REVIEWS

Antioxidant enzyme responses of plants to heavymetal stress

Anwesha M. Bhaduri • M. H. Fulekar

Published online: 29 October 2011

� Springer Science+Business Media B.V. 2011

Abstract Heavy metal pollutions caused by natural

processes or anthropological activities such as metal

industries, mining, mineral fertilizers, pesticides and

others pose serious environmental problems in present

days. Evidently there is an urgent need of efficient

remediation techniques that can tackle problems of

such extent, especially in polluted soil and water

resources. Phytoremediation is one such approach that

devices effective and affordable ways of engaging

suitable plants to cleanse the nature. Excessive accu-

mulation of metal in plant tissues are known to cause

oxidative stress. These, in turn differentially affect

other plant processes that lead to loss of cellular

homeostasis resulting in adverse affects on their growth

and development apart from others. Plants have limited

mechanisms of stress avoidance and require flexible

means of adaptation to changing. A common feature to

combat stress factors is synchronized function of

antioxidant enzymes that helps alleviating cellular

damage by limiting reactive oxygen species (ROS).

Although, ROS are inevitable byproducts from essen-

tial aerobic metabolisms, these are needed under sub-

lethal levels for normal plant growth. Understanding

the interplay between oxidative stress in plants and role

of antioxidant enzymes can result in developing plants

that can overcome oxidative stress with the expression

of antioxidant enzymes. These mechanisms have been

proving to have immense potential for remediating

these metals through the process of phytoremediation.

The aim of this review is to assemble our current

understandings of role of antioxidant enzymes of plants

subjected to heavy metal stress.

Keywords Phytoremediation � Antioxidant

enzymes � Oxidative stress � Heavy metal �Reactive oxygen species

Abbreviations

CAT Catalase

GR Glutathione reductase

GSH Glutathione

NADPH Nicotinamide adenine dinucleotide

phosphate

POD Guaiacol peroxidase

POX Peroxidase

PPO Polyphenoloxidase

ROS Reactive oxygen species

SOD Superoxide dismutase

1 Introduction

Environmental pollution is posing an ever increasing

stress in all forms of life. Each source of contamina-

tion has its own damaging effects on plants, animals

A. M. Bhaduri � M. H. Fulekar (&)

Environmental Biotechnology Laboratory, Department of

Life Sciences, University of Mumbai, Kalina Campus,

Santacruz (E), Mumbai 400098, India

e-mail: mhfulekar@yahoo.com

123

Rev Environ Sci Biotechnol (2012) 11:55–69

DOI 10.1007/s11157-011-9251-x

and ultimately on human health but those that add

heavy metals to soils and water are of serious concern

due to their persistence in the environment. They

cannot be destroyed biologically, but are only trans-

formed from one oxidation state or organic complex to

another (Garbisu and Alkorta 2001; Gisbert et al.

2003). Current technology does not eradicate the

problem it merely transfers it to future generations.

Visibly, there is an urgent need for alternative,

affordable and efficient methods to clean up heavily

contaminated industrial areas. This could be achieved

by a relatively new technology known as Phytoreme-

diation. Plant based bioremediation technologies have

been collectively termed as phytoremediation; this

refers to the use of green plants and their associated

micro biota for in situ treatment of contaminated soil,

sediments and ground water. Biologically based

remediation strategies, including phytoremediation,

have been estimated to be four to 1,000 times cheaper,

on a per volume basis, than current non-biological

technologies (Sadowsky 1999).

Phytoremediation is actually a generic term for

several ways in which plant can be used to clean

contaminated soil and water by breaking down or

degrading organic pollutants or by removing and

stabilizing metal contaminants. This may be done

through one of or a combination of the methods.

Results of research and development into phytoreme-

diation processes and techniques report it to be

applicable to a broad range of contaminants including

numerous metals and radionuclides, various organic

compounds (such as chlorinated solvents, BTEX,

PCBs, PAHs, pesticides/insecticides, explosives,

nutrients, and surfactants (Miller 1996).

Numerous plant species have been identified and

tested for their traits and mechanisms of metal uptake

(Lone et al. 2008). Uptake of phytotoxic metals in

higher amounts by plants or algae can result in

inhibition of several enzymes or increase in activity in

others (Van Assche and Clijsters 1990). These

biochemical attributes such as different enzymes,

stress protein, phytochelatins serve as an index of

metal sensitivity or tolerance in different groups of

plants (Li et al. 2006; Srivastava 1999).

One possible mechanism via which elevated con-

centrations of heavy metals may damage plant tissues

is the stimulation of free radical production by

imposing oxidative stress (Foyer et al. 1997). Accu-

mulation of reactive oxygen species (ROS) activates

antioxidative defense mechanisms in plants. ROS are

partially reduced forms of atmospheric oxygen and

under normal conditions their production in plant cells

is tightly controlled by the scavenging system. ROS

can oxidize biomolecules such as DNA, proteins and

lipids, creating oxidative injury that results in a

reduction of plant growth and development (Hernan-

dez-Jimenez et al. 2002). Since the half-lives of ROS

are extremely short, their stable end products of

oxidative damage to cellular macromolecules can be

used for oxidative stress monitoring (Orhanl et al.

