cd within plant organs of different plants……………..53
Fig 13 a-b: GPOXs activity in leaves of contaminated and control plants………….. 55
Fig 14 a-b: GPOXs activity in stem of contaminated and control plants……….……56
Fig 15 a-b: GPOXs activity in the roots of contaminated and control plants………...57
Fig 16 a-b: The CATs activity in the leaves of contaminated and control plants…….58
Fig 17 a-b: The CATs activity in stems of contaminated and control plant………….58
Fig 18 a-b: The CATs activity in the roots of contaminated and control
plants……………….………………………………………………………………….59
Fig 19 a-b: The SODs activity in leaves of contaminated and control plants………...60
xiii
Fig 20 a-b: The SODs activity in stems of contaminated and control plants…………60
Fig 21 a-b: The SODs activity in roots of contaminated and control plants……........ 61
xiv
LIST OF TABLES
Table1: Bioaccumulation coefficient and translocation factor of contaminated
plants………………………………………………………………….…… 54
xv
LIST OF ABBREVIATIONS
RA Rumex acetosa
CS Cannabis sativa
PH Parthenium hysterophorus
CC Conyza canadensis
BAC Bioaccumulation coefficient
TF Translocation factor
mg/ L Milligram per liter
l Microliter
mg/ kg Milligram per kilogram
xvi
Chapter 1
Introduction
xvii
Heavy metal is term used for any metallic element having high density and high
toxicity affect even in small amount. In general, heavy metals refer to group of metals
and metalloids having density greater than 4gm/cm3 or 5 times/ more, greater than
water. But, as compared to density, the chemical properties of heavy metals are more
influencing factors. Heavy metals include Lead(Pb), Cadmium(Cd), Nickel(Ni),
Zinc(Zn), Cobalt(Co), Manganese(Mn), Chromium(Cr), Copper(Cu), Arsenic(As),
Iron(Fe) and Platinum group (Nagajyoti et al., 2010).
In 19th and 20th century, heavy metal pollution level has been increased. Lead (Pb),
Cadmium (Cd) and Mercury (Hg) are highlighted as most hazardous metals present in
environment. The toxic metal elements such as Cd and Pb enter into the environment
as the result of anthropogenic activities. Since most of the activities involving these
metals are essential for today’s life so environmental contamination due to heavy
metals pollution is not likely to be ended in future (Dey et al., 2007). A range of
transition metals is required by plants as essential micronutrients for normal growth
and development. As, it is known that these metals are essential for most redox
reactions which, in turn, are fundamental to cellular function (Hall and William, 2003).
With the mode of entrance to plants effects of toxic heavy metals vary, i.e., via roots or
foliar. It has been shown that plants can be influenced differently by specific applied
levels of heavy metals which depends on what part/organ of the plant is directly
exposed to the metals (Shahid et al., 2016). Plants that have been grown on soil, rich in
heavy metals or industrial effluents are known to absorb heavy metals in the quantity
that may be toxic to plant growth and metabolism (Pandey et al., 2009). Metals in the
soil are strongly held by the soil particles and a little removal of metals occur either by
plant uptake or movement down the soil profile. Large amounts of metals may be lost
from the soil by soil erosion. Generally, heavy metals are less mobile and therefore less
bioavailable in soils with higher organic matter contents and clay contents. Though,
they are more mobile in acid soils and therefore, more available for plant uptake but
their availability decreases as the pH is raised (McGrath et al., 1995). In the UK,
Sweden, Germany and the USA, controlled field experiments with sewage sludge
existed and the data of which is presented here are from those long-term field
experiments only. Metals concentration had adversely affected the microbial activity,
1
populations of cyanobacteria, Rhizobium leguminosarum bv. trifolii, mycorrhizae and
the total microbial biomass in soil while in some cases it had been affected by metals
which were present below the European Community's maximum allowable
concentration limits in sludge-treated soils. For example, Nitrogen fixation inhibition
by free living heterotrophic bacteria was found at soil metal concentrations of (mg/kg):
127 Zn, 37 Cu, 21 Ni, 3.4 Cd, 52 Cr and 71 Pb. 50% Nitrogen fixation reduction by
free-living cyanobacteria was found at metal concentrations of (mg/kg): 114 Zn, 33 Cu,
17 Ni, 2.9 Cd, 80 Cr and 40 Pb. Reduction in Rhizobium leguminosarum bv. trifolii
numbers occur by several orders of magnitude at soil metal concentrations of (mg kg-
1
): 130-200 Zn, 2748 Cu, 11-I5 Ni, and 0.8-1.0 Cd. The concentrations at which
toxicity occurred to both microorganisms and plants were found to be influenced by
soil texture and pH. Metal toxicity is considerably reduced at higher pH, and increased
contents of clay and organic carbon. Adverse effects on soil microbial parameters were
generally found at surprisingly modest concentrations of metals in soils as suggested
by evidence. Hence, it is concluded that prevention of adverse effects on soil microbial
processes and ultimately soil fertility, should be a factor which effects soil protection
legislation (McGrath et al., 1995).
Up till now, it has been shown that more than 400 hyper-accumulating plant species,
among them Thlaspicaerulescens, Silena vulgaris, and Brassica juncea are involved in
removal of Cd, Cu, Ni, Pb and Zn from metal-contaminated soils (Salt et al., 1998).
Successful phytoextraction not only depends on the plant itself but also on the
interaction of the plant roots with the rhizosphere bacterial community (Whiting et al.,
2001). It has been suggested that heavy metal mobilization and uptake by plants might
be improved by soil microorganisms and in particular the active rhizosphere bacteria.
Although the detail investigation of the mechanisms is needed to be understood, the
accumulation of selenium and mercury in wetland plants were shown to be promoted
by rhizosphere bacteria (De Souza et al., 1999) and also the dissolution of Zn from the
non-labile phase in soil increases (Whiting et al., 2001). Metal accumulating plant as
compared to non-accumulating planta species can concentrate heavy metals like Cd,
Zn, Co, Mn, Ni, and Pb up to 100 or 1000 times greater and are termed as excluder
plants (Erdei et al., 2005).
2
Uptake of high amount of metals ions is toxic to plants. Normally, root inhibition is
observed at micro-level concentrations of heavy metals. Phytotoxicity of heavy metals
can be attributed to accumulation of heavy metals specifically in plasmatic
compartment of cells such as cytosol and chloroplast stroma. Metals induced
development changes are caused by either direct and immediate impairment of
metabolism or signaling process that initiate adaptive or toxicity responses that need to
be consider as active processes of organisms. Transport processes are recognized as a
central mechanism of metal detoxification and tolerance. Some metals like Cu and Zn
play important role in plant growth and development as they are the part of structural
and functional part of protein. Other metals like Cd and Pb have no function in plant
(Sharma and Dietz, 2006).
During oxidative stress ROS are commonly produced. The high level of ROS can
damage macro molecules in plants, thus ROS is needed to be strictly controlled by
complex mechanism in plants which includes antioxidants enzymes like SODs, POXs
and CAT (Feigl et al., 2015). Increased production of reactive oxygen species ROS is
enflamed by both natural and stress situations. The normal functions of plant can be
disrupt through oxidative damage to cellular component by these highly cytotoxic
species. Plants for their protection against ROS are well equipped with non-enzymes
antioxidant such as ascorbate and glutathione and oxygen radical detoxifying enzymes
such as CAT, POXs and (Glutathione reductase) GR (Chaoui et al., 1997). Plants have
been known to develop both enzymatic and non-enzymatic defense systems to
scavenge and detoxify ROS (Li and Luo, 2012).
1.1 Heavy Metals
The term heavy metals generally refers to the group of metals and metalloids having
density greater than 5gm/cm3 and are related to pollution and toxicity although some
elements (essential metals) are required by living organisms at low concentrations
(Adriano, 2001). Plants, algae and other microorganisms require several transition
metals for their growth. The list of transition elements comprises of Vanadium to Zinc
in the first row series and molybdenum in the second row series of the periodic table.
Because of their chemical properties such as redox- activity under physiological
conditions (Cu, Fe) and Lewis acid strength (Zn), these elements have been required in
3
the course of evolution (Da Silva and Williams, 2001). The above mention properties
not only made transition metal elements important for life but also made them toxic
when the permissible limit exceeds. Redox metal ions activate the formation of
hydroxyl radicals by participating in Habier-Weiss and Fenton reactions (Halliwell and
Gutteridge, 1990). Inactivation and damage to biomolecules can occur, because of
uncontrolled high affinity binding to sulfur-, nitrogen- and oxygen-containing
functional groups in biological molecules. Extremely narrow and tightly controlled
metal homeostasis network is needed to adjust to fluctuations in micronutrient
availability which is the necessity for all organisms as the physiological range of heavy
metals between deficiency and toxicity is constricted (Clemens, 2006).
Majority of metals in soils are bound to organic humus soil constituents, inorganic clay
soil constituents or on the other hand are present as insoluble precipitates. For plants, in
order to accumulate these soil bound metals, firstly they should be mobilize in to the
soil solution. Number of different ways are adopted to achieve this mobilization of soil
bound metals. Firstly, phytosiderophores which are metal chelating molecules will be
secreted in to the rhizosphere to chelate and solubilize soil bound metals such as
mugineic acid, avenic acid and nicotine amine of graminaceous species (Kinnersely,
1993). The phytosiderophores are secreted in reaction to Fe and Zn deficiency plus Cu,
Zn and Mn can also be mobilized from the soil (Romheld, 1991). Metal chelating
proteins belonging to metallothioneins (kay et al., 1991) or y-glutamylcysteinyl
isopeptides (3’EC-isopepticles) (Rauser, 1990) may also act as siderophores in plants.
Secondly, specific plasma membrane bound reductase will help the root to reduce soil
bound metal ions. Pea plants lacking in Fe and Cu have great ability to reduce Fe (III)
and Cu (II) that is linked with increase uptake of Cu, Mg, Fe and Mn (Welch et al.,
1993). Thirdly, heavy metals can be solubilized by the plant roots by acidifying their
soil environment with protons extruded from roots. Metal precipitates are solubilized
by lower pH and soil bound metal ions are released into the soil. In some Fe deficient
dicotyledonous plants, similar mechanism has been observed for Fe mobilization
(Crowley et al., 1991).
It should be cleared that all of three above mention processes are performed by either
mycorrhizal fungi or root colonizing bacteria. Hence, it is difficult to evaluate the
4
individual contribution of root cells and rhizosphere microorganisms in metal
mobilization carried out by plant. The solubilized metal ions will enter in to the roots
either by apoplastic (extracellular) or symplastic (intracellular) pathways. High cation
exchange capacity of cell walls limit the apoplastic transport unless metal ions are
transported as non-cationic metal chelate. Symplastic transport requires the movement
of metal ions across the plasma membrane that mostly has large negative resting
potential of 170mV (negative charges resides inside the membrane). Strong
electrochemical gradient for inward movement of metal ions is provided by this
membrane potential. Energy dependent saturable process either specific or generic
metal ion carriers or channels promote the metal ions to enter in to plant cells
(Clarkson and Luttege, 1989).
Non-essential heavy metals may strongly strive for the same trans-membrane carriers
as used by essential heavy metals. Why non-essential heavy metals can enter cells even
against concentration gradient can partially be explained by this relative lack of
selectivity in transmembrane ion transport. For example, kinetic data illustrated that
essential Cu2+ and Zn2+ and non-essential Ni2+ and Cd2+ strive for same transmembrane
carrier. Also the transfer of metal chelate complexes occur across the plasma
membrane by the mean of specialized carriers like in the case for Fe-phytosiderophore
transport in graminaceous species. Metal ions can either be stored or exported to shoots
after their entrance into the roots. The vacuole plays vital role in metal ion storage
(Raskin et al., 1994). At time, when the level of ROS exceeds the defense mechanism
then cell is said to be in state of “oxidative stress”. During environmental stress, the
enhanced production of ROS can pose a threat to cell by causing peroxidation of lipid,
oxidation of proteins, damage to nucleic acid, enzyme inhibition, activation of
programmed cell death (PCD) pathway and ultimately leading to cell death. In spite of
ROS destructive activity, ROS are described as second messengers in variety of
cellular processes including tolerance to environmental stresses. The action of ROS
whether its damaging or signaling molecule depends on delicate equilibrium between
ROS production and scavenging. As the result of multifunctional roles of ROS, it
becomes essential for cells to control the level of ROS tightly to avoid any oxidative
injury and not to eradicate them completely (Sharma et al., 2012). Scavenging or
5
detoxification of excess of ROS is achieved by an efficient oxidative system
comprising of non enzymic as well as enzymic antioxidant (Noctor and Foyer, 1998).
Superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), enzymes of
ascorbate glutathione (AsA-GSH) cycle such as ascorbate peroxidase (APX),
monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR)
serve as enzymic antioxidants. Non enzymic antioxidants in the cell include Ascorbate
(AsA), glutathione (GSH), carotenoids, tocopherols and phenols. Different workers
have reported the increased activities of many enzymes of the antioxidant defense
system in plants to combat oxidative stress induced by various environmental stresses.
Maintenance of high antioxidant capacity to scavenge the toxic ROS has been linked to
increased tolerance of plants to these environmental stresses (Zaefyzadeh et al., 2009;
Chen et al., 2010).
Plants constitute the important component of ecosystem and they transfer the
heavy metal elements from biotic to abiotic environments through air, water and soil
(Hamilton, 1995). Arsenic, Lead, Cadmium and Mercury are considered as most
important elements in term of food chain contamination. While some micronutrients
like Copper, Chromium, Nickel and Zinc may be toxic to plants and animals at high
concentration (McLaughlin et al., 1999). The bioavailability of the elements to the
plant is influenced by many factors associated with the soil, climatic conditions, plant
genotype, agronomic management including active/passive transfer processes,
sequestration, speciation, redox states, the type of plant roots system and the response
of plant to elements in relation to the seasonal cycles (Chojnacka et al., 2004).
1.2 Lead
Lead is one of the most hazardous pollutants. Lead is of ecological concern due to its
impact on human health and environment. Lead is an abundant heavy metal and has
gained importance as effective paint, gasoline and explosive as well as from the
disposal of municipal sewage sludge enriched in lead (Chaney and Ryan, 1994). In
order to limit the lead input in the country, different countries have adopted different
regulatory measures but in spite of that it continues to be one of major global
environmental and human threat. Many of the lead pollutants are important for human
life therefore soil contamination with lead is not likely to be decreased in near future
6
(Yang et al., 2000). It has been observed that lead content has been significantly
increased in the cultivated soils near the industrial area. Accumulation of lead occur
near the surface ground layer and its concentration decrease along with soil depth (de
Abreu et al., 1998).
Lead accumulation occur in different organs of plant, after it is taken up by the plant
from soil. Lead, itself is protoplasmic poison and is cumulative, slow acting and
refined. Lead contamination in soil results in decrease in crop productivity and hence
presenting a serious problem for agriculture (Johnson and Eaton, 1980).
1.2.1 Chemical properties of lead
Lead is soft metal, gray in colour and occurs naturally in soil. It belongs to group XIV
b of the periodic table and has atomic weight of 207.2. It is found in traces in common
rock forming and readily weatherable minerals and form major part of sulphide,
sulphate, oxide, carbonate and silicate minerals (Reimann and deCaritat, 1998).
Different oxidation states of Pb exists like Pb, Pb+2 and Pb+4. The Pb+2 cation is the
acid in Lewis sense which can bind to several ions in medium (Sposito et al., 1982).
1.2.2 Sources of lead
A major pollutant in both the terrestrial and aquatic ecosystem is lead. Natural
weathering process is the main source of lead along with it other sources are exhaust
fumes of automobiles, chimney of factories using lead, effluents from the storage
battery, industry, mining and smelting of lead ores, metal plating and finishing
operations, fertilizers, pesticides and additive in pigments and gasoline (Sharma and
Dubey, 2005).
1.2.3 Lead uptake
Generally, plants uptake the Pb through the root system via passive diffusion (Tung
and Temple, 1996). It has been observed that Pb is transported chiefly in apoplast
through intercellular spaces following the water movement within plant (Lane and
Martin, 1977). It is also important to note that beside roots that are involved in uptake
of heavy metals, leaves could also be involved in absorption of Nickel, Cadmium and
Lead (Amari, 2017). The heavy metals uptake from the soil solution is directly
dependent on species, form, concentration of metals, the soil or nutrient solution
acidity and organic matter composition (Yanai et al ., 2006; Chen et al., 2009). The
7
absorption of Pb increases with increase in pH up to 8.5 (Lee et al., 1998). At pH
ranging from 5.5 -7.5, the solubility of Pb in soil is limited by phosphate or carbonate
ions precipitates so small quantity of Pb is available to plants (Blaylock, 1997). The
heavy metal uptake from the soil solution is strongly affected by Calcium. Ca+2
addition to the growth medium significantly inhibited the root absorption of lead and
nickel in Alyssum bertolonii (Kim et al., 2002) and Ni absorption is promoted in
Berkheya coddii (Boyd and Martens, 1998; Verbruggen et al., 2009).
1.2.4 Lead transport
As the metals are taken up by the roots, they can be either stored in roots or exported to
shoots. Ni, Cd and Pb ions are transported to various plant organs through the
transpiration stream or via xylem (Yang et al., 1996; Tung and Temple, 1996; Peralta-
Videa et al., 2002). It has been observed that also amino acids and peptides like
Histidine, Nicotianamine, metallothioneins are the potential metal ligands that
specifically bind metal in cytosol or in subcellular compartments for transport,
translocation and accumulation with in plant cell (Clemens, 2001; Douchkov et al.,
2005). Phytochelatins (PCs) are the lower molecular weight polypeptides and are
closely involved in root to shoot transport of Cd and Pb (Clemens, 2006; Liu et al.,
2010). The important class of heavy metals binding lignads in plants are the PCs
(Cobbett, 2000).
Metals are transported in plants from roots to shoot through xylem which may involve
several types of transport proteins including P1B- type ATPases, MATESs and OPTs
(Dalcorso et al., 2013). Although P1B- ATPases are oftenly reported to be the potential
transporter of Cd and Pb and to participate in cytosolic efflux of these metals (Mills et
al., 2003; Verret et al., 2004). Different distribution patterns were observed with in the
cell. Distribution of Ni in plant tissue is different from that Cd and Pb where
endodermis limit their movement into the central cylinder (Tung and Temple, 1996;
Seregin and Ivanov, 1998). When heavy metals like Cd, Ni and Pb enter into the cell at
high amount, they induce a high range of toxic effects to plants at morphological,
physiological and biochemical levels.
8
1.2.5 Lead toxicity
The lead has shown to cause adverse effects on both animals and plants. In plants, lead
effects metabolic activities in different cell compartments. Reduction in cell division,
inhibition of photosynthesis, disruption of mineral nutrition and decrease in dry
biomass of roots and shoots, seed germination and growth are the results of lead
toxicity. Lead has been reported to produce ROS and enhance the antioxidant enzyme
activity in plant. The ROS produce as the result of oxidative stress cause adverse
effects in plant cells like inhibition of ATP production, inhibition of photosynthetic
activity, lipid peroxidation and DNA damage (Malar et al., 2014). The first functional
content of the plant cell that comes in direct content with heavy metals is the plasma
membrane. Cd, Ni and Pb have been reported to adversely affect the membrane
functionality and provoke a strong change in lipid composition of various cell
membranes (Fodor et al., 1995; Gonnelli et al., 2001). Generation of reactive oxygen
species is induced by Cd, Pb and Ni which result in oxidative damage to different
biomolecules including proteins (Ahamed and Siddiqui, 2007; Gill and Tuteja, 2010).
Pb is involved in the production of reactive oxygen species and increase antioxidant
enzyme activity in plants (Mishra et al., 2006).
Various harmful effects are caused in plant cells by ROS which are produced as result
of oxidative stress such as inhibition of photosynthetic activity, inhibition of ATP
production, lipid peroxidation and DNA damage (Ruley, 2004). Pb toxicity result in
increased production of ROS which damages the cell membrane, nucleic acids and
chloroplast pigments (Tewari et al., 2002). Production of excess ROS in heavy metal
stress plants may be the significance of distribution of balance between their
production and antioxidants activity consisting of enzymic antioxidants such as SOD,
CAT, POX and APX (Zhang et al., 2007; Jiang et al., 2010). Oxidative stress, a part of
general stress that may arise when an organism experiences different external or
internal factors changing its homeostasis and in response, an organism will either aim
to maintain the previous status by activation of corresponding protective mechanisms
or goes to a new stable state (Yan et al., 2008). Antioxidants and antioxidant enzymes
form a defense system and function to interrupt the cascades of uncontrolled oxidation.
9
These defense systems remove, neutralize or scavenge oxy-radicals and their
intermediates (Marques and Soares, 2011).
The superoxide radical -O2 is scavenged in stress plant by superoxide dismutase which
converts superoxide into hydrogen peroxide (H2O2) (Reddy et al., 2005). H2O2 is
scavenged by catalase and converted to water and oxygen (O2). Peroxides such as
ascorbate peroxidase and peroxidase also scavenge H2O2 indirectly by combining with
antioxidant compounds such as ascorbate (Yang et al., 2011). Plants have divergent
mechanisms for modulating heavy metal levels to adapt to change in the concentration
of metals in polluted environment. If the accumulation level of heavy metals exceeds
the detoxification capacity of plant tissues than it become toxic (Zhang et al., 2007).
1.3 Cadmium
Cadmium is a nonessential element and higher concentration of it may negatively
affects plant growth and development (Hashem, 2013). Cadmium is assumed as one of
the major contaminant of ecosphere. Mining and smelting of Cadmium containing ores,
municipal wastes, pesticides, trace emissions, burning fossil fuels, and fertilizers are
the major sources of Cadmium. In plants, roots have high concentration of Cadmium as
compared with its aerial parts as roots are in direct contact with soil having toxic metal
ions. Cd is non-essential heavy metal as compared with other metals like Cu and Zn.
The Cd at low concentration is nontoxic and highly toxic at higher concentration. It
manifests its toxicity by inhibiting plant growth, changing plant nutrient contents and
by antagonizing the effect on essential elements and several enzyme activities. Cd
induce changes in plants at genetics, physiological and biochemical level leading to
phytotoxicity, whose indicators are leaf rolls, chlorosis, reduction of roots and stem
growth, limiting respiratory and photosynthetic activities and generation of oxidative
stress (Muradoglu et al., 2015).
Cadmium is considered as highly toxic trace element which is present in environment
as the result of human activities. Cadmium is taken up by the roots which are essential
metal transporters and after prolonged exposure to cadmium, partly translocated to the
shoot. Cadmium affect various aspects of plant physiology and hence decreases the
plant growth rate. It has been shown that Cadmium decreases carbon assimilation, can
10
generate oxidative stress and eventually result in wilting, in different plant species
(Forestier et al., 2002).
1.3.1 Chemical properties of cadmium
Cadmium is silver white, slightly bluish metal. It is the member of transition elements
series and has 8 stable natural isotopes. It has atomic weight of 112.4. It exists in cation
form of Cd+2 (Tricot, 1999). It has similar chemical properties as that of zinc and
calcium. Hence it can cross the biological barrier and accumulate in tissues (Amari et
al., 2017).
1.3.2 Sources of cadmium
Cadmium enter in to the environment by anthropogenic pathways which include
electroplating, manufacturing of plastics, mining, paint pigments, alloy preparation,
alloy preparation, batteries that contain Cadmium, household appliances, automobiles
and trucks, agriculture implements, airplane parts, industrial tools, hand tools and
fasteners of all types are commonly coated with Cadmium. Heavy metals may enter in
to the soil through sludge, compost or fertilizers addition (Kirkham, 2006). Heavy
metals are found in soil naturally as rare elements. High concentrations are confined
chiefly to certain minerals usually present in forms which are not easily available. Both
natural and manmade ecosystems may be damage by the release of heavy metals as
result of anthropogenic activities (Tyler et al., 1989).
The chemical form of heavy metals in soil solution is directly dependent on the metal
element concerned, pH and presence of other ions etc. Toxic actions of heavy metal
ions are applied on the enzymes. The exposure of soils to heavy metals for long time
may result in manifest decreases in soil enzyme activity (Tyler et al., 1989). Cadmium
has been considered as significant pollutant due to its high toxicity and greater
solubility in water which determines wide distributions in aquatic ecosystems
(Lockwood, 1976).
1.3.3 Cadmium uptake and transportation
Due to the high mobility of Cd and small concentration at which its effects on plants
begin to show, Cd is regarded as one of the most dangerous element (Barcelo and
Poschenfieder, 1992). It has been shown by the research that the root of lettuce released
much more of their absorbed Cd for translocation to shoot than other crops. Active
11
transport or lack of metal absorption to fixed or soluble chelators in the root or may be
due to exchange with Ca, Mn and Zn moving through the roots result in greater
translocation (John, 1976). It has been reported by Moral et al., 1994 that Cd was
transported to the aerial parts of tomato plant and was absent in fruits. Hinsely et al.,
(1984) stated that pH of soil had large influence on Cd transportation in corn. The
highest grain Cd concentration is found at soil pH about 6. The uptake of Cd by corn
was less from the most acid soil which possess the highest content of organic matter
(Street et al., 1977). The pH over the range of 6-8.5 slightly affects the ratio of
complexed to uncomplexed Cd and is independent of Cd concentration. The uptake of
heavy metals by plants is influenced by many factors in soil (Das et al., 1997). Cd
uptake is increased as the soil pH decreases (Lager-werff, 1971; Miller et al., 1976)
and it is decreased with increase in soil cation exchange capacity (Haghiri, 1974).
Cd is absorbed passively (Cutler and Rains, 1974) and is translocated freely (Jarvis et
al., 1976). Cadmium uptake can be influenced by chelators in nutrient solutions
(Francis and Rush, 1974). In Cd uptake and translocation pronounced interaction
between Zn and Cd occur (Chaney et al., 1976; Chaney and Harnick, 1978). Actually,
Cd interference in a Zn dependent process resulted in a part of Cd toxicity (Falchuk et
al., 1975). The differences in the root morphology of plant has been linked to the
differences in ability of plants to accumulate heavy metals (Hemphill, 1972; Schierup
and Larsen, 1981). It has been proposed that plant with numerous thin roots would
accumulate more metals as compared to one with few thick roots (Schierup and Larsen,
1981). Chukwuma (1993) had related the accumulation of Cd, Pb and Zn in cultivated
and wild plant species in the abandoned lead-zinc mine.
Chukwuna (1993) had compared the accumulation level of Cd, Pb and Zn in cultivated
and wild plant species in the abandoned lead-zinc mine. It was found that the overall
reduction in the potential toxicity of Cd by Zn through simple mass action effect
specifically for cultivated plants while other additional tolerant or adaptive mechanisms
might be operative in wild plants. McKenna et al., (1993) had stated the interactions
between Zn and Cd in nutrient solution and their effects on the accumulation of Zn and
Cd in plant roots and leaves. Higher Cd concentration had been found in older than in
younger leaves of lettuce and spinach. It is not necessary that the potential higher Cd
12
concentration in old leaves is due to transpiration rate. In tobacco, metal binding
peptides were found in higher amount in older leaves than in younger leaves and Cd
was transported into vacuoles as a mean of detoxification (Vogeli-Lange and Wagner,
1990). Cadmium was found at higher concentration in roots than in shoots (Cataldo et
al., 1981; Rauser, 1986).
As per recommendation of the National Research Council of the United States (1980),
the Cd content of forage crops should not be more than 0.5 µgg-1 in order to limit the
concentration of Cd in liver and kidney of animals who feed on these crops to protect
humans against Cd toxicity. Cd is known to produce oxidative stress (Hendry et al.,
1992; Somashekaraiah et al., 1992), but as compared with other metals it does not
seem to act directly on the production of ROS via Fenton and/or Haber Weiss
reactions; Salin, 1988). Cd ions inhibit or sometimes stimulate the activity of several
antioxidant enzymes. Cd increased lipid peroxidation, increased lipoxygenase activity
and decreased the activity of the following antioxidative enzymes: superoxide
dismutase, catalase, ascorbate peroxidase, glutathione reductase and dehydroascorbate
reductase in Helianthus annuus leaves (Gallego et al., 1996).
1.3.4 Toxicity of cadmium
Cadmium toxicity symptoms are easily recognizable. Stunting and chlorosis are the
general symptoms in plants. Iron (Fe) deficiency may result in chlorosis and study
regarding to the interaction of toxic metals and Fe have been made for many years.
Chlorosis resulting from excess of Cd seems to be due to a direct or indirect interaction
with foliar iron. The Fe uptake in plant is suppressed by high Cd content in the growing
medium (Haghiri, 1973). Change in Fe:Zn ratios may be the cause of Cd induced
chlorosis in corn leaves (Root et al., 1975). Cd toxicity seemed to induce phosphorous
deficiency or reduce manganese transport problems (Godbold and Huttermann, 1985).
A single plant species or variety has been involved in much of physiological research
on the mechanism of Cd toxicity. Generally, Cd has been shown to restrict the uptake,
transport, use of several elements (Ca, Mg, P and k) and water by plants. The
interactions between metals are affected by many factors as reported earlier in
biological systems (Sharma et al., 1985).
13
1.3.5 Cytogenetic effects of cadmium
Cadmium; a non-essential heavy metal and powerful enzyme inhibitor (Lockwood,
1976) has been considered highly substantial pollutant because of its high toxicity and
solubility in water. When three species of green algae are exposed to CdCl 2, it result in
the formation of intra-mitochondrial granules containing Cd (Silverberg, 1976).
It was observed that the swelling, vacuolization and degeneration of mitochondria
implied significant cytotoxicity. Rosas et al., 1984 has reported that Cd inhibited cell
proliferation and when Eichhornia crassipes was exposed to Cd solution, it showed
low mitotic index. In onions, chromosomal aberrations have been found (Avanzi,
1950), it has also been found in beans (Oehlkers, 1953), peas (Von Rosen, 1954) and
barley (Degreave, 1971). Earlier many workers have reported the cytotoxic action of
Cd in plants (Levan, 1945; Avanzi, 1950; Von Rosen, 1954; Jacobs et al., 1956; Hecht
and Bock, 1971; Siegel, 1977).
When plants were exposed to Cd for 24 hrs, Cd penetrated into the cells thus inducing
physiological and genetical damages especially at concentration of 1.5 to 10 mg/L.
They also stated that Cd inhibited cell division and altered the chromosomes. The
inhibition of cell proliferation which had been shown by low mitotic index was directly
proportional to the concentration and time of exposure. The cytotoxic action of
Cadmium has been confirmed by the induction of pycnosis in root cells. The same
effect has also been reported in protoplast of Nicotiana tabacum (Siegel, 1977).
1.3.6 Differential cadmium tolerance in plants
The interest of plant ecologists, physiologists and evolutionary biologists has been
attracted by the phenomena of heavy metal tolerance in plants (Baker, 1987). The
harmful effects of excessive exposure to heavy metal ions has been reduce by the
development of metal tolerance (Tyler et al., 1989). The restricted uptake of these
elements by the roots does not signify the tolerance of vascular plants to the heavy
metals. It means that tolerant plants having high capacity of immobilizing heavy metal
ions in their root tissues have potency to renew the most active parts of their below
ground biomass often than nontolerant plants growing in normal soils. The tolerance to
excess cadmium in growth medium varies widely with plant species and varieties
(Hertstein and Jager, 1986).
14
These differences are genetically controlled in several species. The valuable tools for
studying the physiological mechanism of toxicity or tolerance are closely related
genotypes. Various authors had reported the inter-population variation of the plant
species tolerant to Cadmium (Bradshaw, 1984; Symeonidis et al., 1985). Element
present in excess could obstruct with metabolism through competition for uptake,
inactivation of enzymes, displacement of essential elements from functional sites or
alteration of the structure of water.
Modification of membrane structure and function is affected by many of these.
Debates regarding to the physiological mechanism of Cd toxicity or tolerance are
carried out, this mechanism may vary with plant species and varieties which are
controlled by different genes through biochemical pathways. It is cleared that Cd
tolerant plants must be able to prevent absorption of excess Cd or detoxify Cd after it
has been absorbed. A large number of physiological factors are associated with
differential Cd tolerance e.g. pH and other factors. In agreement with Antonovics et al.,
1971 and Woolhouse, 1983 it may be assumed that with in each population that are
related to the contamination of their original habitat, there exist different specific
mechanisms of tolerance which are related to contamination of their original habitat.
Hertstein and Jager (1986) showed that a principal mechanism for Cd tolerance in
tolerant populations of Agrostis tenuis and Arrhenatherum elatius was linked after the
protection of shoots from excess cadmium. They have also reported the changes in the
enzyme activities like peroxidase and acid phosphate to show the metabolic responses
of plants to stress conditions. Ernst, 1980 stated that the activity of acid phosphatase
and peroxidase increased in Cd tolerant plants. Foy et al., (1978) and Jager et al.,
(1983) described that in plants, roots and shoots had different concentration of Cd.
Metal tolerant plants showing a lessen accumulation of the respective metal in shoots
have been found to accumulate higher amounts in roots as equated to non-tolerant ones
of the same species (Baker, 1984; Coughtrey and Martin, 1979; Woolhouse, 1983). It
had been reported that high concentration of Cd accumulate in different species of
aquatic macro and micro flora (Kumada et al., 1973; Stokes et al., 1973; Ravera,
1984).
15
1.3.7 Phytotoxicity of cadmium and their interactions with nutrients
In ideal conditions, phytotoxicity cause by metals would result in some characteristic
symptoms that would let its diagnosis; moreover symptoms would be visible before
significant economic or ecological damages occurred (Hewitt, 1966). The common
symptoms are stunting and chlorosis. The chlorosis may be result from Fe deficiency
and interactions of toxic metals and Fe have been studied for many years. The direct
and indirect interaction of Cd with foliar Fe content with excess of Cd may result in
chlorosis. When soils or solutions had contained high Cd, the analysis of chlorotic
young foliage showed low foliar Fe (Haghiri, 1973; Garty et al., 1992). Chancy et al.,
described that Cd caused chlorosis that has been observed at low pH or neutral soils
and low availability of iron.
Higher concentration of iron has been found in symptoms bearing leaves than in
normal leaves (Root et al., 1975; White, 1976). Changes in Fe and Zn ratios may result
in Cd- induced chlorosis in corn leaves (Root et al., 1975). In case of bean plant,
curling of stems has been induced by Cd (Imai and Siegel, 1973). Decrease in Ca
concentration is found to reduce Cd absorption in plant (Christensen, 1984).
1.4 Zinc
Zinc is another significant heavy metal which is known to cause abiotic stress in plant.
Zinc, essential micronutrient is required by plant for normal growth and development
but excess of induce toxicity in plant. The elevated Zinc is found in contaminated soil
and it may result in phytotoxicity and inhibit the plant metabolic functions thus
resulting in growth retardation and senescence (Kumchai et al., 2013).
1.4.1 Sources of zinc
Mining, waste disposal, electroplating and smelting are the most important sources of
zinc pollution in environment. As zinc is the essential micronutrient so it plays vital
role as a cofactor in numerous enzymatic reactions involved in protein synthesis,
carbohydrates, nucleic acid and lipid metabolism. Zn, if present in excess may have
negative effect on plants which includes seed germination, plant growth inhibition,
changes in root development loss of membrane integrity, cell death. The Zn toxicity
mechanism is not completely understood, competition for catalytic sites or transporters,
16
evidence for Zn induced micronutrient deficiency or induction of oxidative stress has
been provided. Non redox active heavy metals like Zn because of their ability to bind
strongly to oxygen, nitrogen or Sulphur atoms may result in oxidative stress by
blocking essential functional groups in biomolecules thus inactivating enzymes by
binding to their cysteine residues or by replacing other essential metal ions in their
catalytic sites. Reactive oxygen species (ROS) such as superoxide anions (O2.-),
hydrogen peroxide (H2O2) and hydroxyl radicals (-OH) are commonly produced.
Macromolecules are damaged by high level of ROS. Therefore ROS level is needed to
be strictly controlled by complex mechanism in plants which include several enzymes
such as ascorbate peroxidase(APX), glutathione reductase(GR), catalase(CAT),
superoxide dismutase(SOD), and non-enzymatic soluble antioxidants such as
glutathione and ascorbate (Feigl et al., 2015). Zinc has a extensive range of functions
in plants and it is required for protein synthesis, gene regulation, structure and integrity
of bio-membranes, the protection of cells from oxidative damage (Bell and Dell, 2008).
During zinc deficiency, protein synthesis is lowered because of low levels of RNA, as
zinc plays an essential role in RNA polymerase. The role of zinc portions in DNA
transcription and gene relation also displays the importance of zinc in protein synthesis.
On account of the importance of zinc in protein synthesis, it marks in high zinc
requirements in meristematic tissues of plants (Bell and Dell, 2008).
Photosynthesis is reduced as the result of zinc deficiency, but the precise cause is
unknown. In plants for example maize, the zinc dependent enzyme carbonic anhydrase
is essential for photosynthesis to provide HCO3- as a substrate for phosphoenal
pyruvate carboxylase. In structure and function of bio-membranes of plants zinc plays
important role. Zinc, a micronutrient also plays an important role in the generation and
detoxification of reactive oxygen species (Cakmak, 2000). Some of the symptoms of
zinc deficiency in plants are produced by the oxidative degradation of the growth
hormone, auxin (Bell and Dell, 2008).
1.4.2 Uptake, translocation and functions of zinc in plants
The uptake of zinc by plant root arises predominantly through the absorption of Zn2+
from the soil solution (Alloway, 2004). Regarding to the uptake of Zn whether it is
active or passive, there exists considerable disagreement in the literature (Mengel and
17
Kirkby, 1987). Kochian (1993) proposed that the process of transport of zinc during
uptake is thermodynamically passive as it takes place across the plasma membrane
towards a large negative electrical potential. In grasses, the non-protein amino acid
called phytosiderophores form a complex with zinc and transfer it to the outer face of
the root cell plasma membrane. These phytosiderophores are released from the roots
during zinc deficiency (Kochian, 1993). Yet, the uptake of zinc is inhibit by other
metal cations which include Cu2+, Fe2+ and Mn2+ possibly because of competition for
the same carrier site in the casparian bands or plasmalemma. The antagonistic effect is
especially predominant with Cu2+ and Fe2+ (Havlin et al., 1999). Low temperature and
metabolic inhibitors also reduce the Zinc uptake (Bowen, 1969).
The form zinc which is translocated from the roots to the upper part of the plant is
unknown (Mengel and Kirkby, 1987). Tiffen (1967) stated that zinc is slightly cathodic
in tomato exudates and established that it is not translocated as citrate, as zinc citrate
complexes are anodic. The zinc translocation in plants is not great (Mengel and Kirkby,
1987) and when the zinc supply is great, zinc stores in root tissue. Zinc becomes very
immobile in older leaves (Rinnie and Langston, 1960). In zinc deficient plants, the rate
of transport to younger tissues is particularly inhibited (Lonergan, 1975).
There is little knowledge about the mechanisms by which zinc is translocated from the
vegetative parts of plants to their seed in the reproductive phase (Martens &
Westermann, 1991). However, increased in zinc levels in grain of maize and wheat
takes place either by soil or foliar applications of zinc (Yilmaz et al., 1997; Cakmak,
2008; Wang et al., 2012; Velu et al., 2014).
The micronutrient zinc has an vital function in the enzyme systems of plants. In some
enzyme systems, Zinc as Zn2+ resembles Mn2+ and Mg2+,that brings the binding and
conformation between enzyme and substrate (Mengel and Kirkby, 1987). Up till now,
the only authentic enzyme specifically activated by Zn2+ was carbonic anhydrase. This
enzyme stimulates hydrolysis and hydration reactions involving carbonyl groups
(Sandmann and Goger, 1983).
Alcohol dehydrogenase, superoxide dismutase and RNA polymerase are the other
important enzymes containing zinc (Vallee and Wacker, 1970). Zinc is thoroughly
involved in the nitrogen metabolism of plants. Protein synthesis and protein levels are
18
severely reduced and amino acids and amides are accumulated, during zinc deficiency
in plants (Mengel and Kirkby, 1987).
In the interveinal areas of the leaf, plants suffering from zinc deficiency frequently
show chlorosis. These areas are mostly pale green, yellow or white. Chlorotic bands
are formed on either side of the midrib of the leaf, in maize (Mengel and Kirkby,
1987). The inhibition of RNA synthesis is directly related to Zn deficiency. Prevention
of the normal development of chloroplast grana occur and vacuoles are developed in
them (Thomson and Weier, 1962). Short internodes and chlorotic areas in older leaves
characterizes the zinc deficiency (Mengel and Kirkby, 1987) which result in reduction
of crop yields.
Many researchers stated that high levels of soil phosphate are one of the most common
causes of zinc deficiency in crops (Alloway, 2004). The plant uptake of zinc decreases
sharply due to the growth enrichment from high phosphorus levels. Yet, by a high
phosphorus supply, extractable zinc in soil is either not at all or slightly decreased
(Marschner, 1993). Phosphorus applies an inhibiting effect on the absorption of zinc by
roots and the translocation of zinc from roots to shoots (Alloway, 2004). Four possible
mechanisms can explain the reduce uptake of zinc by plant roots:
i. High concentration of phosphorus suppresses the infection of roots with

