1.
Journal of Endocytobiosis and Cell Research (2014) 33-41 | International Society of Endocytobiology
zs.thulb.uni-jena.de/content/main/journals/ecb/info.xml
Journal of Endocytobiosis and Cell Research VOL 25 | 2014 33
Journal of
Endocytobiosis and
Cell Research
Heavy metal induced adaptation strategies and repair mecha-
nisms in plants
Tayyaba Komal1
, Midhat Mustafa1
, Zeshan
Ali2
and Alvina G. Kazi1*
1Atta‐ur‐Rahman School of Applied Biosciences, National
University of Sciences and Technology (NUST), Islamabad,
Pakistan; 2Ecotoxicology Research Institute, National Agri‐
cultural Research Centre, Islamabad, Pakistan;
*correspondence to: alvina_gul@yahoo.com
Increasing environmental pollution is a consequence of
rapidly expanding population and elevated anthropo‐
genic activities. The fastest growing industry nowadays
with inappropriate management and improper dispos‐
al of toxic waste is causing serious damages not only to
the environment but to its inhabitants as well. Enor‐
mous amounts of liquid and solid wastes are produced
each day by industries and other practices like agricul‐
ture and human activities. These all result in the accu‐
mulation of toxic metals into soils ultimately incorpo‐
rating into food chains causing serious problems in
ecological system. Among different pollutants, heavy
metals are the lethal ones with persistent nature. They
are generated not only by different activities but also
by the natural causes. Air, water and soil compart‐
ments are receiving an ever‐increasing metal load due
to diverse human related activities, which lead to seri‐
ous human and environmental health risks. Soil con‐
tamination with heavy metals is important because it
not only serves as a sink to every anthropogenic activi‐
ty but also transfers hazardous metals to food chain.
Journal of Endocytobiosis and Cell Research (2014) 33‐41
Category: Review
Keywords: heavy metals, stress, signal transduction, glutha‐
tione, phosphorylation, kinase, metal complexes
Accepted: 22 September 2014
____________________________________________________________________
Introduction
Municipal/industrial activities, automobile exhaust fumes,
atmospheric deposition and wastewater/agrochemicals
utilization in agriculture have resulted in significant deteri‐
oration of soil. Soil is a major reservoir of chemicals as it
has the ability to chelate a variety of metals. The presence
of heavy metals in food chain depends on bioavailability of
metal ions in soil. Scarcity of irrigation water and reliance
on municipal/industrial wastewater for irrigation coupled
with other factors has increased metal loads in soils. Heavy
metals are not only toxic to soil flora/fauna but also influ‐
ence the uptake/biodegradation of other contaminants like
mercury, arsenic, lead and cadmium. Wastewater irrigation
is a potential source of heavy metal built‐up in agricultural
soils (Olaniran et al. 2011). Treated wastewater can be a
good alternative of irrigation for barren (arid) and semi‐
barren (arid) lands with insignificant risks of contamina‐
tion (Ben Fredj et al. 2012). Heavy metal contaminated soils
pose a difficult challenge for remediation using plants or
other organisms, with less effective methods for proper
cleanup of the soil. Effective remediation measures using
plants and other organisms are gaining increasing attention
nowadays because of the cost effective accumulation of
metals (Farid et al. 2014). Plants with their rapid develop‐
ment, extensive root system and effective translocation of
metals from roots toward aerial parts make them the best
candidates for such cleanup processes, where optimum
plant growth is a critical requirement for efficient cleanup
(De Paolis et al. 2011). One of the key sources of heavy
metals in soils includes the municipal solid waste. Metals
from municipal wastes leach into the soil that not only
further contaminates the surrounding area but also the
underneath aquifers (Wang et al. 2011). Anthropogenic
events like poor management of industrial/sewage efflu‐
ents, transportation, factory emissions and volcanic activi‐
ties result in soil contamination leading to entire food chain
contamination and serious health risks (Ferrante et al.
2013).
Heavy metals and factors affecting their concen‐
trations
According to a recent study, the most common cationic
metals found in soil are mercury, lead, nickel, cadmium,
zinc, chromium and manganese whereas anionic toxic
metals include arsenic, selenium, boron and molybdenum.
Metals in limited concentrations are natural components of
rock, water, air and living organisms but cause several
problems when their amount exceeds the normal levels
(Besada et al. 2011). The concentration of heavy metal in
soils is correlated with geochemical and biological cycles,
anthropogenic factors such as industrial practices, agricul‐
tural practices and wastewater treatments (Buccolieri et al.
2010). Heavy metals are released in the environment by
both anthropogenic and natural sources. Mining and smelt‐
ing are important anthropogenic sources of soil metal con‐
tamination. It was found in a study that soils over 4000 km2
in the vicinity of mining and smelting areas in England
were contaminated by heavy metals (Thornton 1980).
Metalliferous mining areas result into accumulation of toxic
metals. For example, in a study in Korea on metal disper‐

2. Heavy metal induced adaption in plants, Komal T et al.
34 Journal of Endocytobiosis and Cell Research VOL 25 | 2014
sion in soils and plants in the vicinity of Dalsung copper‐
tungten mine, the peak concentrations of Cu, Cd, Pb and Zn
were found in approximately 100~300 meters in the area
surrounding mine and decreased with the distance. The site
of the mine is greatly influenced by volcanic activities. The
ore minerals of the mine are chalcopyrite (CuFeS2), wolf‐
ramite ([FeMn]WO4) and pyrite (FeS2) (Jung 2008). The
metal binding to soil in a particular area is affected by three
parameters of soil i.e. pH, organic matter and clay minerals
(Li et al. 2009). Increase in pH increases the net negative
charge on soil resulting in more affinity of the toxic ions.