2004). Plants possess a number of antioxidant mole-

cules and enzymes that protect them against the

oxidative damage to control the level and effects of

ROS. They regenerate the active form of antioxidants

and eliminate or reduce the damage caused by ROS

(Alscher et al. 1997). The protection against oxidative

stress is achieved by the production of enzymatic

antioxidants comprised of superoxide dimutase

(SOD), peroxidase (POD) and catalase (CAT) while

glutathione, carotenoids and ascorbate represent non

enzymatic components (Hall 2002; Caregnato et al.

2008). Above all, interplay between different ROI-

producing and ROI scavenging mechanisms can

change drastically depending upon the physiological

condition of the plant and the integration of different

environmental, developmental and biochemical stim-

uli (Mittler 2002).

Therefore, in the present review, we aim to identify

few useful antioxidant enzymes and their strategies in

defense mechanism in plants during phytoremediation

of different heavy metals.

2 Generation of ROS in plants during heavy

metal stress

Exposure to heavy metals is a common phenomenon

due to their environmental pervasiveness conse-

quently causes potential ecological risk. Although

many metals are essential, all metals are toxic at higher

concentrations, because they cause oxidative stress by

formation of free radicals. Another reason why metals

may be toxic is that they can replace essential metals in

pigments or enzymes disrupting their function. Thus,

metals render the land unsuitable for plant growth and

destroy the biodiversity (Ghosh and Singh 2005). High

concentrations of heavy metals in soil can negatively

affect crop growth, as these metals interfere with

56 Rev Environ Sci Biotechnol (2012) 11:55–69

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metabolic functions in plants, including physiological

and biochemical processes, inhibition of photosynthe-

sis, and respiration and degeneration of main cell

organelles, even leading to death of plants (Garbisu and

Alkorta 2001; Schmidt 2003). Based on their solubility

under physiological conditions, 17 heavy metals may

be available for living cells and of importance for

organism and ecosystems (Weast 1984). Among these

metals, Fe, Mo and Mn are important as micronutri-

ents. Zn, Ni, Cu, V, Co, W, and Cr are toxic elements

with high or low importance as trace elements. As, Hg,

Ag, Sb, Cd, Pb, and U have no known function as

nutrients and seem to be more or less toxic to plants and

micro-organisms (Breckle 1991; Nies 1999).

Phytoremediation is an effective and affordable

technological solution used to extract or remove

inactive metals and metal pollutants from contami-

nated soil and water. This technology is environmental

friendly and potentially cost effective (Tangahu et al.

2011). At the end of the ninetieth century, Thlaspi

caerulescens and Viola calaminaria were the first plant

species documented to accumulate high levels of

metals in leaves (Hartman 1975). The idea of using

plants to extract metals from contaminated soil was

reintroduced and developed by Utsunamyia (1980) and

Chaney (1983), and the first field trial on Zn and Cd

phyto extraction was conducted by Baker et al. (1991).

Metal uptake is subjective to the availability of metals,

which is in turn determined by both external (soil

associated) and internal (plant associated) factors.

In 2003 Cho et al. reported that the sensitivity of

plants to heavy metals and potential of plant for

accumulation depends on an interrelated network of

physiological and molecular mechanisms such as:

1. Uptake and accumulation of metals through

binding to extracellular exudates and cell wall

constituents;

2. Efflux of heavy metals from cytoplasm to extra-

nuclear compartments including vacuoles.

3. Complexation of heavy metal ions inside the cell

by various substances, for example, organic acids,

amino acids, phytochelatins and metallothioneins;

4. Accumulation of osmolytes and osmoprotectants

and induction of antioxidative enzymes, and

5. Activation or modification of plant metabolism to

allow adequate functioning of metabolic path-

ways and rapid repair of damaged cell structures

(Cho et al. 2003).

A growing amount of data provide evidence that

metals are capable of interacting with nuclear proteins

and DNA causing oxidative deterioration of biological

macromolecules (Leonard et al. 2004). Metal coordi-

nation chemistry and redox properties have provided

them with an added advantage that these metals could

escape out of the control mechanism such as transport,

homeostasis, compartmentalization and binding to

designated cell constituents. Although, this process

does not occur on a regular basis but such an action by

metals could lead to malfunctioning of cells and

eventually toxicity (Flora et al. 2008). Heavy metal

toxicity comprises inactivation of biomolecules by

either blocking essential functional groups or by

displacement of essential metal ions (Goyer 1997).

In addition, autoxidation of redox-active heavy metals

and production of reactive oxygen species (ROS) by

the Fenton reaction causes cellular injury (Stohs and

Bagchi 1995), when metal toxicity stress point is

reached at the toxic threshold level of the metal in the

tissues of the plants.

In-depth studies in the past few decades have shown

metals like iron, copper, cadmium, mercury, nickel,

lead and arsenic possess the ability to generate reactive

radicals, resulting in cellular damage like depletion of

enzyme activities, damage to lipid bilayer and DNA

(Stohs and Bagchi 1995). These reactive radical

species include wide variety oxygen-, carbon-, sulfur-

and nitrogen- radicals, originating not only from

superoxide radical, hydrogen peroxide, and lipid

peroxides but also in chelates of amino-acids, pep-

tides, and proteins complexed with the toxic metals.

The unifying factor in determining toxicity for all

these metals is the generation of reactive oxygen and

nitrogen species. The toxic manifestations of these

metals are caused primarily due to imbalance between

pro-oxidant and antioxidant homeostasis which is

termed as oxidative stress (Flora et al. 2008).