vesicular arbuscular mycorrhizae
ii. Zinc absorption from the soluble fraction may be inhibited by cations added
with phosphate salts
iii. Zinc absorption may be inhibited by hydrogen ions generated by phosphate
salts and
iv. The adsorption of zinc onto soil constituents is enhanced by phosphorus
(Alloway, 2004).
Several possible mechanisms can explain the reduced translocation of zinc by
phosphorus in plants which include:
i. The inhibition of translocation of zinc from roots to shoots.
ii. Reduction in the amount of soluble zinc.
iii. Binding of zinc by phosphorus containing phytate.
19
iv. Leakage of phosphorus from membranes (Loneragan and Webb, 1993).
It is often proposed that the physiological availability of zinc in plant tissue is affected
by phosphate (Mengel and Kirkby, 1987). The zinc status of crops is affected by
nitrogen therefore promoting plant growth and changing the pH of the root
environment. Nitrogen is one of the major limiting factor influencing plant growth, in
most soils. Crops do not respond to zinc alone but often respond to zinc and nitrogen
application together. Different nitrogen fertilizers influence zinc availability as they
differ in their influence on soil pH. Several macronutrients such as calcium,
magnesium, potassium and sodium are known to inhibit zinc absorption in solution
culture experiments but in soil these nutrients main effect seems to be through their
influence on soil pH. With low levels of calcium it was found that potassium and
magnesium inhibit the absorption of zinc but the effect disappeared with the increase of
the calcium concentration (Alloway, 2004).
The fundamental interaction between zinc and other micronutrients is those with
copper, iron, manganese and boron. Through the competitive inhibition of absorption,
an interaction between zinc and copper occur. This is because of the sharing of the
same site for root absorption. The redistribution of zinc in plants is affected by copper.
Increased zinc uptake by plants and hence the zinc concentration in shoots can be
considerably increased, under conditions of iron deficiency. An increasing iron
concentration in the shoots of plants is shown by zinc deficiency. It has been found that
the absorption of zinc by rice in flooded soils may be inhibited by high levels of
manganese in combination with high iron and enhance zinc deficiency. These plants
absorb high concentrations of boron due to the impaired membrane function in the
roots of zinc deficient plants (Alloway, 2004).
Climate conditions may also be related to the occurrence of zinc deficiency symptoms.
Zinc deficiency may occur in areas with cool and wet spring seasons (Lucas and
Knezek, 1972). This can be explained on the basis of restricted root development in
cool soils and the inhibition of microbiological activity that directly effects the release
of zinc from organic material.
Zinc toxicity is rarely run into practice. The major problems are found near deposits of
zinc ore, ore mines and lead or zinc smelting plants, where the stack gasses contain
20
considerable amounts of zinc (Bergmann, 1992). The zinc content of the soil can also
be raised by rainwater collected and stored in galvanized roofs and gutters, if used for
watering. Zn level can also be raised by heavy applications of slurry from piggeries,
repeated application of sewage sludge and composted domestic wastes to improve soil
structure. High zinc levels have also been reported in soils near roads. By raising the
pH, soils with high levels of zinc can be cured. To avoid toxicity in plants, liming is the
most economical approach.
Total zinc concentration in soil usually falls in the range of 10 - 300 mg kg-1, with
concentrations above 150 mg kg-1 regarded as high and this may cause reduced plant
growth (Landon, 1991). Levels of 150 to 200 mg kg-1 in dry matter of plant tissue are
considered as toxic as stated by Sauerbeck (1982).
1.4.3 Zinc toxicity
Oxidative stress can be caused by zinc (a Non-redox active heavy metal) which blocks
essential functional groups in biomolecules because of their ability to bind strongly to
oxygen, nitrogen or sulphur atoms, thus resulting in the inactivation of enzymes by
binding to their cysteine residues (Nieboer and Richardson, 1980) or by replacing other
essential metal ions in their catalytic sites (Schu¨tzendu¨belandPolle, 2002). Reactive
oxygen species (ROS) such as superoxide anion, hydrogen peroxide (H2O2) and
hydroxyl radicals (-OH), are generated during oxidative stress. ROS concentrations
need to be strictly controlled by complex mechanisms in plants as the high level of
ROS damages macromolecules (Apel and Hirt, 2004) which comprises of several
enzymes such as ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase
(GR; EC 1.6.4.2), catalase (CAT; EC 1.11.1.6) and superoxide dismutase (SOD; EC
1.1.5.1.1), and non-enzymatic soluble antioxidants such as glutathione and ascorbate,
among others. (Feigl et al., 2015).
1.5 Antioxidants
The production of reactive oxygen species is the consequence of uncontrolled aerobic
metabolism. Free radicals like superoxide anions (O2-) and hydroxyl radicals (•OH)
plus non radical like hydrogen peroxide (H2O2) and singlet oxygen (1O2) are included
in reactive oxygen species. The production of highly reactive ROS takes place by
21
stepwise reduction of molecular oxygen with the help of high energy exposure or
electron transfer chain. ROS are formed in plants either by unavoidable leakage of
electron on to the oxygen from electron transport activities of the chloroplast,
mitochondria and plasma membrane or as a byproduct of various metabolic pathways
localized in different cellular compartments. Different environmental stresses like
drought, salinity, chilling, metal toxicity, UV-B radiation and pathogens attack direct
the increase production of ROS in plants because of the disturbance of cellular
homeostasis. High concentration of ROS is harmful for living organisms. When the
level of ROS exceeds defense mechanism, a cell is said to be in state of “oxidative
stress”. During environmental stress, the increase production of ROS can create a threat
to cells by causing peroxidation of lipids, oxidation of proteins, damage to nucleic
acids, enzyme inhibition, activation of programmed cell death(PCD) thus leading to
cell death (Sharma et al., 2012).
ROS are considered second messenger in the variety of cellular processes such as
tolerance to environmental stresses, regardless of their destructive activity (Desikan et
al., 2001; Neill, 2002; Yan et al., 2007). The function of ROS whether it would act as a
damaging or signaling molecule depends on a delicate equilibrium between ROS
production and scavenging. As the ROS has multifunctional role so it is necessary for
the cells to control the level of ROS tightly, in order to avoid any oxidative injury and
not to eradicate them completely.
An efficient antioxidant system comprising of non enzymic as well as enzymic
antioxidants will help to achieve the scavenging or detoxification of excess of ROS
(Noctor and Foyer, 1998). Superoxide dismutase (SOD), catalase (CAT), guaiacol
peroxidase (GPX), enzymes of ascorbate glutathione (AsA-GSH) cycle such as
ascorbate peroxidase (APX), mono de hydro ascorbate reductase (MDHAR), de hydro
ascorbate reductase (DHAR) and glutathione reductase are included in enzymic
antioxidants (Noctor and Foyer, 1998). On the other hand non enzymic antioxidants
with in cell include Ascorbate (AsA), glutathione (GSH), carotenoids, tocopherols and
phenolic.
It has been reported by various scientists that increased activities of enzymes of
antioxidant defense system in plant is associated with the combating of the oxidative
22
stress induced by various environmental stresses. Up holding of high antioxidant
capacity to scavenge the toxic ROS has been associated with the increased tolerance of
plant to these environmental stresses (Zaefyzadeh et al., 2009; Chen et al., 2010).
Remarked progress has been made by developing transgenic lines with altered levels of
antioxidants for improving stress induced by oxidative stress tolerance in crop plants
(Allen et al., 1997; Faize et al., 2011).
For developing transgenic plants with enhanced tolerance to multiple environmental
stresses, it has been assumed that simultaneous expression of multiple antioxidant
enzymes are more valuable than single or double expression (Lee et al., 2007).
1.5.1 Reactive oxygen species, sites of production and their effects
ROS are described as the group of free radicals, reactive molecules and ions which
have been derived from oxygen. Approximately 1% of oxygen consumed by plants is
side tracked to produce ROS in various sub cellular loci like chloroplast, mitochondria
and peroxisomes (Asada and Takahashi, 1987). Depending on their concentration in
plants, ROS are recognized for playing dual role as both deleterious and beneficial
species. ROS cause damage to biomolecules at high concentration, on the other hand at
low or moderate concentration it acts as second messenger in intracellular signaling
cascades which intervene several responses in cell of plants.
23
Figure 1: Sites of production of reactive oxygen species (ROS) in plants. ROS are
produced at several locations in the cell-like chloroplast, mitochondria, plasma
membrane, peroxisomes, apoplast, endoplasmic reticulum, and cell wall. Adapted from
Sharma et al., 2012.
Effects of ROS
The following diagrams show effects of ROS:
24
Figure 2: Reactive oxygen species (ROS) as second messengers in several plant
hormone responses, including stomatal closure, root gravitropism, seed germination,
lignin biosynthesis, programmed cell death, hypersensitive responses, and osmotic
stress. Adapted from Sharma et al., 2012.
25
Figure 3: Reactive oxygen species (ROS) induced oxidative damage to lipids,
proteins, and DNA. Adapted from Sharma et al., 2012.
1.5.2 Types of ROS

The common types of ROS are 1O2, O2.−, H2O2, and •OH. O2 itself is harmless molecule
as it has two unpaired electrons in its ground state with parallel spin which make it
paramagnetic and therefore it participate in reactions with organic molecules until it is
activated (Apel and Hirt., 2004). Two different mechanisms are involved for activation
of O2:
i. Absorption of energy to reverse the spin on one of unpaired electron
ii. Stepwise monovalent reduction.
In first case 1O2 is formed on the other hand in latter case O2 is sequentially reduced to
O2•−, H2O2, and •OH. Electrons have parallel spin in bi-radical form of oxygen. The
spin of one of its unpaired electron is reversed by absorption of sufficient energy
26
leading to formation of singlet state in which two electrons have opposite spin. Spin
restriction is overcome by this activation and 1O2 participation occur in reaction
involving transfer of two electrons that is divalent reduction (Apel and Hirt., 2004). In
the presence of light, production of highly reactive oxygen can take place through the
formation of triplet chlorophyll in the antenna system and in the reaction center of
photosystem II (Krieger-Liszkay, 2004). Formation of chlorophyll triplet state occur as
the result of insufficient energy dissipation during photosynthesis, in antenna, on the
other hand in the reaction center its formation occur by charge recombination of light
induced charge pair (Krieger-Liszkay, 2004).
Highly destructive ROS 1O2 can be formed as the result of reaction of Chlorophyll
triplet state with 3O2.
Chl 3Chl
3Chl+ 3O2 Chl+ 1O2
The formation of 1O2 is favored during environmental stresses such as salinity and
drought due to limited CO2 availability because of closure of stomata (Hatz et al.,
2007; Hackbarth et al., 2010). Formation of O2.-, H2O2 and .OH occur as result of
stepwise monovalent reduction of O2. On the other hand energy transfer to O2 leads to
formation of 1O2. O2.- is easily dismutated to H2O2 either nonenzymatically or by
Superoxide dismutase (SOD) catalyzed reaction to H2O2. H2O2 is converted to H2O by
catalase (CAT), guaiacol peroxidase( GPX) and ascorbate peroxidase (APX).
27
Figure 4: Schematic presentation of generation of reactive oxygen species in plants.
Activation of O2 occurs by two different mechanisms. Stepwise monovalent reduction
of O2 leads to formation of O2•−, H 2O2, and •OH, whereas energy transfer to O2 leads
to formation of 1O2. O2•− is easily dismutated to H2O2 either non enzymatically or by
superoxide dismutase (SOD) catalyzed reaction to H2O2. H2O2 is converted to H2O by
catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX). Adapted
from Sharma et al., 2012.
1.5.3 Antioxidative Defense System in Plants

Plants possess complex antioxidative defense system to scavenge ROS. Specific ROS
producing and scavenging systems are found in plant cells with in different organelles
such as chloroplast, mitochondria and peroxisomes. From different cellular
compartments ROS scavenging pathways are coordinated (Pang and Wang, 2008).
Potential toxic metabolites are generated at a low level and there is appropriate balance
between production and quenching of ROS, under normal conditions. A number of
28
adverse environmental factors may disturb the balance between production and
quenching of ROS thus giving rise to rapid increase in intracellular ROS levels,
resulting in oxidative damage to lipids, proteins and nucleic acids. Higher plants have
raised the level of endogenous antioxidant defense to avoid the oxidative damage
(Sharma et al., 2010).
1.5.4 Non enzymatic components of antioxidative defense system

The major cellular redox buffers ascorbate (AsA) and glutathione (γ-glutamyl-
cysteinyl-glycine, GSH) as well as tocopherol, carotenoids and phenolic compounds
are included in non-enzymatic components of antioxidative defense system. In addition
to crucial role in defense, they interact with numerous cellular components and as
enzyme cofactors these antioxidants influence plant growth and development by
modulating processes from mitosis, cell elongation to senescence and cell death (Pinto
and Gara, 2004). The mutants are shown to be hypersensitive to stress with decreased
non enzymic antioxidant contents (Gao and Zhang, 2008; Semchuk et al., 2009).
1.5.5 Enzymatic components

Several antioxidant enzymes such as superoxide dismutase(SOD), catalase(CAT),
guaiacol peroxidase(G;PX), enzymes of ascorbate glutathione(AsA-GSH) cycle,
ascorbate peroxidase(APX), monodehydro ascorbate reductase(MDHAR), dehydro
ascorbate reductase(DHAR) and glutathione reductase(GR) comprise the enzymatic
components of antioxidative defense system (Noctor and Foyer, 1998). These enzymes
respond when cells are exposed to oxidative stress and operate in different subcellular
compartments.
29
1.5.6 Superoxide Dismutase (SOD)
Central role in defense against oxidative stress is played by Superoxide dismutase

(SOD, EC 1.15.1.1) in all aerobic organisms (Scandalios, 1993). The enzyme SOD
catalyzes the dismutation of O2.- to O2 and H2O2, enzyme SOD belongs to the group of
metalloenzymes. It is existing in majority of subcellular compartments that produce
activated oxygen. In plants, three isozymes of SOD namely copper/zinc SOD (Cu/Zn-
SOD), manganese SOD (MnSOD), and iron SOD (FeSOD) have been reported
(Fridovich, 1989; Racchi et al., 2001).
All the types of SOD are nuclear encoded and targeted to their respective subcellular
compartments by amino terminal targeting sequence. Cu/Zn-SOD is found sin three iso
forms which are present in the cytosol, chloroplast, peroxisome and mitochondria
(Bowler et al., 1992). MnSOD is present in mitochondria. Moreover, FeSOD is present
in chloroplast (Jackson et al., 1978). Eukaryotic Cu/Zn-SOD is cyanide sensitive and
found as dimer. On the other hand, the remaining two (Mn-SOD and Fe-SOD) are
cyanide insensitive and may be found as dimer or tetramers (Scandalios, 1993; Del Rio
et al., 1998). In plants exposed to various environmental stresses (like drought and
metal toxicity), it is reported that SOD activity has been increased (Sharma and R.S.
Dubey, 2005; Mishra et al., 2011). It has been notice that increase activity of plant is
often correlated with increase tolerance of plant against environmental stresses.
Moreover, it was suggested that SOD can be used as an indirect selection criterion for
screening drought-resistant plant materials (Zaefyzadeh et al., 2009). However in
plants, overproduction of SOD has been reported to result in improved oxidative stress
tolerance (Gupta, 1993).
30
1.5.7 Catalase (CAT)
Catalase (CAT, EC 1.11.1.6) was the first enzyme to be discovered and characterized.
It is universal tetrameric heme-containing enzyme that catalyzes the dismutation of two
molecules of H2O2 into water and oxygen. It is highly specific for H2O2 and has weak
activity against organic peroxidase. Several types of H2O2 degrading enzymes are
found in plants, but CAT are unique as cellular reducing equivalent is not required by
them. CAT cut off a very fast turnover rate but has a much lower affinity for H2O2 as
compared to APX. The major sites of H2O2 production are peroxisomes. Basically,
CAT scavenges H2O2 produced in these organelles during photorespiratory oxidation, ,
β-oxidation of fatty acids, and other enzyme systems such as XOD coupled to SOD
(Luis et al., 2006 ; Scandalios et al., 1997 ; Corpas et al., 2008). It has been reported
that CAT is present inn cytosol, chloroplast and mitochondria but the presence of
significant Cat activity in these is well is less established (Mhamdi et al., 2010). Till
now, all angiosperm species studied contain three CAT genes. Classification of CAT
based on expression profile of tobacco genes has been proposed. Class I CATs are
being expressed in photosynthetic tissues and are regulated by light. Class II CATs are
being expressed at high level in vascular tissues. Class III CATs are found abundantly
in seeds and young seedlings (Willekens et al., 1995).
H2O2 has been involved in many stress conditions. When the cells are under energy
stress, they start generating H2O2 through catabolic processes rapidly. In an energy
efficient manner, H2O2 is degraded by CAT (Mallick and Mohn, 2000). Enhancement
or degradation of CAT activity is caused by environmental stresses depending on the
intensity, duration and type of stress. In general, it has been observed that stresses that
reduce rate of protein turnover also reduce CAT activity. Hence, Stress analysis
revealed increased susceptibility of CAT-deficient plants to paraquat, salt and ozone
but not to chilling. CAT activity showed accumulation of GSSG and 4-fold decrease in
AsA, in transgenic tobacco plants having 10% wild-type thus indicating CAT is critical
for maintaining the redox balance during oxidative stress (Willekens et al., 1997). Over
31
expression of a CAT gene from Brassica juncea introduced into tobacco improved its
tolerance to Cd induced oxidative stress (Guan et al., 2009).
1.5.8 Guaiacol Peroxidase (GPX)
Guaiacol peroxidase (GPX, EC 1.11.1.7) is a heme containing protein and if at all

possible oxidizes aromatic electron donor such as guaiacol and pyragallol at the cost of
H2O2. It is usually found in animals, plants and microbes. Moreover, these enzymes
have four conserved disulfide bridges and contain two structural Ca2+ ions (Schuller et
al., 1996). Many isoenzymes of GPX are localized in vacuoles, cell wall and cytosol of
plant tissue (Asada, 1992). Many important biosynthetic processes including
lignification of cell wall degradation of IAA, biosynthesis of ethylene, wound healing
and defense against biotic and abiotic stresses are associated with GPX (Kobayashi et
al., 1996). GPXs are globally accepted as stress enzyme. Under stressed conditions,
GPX can function as Effective quencher of reactive intermediary forms of O2 and
peroxy radicals (Vangronsveld and Clijsters, 1994). The activity of GPX has shown to
be increased by various stressful conditions of environment. The increased activity of
GPX is correlated to oxidative reactions under metal toxicity conditions and suggested
its potential as biomarker for subslethal metal toxicity in plants (Radotic et al., 2000).
Tayefi-Nasrabadi and coworkers have determined that greater protection of salt tolerant
safflower plants from salt-induced oxidative damage results, at least in part, through
the increase of GPX activity, catalytic efficiency and induction of specific isoenzymes
compared to salt-sensitive cultivar (Tayefi-Nasrabadi et al., 2011).
1.5.9 Enzymes of Ascorbate-Glutathione cycle
The change in ratio of AsA to DHA and GSH to GSSG is critical for the cell, inorder to
sense oxidative stress and respond. The AsA-GSH cycle is also reffered to as halliwell-
Asada pathway is the recycling pathway of AsA and GSH regeneration which therefore
also detoxify H2O2. Successive oxidation and reduction of AsA, GSH and NADPH
32
catalyzed by the enzymes APX, MDHAR, DHAR and GR are involved in AsA-GSH
cycle. The presence of AsA-GSH cycle is found at least at four different subcellular
locations including cytosol, chloroplast, mitochondria and peroxisomes (Jimenez et al.,
1997). The important role in combating oxidative stress induced by environmental
stresses is played by AsA-GSH cycle (Pallanca and Smirnoff, 2000).
1.5.10 Ascorbate Peroxidase (APX)
The central component of AsA-GSH cycle is Ascorbate peroxidase (APX, EC 1.1.11.1)

and it plays an important role in control of intracellular ROS levels. Two molecules of
AsA are used by APX to reduce H2O2 to water with a simultaneous generation of two
molecules of MDHA. APX belongs to Class I super family of heme peroxidases and
redox signals plus H2O2 regulate it (Welinder, 1992; Patterson and Poulos, 1995). Five
chemically and enzymatically distinct isoenzymes of APX which include cytosolic,
stromal, thylakoidal, mitochondrial and peroxxisomal isoforms have been found at
different subcellular localization in higher plants based on amino acid sequences
(Madhusudhan et al., 2003). H2O2 produce with in organelles is scavenged by APX
present in organelles. On the other hand, H2O2 produced in the cytosol, apoplast or
that diffused from organelles is eliminated by cytosolic APX (Mittler and Zilinskas,
1992). As a electron donor, the chloroplastic and cytosolic APX isoforms are specific
for AsA and as compared to chloroplastic isoenzymes including stromal and thylakoid
bound enzymes, the cytosolic isoenzymes are less sensitive to depletion of AsA
(Sharma and Dubey, 2004; Ishikawa et al., 1998). One of the most widely distributed
antioxidant enzymes found in plant cell is APX and isoforms of APX have much
higher affinity for H2O2 than CAT, thus making APXs efficient scavengers of H2O2
under stress (Wang et al., 1999). In response to abiotic stresses such as drought,
salinity, chilling, metal toxicity and UV irradiation enhanced activity of APX have
been reported by many workers (Boo and Jung, 1999). Over expression of a cytosolic
APX-gene derived from pea improved oxidative injury induced by chilling and salt
stress (Wang et al., 2005). Similarly, tolerance to oxidative stress have been increased
33
by over expression of the tAPX gene in either tobacco or in Arabidopsis (Yabuta et al.,
2002).
1.5.11 Monodehydroascorbate Reductase (MDHAR)
MDHA radical has a short life time, it catalyzed reaction and is produced in APX. If it
is not rapidly reduced, disproportionates to AsA and DHA (Ushimaru et al., 1997).
Monodehydroascorbatereductase (MDHAR, EC 1.6.5.4) is a FAD enzyme. It catalyzes
the regeneration of AsA from the MDHA radical using NAD(P)H as the electron donor
(Hossain and Asada, 1985). Organic radical (MDA) is used as a substrate by this only
known enzymes and MDA is also capable of reducing phenoxyl radicals which are
generated by horseradish peroxidase with H2O2 (Sakihama et al., 2000). In plants,
MDHAR activity is widespread. It has been reported that the isoenzymes of MDHAR
are present in several cellular compartments such as chloroplast, cytosol, mitochondria
and peroxisomes (Hossain et al., 1984; Dalton et al., 1993). The two physiological
functions of MDHAR have been found in chloroplast:
i. The regeneration of AsA from MDHAR
ii. The mediation of the photoreduction of dioxygen to O2 when the substrate
MDHA is absent (Miyake et al., 1998).
Hence, the characterization of membrane polypeptides from pea leaf peroxisomes also
revealed MDHAR to be involved in O2.- generation. The increased activity of MDHAR
in plants is subjected to environmental stresses as shown by several studies. Increased
tolerance to salt and polyethylene glycol stresses is consulted with overexpression of
Arabidopsis MDHAR gene in tobacco (Eltayeb et al., 2007). Overexpression of
intransgenic is caused by tomato chloroplastic MDHAR, improved tolerance to
temperature-Arabdopsis and methyl viologen-mediated oxidative stresses (Li et al.,
2010).
34
1.5.12 Dehydroascorbate Reductase (DHAR)
Dehydroascorbate reductase (DHAR, EC 1.8.5.1), it catalyzes the reduction of DHA to