Metals have high affinity for thiol groups contain-

ing enzymes and proteins, which are responsible for

normal cellular defense mechanism (Flora et al. 2008).

Above certain point of tolerance, the physiological

state of the cells of plant will be irreversibly changed.

This change is reflected by an increase in activity of

certain enzymes defined as enzyme induction or

inhibition of enzyme activity in the plants as result

of tolerance or protective mechanism.

Two mechanisms of enzyme inhibition predomi-

nate during the process of metal uptake:

Rev Environ Sci Biotechnol (2012) 11:55–69 57

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1. Binding of the metal to sulphydryl groups,

involved in the catalytic action or structural

integrity of enzymes, and

2. Deficiency of an essential metal in metallopro-

teins or metalprotein complexes eventually com-

bined with substitution of the toxic metal for the

deficient element (Babaoglu et al. 2009).

Different plants absorb toxic and non-toxic metals

from soil and water to varied extent and accumulate in

different body parts (Chambers and Sidle 1991).

Although the morpho-physiological responses of

plants may vary according to the nature and the dose

of the pollutant and the species but at biochemical and

molecular levels, there appears to be a similarity

among different pollutants and also among most of the

environmental stresses which can be classified as

(Srivastava 1999).

1. Increase in antioxidant enzymes and metabolites

and

2. Induction of protection-related secondary metab-

olite genes. If the plants are able to express these

responses adequately, pollution-induced ‘visible’

or ‘hidden’ damages do not occur. However, if

these protective responses are inadequate and are

unable to cope with the incidence and the dose of

the pollutant, the injury occurs. These responses

therefore, can be compared to immune responses

in animals, which of course are evoked in

response to pathogens (Srivastava 1999).

It is observed that an active antioxidative metab-

olism does not represent a metal tolerance mechanism

but many a times it is beneficial for plant performance

under heavy metal stress. Dietz et al. (1999) and Sahw

et al. 2004 reported that heavy metal induce oxidative

stress in cells and tissues in the following ways:

• They transfer electrons directly in single-electron

reactions, which generate free radicals. The so-

called transition metals (Fe, Cu, Mn, etc.), which

have unpaired electrons in their orbitals, accept

and donate single electrons, thus promoting mono-

electron transfers to O2 and generally ROS inter-

conversion and oxireduction phenomena,

• Metals disturb metabolic pathways, especially in

the thylakoid membrane, which also results in

increased formation of free radicals and reactive

oxygen species,

• In addition, heavy metals mainly inactivate the

antioxidant enzymes (peroxidases, catalases,

superoxide dismutases) responsible for free radical

detoxification, although peroxidases also may be

activated due to metal stress,

• Finally, heavy metal accumulation results in the

depletion of low molecular weight antioxidants,

such as glutathione, which is consumed under

phytochelate formation.

• The activation of the cellular antioxidant metab-

olism belongs to the general stress responses

induced by heavy metals (Dietz et al. 1999).

Irrespective of the production pathway, ROS are

highly cytotoxic and their level within plant cells

must be controlled by enzymatic and non-enzy-

matic antioxidant defense systems. Close contact

between the pollutant and the detoxifying enzymes

of plants that are localized in the cytosol of living

cells are the necessary prerequisite for successful

uptake by plants. The presence and activity of

these enzymes is crucial for a potential metaboli-

zation and further degradation of the chemicals

under consideration. Conjugation to biomolecules

is regarded as a beneficial detoxification reaction.

3 Interaction of antioxidant enzyme

and oxidative stress

A variety of environmental stresses like soil salinity,

drought, extremes of temperature and heavy metals are

known to cause oxidative damage to plants either

directly or indirectly by triggering an increased level of

ROS (Malecka et al. 2001; Shah et al. 2001). A common

feature among the different ROS types is their capacity to

cause oxidative damage to proteins, DNA, and lipids.

These cytotoxic properties of ROS explain the evolution

of complex arrays of nonenzymatic and enzymatic

detoxification mechanisms in plants (Apel and Hirt

2004). Oxidative stress is essentially a regulated process,

the equilibrium between the oxidative and antioxidative

capacities determine the fate of the plant. Under non

stressful conditions the antioxidant defense system

provides adequate protection against active oxygen and

free radicals. In response, the capacity of the antioxida-

tive defence system is increased (Srivastava 1999). The

term antioxidant generally refers to a broad class of

compounds that protect cells from damage otherwise

caused by exposure to certain highly reactive compounds

58 Rev Environ Sci Biotechnol (2012) 11:55–69

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(Salt 2004). An antioxidant is a molecule capable of

inhibiting the oxidation of other molecules. Oxidation is

a chemical reaction that transfers electrons from a

substance to an oxidizing agent. Oxidation reactions can

produce free radicals. In turn, these radicals can start

chain reactions that damage cells. Antioxidant enzymes

terminate these chain reactions by removing free radical

intermediates, and inhibit other oxidation reactions

(Fig. 1). They do this by being oxidized themselves, so

antioxidants are often reducing agents such as thiols,

ascorbic acid or polyphenols (Sies 1997).

4 Different types of antioxidant enzymes

The enzymatic components associated with defense

against ROS include, superoxide dismutase, catalase,

peroxidase and metabolites like glutathione, ascorbic

acid, a-tocopherol, carotenoids (Sairam et al. 2000)

and reduced glutathione which remove, neutralize and

scavenge the ROS.