AsA using GSH as the reducing substrate and therefore DHAR plays an important role
in maintaining AsA in its reduced form (Ushimaru et al., 1997). Mostly, enzymic and
non enzymic regeneration of AsA occur directly from MDHA though some DHA is
also produced when AsA is oxidized in leaves and other tissues.
DHA can either be hydrolyzed to 2, 3-diketogluonic acid, the reaction is irreversible or
recycled to AsA by DHAR. DHA is short-lived chemical. AsA content is reported to
be increased by over expression of DHAR in tobacco leaves, maize an tobacco, thus
suggesting that vital role is played by DHAR in determining the pool size of AsA
(Chen et al., 2003 ; Qin et al., 2011). DHAR, monomeric thiol enzyme mostly found in
dry seeds, roots and etiolated as well as green shoots. Purification of DHAR occur
from chloroplast and non-chloroplast sources of several plant species including spinach
leaves and potato tuber (Dipierro and Borraccino, 1991). The activity of DHAR in
plants is increased by environmental stresses such as drought, metal toxicity and
chilling (Yoshida et al., 2006).
In L. japonicas, the consistent upregulation of gene encoding cytosolic DHAR was
found, which was more tolerant to salt stress than other legumes. This upregulation of
DHAR was linked to its role in AsA recycling in the apoplast (Rubio et al., 2009). The
higher tolerance to herbicide, drought and salt stresses was shown by transgenic potato
overexpressing Arabidopsis cytosolic AtDHAR1 (Eltayeb et al., 2011).
1.5.13 Glutathione Reductase (GR)
GSH is oxidized to GSSG while acting as an antioxidant by participating in enzymic as

well as non enzymic oxidation reduction cycles. GSH is oxidized in a reaction
catalyzed by DHAR in AsA-GSH cycle. Glutathione reductase (GR, EC 1.6.4.2) is a
NAD(P)H- dependent enzyme. It catalyzes the reduction of GSSG to GSH and
maintains high cellular GSH/GSSG ratio.GR is member of flavor-enzymes group and
possess essential disulfide group. Two steps are involved in catalytic mechanism:
35
(1) the flavin moiety is reduced by NADPH, the flavin is oxidize and a redox active
disulfide bridge is reduced to produce a thiolate anion and a cysteine.
(2) the reduction of GSSG via thiol disulfide interchange reactions (Ghisla and Massey,
1989).
If the reoxidation of reduced enzyme does not occur by GSSG than it may suffer a
reversible inactivation. GR is present in chloroplasts, cytosol, mitochondria and
peroxisomes and about 80% of GR activity in photosynthetic tissues is accounted for
by chloroplastic isoforms (Edwards et al., 1990). GSH and GR are involved in
detoxification of H2O2 which is generated by Mehler reaction with in chloroplast.
Under environmental stresses the increased activity of GR has been reported by various
authors. The correlation between the oxidative stress resistance and activity of GR has
been observed by Pastori and Trippi which suggested that oxidative stress caused by
paraquat or H2O2 could stimulate GR de novo synthesis, most likely at the level of
translation by preexisting mRNA (Pastori and Trippi, 1992). Increased susceptibility to
chilling stress has been shown by antisense-mediated depletion of tomato chloroplast
GR (Shu et al., 2011). Higher foliar AsA contents and improved tolerance to oxidative
stress has been resulted from overexpression of GR in N. tabacum and Populus plants
(Foyer et al., 1995; Aono et al., 1993). As the result of complexity of ROS
detoxification system, the capacity of pathway as a whole may or may not be changed
as a whole by overexpressing the one component of antioxidative defense system
(Tseng et al., 2008; Lee et al., 2009).
Overexpression of combinations of antioxidant enzymes in transgenic plants has
synergistic effect on stress tolerance as shown by several studies (Aono et al., 1995;
Kwon et al., 2002). Simultaneous expression of Cu/Zn-SOD and APX genes in tobacco
chloroplasts increased tolerance to methyl viologen (MV) stress compared to
expression of either of these genes alone as demonstrated by studies. By simultaneous
overexpression of genes of SOD and APX in the chloroplast, SOD and CAT in cytosol
and SOD and GR in the cytosol increased tolerance to multiple environmental stresses
(Lim et al., 2007; Kwak et al., 2009; Aono et al., 1995). Moreover, instantaneous
expression of multiple antioxidant enzymes such as Cu/Zn-SOD, APX and DHAR in
chloroplast has shown to be more effective than single or double expression for
36
developing transgenic plants with increased tolerance to multiple environmental
stresses (Lee et al., 2007). Hence, in order to gain tolerance to multiple environmental
stresses enhanced emphasis is now being given to produce transgenic plants
overexpressing multiple antioxidants.
1.6 Phytoremediation
Phytoremediation is defined as the use of green plants to remove pollutants from the
environment or render them harmless (Raskin et al., 1997). In order to tackle heavy
metal problems, the use of green plants to remediate a contaminated soil seems as an
attractive approach. Because of number of limitations, the traditional phytoremediation
techniques lack large scale background. Hyper-accumulator plant species that are
produced naturally are either slow growing produce low above the ground level or they
are not well adapted to variety of environmental conditions. Certain limitations are
faced by traditional phytoremediation approaches like,
i. Long time is required by them to remediate the contaminated soil.
ii. Due to low above ground biomass production, the photo-extraction ability of
hyper-accumulator plants is limited.
iii. A very small amount of metals is bioavailable and concentration of bioavailable
metals vary with pH, organic matter, competitive cations and calcareousness
etc.
iv. Can be applied to sites with low or moderate contamination.
v. Lacking knowledge about agronomy, breeding potential, insect pest and disease
spectrum.
vi. Food chain contamination may be resulted because of any mismanagement or
carelessness.
Researches are compelled to modify the traditional approaches of phytoremediation, in
order to minimize these limitations and to ensure large scale application of
phytoremediation (Sarwar et al., 2016).
The plant used in phytoremediation technique must have a considerable capacity of
metal absorption, its accumulation and strength to decrease the treatment time. Certain
plants absorb these toxic metals and help to clean up them from soils, these plants are
37
termed as hyper accumulators. These plants have been shown to be resistant to heavy
metals and are capable of accumulating them into their roots and leaves and
transporting these soil pollutants to high concentrations. Thus careful investigation of
the mechanism of tolerance of heavy metals at physiological as well as genetic level is
essential. The aim of study is to determine heavy metals induced toxicity and response
of antioxidant enzymes in different plants which includes Cannabis sativa, Conyza
canadensis, Parthenium hysterophorus and Rumex acetosa of both contaminated and
non-contaminated areas of Hattar industrial estate.
38
1.7 Statement of the Problem
Plants which are grown on soil contaminated with heavy metals are known to absorb
heavy metals in quantities that may adversely affect the plant growth and metabolism.
The comparison of the relative oxidative damage and antioxidant responses caused by
heavy metals stress in industrial area of Hattar has not been explored, yet.
1.8 Purpose of the Study

In most of the studies, the antioxidant efficiency under metal stress has documented in
the different parts mainly roots or shoots of a plant. Reports on changes in antioxidant
enzymes activities in roots, shoots and leaves of plants due to Lead, Cadmium and Zinc
of Hattar industrial zone are deficient. In this study, attempt has been made to study the
heavy metals Lead, Cadmium and Zinc induced toxicity and antioxidant enzymes
levels in different plants of Hattar industrial area.
1.9 Objectives
i. To analyze metals content of different plant species as well as their adhered
soil.
ii. To determine Antioxidant enzymes levels to elaborate different plant defense
mechanism in response to heavy metals induced toxicity.
39
Chapter 2
Materials & Methods
40
2.1 Sample collection
Plants and soil samples were collected from Hattar Industrial Estate (near volta battery
industries), Haripur on 17 December 2016. The different plants were randomly
collected. The adhered soil samples were also collected from 0-25 cm depth (Fig.5).
Figure 5: Site view and sample collection
41
2.2 Sample storage
The leaves, roots and stems of plants were segregated. All samples were kept in airtight
zipper bags, given specific codes and stored in freezer at -20°C.
2.3 Sample preparation
Each sample was divided into three parts and one part of sample was used for heavy
metals detection. Selected part of sample was placed in oven at 65°C for 48 hrs. The
moisture content of each sample was measured. Dried samples were grinded in to fine
particles.
2.4 Heavy metals analysis
The approach adopted was partly modified from that of Zheljazkov and Nielson (1996)
as illustrated in Fig. 6. 0.5g of dried samples were placed in conical flask and 5 ml of
concentrated nitric acid was added. The mixture was heated at magnetic stirrer hot
plate for one and half hours. Concentrated nitric acid was added to the sample (2.5 ml
was added at least three times) during heating and digestion occurred till the volume
was reduced to about 1 ml. A little distilled water was also added to wash the interior
walls of conical flask and to prevent the loss of sample. 2.5 ml of 1% nitric acid was
added to the sample, after cooling. Finally, solution was filtered with Whatmans filter
paper and sent to instrumental laboratory for analysis by atomic absorption
spectrometer.
Figure6: Different steps involved in heavy metals analysis
42
2.5 Extraction and antioxidant assays
It was done according to amended method of (Fu and Huang, 2001) as depicted in
Fig.6. Approximately 0.2 g of plant tissue was placed in freezer at -80°C for an hour.
The plant tissue was homogenized in pre cooled mortar with 5ml of 50mM/L
phosphate buffer saline (pH = 7.3). The homogenate was first placed in shaker for 30
mins and then centrifuged at 11000 rpm for 20min at 4°C. The supernatant was used
for the determination of antioxidant enzyme activities [superoxide dismutase (SOD),
peroxidase (POD) and catalase (CAT)].
For POD, it was done according to amended method of Zhang et al., (1995). 2.2 ml of
the reaction mixture containing 1.6ml of 100mM of phosphate buffer saline solution
(pH = 7.3), 200 µl of 40mM Hydrogen peroxide, 200 µl of 10% guaiacol and 200µl of
enzyme extract was used. The reaction started on the addition of enzyme extract. The
oxidation of guaiacol was measured at 470nm by increased in absorbance at 470nm.
After 15 minutes the solution colour changes from colorless to bright orange. The
enzyme activity was calculated by using the extinction coefficient 25.5mM-1cm-1 and
expressed in units i.e. mg/protein. One unit of enzyme was the amount necessary to
decompose 1 µ mol of substrate per minute.
CAT analysis done according to modified method of Beer and Sizer (1952). CAT
activity was measured by quantifying the decomposition of hydrogen peroxide. The
CAT analysis was performed with the 3 ml of reaction mixture containing 2 ml of 100
mM phosphate buffer saline, 500 µl of 40mM hydrogen peroxide and 500 µl of
enzyme extract. The reaction started with addition of enzyme extract and absorbance
was measured at 240 nm. After 10 minutes, sample color changed from colorless to
light yellow. The activity was calculated by using an extinction coefficient of 39.04
mM-1 cm-1. One unit of CAT activity was defined as the amount required for
decomposing 1 µmol of hydrogen peroxide/min/mg protein under assay conditions.
The activity of SOD was quantified by measuring its ability to inhibit the
photochemical reduction of nitroblue tetrazolium (NBT) (Beauchamp and Fridovich,
1971). For SOD, 1.7 ml of reaction mixture containing 500 µl of phosphate buffer
saline (pH= 7.3), 20mM of 200 µl of methionine, 2mM of 100 µl NBT, 0.2% of 200 µl
43
Triton X, 6mM of 100 µl riboflavin, 500 µ l of water and 100 ml of enzyme extract
was used. Then, the content in cuvette were illuminated to light for 15 minutes.
Enzyme extract kept in dark served as blank while the buffer with no enzyme extract
kept in light worked as control. The absorbance was measured at 560nm against blank
using UV- visible spectrophotometer. NBT reduction in light was measured in the
presence and absence of enzyme extract. The activity of SOD was calculated as the
absorbance of control minus the absorbance of sample, giving the total inhibition. One
unit of activity was the amount of enzyme required for 50% reduction in color and
expressed in units of the enzyme (mg/protein/h) (Fig.7).
GPOXs CATs SODs
Figure 7: Antioxidants extraction and assays
44
Chapter 3
Results
45
3.1 Analysis of lead in contaminated plants
The accumulation of lead with in plants as well as soils of contaminated area is shown
in Figure 8a-d. The level of lead concentration among leaves of different contaminated
plants revealed that its accumulation was significantly higher in RA (10.11mg/kg)
followed by CS (4.93mg/kg), CC (1.24mg/kg) and PH (0.96mg/kg). Maximum lead
accumulation was found in stem of RA (6.84mg/kg) whereas its minimum
accumulation occur in CC (0.34mg/kg), the CS (2.9mg/kg) and PH (0.75mg/kg) lies in
between of the two extreme values. While the roots of PH (10.08mg/kg) showed
increased level of lead uptake afterwards CS (8.51mg/kg), the lead found in roots of
CC (2.63mg/kg) and RA (0.17mg/kg) was relatively less. The accumulation of lead in
four plants of contaminated area followed the trend as RA>CS>PH>CC. The quantity
lead in soil of contaminated area ranged from 21.31mg/kg to 20.65mg/kg which
showed that lead contamination in soil of Hattar Industrial Estate was very high.
The level of lead accumulation in leaves, stem and roots of four the plants of control
area as well as their adhering soil is depicted in fig 9 a-d. The leaves of contaminated
CS possessed lead 27 times higher than its control plant leaves, stem contained 9.6
times higher whereas roots had 51.3 times higher value. In case of CC, the
contaminated leaves had 7 times elevated value whereas stem had 3.5 times and roots
had shown 2.5times higher addition of lead. In present study, the level of lead
contamination in PH leaves was found to be 6 times higher than that of its control. The
stem of contaminated PH possessed 16 times and roots had 96 times higher value as
compared to control. The lead accumulation in leaves of contaminated RA was 79.5
times amplified. Lead amount in stem of contaminated RA was elevated 87 times and
in roots it had shown 1.3 times increased. Except in RA and CC, in all others plant the
roots had amplified more lead accumulation. The RA and CC showed high level of
lead addition in leaves. Only in RA, stem of plant along with leaves had shown
intensified level of lead.
46
Figure 8 a-d: Concentration of Lead with in different plant organs of contaminated
plants. (a) Absorption of lead in CS: roots>leaves>stems (b) Absorption of lead in CC:
roots>leaves>stems (c) Absorption of lead in PH: roots>leaves>stems (d) Absorption
of lead in RA: leaves> stem>roots.
47
Figure 9 a-d: Concentration of lead with in different plant organs of control plants. (a)
Absorption of lead in CS: stem>leaves>roots (b) Absorption of lead in CC: leaves>
roots>stem (c) Absorption of lead in PH: leaves>roots>stem (d) Absorption of lead in
RA: roots>leaves>stem.
48
3.2 Analysis of zinc in contaminated plants
The amount of zinc in contaminated plants and their adhering soil as depicted in Figure
10 a-d. The leaves of four different plants of contaminated area showed the different
zinc accumulation as CS (0.417mg/kg), PH (0.298mg/kg), CC (0.279mg/kg) and RA
(0.246mg/kg). The stem of PH (0.228mg/kg) show maximum amount of zinc
accumulation while the stem of CC (0.073mg/kg) show minimum amount of zinc. The
stem of RA (0.199mg/kg) and CS (0.197mg/kg) lies in the middle of the range. PH
roots show high level of zinc accumulation 0.192mg/kg. The low level zinc
accumulation was found in RA which value was 0.033mg/kg whereas the roots of CS
and CC contain 0.177mg/kg and 0.086mg/kg amount of zinc. The accumulation of zinc
in plant of contaminated area follows the trend as CS>PH>RA>CC. The amount of
zinc in the soil of contaminated area ranges from 0.427 to 0.3 mg/kg.
Figure 11 a-d shows the levels of zinc accumulated in different organs of plants in
control area. The amount of zinc in leaves of CS in contaminated area was 10.5 times
higher than that of control leaves of CS whereas in stem it had been increased 5.24
times. The roots of CS showed 5.6 times higher value. The level of zinc accumulated in
leaves of CC was elevated 4.08 times. Stems of contaminated CC showed zinc 1.03
times in correspondence to control stem of same plant. The value of zinc in CC roots
was raised 3.75 times. PH leaves had shown 7.85 times increased in zinc as compared
to that of control. The stem of PH had shown 6.8 times elevated zinc level and in roots
it was amplified 14.7 times. The level of zinc in leaves of contaminated RA showed
that it had been 4.74 times higher than its control leaves. Zinc contamination in the
stem of RA was 3 times greater. The roots of RA had 1.87 times developed value of
zinc. The level of zinc had shown greater amplification in leaves of CS, CC and RA
and roots of PH.
49
Figure 10 a-d: Concentration of zinc within plant organs of different plants. (a)
Absorption of Zn in CS: leaves>stem>roots (b) Absorption of Zn in CC:
leaves>roots>stem (c) Absorption of Zn in PH: leaves>stem>roots (d) Absorption of
Zn in RA: leaves>stem>roots.
50
Figure 11 a-d: Concentration of Zn within plant organs of different plants. (a)
Absorption of Zn in CS: leaves>stem>roots (b) Absorption of Zn in CC:
stem>leaves>roots (c) Absorption of Zn in PH: leaves>stem>roots (d) Absorption of
Zn in RA: stem>leaves>roots.
51
3.3 Analysis of cadmium in contaminated plants
The figure 12 a-d represents the level of cadmium accumulation in plant organs of
different plants in contaminated area. The highest amount of cadmium found in the
leaves of PH was 0.015mg/kg while CS leaves had shown low addition of Cd that was
0.0008mg/kg. The quantity of cadmium found in the leaves of CC, RA was
0.0105mg/kg and 0.0108mg/kg respectively which stood in the middle of two ends.
The absorption of cadmium found only in the stem of RA which was 0.0005mg/kg. PH
roots had shown maximum accumulation of cadmium which was 0.0047mg/kg and its
lowest gathering was observed as 0.0015mg/kg in roots of RA. The quantity of
cadmium in roots of CS and CC was found as 0.003mg/kg, 0.0014mg/kg
correspondingly. The cadmium concentration in soil of contaminated area ranges from
0.015 to 0.0052 mg/kg. The accumulation of cadmium in the plants of contaminated
area follows the trend as: PH>RA>CC>CS except CS, leaves of all three contaminated
plants had shown high accumulation of cadmium.
52
Figure 12 a-d: Concentration of Cd within plant organs of different plants. (a)
Absorption of Cd in CS: roots>leaves (b) Absorption of Cd in CC: leaves >roots (c)
Absorption of Cd in PH: leaves>roots (d) Absorption of Cd in RA: leaves>roots>stem.
Analysis of cadmium in control plants

No Cd was found with in plant organs of different control plants.
53
3.4 Bioaccumulation Coefficient (BAC) and translocation factor (TF)
The Bioaccumulation Coefficient (BAC) is the ratio between the concentrations of a
given metal in the whole plant to its concentration in soil. (Zayed et al., 1998).
BAC= conc. of metal in plant / conc. of metal in soil
Translocation factor (TF) was calculated agreeing to the method of Marchiol et al.
2004, as:
TF = metals conc. in shoots/ metal concentration in roots
Plants BAC TF
CS 0.813 0.36
CC 0.22 0.15
PH 0.582 0.10
RA 0.814 34.42
Table 1: Bioaccumulation coefficient and translocation factors of contaminated plants.
54
3.5 Antioxidants assay
Within organs of different plants of contaminated area, the amount of GPOXs activity
was found maximum in roots and leaves of CS, PH and CC while in case of RA, it was
found higher in leaves and stem. The CATs activity was found in maximum amount in
the leaves and stem of all contaminated plants except PH (stem and roots). The SODs
activity was found maximum in the leaves and stems of all plants except PH.
3.5.1 Analysis of GPOXs activity

In the leaves of contaminated plants, heavy metals contamination was high which
resulted in the increase production of GPOXs as shown in Fig. 13 a-b. As compared
with control, GPOXs in leaves of RA, CS, PH, CC plants were 5, 4.5, 1.94 and 1.56
times higher, respectively. Maximum production of GPOXs had been reported in
leaves of contaminated plants of RA and CS as compared to control.
GPOXs activity in leaves of contaminated GPOXs activity in leaves of control plants
55
plants
Figure 13 a-b: GPOXs activity in leaves of contaminated and control plants.
In correspondence to control, the concentration of GPOXs in the stems of RA, CS, PH

and CC of contaminated area had been elevated 4.02, 2.1, 1.5 and 1.06 times (Fig. 14
a-b). Highest increase in folds of GPOXs activity was observed in the stem of RA.
GPOXs activity in stem of contaminated plants GPOXs activity in stem of control plants
Figure 14 a-b: GPOXs activity in stem of contaminated and control plants.
The roots of contaminated CS had shown maximum production of GPOXs i.e. 5 times
higher as compared to control. In correspondence to the control, roots of PH and CC
showed increased GPOXs activity as 3.5 and 3.2 times. As compared to control, the
maximum increased in GPOXs activity in RA roots (2.9 times) was observed (Figure
15 a-b).
56
GPOXs activity in roots of contaminated plants GPOXs activity in roots of control plants
Figure 15 a-b: GPOXs activity in the roots of contaminated and control plants.
3.5.2 Analysis of CATs activity

The amount of CAT produced in the leaves of contaminated plants increases as
illustrated in Figure 16 a-b. The concentration of CATs in the leaves of contaminated
plant of RA, CS, CC and PH as compared to control increased as 1.71, 1.22, 1.1 and
1.9 times, respectively. The maximum increase in folds of CATs activity was shown in
PH and RA leaves.
57
CATs activity in leaves of contaminated plants CATs activity in leaves of control plants
Figure 16 a-b: The CATs activity in the leaves of contaminated and control plants.
The CATs activity in the stems of contaminated and control plants has been shown in
Fig. 17 a-b. The quantity of CATs produced in stems of RA, CS, PH and CC was
amplified 1.7, 1.82, 1.54 and 1.12 times (than control). The stem of CS and RA showed
highest increase in the fold of CATs activity in correspondence to the control.
58
CATs activity in stem of contaminated plants CATs activity in stem of control plants
Figure 17 a-b: The CATs activity in stems of contaminated and control plant.
The concentration of CATs in roots of different plants of contaminated area is shown
in Fig .18 a-b. As compared to control, the roots of contaminated PH, CS, CC and RA
showed increased in CATs activity i.e. 1.13, 1.5, 1.51 and 1.47 times. The roots of CS
and CC showed maximum increased in folds of CATs activity as compared to control.
CATs activity in roots of contaminated plants CATs activity in roots of control plants
Figure 18 a-b: The CATs activity in the roots of contaminated and control plants.
59
3.5.3 Analysis of SODs activity
The SODs activity in the leaves of contaminated RA, CS, CC and PH was found to be
1.67, 2.08, 1.45 and 1.82 times increased as compared to the control (Fig 19 a-b). CS
and PH had shown maximum increased in folds of SODs activity as compared to
control.
SODs activity in leaves of contaminated plants SODs activity in leaves of contaminated plants
Figure 19 a-b: The SODs activity in leaves of contaminated and control plants.
The Figure 20 a-b illustrates that the stems of RA, CS, PH and CC had shown increase
in SODs activity as 1.9, 1.82, 2.81 and 4.2 times (greater than control). The highest
increased in the folds of SODs activity was found in stem of CC and PH as compared
to control.
60
SODs activity in stem of contaminated plants SODs activity in stem of control plants
Figure 20 a-b: The SODs activity in stems of contaminated and control plants.
It has been shown that in Fig. 21 a-b that the level of SODs found in the roots of
contaminated CS, PH, CC and RA was 1.9, 1.72, 1.7 and 1.45 times higher as
compared to control. CS and PH roots had shown maximum increased in folds of
SODs activity as compared to control.
61
SODs activity in roots of contaminated plants SODs activity in roots of control plants
Figure 21 a-b: The SODs activity in roots of contaminated and control plants.
62
Chapter 4
Discussion
63
It has been observed that some species of plants belonging to family caryophyllaceae,
brassicaceae, cyperaceae, poaceae, fabaceae and chenopodiaceae can tolerate higher
concentrations of trace elements (Gonzalez and Gonzalez-chavez, 2006). These plants
are called as excluders as they can adopt and survive in the contaminated soil
containing higher concentrations of heavy metals for example Silene vulgaris and Zea
may have been termed to be nickel excluder while Hyparrhenia hirta has been termed
as copper excluder (Kabata - Pendias and Pendias 2001; Fischerova et al., 2006;
Wenzel et al., 2003). Low solubility of metals such as Pb and Cr is shown in soil and
presents a strong barrier even in case if they are accumulated in the root and are mostly
not translocated to leaves, fruits and seeds (Deng et al., 2004). Generally, the following
trend is followed by Pb in various plant organs: roots >leaves >stem. Though, this
order can vary depending on plant species (Antosiewicz, 1992). Roots of dicot plants
accumulate more Pb as compared to monocots (Huang and Cunningham, 1996). Fig
8a-d illustrates the lead accumulation in leaves, stems and roots of four different plants
of contaminated area. In CS and PH roots had shown maximum amplification of lead
i.e. 51.3 and 96 times (greater than control) as illustrated in Fig. 8 a, c. RA; as
compared to control, in stem had shown 87 times high amplification of lead followed
by leaves and roots in which it was 79.5 and 1.3 times increased (fig.8d). In CS as
shown in Fig.8b, after roots increase in folds of lead accumulation was shown in leaves
(27times) and stem (9.6 times) of contaminated plants in correspondence to control. In
case of CC, the contaminated raised in lead amount leaves (7 times), stem (3.5 times)
and roots (2.5times) as compared to control is demonstrated in Fig.8b. In present study
expressed that after roots, PH stem had shown 16 times increased in amount of lead
followed by its leaves which it was raised 6 times higher than control (Fig. 8c). The
amount of lead in soil of Hattar Industrial Estate had ranged from 21.31 to 20.65 mg/kg
which showed the high contamination of lead in soil. The trend followed by
accumulation of lead in four plants of contaminated area was as RA>CS>PH>CC.
Roots of CS and PH, leaves of RA and CC and stem of RA had shown high
amplification of lead as compared to control (Fig. 9 a-d).
Zinc is an important element for plant growth and development. It has been shown by
the studies that with the increase in zinc supply in both tolerant and non-tolerant
64
genotype plants rise in total zinc concentration of plant tissues take place (Murray et
al., 2000). The level of zinc found in leaves, stem and roots of four different
contaminated plants is shown in (Fig.10 a-d). In contaminated area, the level of zinc in
leaves, stems and roots of CS as illustrated in Fig.10 a was 10.5, 5.24 and 5.6 times
higher (than control). In CC, as compared to control, the amount of zinc in leaves, stem
and roots (Fig. 10b) had increased 4.08, 1.03 and 3.75 folds. Contaminated PH leaves,
stems and roots as shown in Fig. 10c had shown 7.85, 6.8 and 14.7 times greater
amount of zinc than control. Fig. 10d shows that the zinc contamination found in case
of RA was found to be 4.74, 3 and 1.87 times higher in leaves, stem and roots,
respectively.
The amount of zinc had shown greater amplification in leaves of CS, CC and RA and
roots of PH of contaminated area. The amount of zinc in the contaminated soil ranges
from 0.427 to 0.3 mg/kg. Contaminated CS plant had shown high accumulation of zinc,
lowest was found in CC whereas PH and RA lies in between in correspondence to
control (Fig. 11 a-d).
Cadmium poses threat to animals and plants. It does not play any role in biological
function but its high concentration has adverse effects. The production of stress
ethylene in different species of plant and induction of synthesis of phytochelatins in
plants is promoted by Cd (Nabulo et al., 2011; Mohammad et al., 2009). The variation
in uptake and distribution of trace metal especially cadmium occur from species to
species and this may be linked to differences in ability of plant to control the
movement of trace metals from xylem to phloem and via the phloem to other parts of
the plant (Singh et al., 2011).
The Fig. 12a-d illuminates that maximum accumulation of Cd was found in leaves of
four contaminated plants followed by stem whereas no cadmium was found in control
plants. The cadmium ranges from 0.015 to 0.0052 mg/kg in soil of contaminated area.
In contaminated area, PH shows maximum accumulation of cadmium followed by RA
then CC and CS. The plants of control area did not accumulate any amount of
cadmium.
According to Godzik (1993), the ability of leaves to accumulate the lead differs
depending on age. Maximum concentration of lead is found in senescing leaves and
65
minimum in young leaves. As stated by Dubey and Sharma (2005), with the increase in
distance from root, the amount of heavy metals decreases in aerial parts of plants which
is probably due to greater localization of lead in the cell wall of roots than in other
plant’s parts. Moreover, binding of lead take place more in lignified tissues than in
non-lignified.
It had been found by Tomasevic et al., (2005) that the amount of heavy metals particles
deposited on leaves was reliant on the type of plants species owing to different
characteristics of epidermis. The level of lead among leaves of four different
contaminated plants as shown in fig. 8 a-d it was found to be RA (10.11mg/kg), CS
(4.93mg/kg), CC(1.24mg/kg) and PH(0.96mg/kg). Fig. 10a-d elucidates that Zinc
accumulation found in leaves of contaminated plants were as CS(0.417mg/kg),
PH(0.298mg/kg) ,CC(0.279mg/kg) and RA(0.246mg/kg).Similarly, the accumulation
of cadmium also varied in leaves of contaminated plants (Fig. 12 a-d). The amount of
cadmium found in the contaminated leaves of PH was 0.015mg/kg while CS leaves it
was 0.0008mg/kg (Fig.12 c, a). The quantity of cadmium found in the contaminated
leaves of CC, RA was 0.0105 and 0.0108mg/kg (fig. 12b, d).
Similarly; in case of contaminated plants, maximum lead had been retained in stem of
RA (6.84mg/kg) (Fig. 8d) whereas minimum amount of lead was found in stem of CC
(0.34mg/kg) (Fig. 8b). While the quantity of lead in stems of CS (2.9mg/kg) and PH
(0.75mg/kg) stood in of the middle of two extreme value as shown in Fig 8a, c.
The stem of PH (0.228mg/kg) showed high amount of zinc accumulation while the
stem of CC (0.073mg/kg) had shown low amount of zinc as illustrated in Fig.10 b, c.
The zinc found in stem of RA and CS was 0.199 and 0.197mg/kg (Fig. 10 a, d). In
plants of contaminated area, the absorption of cadmium found only in the stem of RA
which was 0.0005mg/kg (Fig. 11 d).
In accordance to Suchodoller (1967), lead was mainly reserved in epidermis of roots
while a small amount of it could be identified in vascular tissues. He also suggested
that with different species type, the extent of localization of lead in different plant
tissues could varied.
66
The fig. 8 a-d illustrates that in the plants of contaminated area, increased level of lead
uptake was shown by roots of PH (10.08mg/kg) and afterwards CS (8.51mg/kg).
Comparatively less amount of lead was found in roots of CC (2.63mg/kg) and RA
(0.17mg/kg).
The fig.10 c, d demonstrates that PH had showed high level of zinc accumulation in
roots i.e. 0.192mg/kg and the low level was found in RA which value was 0.033mg/kg.
The roots of CS and CC had 0.177mg/kg and 0.086mg/kg amount of zinc as shown in
Fig. 10 a, b.
Highest accumulation of cadmium in roots of PH was 0.0047mg/kg and its lowest
gathering was observed as 0.0015mg/kg in roots of RA as clarified in fig.12 c, d. The
quantity of cadmium in roots of CS and CC was 0.003mg/kg, 0.0014mg/kg (Fig. 12 a,
b).
Generally, heavy metals result in production of oxidative stress in plants which affect
several metabolic processes (Mittler, 2002). Yet the precise mechanisms by which
heavy metals cause toxicity to plants are not fully understood. There exist array of
different layers of defense mechanism which results in the reduction of levels of heavy
metals toxicity and the cooperative function of all these different mechanisms
determine the tolerance capacity of each plant which include the induction of both
enzymatic and non-enzymatic substances (Mourato et al., 2012). Several enzymes that
work together to avoid the deleterious effects of ROS and other toxic species are
included in enzymatic mechanism. Superoxide dismutase (SOD, EC 1.15.1.1), forms a
primitive defense against oxidative stress, which converts O2·− species into H2O2 that
can then be converted to water by peroxidases and catalase (Mourato et al., 2012). In
fig. 19-21, we can see that there is a general trend for the increase in SOD activity in
different plants and their parts of contaminated area. Both guaiacol peroxidase (GPOD,
EC 1.11.1.7) and catalase (CAT, EC 1.11.1.6) catalyze the breakdown of H2O2 to
water, though at different rates with different affinities and in different
organelles(Mittler, 2002) and activities of these enzymes had been illustrated in fig 13-
18. In roots the enhanced GPOD activity had been reported in some studies (Armas et
al., 2015; Pinto et al., 2009) that could also be related to increased lignin synthesis
which could have to be assumed a protective role resulting in cell wall stiffening.
67
Important component of plant antioxidant defense are Ascorbate and glutathione as
they are part of the ascorbate-glutathione cycle, which is an important metabolic
pathway for the removal of ROS. Glutathione is necessary for the synthesis of
phytochelatins, a family of important sequesters for heavy metals and both glutathione
and ascorbate themselves are powerful antioxidants (Potters et al., 2002). Mostly
authors has reported increases in these enzymes activities though in certain cases a
decrease in the activity is reported. Hence, the exact mechanisms through which plants
get rid of excess hydrogen peroxide is highly dependent not only of the species
nevertheless on all the different exogenous factors affecting the experiment though at
least one of these enzymes is usually found to increase its activity. A decline in
enzymatic activities at higher concentrations or at extended periods of time after
contamination is also often described, signifying that the toxic effects have probably
overcome the antioxidant defense capacity of the plant. In fact, the increase in the
activity of antioxidative defense enzymes can logically be attributed to their generation
due to the heavy metal toxicity, its decrease has also been justified by the disruption of
the anti-oxidative mechanisms due to the same toxicity. Yet, the threshold levels
between both effects are dependent on a large number of factors and are difficult to
determine. Large number of studies regarding the effect of metal toxicity has reported
quantitative results for antioxidative enzymes and there are fewer studies with more
specific gene expression measurements (Bernard et al., 2015).
In many literatures or articles phytoremediation techniques are briefly shown. The
generic term “phytoremediation” is the combination of the Greek prefix phyto (plant),
attached to the Latin root remedium (to correct or remove an evil) (Erakhrumen and
Agbontalor, 2007; U. S. Environmental Protection Agenc, 2000). To estimate a plant’s
potential for phytoremediation purpose, both bioconcentration factors (BCF) and
translocation factors (TF) have a wide use (Yoon et al. 2006). Enrichment takes place
when a contaminant taken up by a plant is retained in the plant instead of getting
degraded. The translocation Factor (TF) measures the ability of the plant to transport
the metal eventually accumulated in the roots to the aerial part (Trebolazabala, 2017).
Table. 1 illustrates that RA(35) had the maximum value of translocation factor
followed by CS (0.36), than CC(0.15) and PH(0.1). The translocation of heavy metals
68
to the easily harvestable plant parts, i.e. shoot is required for phytoextraction
(Chowdhury and Maiti, 2016). The Bioaccumulation Coefficient (BAC) is the ratio
between the concentrations of a given metal in the whole plant to its concentration in
soil. Four categories are defined in accordance to the BAC value obtained. BAC values
of < 0.01, 0.01-0.1, 0.1-1 and 1-10 classes plants in four different categories, e.g., non
accumulator, low accumulator, moderate accumulator and high accumulator/
hyperaccumulator, respectively (Zayed et al., 1998).The value of bioaccumulation
coefficient in the four different plants of contaminated area was ranged from 0.22 to
0.814 and they had fallen in the class of moderate accumulator.
In the present studies, the best accumulating plant among four plants of contaminated
area was RA whose maximum BAC and TF value were 34.42 and 0.814. Among all
four plants, the increased amount of heavy metals was found in leaves and stem of RA.
The levels of three different antioxidants in RA varied; GPOXs found in the leaves,
stem and roots of contaminated RA were 5, 4.02 and 2.9 times higher as compare to
control.
RA had CATs in leaves, stem and roots as 1.71, 1.7 and 1.47 times (increased than
control). SODs found in RA leaves, stem and roots as compared to control had shown
1.67, 1.9 and 1.45 times increased, respectively.
The CS was the second best plant for metal removal from soil. The BAC and TF values
of CS were 0.36 and 0.812. The roots and leaves of CS had shown maximum
accumulation of heavy metals. The quantity of GPOXs, CATs and SODs found in CS
were varying. Leaves, stem and roots of CS had shown 4.5, 2.1 and 5 times GPOXs
increased as compared to control. The CATs measured in the leaves (1.22 times),
stem(1.82 times) and roots (1.5 times) were higher than control. CS leaves, stem and
roots had shown increased in SODs amount by 2.08, 1.82 and 1.9 times.
PH stood at the third number as the phytoremidiating plant. PH, BAC and TF values
were found to be 0.58 and 0.1. After RA and CS, the roots and leaves of PH had shown
maximum accumulation of heavy metals as compared to its stem. The levels of
antioxidants in PH were as; GPOXs in leaves, stem and roots had 1.94, 1.5 and 3.5
times (increased than control).
69
In correspondence to control, the CATs found in PH leaves, stem and roots were 1.9,
1.54 and 1.13 times higher. The amount of SODs that was present in the leaves, stem
and roots of PH as compared to control had increased by 1.82, 2.81and 1.72 times.
This study shows that CC was found as a low phtyoremediating plant as compared to
other three plants of contaminated area with BAC and TF value of 0.22 and 0.15. The
maximum amount of heavy metals were accumulated in roots and leaves of plant. The
different levels of three antioxidants were found in CC. The production of GPOXs in
the leaves, stem and roots were 1.56, 1.06 and 3.2 times amplified than control. The
CATs production in CC leaves, stem and roots were 1.1, 1.12 and 1.51 times (increased
than control), respectively. The amount of SODs in CC as to control had increased
1.45, 4.2 and 1.7 times.
According to Baker and Whiting (2002), much attention is being paid to metal
accumulating plants that may be used for the phytoremediation of contaminated soils
(Baker and Whiting, 2002). Some plants can accumulate remarkable levels of metals –
100–1000 fold the levels normally accumulated in most species and are termed as
hyper accumulators. Up till, metal hyper accumulating species in at least 45 plant
families and individual species (or even populations) that can accumulate different
metals such Zn, Cd, Cu, Co, Ni, Se and As or particular combinations of these have
been identified (Reeves and Baker, 1999). Identification of new metal hyper
accumulating species or populations is continued (Kraemer, 2003).
70
Chapter5
References
71
1) Adriano, D.C. (2001). Trace elements in terrestrial environments.
Biochemistry, bioavailability and risks of metals. Springer-Verlag, New
York. 2, 10-12.
2) Ahamed, M. & Siddiqui, M. K. J. (2007). Low level lead exposure and
oxidative stress: current opinions. Clinica Chimica Acta. 383(1), 57-64.
3) Allen, R. D., Webb, R. P. & Schake, S. A. (1997). Use of transgenic plants to
study antioxidant defenses. Free Radical Bio. Med. 23(3), 473-479.
4) Alloway, B.J. (2004). In: Zinc in soil and crop nutrition. Second edition. pp.
78-84. International Zinc Association, Belgium.
5) Amari, T., Ghnaya, T. & Abdelly, C. (2017). Nickel, cadmium and lead
phytotoxicity and potential of halophytic plants in heavy metal extraction. S.
Afr. J. Bot. 111, 99-110.
6) Antonovics, J., Bradshaw, A. D. & Turner, R. G. (1971) Heavy metal
tolerance in plants. Adv. Ecol. Res. 7, 1-85.
7) Antosiewicz, D. M. (1992). Adaptation of plants to an environment polluted
with heavy metals. Acta. Soc. Bot. pol. 61(2), 281-299.
8) Aono, M., Kubo, A., Saji, H., Tanaka, K. & Kondo, N. (1993). Enhanced
tolerance to photooxidative stress of transgenic Nicotiana tabacum with high
chloroplastic glutathione reductase activity. Plant Cell Physiol. 34(1), 129-
135.
9) Aono, M., Saji, H., Sakamoto, A., Tanaka, K., Kondo, N. & Tanaka, K.
(1995). Paraquat tolerance of transgenic Nicotiana tabacum with enhanced
activities of glutathione reductase and superoxide dismutase. Plant Cell
Physiol. 36(8), 1687-1691.
10) Apel, K. & Hirt, H. (2004). Reactive oxygen species: metabolism, oxidative
stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373-399.
11) Armas, T., Pinto, A.P., de Varennes, A., Mourato, M.P., Martins, L.L.,
Gonçalves, M.L.S. & Mota, A.M. (2015). Comparison of cadmium-induced
oxidative stress in Brassica juncea in soil and hydroponic cultures. Plant Soil.
388(1-2), 297-302.
72
12) Asada, K. (1987). Production and scavenging of active oxygen in
photosynthesis. Photoinhibition. 2, 45-48.
13) Asada, K. (1992). Ascorbate peroxidase-a hydrogen peroxide‐scavenging
enzyme in plants. Physiol. Plant. 85(2), 235-241.
14) Avanzi, M. (1950) Observazioni sull'attivita citologica di alcuni composti
chimic. Caryologia. 3, 234-248.
15) Halliwell, B. & Gutteridge, J. M. (1990). Role of free radicals and catalytic
metal ions in human disease: an overview. Methods Enzymol. 186, 1-85.
16) Baker, A. J. & Whiting, S. N. (2002). In search of the Holy Grail–a further
step in understanding metal hyperaccumulation? New Phytol. 155(1), 1-4.
17) Baker, A. J. M. (1984). Environmentally-induced cadmium tolerance in the
grass Holcuslanatus L. Chemosphere. 13(4), 585-589.
18) Baker, A. J. M. (1987). Metal Tolerance. New Phytol. 106, 93-111.
19) Barcelo, J. & Poschenrieder, C. (1992). Plant responses to heavy metal
contamination. Suelo y Planta (Spain). 2, 215-224.
20) Beauchamp, C. & Fridovich, I. (1971). Superoxide dismutase: improved
assays and an assay applicable to acrylamide gels. Anal. Biochem. 44(1), 276-
287.
21) Beers, R. F. & Sizer, I. W. (1952). A spectrophotometric method for
measuring the breakdown of hydrogen peroxide by catalase. J. Biol.
Chem. 195(1), 133-140.
22) Bell, R.W. & Dell, R.W. (2008). Micronutrients for sustainable food, feed,
fiber and bioenergy production. Second edition. pp 135-142. IFA, Paris.
France.
23) Bergmann, W. (1992). Nutritional disorders of plants. Development, visual
and analytical diagnosis. Gustav Fischer Verlag Jena Stuttgart, New York.
33(4), 410-411.
24) Bernard, F., Brulle, F., Dumez, S., Lemiere, S., Platel, A., Nesslany, F., Cuny,
D., Deram, A. & Vandenbulcke, F. (2015). Antioxidant responses of annelids,
Brassicaceae and fabaceae to pollutants: A review. Ecotoxicol. Environ. Saf.
114, 273–303.
73
25) Blaylock, M. J., Salt, D. E., Dushenkov, S., Zakharova, O., Gussman, C.,
Kapulnik, Y., Burt D. E. & Raskin, I. (1997). Enhanced accumulation of lead
in Indian mustard by soil-applied chelating agents. Environ. Sci.
Technol. 31(3), 860-865.
26) Boo, Y. C. & Jung, J. (1999). Water deficit—induced oxidative stress and
antioxidative defenses in rice plants. J. Plant Physiol. 155(2), 255-261.
27) Bowen, J.E. (1969). Adsorption of copper, zinc and manganese by sugarcane
tissue. Plant Physiol. 44, 255-261.
28) Bowler, C., Montagu, M. V. & Inze, D. (1992). Superoxide dismutase and
stress tolerance. Annu. Rev. Plant Biol. 43(1), 83-116.
29) Boyd, R. & Martens, S. (1998). Nickel hyperaccumulation by Thlaspi
montanum var. montanum (Brassicaceae): a constitutive trait. Am. J. Bot.
85(2), 259-261.
30) Bradshaw, A. D. (1984). Adaptation of plants to soils containing toxic metals-
--a test for conceit. In: Origins and Development of Adaptation. CIBA
Foundation Symposium. (eds. D. E. Vered and G. M. Collins), pp. 4-19.
Pitman, London.
31) Cakmak, I. (2000). Possible roles of zinc in protecting plant cells from
damage by reactive oxygen species. New Phytol. 146, 185-205.
32) Cataldo, D. A., Garland, T. R. & Wildung, R. E. (1981). Cadmium
distribution and chemical fate in soybean plants. Plant Physiol. 68, 835-839.
33) Chaney, R.L. & Ryan J.A. (1994). Risk based standards for arsenic, lead and
cadmium in urban soils. Dechema. Frankfurt, Germany. 2,130-136.
34) Chaney, R. L., & Hornick, S. B. (1978). Accumulation and effects of
cadmium on crops. In Edited Proc. First International Cadmium Conference
(pp. 125-140).
35) Chaney, R. L., White, M. C., & Tienhoven, M. V. (1976). Interaction of Cd