4.1 Role of antioxidant enzymes

The phyto remedial potential of plants can be assessed

with tolerance mechanisms for toxic metals that

allows plant to combat increased ROS levels during

abiotic stress conditions. Moreover in other circum-

stances plants appear to purposefully generate ROS as

signaling molecules to control various processes

including pathogen defense, programmed cell death,

and stomatal behavior (Apel and Hirt 2004).

Plant damage occurs when the capacity of antiox-

idant processes and detoxification mechanisms are

lower than the amount of ROS production. In plants,

ROS are continuously produced predominantly in

chloroplasts, mitochondria and peroxisomes. There-

fore production and removal of ROS must be strictly

controlled (Apel and Hirt 2004).

The oxidative damage to cellular components is

limited under normal growing conditions due to

efficient processing of ROS through several enzymes

and redox metabolites (Fig. 2). The major ROI-

scavenging pathways of plants include SOD, found

Fig. 1 Antioxidant enzymes confiscating the free radical chain

reaction

Fig. 2 Mechanism of

antioxidant enzymes in

combating oxidative stress

Rev Environ Sci Biotechnol (2012) 11:55–69 59

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in almost all cellular compartments, the water–water

cycle in chloroplasts, the ascorbate–glutathione cycle

in chloroplasts, cytosol, mitochondria, apoplast and

peroxisomes, glutathione peroxidase (GPX); and CAT

in peroxisomes. The finding of the ascorbate–gluta-

thione cycle in almost all cellular compartments tested

to date, as well as the high affinity of APX for H2O2,

suggests that this cycle plays a crucial role in

controlling the level of ROIs in these compartments.

By contrast, CAT is only present in peroxisomes, but it

is indispensable for ROI detoxification during stress,

when high levels of ROIs are produced. In addition,

oxidative stress causes the proliferation of peroxi-

somes. The balance between SOD and APX or CAT

activities in cells is crucial for determining the steady-

state level of superoxide radicals and hydrogen

peroxide. This balance, together with sequestering of

metal ions, is thought to be important to prevent the

formation of the highly toxic hydroxyl radical via the

metal-dependent Haber–Weiss or the Fenton reactions

(Mittler 2002).

The Haber–Weiss reaction generates •OH Hydro-

xyl Radicals from H2O2 and superoxide.

First Step:

Fe3þ þ� O�2 ! Fe2þ þ O2

The second step is the Fenton Reaction:

Fe2þ þ H2O2 ! Fe3þ þ OH� þ� OH

Net reaction:

�O�2 þ H2O2 !� OHþ OH� þ O2

(Michalak 2006).

The coordinated function of antioxidant enzymes

such as SOD, APX, catalase and GR helps in

regeneration of redox ascorbate and glutathione

metabolites (Asada 1996; Foyer and Nector 2000).

In 2005Smeets et al. reported that the affinity of

heavy metals such as Cd to bind to GSH, forming

metal-thiolate compounds, suggesting that GSH

(Fig. 3) might be involved in the synthesis of

phytochelating which could detoxify Cd ions (Smeets

et al. 2005). Potentially toxic heavy metal ions are

firstly chelated by GSH and then transferred to PCs for

eventual sequestration. However, GSH act as a first

line of defense against metal toxicity by complexing

metals before the induced synthesis of PCs arrives at

effective levels (Freedman et al. 1989). Glutathione

(GSH) directly reduces most active oxygen species, is

a major water soluble antioxidant in plants cells

having low-molecular weight thiol tripeptide,

involved in cellular defense against the toxic action

of xenobiotics, oxyradicals as well as of metal cations

(Meister and Anderson 1983). Owing to its redox

active thiol group, GSH is involved in the redox

regulation of the cell cycle (Sanchez-Fernandez et al.

1997) and has often been considered to play an

important role in defense of plants and other organ-

isms against oxidative stress (Grant et al. 1996).

Various free radicals and oxidants are able to oxidize

GSH to GSSG (Noctor and Foyer 1998), hence this

reaction maintains a proper GSH/GSSG concentration

ratio in cells (Rendon et al. 1995). It is also able to

modify metal toxicity by chelating metal ions in cells

and plays a key role in protecting macromolecules

from damage by free radicals by trapping them in an

aqueous phase (Freedman et al. 1989). Glutathione

reductase contains a highly conserved disulphide

bridge between Cys76 and Cys81 (Creissen et al.

1992; Lee et al. 1998), which may undergo cleavage

by heavy metals. Heavy metal-induced loss in gluta-

thione reductase has frequently been observed: in pea

by Zn, Cu and Fe (Bielawski and Joy 1986), in

sunflower by Fe, Cu and Cd (Gallego et al. 1996), in

Lemna minor by Cu (Teisseire and Guy 2000).

The ascorbate- glutathione cycle seems to be a

mechanism of great importance in controlling the

cellular redox status especially after application of

heavy metals such as copper, zinc and cadmium

(Cuypers et al. 2000; Smeets et al. 2005). Ascorbic

acid controls the concentration of oxygen and its

derivatives (OH (AsA) and glutathione (GSH) in the

oxidized and reduced forms are among the most

important non-enzymatic cellular antioxidant defense

compounds by quenching ROS and are important in

controlling the metal.

Another enzymes in the family of enzymes is

‘‘Peroxidases’’ (Fig. 4) which are widely accepted asFig. 3 Structure of Glutathione

60 Rev Environ Sci Biotechnol (2012) 11:55–69

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‘stress enzymes’ (Gaspar et al. 1991). Peroxidases

may can contain a heme cofactor in their active sites,

or redox-active cysteine or selenocysteine residues.

Changes in peroxidase activity have been associated

with wide array of physiological processes involved

with auxin function and cell wall synthesis. The

association with auxin and lignifications made Per-

oxidase analysis informative in response to external

stimuli such as light, temperature, irritation and

wounding, parasites and pathogens and variation in

ion status. Peroxidases are heme containing proteins

that utilize H2O2 in the oxidation of various organic

and inorganic substrates (Asada 1994). Peroxidases

utilizing guaiacol as electron donor in vitro are

guaiacol peroxidases and participate in developmental

processes, lignification, ethylene biosynthesis,

defense, wound healing, etc. Peroxidase which par-

ticipate in lignin biosynthesis might built up a

physical barrier against poisoning of heavy metals

(Rai et al. 2004). The other group of peroxidases

scavenge H2O2 in cell and utilize glutathione, Cyt c,

pyridine nucleotide and ascorbate as electron donors

in vitro (Verma and Dubey 2003). Guaiacol peroxi-

dases are glycoproteins, located in cytosol, vacuole,

cell wall and in extracellular space, while the other

group is non glycosylated and localized in chloro-

plasts and cytosol (Asada 1992). Peroxidase activity

and photosynthetic pigments are sensitive indicators

of heavy metal stress and can be used to anticipate

events on the organism level (Wu et al. 2003; Mac

Farlane and Burchett 2001).

CAT is universally present oxidoreductase and an

important heme-containing enzyme that catalyses the

dismutation of H2O2 to H2O and oxygen and is

localized in the peroxisomes. CAT is found in most

plant and animal cells that functions as an oxidative

catalyst and an indispensable enzyme required for ROS

detoxification in plants. Catalase decomposes H2O2 to

water and molecular oxygen and it is one of the key

enzymes involved in removal of toxic peroxides (Lin

and Kao 2000). Catalases are involved in scavenging

H2O2 generated during the photo-respiration and

b-oxidation of fatty acids (Morita et al. 1994).

While the complete mechanism of catalase is not

currently known, the reaction is believed to occur in

two stages:

H2O2 þ Fe IIIð Þ � E! H2Oþ O ¼ Fe IVð Þ � E �þð Þ

H2O2 þ O ¼ Fe IVð Þ � E �þð Þ! H2Oþ Fe IIIð Þ � Eþ O2

Here Fe (IV)-E represents the iron center of the heme

group attached to the enzyme. Fe(IV) - E(�?) is a

mesomeric form of Fe(V) - E, meaning that iron is

not completely oxidized to ?V but receives some

‘‘supporting electron’’ from the heme ligand. This

heme has to be drawn then as radical cation (�?) (Boon

et al. 2007).

Ascorbate peroxidase is one of the important

peroxidases, of ubiquitous occurrence in plants. It is

regarded as a universal housekeeping protein in the

cytosol and chloroplasts of plant cells. In the cytosol.

Ascorbate peroxidases use ascorbate as a substrate and

are believed to scavenge excess of H2O2 formed in

plant cells under both normal and stress conditions

(Asada 1992). The product of ascorbate oxidation by

ascorbate peroxidase is an ascorbate-free radical

which is reduced back to dehydroascorbate by the

enzyme monohydroascorbate reductase with NADPH

as the electron donor (Asada et al. 1996). Increase in

ascorbate peroxidase activity in response to air

pollutants specially with O3 has been demonstrated

in several species such as in wheat (Bender et al. 1994)

spinach (Tanaka et al. 1985), pumpkin (Ranieri et al.

1994), and Picea abies (Sehmer et al. 1998). The

different affinities of APX (mM range) and CAT (mM

range) for H2O2 suggest that they belong to two

different classes of H2O2-scavenging enzymes: APX

might be responsible for the fine modulation of ROIs

for signaling, whereas CAT might be responsible for

or the removal of excess ROIs during stress (Mittler

2002).

Fig. 4 Structure of Peroxidase

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Superoxide dismutase (SOD) is an enzyme com-

posed of metal-containing proteins that convert super-

oxide radicals into less toxic agents. It constitutes the

main enzymatic mechanism for clearing superoxide

radicals from the plants. It represents a group of

multimeric metalloenzymes catalyzing the dispropor-

tionation of superoxide free radicals, generated by

univalent reduction of molecular oxygen to H2O2 and

O2 in different cellular compartments (Fridovich

1989)

The reaction as follows:

M nþ1ð Þþ � SODþ O�2 ! Mnþ � SODþ O2

Mnþ � SOD þ O�2 þ 2Hþ

! M nþ1ð Þþ � SOD þ H2O2:

Here, M = Cu (n = 1), Mn (n = 2), Fe (n = 2),

Ni(n = 2).

In this reaction the oxidation state of metal ion

oscillates between n, n ? 1(online dictionary.org).

The hydrogen peroxide molecule, which is still a

danger to cells, is then further processed to nontoxic

by-products. In an aqueous environment, the enzyme

catalase degrades the hydrogen peroxide as follows:

2H2O2 ! 2H2Oþ O2

H2O2 is relatively stable and not very reactive,

electrically neutral ROS, but is very dangerous

because it can pass through cellular membranes and

reaches cell compartments far from the site of its

formation.