and Zn in phytotoxicity to and uptake by soybean. In Agron. Abstr .68, 21-26.
74
36) Chaoui A. and Ferjani E.E. (1997). Cadmium and Zinc induction of lipid
peroxidation and the effect on antioxidant enzyme activities in bean. Plant
Sci. 127, 139-147
37) Chen, C., Huang, D. & Liu, J. (2009). Functions and toxicity of nickel in
plants: recent advances and future prospects. Clean–Soil, Air, Water. 37(5),
304-313.
38) Chen, Q., Zhang, M. & Shen, S. (2010). Effect of salt on malondialdehyde
and antioxidant enzymes in seedling roots of Jerusalem artichoke (Helianthus
tuberosus L.). Acta Physiol. Plant. 33(2), 273-278.
39) Chen, Z., Young, T. E., Ling, J., Chang, S. C. & Gallie, D. R. (2003).
Increasing vitamin C content of plants through enhanced ascorbate
recycling. P. Natl. Acad. Sci. 100(6), 3525-3530.
40) Chojnacka, K., Chojnacki, A., Gorecka, H. & Gorecki, H. (2005).
Bioavailability of heavy metals from polluted soils to plants. Sci. Total
Environ. 337(1), 175-182.
41) Chowdhury, A. & Maiti, S. K. (2016). Identification of metal tolerant plant
species in mangrove ecosystem by using community study and multivariate
analysis: a case study from Indian Sunderban. Environ Earth Sci. 75(9), 1-8.
42) Christensen, T. H. (1984). Cadmium soil absorption at low concentrations: I.
Effect of time, cadmium load, pH and calcium. Water, Air and Soil Pollution.
21, 105-114.
43) Chukwuma Sr, C. (1993) Comparison of the accumulation of Cd, Pb and Zn
in cultivated and wild plant species in the derelict Enyigba lead-zinc mine.
Toxicol. Environ. Chem. 38(4), 167-173.
44) Clarkson, D. T., & Luttge, U. (1989). Mineral nutrition: divalent cations,
transport and compartmentation. In Progress in botany. Springer Berlin
Heidelberg.51, 93-112.
45) Clemens, S. (2001). Molecular mechanisms of plant metal tolerance and
homeostasis. Planta. 212(4), 475-486.
75
46) Clemens, S. (2006). Toxic metal accumulation, responses to exposure and
mechanisms of tolerance in plants. Biochimie. 88(11), 1707-1719.
47) Cobbett, C. S. (2000). Phytochelatin biosynthesis and function in heavy-metal
detoxification. Curr. Opin. Plant Biol. 3(3), 211-216.
48) Corpas, F. J., Palma, J. M., Sandalio, L. M., Valderrama, R., Barroso, J. B. &
Luis, A. (2008). Peroxisomal xanthine oxidoreductase: characterization of the
enzyme from pea (Pisum sativum L.) leaves. J. Plant Physiol. 165(13), 1319-
1330.
49) Coughtrey, P. J. & Martin, M. H. (1979). Cadmium, lead and zinc interactions
and tolerance in two populations of Holcus ianatus L. grown in solution
culture. Environ. Exp. Bot. 19, 285-290.
50) Crowley, D. E., Wang, Y. C., Reid, C. P. P. & Szaniszlo, P. J. (1991).
Mechanisms of iron acquisition from siderophores by microorganisms and
plants. Plant and soil. 130(1), 179-198.
51) Cutler, J. M. & Rains, D. W. (1974). Characterization of cadmium uptake by
plant tissue. Plant Physiol. 54, 67-71.
52) Da Silva, J. F., & Williams, R. J. P. (2001). The biological chemistry of the
elements: the inorganic chemistry of life. pp 26-32. Oxford University Press.
53) DalCorso, G., Manara, A. & Furini, A. (2013). An overview of heavy metal
challenge in plants: from roots to shoots. Metallomics. 5(9), 1117-1132.
54) Dalton, D. A., Baird, L. M., Langeberg, L., Taugher, C. Y., Anyan, W. R.,
Vance, C. P. & Sarath, G. (1993). Subcellular localization of oxygen defense
enzymes in soybean(Glycine max [L.] Merr.) root nodules. Plant
Physiol. 102(2), 481-489.
55) Das, P., Samantaray, S. & Rout, G. R. (1997). Studies on cadmium toxicity in
plants: a review. Environ. Pollut. 98(1), 29-36.
56) de Abreu, C.A., de Abreu M.F. & de Andrade J.C. (1998). Distribution of
lead in the soil profile evaluated by DTPA and Mehlich- 3 solutions.
Bragantia. 57,185-192
76
57) de Pinto, M. C. & De Gara, L. (2004). Changes in the ascorbate metabolism
of apoplastic and symplastic spaces are associated with cell differentiation. J.
Exp. Bot. 55(408), 2559-2569.
58) De Souza, M.P., Huang, C.P., Chee, N. & Terry, N. (1999). Rhizosphere
bacteria enhance the accumulation of selenium and mercury in wetland plants.
Planta. 209, 259-263.
59) Degreave, N. (1971). Modification des effects du methane sulfonate d'ethyl
au niveau chromosomique. I. Les ions metal- ligues. Rev. Cytol. Biol. Veget.
34, 233-244.
60) del Rı́o, L.A., Pastori, G.M., Palma, J.M., Sandalio, L.M., Sevilla, F., Corpas,
F.J., Jiménez, A., López-Huertas, E. & Hernández, J.A. (1998). The activated
oxygen role of peroxisomes in senescence. Plant Physiol. 116(4), 1195-1200.
61) Deng, H., Ye, Z.H. & Wong, M.H. (2004). Accumulation of lead, zinc,
copper, and cadmium by 12 wetland plant species thriving in metal-
contaminated sites in china. Environ. Pollut. 132, 29-40.
62) Desikan, R., Soheila, A. H., Hancock, J. T. & Neill, S. J. (2001). Regulation
of the Arabidopsis transcriptome by oxidative stress. Plant Physiol. 127(1),
159-172.
63) Dey, S. K., Dey, J., Patra, S. & Pothal, D. (2007). Changes in the
antioxidative enzyme activities and lipid peroxidation in wheat seedlings
exposed to cadmium and lead stress. Braz. J. Plant Physiol. 19(1), 53-60.
64) Dipierro, S. & Borraccino, G. (1991). Dehydroascorbate reductase from
potato tubers. Phytochemistry. 30(2), 427-429.
65) De Abreu, C. A., De Abreu, M. F. & De Andrade, J. C. (1998). Distribution
of Lead in the Soil Profile Evaluated by DTPA and Mehlich-3
Solutions. Bragantia. 2(1), 4-6.
66) Douchkov, D., Gryczka, C., Stephan, U. W., Hell, R. & Baumlein, H. (2005).
Ectopic expression of nicotianamine synthase genes results in improved iron
accumulation and increased nickel tolerance in transgenic tobacco. Plant, Cell
& Environ. 28(3), 365-374.
77
67) Edwards, E. A., Rawsthorne, S. & Mullineaux, P. M. (1990). Subcellular
distribution of multiple forms of glutathione reductase in leaves of pea (Pisum
sativum L.). Planta. 180(2), 278-284.
68) Eltayeb, A. E., Kawano, N., Badawi, G. H., Kaminaka, H., Sanekata, T.,
Shibahara, T., Inanaga, S. & Tanaka, K. (2007). Overexpression of
monodehydroascorbate reductase in transgenic tobacco confers enhanced
tolerance to ozone, salt and polyethylene glycol stresses. Planta, 225(5),
1255-1264.
69) Eltayeb, A. E., Yamamoto, S., Habora, M. E. E., Yin, L., Tsujimoto, H., &
Tanaka, K. (2011). Transgenic potato overexpressing Arabidopsis cytosolic
AtDHAR1 showed higher tolerance to herbicide, drought and salt
stresses. Breeding Sci. 61(1), 3-10.
70) Erdei, L., Mezôsi, G., Mécs, I., Vass, I., Fôglein, F., & Bulik, L. (2005).
Phytoremediation as a program for decontamination of heavy-metal polluted
environment. Acta Boil. Szege. 49(1-2), 75-76.
71) Ernst, W. H. O. (1980). Biochemical aspects of cadmium in plants. In:
Cadmium in the Environment. Part 1, (ed. J. O. Nriagu). pp. 639-653. John
Wiley, New York.
72) Faize, M., Burgos, L., Faize, L., Piqueras, A., Nicolas, E., Barba-Espin, G.,
Clemente-Moreno, M.J., Alcobendas, R., Artlip, T. & Hernandez, J.
A. (2011). Involvement of cytosolic ascorbate peroxidase and Cu/Zn-
superoxide dismutase for improved tolerance against drought stress. J. Exp.
Bot. 62(8), 2599-2613.
73) Falchuk, K. H., Fawcett, D. W. & Vallee, B. L. (1975) Competitive
antagonism of cadmium and zinc in the morphology and cell division of
Euglena gracilis. J. Submicr. Cytol. 7, 139-152.
74) Feigl G., Lehotai N., Molnar A., Ordog A., Rodriguez-Ruiz, Palma J.M.,
Corpas F.J., Erdei L. & Kolbert Z. (2015). Zinc induces distinct changes in
the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in
the roots of two Brassica species with different sensitivity to zinc stress. Ann
Bot. 116, 613-625.
78
75) Fischerova, Z., Tlustos, P., Szakova, J. & Sichorova, K. (2006). A
comparison of phytoremediation capability of selected plant species for given
trace elements. Environ. Pollut. 144, 93-100
76) Fodor, E., Szabo-Nagy, A. & Erdei, L. (1995). The effects of cadmium on the
fluidity and H+-ATPase activity of plasma membrane from sunflower and
wheat roots. J. Plant Physiol. 147(1), 87-92.
77) Foy, C. D., Chancy, R. L. & White, M. C. (1978). The physiology of metal
toxicity in plants. Annu. Rev. plant Phys. 29, 511-566.
78) Foyer, C. H., Souriau, N., Perret, S., Lelandais, M., Kunert, K. J., Pruvost, C.,
& Jouanin, L. (1995). Overexpression of glutathione reductase but not
glutathione synthetase leads to increases in antioxidant capacity and
resistance to photoinhibition in poplar trees. Plant physiol. 109(3), 1047-
1057.
79) Francis, C. W. & Rush, S. G. (1974). In: Trace Substances in Environmental
Health. Factors affecting uptake and distribution of cadmium in plants. (ed.
D. D. Hemphill). pp. 75-81. Columbia University, MO.
80) Fridovich, I. (1989). Superoxide dismutases. An adaptation to a paramagnetic
gas. J. Biol. Chem. 264(14), 7761-7764.
81) Fu, J. & Huang, B. (2001). Involvement of antioxidants and lipid
peroxidation in the adaptation of two cool-season grasses to localized drought
stress. Environ. Exp. Bot. 45(2), 105-114.
82) G. Noctor and C. H. Foyer. (1998). “Ascorbate and glutathione: keeping
active oxygen under control ,” Annu. Rev. plant Biol. 49, 249–279.
83) Gallego, S.M., Benavides, M.P. & Tomaro, M.L., 1996. Effect of heavy metal
ion excess on sunflower leaves: evidence for involvement of oxidative stress.
Plant Sci. 121, 151–159.
84) Gao, Q., & Zhang, L. (2008). Ultraviolet-B-induced oxidative stress and
antioxidant defense system responses in ascorbate-deficient vtc1 mutants of
Arabidopsis thaliana. J. Plant Physiol. 165(2), 138-148.
79
85) Garry, J., Karay, Y. & Harel, J. (1992) Effect of low pH, heavy metals and
anions an chlorophyll degradation in the lichen Ramalina duriaei. Environ.
Exp. Bot. 32, 229-241.
86) Ghisla, S. & Massey, V. (1989). Mechanisms of flavoprotein‐catalyzed
reactions. The FEBS Journal. 181(1), 1-17.
87) Gill, S. S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant
machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem.
48(12), 909-930.
88) Godzik, B. (1993). Heavy metals content in plants from zinc dumps and
reference areas. Pol. Botan. Stud. 5, 113-132
89) Goldbold, D. L. & Huttermann, A. (1985) Effect of zinc, cadmium and
mercury on root elongation of P. abies (Karst.) seedlings and the significance
of these metals to forest die-back. Environ. Pollut. 38, 375-381.
90) Gonnelli, C., Galardi, F. & Gabbrielli, R. (2001). Nickel and copper tolerance
and toxicity in three Tuscan populations of Silene paradoxa. Physiol. Plant.
113(4), 507-514.
91) Gonzalez, R.C. & Gonzalez-chavez, M.C.A. (2006). Metal accumulation in
wild plants surrounding mining wastes. Environ. Pollut. 144, 84-92.
92) Guan, Z., Chai, T., Zhang, Y., Xu, J. & Wei, W. (2009). Enhancement of Cd
tolerance in transgenic tobacco plants overexpressing a Cd-induced catalase
cDNA. Chemosphere. 76(5), 623-630.
93) Gupta, A. S., Heinen, J. L., Holaday, A. S., Burke, J. J. & Allen, R. D. (1993).
Increased resistance to oxidative stress in transgenic plants that overexpress
chloroplastic Cu/Zn superoxide dismutase. Proc. Natl. Acad. Sci. 90(4),
1629-1633.
94) Hackbarth, S., Schlothauer, J., Preub, A. & Roder, B. (2010). New insights to
primary photodynamic effects–Singlet oxygen kinetics in living cells. J.
Photochem. Photobiol. 98(3), 173-179.
80
95) Haghiri, F. (1973). Cadmium uptake by plants. J. Environ. Qual. 2, 93-96.
96) Haghiri, F. (1974). Plant uptake of cadmium as influenced by cation exchange
capacity, organic matter, zinc and soil temperature. J. Environ. Qual. 3, 180-
183.
97) Hall, J. L. & Williams, L. E. (2003). Transition metal transporters in plants. J.
Exp. Bot. 54(393), 2601-2613.
98) Hamilton, E. I. (1995). State of the art of trace element determinations in
plant matrices: determination of the chemical elements in plant matrices, an
overview. Sci. Total Environ. 176(3), 3-14.
99) Hashem, H. A. (2013). Cadmium toxicity induces lipid peroxidation and
alters cytokinin content and antioxidant enzyme activities in
soybean. Botany. 92(1), 1-7.
100) Hatz, S., Lambert, J. D. & Ogilby, P. R. (2007). Measuring the lifetime
of singlet oxygen in a single cell: addressing the issue of cell
viability. Photochem. Photobiol. Sci. 6(10), 1106-1116.
101) Havlin, J.L., Beaton, J.D., Tisdale, S.L. & Nelson, W.L. (1999). Soil
Fertility and Fertilizers. An introduction to nutrient management. Sixth
edition. pp 97-141. Upper Saddle River, New Jersey.
102) Hecht, S. M. & Bock, R. M. (1971). Chemical modifications of transfer
RNA species. Metal ion binding to mono-perphthalate-treated RNA. Bio.
Chem. Bio. Phys. Res. Commun. 45, 416—420.
103) Hemphill, D. D. (1972). Availability of trace elements to plants with
respect to soil-plant interaction. Ann. New York Acad. Sci. 199, 46-61.
104) Hendry, G.A.F., Baker, A.J.M. & Ewart, C.F. (1992). Cadmium
tolerance and toxicity, oxygen radical processes and molecular damage in
cadmium-tolerant and cadmium-sensitive clones of Holcus lanatus. Acta Bot.
Neerl. 41, 271–281.
105) Hertstein, U. & Jager, H. J. (1986). Tolerances of different populations
of three grass species to cadmium and other metals. Environ. Exp. Bot. 26,
309-319.
81
106) Hewitt, E. J. (1966). Sand and water culture methods used in the study
of plant nutrition, 2nd edn, Technical Communication No. 22. pp 32-40.
Commonwealth Agricultural Bureax, Farnham Royal, UK.
107) Hinesly, T. D., Redberg, K. E., Pietz, R. I. & Ziegler, E. L. (1984)
Cadmium and zinc uptake by corn (Zea mays L.) with repeated applications
of sewage sludge. Journal of Agricultural and Food Chemistry. 32, 155-163.
108) Hossain, M. A. & Asada, K. (1985). Monodehydroascorbate reductase
from cucumber is a flavin adenine dinucleotide enzyme. J. Agr. Food Chem.
260(24), 12920-12926.
109) Hossain, M. A., Nakano, Y. & Asada, K. (1984).
Monodehydroascorbate reductase in spinach chloroplasts and its participation
in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell
Physiol. 25(3), 385-395.
110) Huang, J. W. & Cunningham, S. D. (1996). Lead phytoextraction:
species variation in lead uptake and translocation. New phytol. 134(1), 75-84.
111) Imai, I. & Siegel, S. M. (1973). A specific response to toxic cadmium
levels in red kidney bean embryos. Physiol. Plant. 29, 118-120.
112) Ishikawa, T., Yoshimura, K., Sakai, K., Tamoi, M., Takeda, T., &
Shigeoka, S. (1998). Molecular characterization and physiological role of a
glyoxysome-bound ascorbate peroxidase from spinach. Plant Cell Physiol.
39(1), 23-34.
113) Da Silva, J. F. & Williams, R. J. P. (2001). The biological chemistry of
the elements: the inorganic chemistry of life. pp 12-14. Oxford University
Press.
82
114) Jackson, C., Dench, J., Moore, A. L., Halliwell, B., Foyer, C. H. & Hall,
D. O. (1978). Subcellular localisation and identification of superoxide
dismutase in the leaves of higher plants. The FEBS Journal. 91(2), 339-344.
115) Jacobs, E. E., Jacob, M., Sanandi, D. R. & Bradley, L. B. (1956)
Uncoupling of oxidative phosphorylation by cadmium. J. Bio. Chem. 223,
147-156.
116) Jager, H. J., Steubing, I. and Grunhage, I. (1983). Okologische
Untersuchungen in cincm violetum Calaminarae westfali- cure. Vesh Gfo. 11,
207-219.
117) Jarvis, S. C., Jones, L. H. P. & Hopper, M. J. (1976) Cadmium uptake
from solution by plants and its transport from roots to shoots. Plant and Soil.
44, 179-191.
118) Jiang, N., Luo, X., Zeng, J., Yang, Z., Zheng, L. & Wang, S. (2010).
Lead toxicity induced growth and antioxidant responses in Luffa cylindrica
seedlings. Int. J. Agri. Biol. 12(2), 205-210.
119) Jimenez, A., Hernandez, J. A., del Rio, L. A., & Sevilla, F. (1997).
Evidence for the presence of the ascorbate-glutathione cycle in mitochondria
and peroxisomes of pea leaves. Plant physiol. 114(1), 275-284.
120) John, M. K. (1976). Interrelationships between plant cadmium and
uptake of some other elements from culture solutions by oats and
lettuce. Environ. Pollut. (1970), 11(2), 85-95.
121) Johnson, M. S. & Eaton, J. W. (1980). Environmental contamination
through residual trace metal dispersal from a derelict lead-zinc mine. J.
Environ. Qual. 9(2), 175-179.
122) Kabata - Pendias, A. & Pendias, H. (2001). In: Trace Elements in Soils
and Plants. 3rd Edition. pp 413-430. CRC Press, Boca Raton, London.
123) Kay, J., Cryer, A., Darke, B. M., Kille, P., Lees, W. E., Norey, C. G., &
Stark, J. M. (1991). Naturally occurring and recombinant metallothioneins:
structure, immunoreactivity and metal-binding functions. Int. J. Biochem.
23(1), 1-5.
83
124) Kim, Y. Y., Yang, Y. Y., & Lee, Y. (2002). Pb and Cd uptake in rice
roots. Physiol. Plant. 116(3), 368-372.
125) Kinnersley, A. M. (1993). The role of phytochelates in plant growth and
productivity. Plant Growth Regul. 12(3), 207-218.
126) Kirkham, M. B. (2006). Cadmium in plants on polluted soils: effects of
soil factors, hyperaccumulation, and amendments. Geoderma. 137(1), 19-32.
127) Kobayashi, K., Kumazawa, Y., Miwa, K., & Yamanaka, S. (1996). ϵ-(γ-
Glutamyl) lysine cross-links of spore coat proteins and transglutaminase
activity in Bacillus subtilis. FEMS microbiology letters. 144(2-3), 157-160.
128) Kochian, L.V. (1993). Zinc absorption from hydroponic solution by
plant roots. In: Zinc in Soils and Plants. (eds. A.D. Robson). pp 366-371.
Kluwer Academic Publishers, Dordrecht.
129) Kraemer U. 2003. Phytoremediation to phytochelatin – plant trace metal
homeostasis. New Phytol. 158, 4–6.
130) Krieger-Liszkay, A. (2004). Singlet oxygen production in
photosynthesis. J. exp. Bot. 56(411), 337-346.
131) Kumada, H., Kimura, S., Yokata, M. & Matida, Y. (1973) Acute and
chronic toxicity, uptake and retention of cadmium in fresh water organisms.
Bull. Fresh Water Fish Res. Lab. Tokyo. 22, 157-164.
132) Kumchai, J., Huang, J. Z., Lee, C. Y., Chen, F. C., & Chin, S. W.
(2013). The induction of antioxidant enzyme activities in cabbage seedlings
by heavy metal stress. In P. World Acad. Sci. Eng. Techn.World Acad. Sci.
Eng.Tech. (WASET). 73,465-472.
133) Kwak, S. S., Lim, S., Tang, L., Kwon, S. Y., & Lee, H. S. (2009). In:
Salinity and Water Stress , Enhanced tolerance of transgenic crops expressing
both superoxide dismutase and ascorbate peroxidase in chloroplasts to
multiple environmental stress. pp. 197-203. Springer Netherlands.
84
134) Kwon, S. Y., Jeong, Y. J., Lee, H. S., Kim, J. S., Cho, K. Y., Allen, R.
D., & Kwak, S. S. (2002). Enhanced tolerances of transgenic tobacco plants
expressing both superoxide dismutase and ascorbate peroxidase in
chloroplasts against methyl viologen‐mediated oxidative stress. Plant, Cell
Environ. 25(7), 873-882.
135) Lagerwerff, J. V. (1971). Uptake of cadmium, lead and zinc by radish
from soil and air. Soil Sci. 111, 129-133.
136) Landon, R.J. 1991. Booker tropical soil manual: a handbook for soil
survey and agricultural land evaluation in the tropics and subtropics. Booker
Tate Ltd; Longman, London. 3(1), 22-28.
137) Lane, S. D. & Martin, E. S. (1977). A histochemical investigation of
lead uptake in Raphanus sativus. New Phytol. 79(2), 281-286.
138) Lee, S. C., Kwon, S. Y. & Kim, S. R. (2009). Ectopic expression of a
cold-responsive CuZn superoxide dismutase gene, SodCc1, in transgenic rice
(Oryza sativa L.). J. Plant Biol. 52(2), 154-160.
139) Lee, S. Z., Chang, L., Yang, H. H., Chen, C. M., & Liu, M. C. (1998).
Adsorption characteristics of lead onto soils. J. Hazard. Mater. 63(1), 37-49.
140) Lee, Y. P., Kim, S. H., Bang, J. W., Lee, H. S., Kwak, S. S., & Kwon,
S. Y. (2007). Enhanced tolerance to oxidative stress in transgenic tobacco
plants expressing three antioxidant enzymes in chloroplasts. Plant cell
reports. 26(5), 591-598.
141) Lee, Y. P., Kim, S. H., Bang, J. W., Lee, H. S., Kwak, S. S., & Kwon, S.
Y. (2007). Enhanced tolerance to oxidative stress in transgenic tobacco plants
expressing three antioxidant enzymes in chloroplasts. Plant cell reports.
26(5), 591-598.
142) Levan, A. (1945). Cytological reactions induced by inorganic salts
solution. Nature. 156, 751-752.
143) Li H. &Luo H. (2012). Antioxidant enzyme activity and gene