At least three types of SODs with several

isoforms are present in plants. These are: (1)

chloroplastic or cytosolic Cu–Zn SOD; the cytosolic

Cu–Zn SOD is referred to as Cu–Zn SOD I and

while chloroplastic one is referred to as Cu–Zn SOD

II. (2) Mitochondrial Mn SOD and (3) chloroplastic

Fe SOD. The amino acid sequence of Fe–SOD and

Mn–SOD proteins are similar, whereas Cu–Zn SOD

is different. The genes for different types of SODs

are also identified. For example, Arabidopsis con-

tains multiple SOD genes encoding at least three

Cu–Zn SODs, three Fe–SODs and one Mn–SOD

(Srivastava 1999) (Fig. 5).

The enzymes metabolites such as monodehydro

ascorbate reductase (MDAR), dehydroascorbate

reductase (DHAR) and glutathione reductase (GR)

also play a significant role in scavenging H2O2 mainly

in chloroplasts and in maintaining the redox status of

the cell (Foyer et al. 1997), where ROS are produced

under unstressed conditions also, it may constitute up

to 1% of the total protein (Dalton 1995).

5 Tolerance mechanism of plants during

heavy metal

Metal phytotoxicity occurs when metals move from

soil to plant roots and are further transported to various

sites in the shoots. The most pronounced effect of

heavy metals when accumulated in plant tissues is on

development and growth inhibition, which is insepa-

rably connected with cell division (Kumar and Rai

2007). When plants are subjected to heavy metal stress

it is possible that heavy metal stress reduce the capacity

of the plants to assimilate carbon, this in turn trigger an

increase in photosynthetic electron flux to molecular

oxygen, resulting in the increased production of

superoxide, hydrogen peroxide, and hydroxyl radical.

Since these reactive oxygen species are damaging to

lipids, proteins and pigments, they are rapidly scav-

enged by antioxidant enzymes. There are evidences

that increased levels of these scavenging enzymes may

play a role in limiting the degree of photo damage to

plants (Hodges et al. 1997; Collen and Davison 1999;

Fig. 5 Structure of Mn–SOD

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Rao and Sresty 2000; Schutzendubel et al. 2001). The

ability of plants to increase antioxidative protection to

combat negative consequences of heavy metal stress

appears to be limited since many studies showed that

exposure to elevated concentrations of redox reactive

metals resulted in decreased and not in increased

activities of antioxidative enzymes. However, the

mechanisms involved in those processes are still not

completely understood. In-depth studies in the past few

decades have shown metals like iron, copper, cad-

mium, mercury, nickel, lead and arsenic possess the

ability to generate reactive radicals, resulting in

cellular damage like depletion of enzyme activities,

damage to lipid bilayer and DNA (Stohs and Bagchi

1995). These reactive radical species include a wide

variety of oxygen-, carbon-, sulfur- and nitrogen-

radicals, originating not only from superoxide radical,

hydrogen peroxide, and lipid peroxides but also in

chelates of amino-acids, peptides, and proteins form-

ing complexes with the toxic metals.

In recent development it is reported that antioxi-

dant, prevents certain types of damage to living cells,

appears to allow some kinds of plants to thrive on

metal-enriched soils that typically kill other plants.

According to recent research in Purdue University,

USA. This finding provides an important new insight

for the development of plants that could be used to

help clean polluted sites. The work also answers a

fundamental question for researchers studying how

certain types of plants tolerate levels of metals in their

tissues that are toxic to most other plants. The

interplay of antioxidant enzymes released during

uptake of heavy metals. Therefore, it is crucial that

plants should maintain the activities of these enzymes

in order to accommodate these oxidative stresses. The

main enzyme determining the resistance to the oxida-

tive stresses might be dependent on the plant species

and the metal toxicity. However, some common

reaction patterns can be found in response to the

heavy metals. In most cases, exposure to heavy metals

initially resulted in a severe depletion of GSH as

reported in various studies (Schutzendubel and Polle

2002) for example (Cd: Rauvolfia serpentina: Grill

et al. 1987; pine: Schutzendubel et al. 2001; carrot: di

Toppi et al. 1999; tobacco: Vogeli-Lange and Wagner

(1996); Cu: Silene cucubalus: De Vos et al. 1992; Cu

or Cd: Arabidopsis: Xiang and Oliver 1998; Ni and

Zn: pigeonpea: Rao and Sresty 2000; Fe, Cu or Cd:

sunflower leaves: Gallego et al. 1996). The toxic

effects of heavy metals are exerted at the plasma

membrane and within the cell two different uptake

routes have been reported across the membrane:

(a) passive uptake, only driven by the concentration

gradient across the membrane and (b) inducible sub-

strate-specific and energy-dependent uptake (Nies

1999; Williams et al. 2000). Since the list of studies

for uptake of metals and defense mechanism of plants

during the process of phytoremediation is very long

that are known to cause oxidative damage, we have

limited our few studies reporting the role of these

enzymes in various plants (Table 1).

5.1 Cadmium

The variation in response of antioxidant enzymes to

Cd stress could be due to the variability of plant

species in producing free radicals (Mazhoudi et al.

1997). For example in roots and leaves of Cd-exposed

Phaseolus vulgaris as well as suspension cultures of

tobacco (Nicotiana tabacum) cells contained elevated

APX activities after Cd exposure (Chaoui et al. 1997).