expression in response to lead stress in Perennial Regrass. J. Amer. Soc. Hort.
Sci. 137(2), 80-85.
85
144) Li, F., Wu, Q. Y., Sun, Y. L., Wang, L. Y., Yang, X. H., & Meng, Q. W.
(2010). Overexpression of chloroplastic monodehydroascorbate reductase
enhanced tolerance to temperature and methyl viologen‐mediated oxidative
stresses. Physiol. Plant. 139(4), 421-434.
145) Lim, S., Kim, Y. H., Kim, S. H., Kwon, S. Y., Lee, H. S., Kim, J. S.,
Cho, K.Y., Paek, K.Y. & Kwak, S. S. Enhanced tolerance of transgenic
sweetpotato plants that express both Cu-ZnSOD and APX in chloroplasts to
methyl viologen-mediated oxidative stress and chilling. Mol. Breeding. 19(3),
227-239.
146) Liu, X., Peng, K., Wang, A., Lian, C., & Shen, Z. (2010). Cadmium
accumulation and distribution in populations of Phytolacca americana L. and
the role of transpiration. Chemosphere. 78(9), 1136-1141.
147) Lockwood, M. P. (1976). Effects of Pollutants on Aquatic Organisms.
Cambridge University Press, New York.
148) Loneragan, J.F. & Webb, M.J. (1993). In: Zinc in Soils and Plants,
Interactions between Zinc and Other Nutrients Affecting the Growth of
Plants. (eds. A.D. Robson). pp 124-126. Kluwer Academic Publishers,
Dordrecht.
149) Lonergan, J.F. (1975). In: Trace elements in soil-plant-animal systems,
The availability and adsorption of trace elements in soil-plant systems and
their relation to movement and concentration of trace elements in plants. (eds.
D.J.D. Nicholas & A.R. Egan). pp 34-51. Academic Press, London.
150) Lucas, R. E., & Knezek, B. D. (1972). Climatic and soil conditions
promoting micronutrient deficiencies in plants. Micronutrients in agriculture.
265-288.
151) Luis, A., Sandalio, L. M., Corpas, F. J., Palma, J. M., & Barroso, J. B.
(2006). Reactive oxygen species and reactive nitrogen species in
peroxisomes. Production, scavenging, and role in cell signaling. Plant
Physiol. 141(2), 330-335.
86
152) Tomasevic, M., Vukmirovic, Z., Rajsic, S., Tasic, M. & Stevanovic, B.
(2005). Characterization of trace metal particles deposited on some deciduous
tree leaves in an urban area. Chemosphere. 61, 753-760
153) Zaefyzadeh, M., Quliyev, R.A., Babayeva, S.M. & Abbasov, M.A.
(2009). The effect of the interaction between genotypes and drought stress on
the superoxide dismutase and chlorophyll content in durum wheat land races.
Turk. J. Biol. 33(1), 1–7.
154) Madhusudhan, R., Ishikawa, T., Sawa, Y., Shigeoka, S. & Shibata, H.
(2003). Characterization of an ascorbate peroxidase in plastids of tobacco
BY‐2 cells. Physiol. Plant. 117(4), 550-557.
155) Malar S., Vikram S.S., Favas P.J.C. & Perumal V. (2014). Lead heavy
metal toxicity induced changes on growth and antioxidative enzymes level in
water hyacinths. Botanical Studies. 2, 53-54.
156) Mallick, N., & Mohn, F. H. (2000). Reactive oxygen species: response
of algal cells. J. Plant Physiol. 157(2), 183-193.
157) Marques T.C.L. & Soares A.M. (2011). Antioxidant system of ginseng
under stress by cadmium. Sci. Agric. (Piracicaba, Braz.). 68(4), 482-488.
158) Marschner, H. (1993). In: Zinc in Soils and Plants, Zinc Uptake from
Soils. (eds. A.D. Robson). pp 34-36. Kluwer Academic Publishers,
Dordrecht.
159) Martens, D.C. & Westermann, D.T. (1991). In: Micronutrients in
Agriculture, Fertilizer applications for correcting micronutrient deficiencies.
(eds. J.J. Mortvedt, F.R. Cox, L.M. Shuman & R.M. Welch). pp 45-48. SSSA
Book Series No. 4. Madison, WI.
160) McGrath, S. P., Chaudri, A. M. & Giller, K. E. (1995). Long-term

effects of metals in sewage sludge on soils, microorganisms and plants. J. Ind.
Microbiol. Biotechnol. 14(2), 94-104.
87
161) McKenna, I. M., Chaney, R. L. & Williams, F. M. (1993). The effects
of cadmium and zinc interactions on the accumulation and tissue distribution
of zinc and cadmium in lettuce and spinach. Environ Pollut. 79, 113-120.
162) McLaughlin, M. J., Parker, D. R. & Clarke, J. M. (1999). Metals and
micronutrients–food safety issues. Field crops research. 60(1), 143-163.
163) Mengel, K. & Kirkby, E.A., 1987. Principles of plant nutrition. 4th edn.
pp 14-18. International Potash Institute, Bern.
164) Mhamdi, A., Queval, G., Chaouch, S., Vanderauwera, S., Van
Breusegem, F., & Noctor, G. (2010). Catalase function in plants: a focus on
Arabidopsis mutants as stress-mimic models. J. Exp. Bot. 61(15), 4197-4220.
165) Miller, J. E., Hasset, J. J. & Koeppe, D. E. (1976). Uptake of cadmium
by soybeans as influenced by soil cation exchange capacity, pH and available
phosphorus. J. Environ. Qual. 5, 157-160.
166) Mills, R. F., Krijger, G. C., Baccarini, P. J., Hall, J. L. & Williams, L. E.
(2003). Functional expression of AtHMA4, a P1B‐type ATPase of the
Zn/Co/Cd/Pb subclass. The Plant J. 35(2), 164-176.
167) Mishra, K. K., Rai, U. N. & Prakash, O. (2007). Bioconcentration and
phytotoxicity of Cd in Eichhornia crassipes. Environmental monitoring and
assessment. 130(3), 237-243.
168) Mishra, S., Jha, A. B. & Dubey, R. S. (2011). Arsenite treatment
induces oxidative stress, upregulates antioxidant system, and causes
phytochelatin synthesis in rice seedlings. Protoplasma. 248(3), 565-577.
169) Mishra, S., Srivastava, S., Tripathi, R. D., Kumar, R., Seth, C. S. &
Gupta, D. K. (2006). Lead detoxification by coontail (Ceratophyllum
demersum L.) involves induction of phytochelatins and antioxidant system in
response to its accumulation. Chemosphere. 65(6), 1027-1039.
170) Mittler, R. (2002). Oxidative stress, antioxidants and stress tolerance.
Trends Plant Sci. 7, 405–410.
171) Mittler, R. & Zilinskas, B. A. (1992). Molecular cloning and
characterization of a gene encoding pea cytosolic ascorbate peroxidase. J.
Biol. Chem. 267(30), 21802-21807.
88
172) Miyake, C., Schreiber, U., Hormann, H., Sano, S. & Kozi, A. (1998).
The FAD-enzyme monodehydroascorbate radical reductase mediates
photoproduction of superoxide radicals in spinach thylakoid
membranes. Plant Cell Physiol. 39(8), 821-829.
173) Mohammad, A., Moheman, A. & Seema, A. (2009). The influence of a
single and multiple soil contamination of cadmium with lead and zinc on
growth, chlorophyll content, uptake and translocation of cadmium in tomato
plants. Arch. Agron. Soil Sci. 55(4), 407-413.
174) Moral, R., Palacios, (3, Gomez, I., Navarro-Pedreno, J. & Mataix, J.
(1994) Distribution and accumulation of heavy metals (Cd, Ni and Cr) in
tomato plant. Fresenius Environ. Bull. 3, 395-399.
175) Mourato, M., Reis, R. & Martins, L. (2012). In: Advances in Selected
Plant Physiology Aspects. Characterization of plant antioxidative system in
response to abiotic stresses: A focus on heavy metal toxicity. (eds.
Montanaro, G. & Dichio, B.). pp. 23–44. InTech.
176) Muradoglu F., Gundogdu M., Ercisli S., Encu T., Balta F., Jaafar H.Z.E.
& Muhammad Zia ul Haq. (2015). Cadmium toxicity affects chlorophyll a
and b content, antioxidant enzyme activities and mineral nutrient
accumulation in strawberry. Biol. Res. 48, 11-16.
177) Murray, P., Ge, Y. & Hendershot, W.H. (2000). Evaluating three trace
metal contaminated sites: a field and laboratory investigation. Environ.
Pollut. 107, 127-135.
178) Nabulo, G., Black, C.R. & Young, S.D. (2011). Trace metal uptake by
tropical vegetable grown on soil amended with urban sewage sludge. Environ.
Pollut. 159, 368-376.
179) Nagajyoti, P.C., Lee, K.D. & Sreekanth, T.V.M. (2010). Heavy metals,
occurrence and toxicity for plants: a review. Environ. Chem. Letters. 8(3),
199-216.
180) Neill, S., Desikan, R. & Hancock, J. (2002). Hydrogen peroxide
signalling. Curr. Opin. Plant Biol. 5(5), 388-395.
89
181) Nieboer, E. & Richardson, D.H.S. (1980). The replacement of the
nondescript term ‘heavy metal’ by a biologically significant and chemically
significant classification of metal ions. Environ. Pollut. B. 1, 3–26.
182) Noctor, G. & Foyer, C. H. (1998). Ascorbate and glutathione: keeping
active oxygen under control. Annu. Rev. Plant Biol. 49(1), 249-279.
183) Oehlkers, F. (1953). Chromosome breaks influenced by chemicals.
Heredity. 6, 95-105.
184) Pallanca, J. E. & Smirnoff, N. (2000). The control of ascorbic acid
synthesis and turnover in pea seedlings. J. Exp. Bot. 51(345), 669-674.
185) Pandey, N., Pathak, G. C., Pandey, D. K. & Pandey, R. (2009). Heavy
metals, Co, Ni, Cu, Zn and Cd, produce oxidative damage and evoke
differential antioxidant responses in spinach. Braz. J. Plant Physiol. 21(2),
103-111.
186) Pang, C. H., & Wang, B. S. (2008). Oxidative stress and salt tolerance
in plants. Prog. in Bot. 231-245.
187) Pastori, G. M. & Trippi, V. S. (1992). Oxidative stress induces high rate
of glutathione reductase synthesis in a drought-resistant maize strain. Plant
Cell Physiol. 33(7), 957-961.
188) Patterson, W. R. & Poulos, T. L. (1995). Crystal structure of
recombinant pea cytosolic ascorbate peroxidase. Biochemistry. 34(13), 4331-
4341.
189) Peralta-Videa, J. R., Gardea-Torresdey, J. L., Gomez, E., Tiemann, K.
J., Parsons, J. G. & Carrillo, G. (2002). Effect of mixed cadmium, copper,
nickel and zinc at different pH upon alfalfa growth and heavy metal
uptake. Environ. Pollut. 119(3), 291-301.
190) Perfus‐Barbeoch, L., Leonhardt, N., Vavasseur, A. & Forestier, C.
(2002). Heavy metal toxicity: cadmium permeates through calcium channels
and disturbs the plant water status. The Plant J. 32(4), 539-548.
191) Pinto, A.P., Alves, A.S., Candeias, A.J., Cardoso, A.I., de Varennes, A.,
Martins, L.L., Mourato, M.P., Goncalves, M.L.S. & Mota, A.M. Cadmium
accumulation and antioxidative defences in Brassica juncea L. Czern,
90
nicotiana tabacum L. In addition, solanum nigrum L. Int. J. Environ. Anal.
Chem. 2009, 89, 661–676.
192) Plants growing on a contaminated Florida site. Sci. Total. Environ. 368,
456-464.
193) Potters, G., de Gara, L., Asard, H. & Horemans, N. Ascorbate and
glutathione: Guardians of the cell cycle, partners in crime? Plant Physiol.
Biochem. 2002, 40, 537–548
194) Q. Chen, M. Zhang, and S. Shen. 2010. Effect of salt on
malondialdehyde and antioxidant enzymes in seedling roots of Jerusalem
artichoke (Helianthus tuberosus L.). Acta Physiol. Plant. 33(2), 273–278.
195) Qin, A., Shi, Q. & Yu, X. (2011). Ascorbic acid contents in transgenic
potato plants overexpressing two dehydroascorbate reductase genes. Mol.
Biol. Reports. 38(3), 1557-1566.
196) Racchi, M. L., Bagnoli, F., Balla, I. & Danti, S. (2001). Differential
activity of catalase and superoxide dismutase in seedlings and in vitro
micropropagated oak (Quercus robur L.). Plant Cell Reports. 20(2), 169-174.
197) Radotic, K., Ducic, T. & Mutavdzic, D. (2000). Changes in peroxidase
activity and isoenzymes in spruce needles after exposure to different
concentrations of cadmium. Environ. Exp. Bot. 44(2), 105-113.
198) Raskin, I., Kumar, P. N., Dushenkov, S. & Salt, D. E. (1994).
Bioconcentration of heavy metals by plants. Curr. Opin. Biotech. 5(3), 285-
290.
199) Raskin, I., Smith, R. D. & Salt, D. E. (1997). Phytoremediation of
metals: using plants to remove pollutants from the environment. Curr. Opin.
Biotechn. 8(2), 221-226.
200) Rauser, W. E. (1986). The amount of cadmium associated with Cd-

binding proteins in roots of Agrostis gigantea maize and tomato. Plant Sci.
43, 85-91.
201) Rauser, W. E. (1990). Phytochelatins. Annu. Rev. of Biochem. 59(1),
61-86.
91
202) Ravers, O. (1984). Cadmium in freshwater ecosystems. Experientia. 40,
1-14.
203) Reddy, A. M., Kumar, S. G., Jyothsnakumari, G., Thimmanaik, S. &
Sudhakar, C. (2005). Lead induced changes in antioxidant metabolism of
horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer
arietinum L.). Chemosphere. 60(1), 97-104.
204) Reeves, R.D. & Baker A.J.M. (1999). . In: Phytoremediation of toxic
metals: using plants to clean-up the environment. Metal accumulating plants
(eds. Ensley B.D. & Raskin, I.). pp 193–229. New York, USA: John Wiley &
Sons.
205) Reimann, C. & De Caritat, P. (1998). Chemical elements in the
environment: factsheets for the geochemist and environmental scientist.
Springer-Verlag, Berlin, Germany. 3, 34-38.
206) Rinnie, R.W. & Langston, R.G., 1960. Effect of growth on
redistribution of some mineral elements in peppermint. Plant Physiol. 35,
210-215.
207) Romheld, V. (1991). The role of phytosiderophores in acquisition of
iron and other micronutrients in graminaceous species: an ecological
approach. Plant and soil. 130(1), 127-134.
208) Root, R. A., Miller, R. J. & Koeppe, D. E. (1975). Uptake of cadmium--
its toxicity and effect on the iron-to-zinc ratio in hydroponically grown corn.
J. Environ. Qual. 4, 473-476.
209) Rosas, I., Carbajal, M. E., Gomez-Arroyo, S., Belmont, R. & Villalogos
Pietrini, R. (1984). Cytogenetic effects on cadmium accumulation on water
hyacinth (Eichornia crassipes). Environ. Res. 33, 386-395.
210) Rubio, M. C., Bustos‐Sanmamed, P., Clemente, M. R. & Becana, M.
(2009). Effects of salt stress on the expression of antioxidant genes and
proteins in the model legume Lotus japonicus. New Phytol. 181(4), 851-859.
211) Ruley, A. T. (2004). Effects of accumulation of lead and synthetic
chelators on the physiology and biochemistry of Sesbania drummondii.
92
212) Sakihama, Y., Mano, J. I., Sano, S., Asada, K. & Yamasaki, H. (2000).
Reduction of phenoxyl radicals mediated by monodehydroascorbate
reductase. Biochem. Biophys. Res. Comm. 279(3), 949-954.
213) Salin, M.L. (1988). Toxic oxygen species and protective systems of the
chloroplasts. Physiol. Plant. 72, 681–689.
214) Salt, D.E., Smith, R.D. & Raskin, I. (1998). Phytoremediation. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 49, 643-668.
215) Sandmann, G. & Goger, P. (1983). In: Inorganic Plant Nutrition. The
enzymological function of heavy metals and their role in electron transfer
processes of plants. (eds. A. Lauchli & R.L. Bieleski). Encycl. Plant Physiol.
Pp 130-134. Springer Verlag Berlin, Heidelberg, New York, Tokyo.
216) Sarwar, N., Imran, M., Shaheen, M. R., Ishaq, W., Kamran, A.,
Matloob, A., Abdur Rehim & Hussain, S. (2016). Phytoremediation strategies
for soils contaminated with heavy metals: Modifications and future
perspectives. Chemosphere. 12-14.
217) Sauerbeck, D. (1982). Which heavy metal concentration in plants
should not exceeded in order to avoid detrimental effects on their growth.
Landw. Forsch. Sonderh. 39, 108-129.
218) Scandalios, J. G. (1993). Oxygen stress and superoxide
dismutases. Plant Physiol. 101(1), 7-12.
219) Scandalios, J. G., Guan, L. & Polidoros, A. N. (1997). Catalases in
plants: gene structure, properties, regulation, and expression. Cold Spring
Harbor Monograph Series. 34, 343-406.
220) Schierup, H. & Larsen, V. J. (1981). Macrophyte cycling of Zn, Cu, Pb
and Cd in the littoral zone of a polluted and a non-polluted lake. I.
Availability, uptake and translocation of heavy metals in Phragmites australis
(Cav.). Trin. Aquatic Bot. 11,179-210.
93
221) Schu¨tzendu¨bel, A. & Polle, A. (2002). Plant responses to abiotic
stresses: heavy metal-induced oxidative stress and protection by
mycorrhization. J. Exp. Bot. 53, 1351–1365.
222) Schuller, D. J., Ban, N., van Huystee, R. B., McPherson, A. & Poulos,
T. L. (1996). The crystal structure of peanut peroxidase. Structure. 4(3), 311-
321.
223) Semchuk, N. M., Lushchak, V., Falk, J., Krupinska, K. & Lushchak, V.
I. (2009). Inactivation of genes, encoding tocopherol biosynthetic pathway
enzymes, results in oxidative stress in outdoor grown Arabidopsis
thaliana. Plant Physiol. Biochem. 47(5), 384-390.
224) Seregin, I. V. & Ivanov, V. B. (1998). The transport of cadmium and
lead ions through root tissues. Russ. J. Plant Physiol. 45(6), 780-785.
225) Shahid, M., Dumat, C., Khalid, S., Schreck, E., Xiong, T. & Niazi, N.
K. (2016). Foliar heavy metal uptake, toxicity and detoxification in plants: A
comparison of foliar and root metal uptake. J. Hazard. Mater. 27-28.
226) Suchodoller, A. (1967). Untersuchungen über den Bleigehalt von
Pflanzen in der Nähe von Straßen und über die Aufnahme und Translokation
von Blei durch Pflanzen. na. 2, 56-60.
227) Sharma S.S. and Dietz K-J. (2006). The significance of amino acids-
derived molecules in plant responses and adaptation to heavy metal stress. J.
Exp. Bot. 57(4), 711-726.
228) Sharma, A., Mukherjee, A. and Talukder, G. (1985) Mod- ification of
cadmium toxicity in biological systems by other metals. Curr. Sci. 54, 539-
549.
229) Sharma, P. & Dubey, R. S. (2004). Ascorbate peroxidase from rice
seedlings: properties of enzyme isoforms, effects of stresses and protective
roles of osmolytes. Plant Sci. 167(3), 541-550.
230) Sharma, P. & Dubey, R. S. (2005). Drought induces oxidative stress and
enhances the activities of antioxidant enzymes in growing rice seedlings.
Plant Growth Regul. 46(3), 209-221.
94
231) Sharma, P. & Dubey, R. S. (2005). Lead toxicity in plants. Brazilian J.
Plant Physiol. 17(1), 35-52.
232) Sharma, P., Jha, A. B., Dubey, R. S. & Pessarakli, M. (2010). Oxidative
stress and antioxidative defense system in plants growing under abiotic
stresses. Handbook of plant and crop stress. 3rd ed. 89-138.
233) Sharma, P., Jha, A. B., Dubey, R. S. & Pessarakli, M. (2012). Reactive
oxygen species, oxidative damage, and antioxidative defense mechanism in
plants under stressful conditions. J. Bot. 2-4.
234) Shu, D. F., Wang, L. Y., Duan, M., Deng, Y. S. & Meng, Q. W. (2011).
Antisense-mediated depletion of tomato chloroplast glutathione reductase
enhances susceptibility to chilling stress. Plant Physiol. Biochem. 49(10),
1228-1237.
235) Siegel, S. M. (1977). The cytotoxic response of Nicotiana protoplast to
metal ions: a survey of the chemical elements. Water, Air and Soil Pollut. 8,
293-304.
236) Silverberg, B. A. (1976). Cadmium-induced ultrastructural changes in
mitochondria of freshwater green algae. Phycologia. 15(2), 155-159.
237) Singh, B.R., Gupta, S.K., Azaizeh, H., Shilev, S., Sundre, D., Song,
W.Y., Martinoia, E. & Mench, M. (2011). Safety of food crops on land
contaminated with trace elements. J. Sci-Food Agric. 91, 1349-1366.
238) Somashekaraiah, B.V., Padmaja, K. & Prasad, A.R.K. (1992).
Phytotoxicity of cadmium ions on germinating seedlings of mung bean
(Phaseolus vulgaris): involvement of lipid peroxides in chlorophyll
degradation. Physiol. Plant. 85, 85–89.
239) Sposito, G., Lund, L. J. & Chang, A. C. (1982). Trace metal chemistry
in arid-zone field soils amended with sewage sludge: I. Fractionation of Ni,
Cu, Zn, Cd, and Pb in solid phases. Soil Sci. Soc. Am. J. 46(2), 260-264.
240) Stokes, P., Hutchinson, T. and Kruter, K. (1973). Heavy metal tolerance
in algae isolated from contaminated lakes near Sudburry, Ontario. Can. J.
Bot. 51, 2155-2168.
95
241) Street, J. J., Lindsay, W. L. & Sabey, B. R. (1977). Solubility and plant
uptake of cadmium in soils mended with cadmium and sewage sludge. J.
Environ. Qual. 6, 72-77.
242) Symeonidis, L., McNeilly, T. & Bradshaw, A. D. (1985). Inter
population variation in tolerance to Cd, Cu, Pb, Ni and Zn in mine
populations of Agrostis capiilaris L. New Phytol. 101, 317-324.
243) Tayefi-Nasrabadi, H., Dehghan, G., Daeihassani, B., Movafegi, A. &
Samadi, A. (2011). Some biochemical properties of guaiacol peroxidases as
modified by salt stress in leaves of salt-tolerant and salt-sensitive safflower
(Carthamus tinctorius L. cv.) cultivars. Afri. J. Biotech. 10(5), 751-763.
244) Tewari, R. K., Kumar, P., Sharma, P. N., & Bisht, S. S. (2002).
Modulation of oxidative stress responsive enzymes by excess cobalt. Plant
Sci. 162(3), 381-388.
245) Thomson, I. & Weier, T.E. (1962). The fine structure of chloroplast
from mineral-deficient leaves of Phaseolus vulgaris. Am. J. Bot. 49, 1047-
1055.
246) Tiffen, L.O. (1967). Translocation of manganese, iron, cobalt and zinc
in tomato. Plant Physiol. 41, 510-514.
247) Trebolazabala, J., Maguregui, M., Morillas, H., García-Fernandez, Z.,
de Diego, A., & Madariaga, J. M. (2017). Uptake of metals by tomato plants
(Solanum lycopersicum) and distribution inside the plant: Field experiments
in Biscay (Basque Country). J. Food Comp. Ana. 59, 161-169.
248) Tricot, A. 1999. le Cadmium Encyclopedia, Universalis, France. (6), 46-
52.
249) Tseng, M. J., Liu, C. W. & Yiu, J. C. (2008). Tolerance to sulfur dioxide
in transgenic Chinese cabbage transformed with both the superoxide
dismutase containing manganese and catalase genes of Escherichia coli. Sci.
hortic. 115(2), 101-110.
250) Tung, G. & Temple, P. J. (1996). Uptake and localization of lead in corn
(Zea mays L.) seedlings, a study by histochemical and electron
microscopy. Sci. Total Environ. 188(2), 71-85.
96
251) Tyler, G., Pahlsson, A. M., Bengtsson, G., Baath, E. & Tranvik, L.
(1989). Heavy metal ecology and terrestrial plants, micro-organisms and
invertebrates: a review. Water, Air and Soil Pollut. 47, 189-215.
252) Ushimaru, T., Maki, Y., Sano, S., Koshiba, K., Asada, K. & Tsuji, H.
(1997). Induction of enzymes involved in the ascorbate-dependent
antioxidative system, namely, ascorbate peroxidase, monodehydroascorbate
reductase and dehydroascorbate reductase, after exposure to air of rice (Oryza
sativa) seedlings germinated under water. Plant Cell Physiol. 38(5), 541-549.
253) Vallee, B.L. & Wacker, W.E.C. (1970). Metalloprotein. In: H. Neurath
(ed). The proteins, 2nd ed. pp 23-30.Academic Press, New York.
254) Vangronsveld, J., & Clijsters, H. (1994). Toxic effects of metals. Plants
and the chemical elements. Biochemistry, uptake, tolerance and toxicity, 150-
177.
255) Velu, G., Ortiz-Monasterio, I., Cakmak, I., Hao, Y. & Singh, R.P.
(2014). Biofortification strategies to increase grain zinc and iron
concentration in wheat. J. Cereal Sci. 59, 365-372.
256) Verbruggen, N., Hermans, C. & Schat, H. (2009). Molecular
mechanisms of metal hyperaccumulation in plants. New Phytol. 181(4), 759-
776.
257) Verret, F., Gravot, A., Auroy, P., Leonhardt, N., David, P., Nussaume,
L., Vavasseur, A. & Richaud, P. (2004). Overexpression of AtHMA4
enhances root-to-shoot translocation of zinc and cadmium and plant metal
tolerance. FEBS letters. 576(3), 306-312.
258) Vogeli-Lange, R. & Wagner, G. J. (1990). Subcellular localization of
cadmium and cadmium-binding peptides in tobacco leaves. Plant Physiol. 92,
1086-1093.
259) Von Rosen, G. (1954). Mutations induced by actions of metal ions in
Pisum hereditas. 40, 258-263.
260) Wang, J., Mao, H., Zhao, H., Huang, D. & Wang, Z. (2012). Different
increases in maize and wheat grain zinc concentration caused by soil and
97
foliar applications of zinc in Loess Plateau, China. Field Crop Res. 135, 89-
96.
261) Wang, J., Zhang, H. & Allen, R. D. (1999). Overexpression of an
Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases
protection against oxidative stress. Plant Cell Physiol. 40(7), 725-732.
262) Wang, Y., Wisniewski, M., Meilan, R., Cui, M., Webb, R. &
Fuchigami, L. (2005). Overexpression of cytosolic ascorbate peroxidase in
tomato confers tolerance to chilling and salt stress. J. Am. Soci. Hortic. Sci.
130(2), 167-173.
263) Welch, R.M., Norvell, W.A., Schaefer, S.C., Shaft, J.E., Kochian,
L.V.(1993). Induction of Iron(III) and Copper(II) Reduction in Pea (Pisum
sativum L.) Roots by Fe and Cu Status: Does the Root-Cell Plasma lemma Fe
(III)-Chelate Reductase Perform a General Role in Regulating Cation Uptake?
Planta. 190, 555-561.
264) Welinder, K. G. (1992). Superfamily of plant, fungal and bacterial

peroxidases. Curr. Opin. Struct. Biol. 2(3), 388-393.
265) Wenzel, W.W., Bunkowski, M., Puschenrerter, M. and Horak, O.
(2003). Rhizosphere characteristics of indigenous growing nickel hyper
accumulation and excluder plant on serpentine soil. Environ. Pollut. 123, 131-
138.
266) Zayed, A., Gowthaman, S. & Terry, N. (1998). Phytoaccumulation of
trace elements by wetland plants: I. Duckweed. Journal of environmental
quality. 27(3), 715-721.
267) White, R. E. (1976). Studies on the mineral ion absorption by plants. III.
The interaction of aluminium phosphate and pH on the growth of Medicago
sativa. Plant and Soil. 46, 195-208.
268) Whiting, S. N., de Souza, M. P., & Terry, N. (2001). Rhizosphere
bacteria mobilize Zn for hyperaccumulation by Thlaspi
caerulescens. Environ. Sci. Techn. 35(15), 3144-3150.
98
269) Willekens, H., Chamnongpol, S., Davey, M., Schraudner, M.,
Langebartels, C., Van Montagu, M., Dirk Inze & Van Camp, W. (1997).
Catalase is a sink for H2O2 and is indispensable for stress defense in C-3
plants. The EMBO J. 16(16), 4806-4816.
270) Willekens, H., Inze, D., Van Montagu, M., & Van Camp, W. (1995).
Catalases in plants. Mol. Breeding. 1(3), 207-228.
271) Woolhouse, H. W. (1983). In: Encyclopedia of Plant Physiology.
Toxicity and tolerance in the response of plants. (eds A. Pirson and M. H.
Zimmermann). New Series. pp. 245-300. Springer-Verlag, Berlin.
272) Y.Yabuta, T. Motoki, K. Yoshimura, T. Takeda, T. Ishikawa & S.
Shigeoka. (2002). Thylakoid membrane bound ascorbate peroxidase is a
limiting factor of antioxidative systems under photo oxidative stress. Plant J.
32(6). 915-925.
273) Yan R., Gao S., Yang W., Cao M., Wang S. & Chen F. (2008). Nickel
toxicity induced antioxidant enzyme and phenylalanine ammonialyase
activities in Jatropha curcas L. cotyledons. Plant Soil Environ. 54 (7), 294–
300.
274) Yan, J., Tsuichihara, N., Etoh, T. & Iwai, S. (2007). Reactive oxygen
species and nitric oxide are involved in ABA inhibition of stomatal
opening. Plant, Cell Environ. 30(10), 1320-1325.
275) Yanai, J., Zhao, F. J., McGrath, S. P. & Kosaki, T. (2006). Effect of soil
characteristics on Cd uptake by the hyperaccumulator Thlaspi
caerulescens. Environ. Pollut. 139(1), 167-175.
276) Yang, Y. Y., Jung, J. Y., Song, W. Y., Suh, H. S. & Lee, Y. (2000).
Identification of rice varieties with high tolerance or sensitivity to lead and
characterization of the mechanism of tolerance. Plant Physiol. 124(3), 1019-
1026.
277) Yang, X., Baligar, V. C., Martens, D. C. & Clark, R. B. (1996).
Cadmium effects on influx and transport of mineral nutrients in plant species.
J. Plant Nutr. 19(4), 643-656.
99
278) Yang, Y., Zhang, Y., Wei, X., You, J., Wang, W., Lu, J., & Shi, R.
(2011). Comparative antioxidative responses and proline metabolism in two
wheat cultivars under short term lead stress. Ecotoxicol. Environ. Saf. 74(4),
733-740.
279) Yilmaz, A., Ekiz H., Torun, B., Gultekin, I., Karanlik, S., Bagci, S.A. &
Cakmak, I., 1997. Effect of different zinc application methods on grain yield
and zinc concentration in wheat grown on zinc-deficient calcareous soils in
Central Anatolia. J. Plant Nutr. 20, 461-471.
280) Yoon, J., Cao, X., Zhou, Q. & Ma, L. Q. (2006). Accumulation of Pb,
Cu, and Zn in native plants growing on a contaminated Florida site. Sci. Total
Environ. 368(2), 456-464.
281) Yoshida, S., Tamaoki, M., Shikano, T., Nakajima, N., Ogawa, D., Ioki,
M., Aono, M., Kubo, A., Kamada, H., Inoue, Y. & Saji, H. (2006). Cytosolic
dehydroascorbate reductase is important for ozone tolerance in Arabidopsis
thaliana. Plant Cell Physiol. 47(2), 304-308.
282) Zaefyzadeh, M., Quliyev, R. A., Babayeva, S. M. & Abbasov, M. A.
(2009). The effect of the interaction between genotypes and drought stress on
the superoxide dismutase and chlorophyll content in durum wheat
landraces. Turk. J. Biol. 33(1), 1-7.
283) Zayed, A., Gowthaman, S. & Terry, N. (1998). Phytoaccumulation of
trace elements by wetland plants: I. Duckweed. J. Environ. Qual. 27(3), 715-
721.
284) Zhang, F. Q., Wang, Y. S., Lou, Z. P. & Dong, J. D. (2007). Effect of
heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves
and roots of two mangrove plant seedlings (Kandelia candel and Bruguiera
gymnorrhiza). Chemosphere. 67(1), 44-50.
285) Zhang, J., Cui, S., Li, J., Wei, J. & Kirkham, M. B. (1995).
Protoplasmic factors, antioxidant responses, and chilling resistance in
maize. Plant Physiol. Biochem. 33(5), 567-575.
286) Zheljazkov, V. D., & Nielson, N. E. (1996). Effect of heavy metals on
peppermint and cornmint. Plant and Soil. 178(1), 59-66.
100
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Heavy Metals Induced Toxicity and Levels of

Antioxidant Enzymes to Different Plants of

Thesis Title Heavy Metals Induced Toxicity and Levels of

COMSATS Institute of Information Technology, Abbottabad

of the requirement for the degree of

A Graduate Thesis submitted to the Department of Environmental Sciences as

partial fulfillment of the requirements for the award of Degree of Master of

Science (MS) in Environmental Sciences.

Name Registration Number

Judat Imran CIIT/FA15-RO7-003/ATD

Dr. Bilal Ahmed Zaffar Amin

This thesis titled

Heavy Metals Induced Toxicity and Levels of

For the COMSATS Institute of Information Technology, Abbottabad

External Examiner: _________________________________________________

Member Supervisory Committee: _______________________________________

Member Supervisory Committee: _______________________________________

Member Supervisory Committee: _______________________________________

I, Judat Imran (CIIT/FA15-R07-003/ATD) hereby declare that I have produced the

Date: 3-07-2017 Signature of the student: __________________

Date:____________________ Supervisor: _______________________

Dr. Bilal Ahmed Zafar Amin

Prof. Dr. Arshid Pervez

To Almighty ALLAH and the Holy Prophet

2 Materials & Methods………………………………………………….………….. 40

2.1 Sample collection …………………...………………………….……… 41

2.2 Sample storage…………………………………………………….…...….42

2.3 Sample preparation………………………………………………..……… 42

2.4 Heavy metals analysis…………………………………………….....…… 42

2.5 Extraction and antioxidant assays……………………………..…………. 43

3.1 Analysis of lead in contaminated plants…………………………………….…..... 46

3.2 Analysis of zinc in contaminated plants…………………………………..……… 49

3.3 Analysis of cadmium in contaminated plants……………………...…………….. 52

3.4 Bioaccumulation Coefficient (BAC) and translocation factor (TF)........................ 54

3.5 Antioxidants assay………………………………………………………………... 55

3.5.1 Analysis of GPOXs activity………………………………………….……....… 55

3.5.3 Analysis of SODs activity …………………………………….……………….. 59

Fig 1: Sites of production of reactive oxygen species (ROS) in plants……………….24

responses, including stomatal closure etc……………………………………….…….25

Fig 4: Schematic presentation of generation of reactive oxygen species in

Fig 5: Site view and sample collection………..………………………………………41

Fig 6: Different steps involved in heavy metals analysis…………………..…………42

Table1: Bioaccumulation coefficient and translocation factor of contaminated

BAC Bioaccumulation coefficient

mg/ L Milligram per liter

mg/ kg Milligram per kilogram

i. High concentration of phosphorus suppresses the infection of roots with

1.5.2 Types of ROS

1.5.3 Antioxidative Defense System in Plants

1.5.4 Non enzymatic components of antioxidative defense system

1.5.5 Enzymatic components

Central role in defense against oxidative stress is played by Superoxide dismutase

1.5.8 Guaiacol Peroxidase (GPX)

Guaiacol peroxidase (GPX, EC 1.11.1.7) is a heme containing protein and if at all

1.5.9 Enzymes of Ascorbate-Glutathione cycle

1.5.10 Ascorbate Peroxidase (APX)

The central component of AsA-GSH cycle is Ascorbate peroxidase (APX, EC 1.1.11.1)

1.5.11 Monodehydroascorbate Reductase (MDHAR)

Dehydroascorbate reductase (DHAR, EC 1.8.5.1), it catalyzes the reduction of DHA to

1.5.13 Glutathione Reductase (GR)

GSH is oxidized to GSSG while acting as an antioxidant by participating in enzymic as

1.8 Purpose of the Study

Figure 5: Site view and sample collection

Date: Supervisor: ___