In Phaseolus aureus Cd induced elevated guaiacol

peroxidase (POD) but decreased CAT activities (Shaw

1995). The increase of APX and GPX in different

plants have been reported with Cd (Ashraf et al. 2003;

Karataglis et al. 1991; Rama Devi and Prasad 1998).

Cadmium causes oxidative stress probably through

indirect mechanisms such as interaction with the

antioxidative defence, disruption of the electron

transport chain or induction of lipid peroxidation.

The activation of lipoxygenase, an enzyme that

stimulates lipid peroxidation, has been reported after

cadmium exposure (Michalak 2006).

5.2 Lead

In a study with rice plants it was seen that an

enhancement in the activity of guaiacol peroxidase

during Pb induced stress, suggesting that this enzyme

serves as an intrinsic defense tool to resist Pb-induced

oxidative damage in rice plants. Glutathione action in

stress management is through its direct participation in

H2O2 reduction catalysed by the enzyme glutathione

peroxidase. Glutathione peroxidase activity and

sequences encoding glutathione peroxidase-like genes

have been demonstrated in several species including

Nicotiana sylvestris, Citrus sinensis, Arabidopsis

thaliana, Avena fatua, and Brassica compestris,

Rev Environ Sci Biotechnol (2012) 11:55–69 63

123

indicating that glutathione peroxidase is present in

plants (Eshdat et al. 1997). Pb toxicity causes oxida-

tive stress in plants and the enzymes peroxidases; SOD

and GR appear to play a pivotal role in combating

oxidative stress in plants (Verma and Dubey 2003).

Most of results suggest that plants possess antioxidant

enzymes which operates either unspecifically (SOD

and APX) or depending on the nature of the contam-

ination (CAT, GPX, GRD). Previous studies have also

found a positive relationship between increased POD

and CAT enzyme activity and amounts of heavy metals

such as Cu, Pb and Zn in plant tissue (Mazhoudi et al.

1997; Mocquot et al. 1996). These enzymes remove

superoxide radicals, which are harmful to cell mem-

branes. Over expression of genes encoding these

enzymes in several transgenic plant species conferring

protection against free radicals has also been demon-

strated (Allen 1995). For example it is reported that Pb

toxicity resulted in oxidative stress in rice plants and the

enzymes peroxidases, SOD and GR appear to play a

pivotal role in combating oxidative stress in plants.

Unlike iron, Pb is not and oxido—reducing metal, the

oxidative stress induced by Pb in growing rice seedlings

appear to be indirect effect of Pb toxicity leading to

production of ROS with simultaneous increase of

antioxidant enzymes (Verma and Dubey 2003).

5.3 Chromium

Like copper and iron, chromium is also a redox metal

and its redox behaviour exceeds that of other metals

like Co, Fe, Zn, Ni, etc. Cr was thought to be a non-

redox metal that could not participate in Fenton

reactions. However, other studies have shown that Cr

can indeed participate in Fenton reactions, proving its

redox character (Shi and Dalal 1989). Cr reactivity can

be considered from its interaction with glutathione,

NADH and H2O2, forming OH- radicals in cell-free

systems (Shi and Dalal 1989; Aiyar et al. 1991).

Production of H2O2, OH- and O2- under Cr stress has

Table 1 Examples antioxidant enzymes interaction with plants during phytoremediation of heavy metals

Metals Plants Enzymes

Cu Alhagi camelorum Fisch PC synthesis induced by metals is accompanied by a rapid depletion of total

GSH in plants (Boojar and Tavakoli 2010)

Hydrilla verticillata (L.f.) Decrease in GSH and an elevation in PC levels (Boojar and Tavakoli 2010)

Carthamus tinctorius. Enhanced production of SOD, POD and CAT (Ahmed et al. 2010)

Ni Alyssum murale The significant induction of SOD activity catalase activity and the significant

reduction in GR activity displayed a typical antioxidative enzyme response

pattern (Babaoglu et al. 2009)

Cd Maize seedlings Enhanced the activities of APX and GPX with variation in CAT activity

(Malekzadeh et al. 2007)

Hg Abelmoschus esculentus L. Increased in activity of SOD, APX and GR but CAT activity decreased with

the increased treatment (Hameed et al. 2011)

Phaseolus aureus Increased in activity of APX, (Shaw 1995)

Cd, Ni, and Pb Pteris vittata L. Catalase activity in the plant increased with the increased concentrations of

the metal, but CAT activity decreased at higher concentration as compared

to lower concentration or control in case of Zn and Ni (Fayigaa et al. 2004)

Zn Jatropha seedlings POD activity in the cotyledons and hypocotyls with the increasing zinc

concentrations gradually increased CAT activity in the cotyledons and

hypocotyls increased with the increasing zinc concentration (Luo et al.

2010)

E. crassipes CAT activity increased in response to increasing concentrations of metals

like Ag, Cd, Cr, Pb, Cu but showed decrease in activity with increase in

concentration in Zn and Hg (Odjegba and Fasidi 2007)

Cu, Pb Sainfoin, (Onobrychis vicifolia) Activity and function of three enzymes SOD, CAT and GPX in the leaves

increased (Beladi 2011)

Zn Maize (Zea mays L.) SOD and POD activity increased but at the same concentrtaion of Zn CAT

activity decreased with increase in metal concentrations (Cui and Zhao

2011)

64 Rev Environ Sci Biotechnol (2012) 11:55–69

123

been demonstrated in many plants, generating oxida-

tive stress leading to damage of DNA, proteins and

pigments as well as initiating lipid peroxidation

(Panda and Choudhury 2005). The redox behaviour

can thus be attributed to the direct involvement of

chromium in inducing oxidative stress in plants.

Chromium can affect antioxidant metabolism in

plants. Antioxidant enzymes like SOD, CAT, POX

and GR are found to be susceptible to chromium

resulting in a decline in their catalytic activities.

Unlike other heavy metals Cr detoxification using

Phytoremediation has been little studied (Panda and

Choudhury 2005).

It must be noted that Cr is an non essential element

and toxic metal for plant and hence plant may not

possess any specific mechanism for its transport. In

most of the studies conducted, a gradual decrease in

CAT activity was observed in plants while in mosses

both alleviation and decline was observed (Panda and

Choudhury 2005). During Cr uptake by plants APX

was found to be more efficient in destroying H2O2 than

was catalase under both speciation of Cr. The reason

for this could be that unlike CAT which is present only

in the peroxisome and has low substrate affinities since

it requires simultaneous access of two molecules of

H2O2, APX is present throughout the cell and has

higher substrate affinity in the presence of ascorbic

acid as a reductant (Shanker et al. 2004)

5.4 Copper

It is seen that in C.tinctorius the stimulation of SOD

activity along with CAT seemed to play a protective

role against membrane damage as Cu is particularly

toxic to membranes (Mazhoudi et al. 1997). Thus, it

can be inferred that production of SOD, POD and CAT

may serve as useful biomarkers for Cu tolerance in

plants.

5.5 Zinc

Zinc (Zn) is an essential micronutrient required by

plants for normal growth and development. Like other

heavy metals, excess Zn invariably shows marked

alterations in electron transport, membrane permeabil-

ity and uptake and translocation of nutrient elements

(Wang et al. 2009). The increase in GR activity in

plants growing in soils with Zn addition may be related

to the maintenance of the intracellular levels of reduced

glutathione which is required for phytochelatins bio-

synthesis (Gomes-Junior et al. 2006). In fact, Zn ions

have been shown to induce the synthesis of these

peptides in plants (Gasic and Korban 2007). GR can

also be involved in H2O2 removal by the activation of

ascorbate–glutathione cycle (Asada 1999), in which

APX also plays a role converting H2O2 to water. APX

can be found in different cells compartments, such as

the cytosol and plastids, possibly participating in the

fine modulation of ROS for signaling. In contrast, CAT

might be responsible for the removal of H2O2 when

accumulated Zn in plants tissues exert stress conditions

(Foyer and Noctor 2005), particularly in the peroxy-

somes. A decline in CAT activity was reported in many

plants grown under excess Zn (Andrade et al. 2009).

The decrease in CAT activity observed in plant

supplemented with excess Zn might be due to inhibi-

tion of enzyme synthesis or a change in the assembly of

enzyme subunits (Radic et al. 2010). However, in

Brassica juncea grown under excess Zn increased CAT

activity have been reported by Prasad et al. 1999. These

inconsistent results regarding CAT activity might be

due to differences in the plant organs studied, the plant

growth conditions, the durations and concentrations of

the metals utilized and the plant species.

The results presented in this review showed that

heavy metals increased or inhibited the activity of

catalase, peroxidase and superoxide dismutase in

plants. Therefore exposure of plants to heavy metals

provoke pronounced responses of antioxidative sys-

tems which protects the plants to some extent against

oxidative damage, but the direction of response was

dependent on the plant species, the metal used for the

treatment and the intensity of the stress.

6 Conclusion

Antioxidant enzymes play a major role during the

stress induced by the heavy metal or during uptake of

heavy metal in the technique termed as phytoremedi-

ation. The tolerance mechanisms induced by the

spontaneous functioning of various antioxidant

enzymes helps in slowing down the oxidation of

biomolecules and block the process of oxidative chain

reactions. This role of antioxidant enzymes enables the

plant to survive in stress condition which is reached

when plants accumulate excessive concentrated met-

als in its organs. It can be inferred from the various

Rev Environ Sci Biotechnol (2012) 11:55–69 65

123

reported studies that antioxidative enzymes protects

the plants to some extent against oxidative damage,

but the direction of response is combination of various

parameters vis plant species, the metal used for the

treatment or the type of stress conditions. The present

review consolidated the various studies regarding the

antioxidant enzyme activities of plants under heavy

metal stress and it is well established from the various

studies that Plants have evolved an efficient defense

system by which the ROS is scavenged by antioxidant

enzymes such as superoxide dismutase, catalase,

peroxidase, and Glutathione Reductase (Joseph and

Jini 2010).

Therefore we need to extend our understanding for

factors influencing the tolerance mechanism or

enzymes inhibition or induction under various stress-

ful environments by carrying out studies with various

plants and varying with different parameters vis

temperature, time, pH and other parameters and

different concentrations of different metals. These

would facilitate us to develop strategies for useful

plants to be well adapted to environmental stress

through manipulation of antioxidant system and

further increase the productivity of the plant in stress

condition also. For example overexpression of genes

encoding these enzymes can be used for designing

transgenic plants which enable phytoremediation

above the toxic threshold of particular metal. Under-

standing of antioxidant enzyme response to heavy

metal stress could also provide vital clue to optimizing

efficient ways of phytoremediation and selection of

the most suitable plants for the same.

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