Electrical conductivity, sodium absorption ratio and cation
exchange capacity are significantly altered due to the metal
built up in soils. These parameters in turn affect the soil
quality and cause serious health harms not only to plants
but also to consumers (Ali et al. 2013a). An increase of
carbonates in soil leads to increased mobility of Pb and Zn.
These salts induce the higher ionic strength of the ions
which promotes a higher release of Cd as compared to
other metals (Acosta et al. 2011). Soil moisture regime like
in paddy fields largely affects the transformation rate of
heavy metals where flooding regime and metal reactivity
are directly related (Zheng and Zhang 2011). Fly ash also
contributes to the accumulation of metals in the soils as the
ash of thermal power plant contributes to the buildup of
metals in different soils. In the vicinity of a thermal power
plant, contamination of soil with heavy metals was found
with the higher concentrations in the wind direction
(Agrawal et al. 2010). These factors contribute towards the
accumulation of metals, mostly toxic in nature, which great‐
ly affects not only the immediate soil but also the surroun‐
ding environment.
Heavy metal toxicity in plants
Heavy metal accumulation results into mutations and reac‐
tive oxygen species (ROS) production in plants. It functions
as a signaling molecule and damages plant to a large extent.
These unstable and highly reactive species are produced by
the incomplete reduction of oxygen and the resulting mole‐
cules includes hydroxyl radical (HO‐), superoxide (O2‐),
single oxygen (O12), hydrogen peroxide (H2O2) and lipid
hydro peroxides. (Kim et al. 2011). ROS on generation
actively initiate lipid oxidation and on reaching a certain
threshold activate PCD (Programmed cell death) in plants
(Jambunathan 2010). Arrest of cell cycle and apoptosis in a
cell are characterized by an increase in the amount of ROS
signaling molecules (Lin et al. 2011). In a cell the redox
state is balanced by a delicate balance of energy with the
decreased production of ROS and performs a proper role in
cellular signaling (Suzuki et al. 2012). Compounds that have
a role in alleviating potential abiotic stresses have been
shown to detoxify the ROS signaling pathways (Jami et al.
2010). Cofactors such as Fe, Mg, Ca, Cu and organic mole‐
cules such as heme, FADand biotin are required by many
enzymes to work properly. Heavy metals cause inhibition
of the enzymes by binding to functional groups of the pro‐
teins (Juknys et al. 2012).
Plant responses against heavy metal stress
Heavy metal toxicity occurs as a result of modifications of
several physiological processes occurring at cellular and
molecular level by inactivating enzymes. Heavy metals
affect metabolically important compounds by blocking
functional groups; they also disrupt membrane integrity by
displacing essential elements. The plant cells are exposed
to ROS stress due to interference of heavy metal ions with
electron transport chain in chloroplast membrane (La
Rocca et al. 2009). Heavy metal toxicity elicits a number of
adaptive mechanisms in plants. These responses lower the
metal uptake and accumulation in plant parts. Some mech‐
anisms are meant to detoxify the metal ions such as chela‐
tion. Most of these mechanisms are explained below.
Heavy metals and signal transduction in plants
To deal with heavy metal stress, different strategies are
used by the plant including compartmentalization, metals
export and many others. This can be achieved when a plant
senses increased heavy metal concentration in its vicinity.
It results into activation of complicated signal transduction
network. Stress signaling molecules and stress related
proteins are synthesized; as a result specific genes are
transcriptionally activated that are specific to the metal
stress hence the response to heavy metals is epigenetically
controlled (Cicatelli et al. 2013). This mechanism requires
the coordination of complex biochemical and physiological
processes. Abiotic and biotic stresses affect differently with
varying degrees of damage like loss of leaves, reduction in
growth or wilting, affecting plant growth, development and
various plant growth stages by reprogramming of tran‐
scription at multiple stages. Generally the signaling path‐
way consists of a sequential strategy that includes percep‐
tion of heavy metal concentration, activation of signaling
molecules like lipids and the modulation of endocytic
pathways (Galvan‐Ampudia and Testerink 2011). Along
with plant developmental processes, stress responses are
also mediated by other signaling molecules like jasmonates
(lipid derived signals), annexins etc. (Wasternack and
Kombrink 2010; Jami et al. 2010).
Different heavy metals induce different responses with
different signaling molecules cascade, but a general signal
transduction pathway involves the following: ROS, signal‐
ing pathway, calcium‐calmodulin system, phosphorylation
cascade, mitogen‐activated protein (MAP) kinase and the
hormones (DalCorso et al. 2010).
Phosphorylation cascades
In many cellular processes, phosphorylation is a major
event (Chen et al. 2013). During stress, different proteins of
thylakoid membranes undergo phosphorylation or dephos‐
phorylation in response to biotic or abiotic stresses e.g.
light intensity (Tikkanen and Aro 2012). In most of the
cases, phosphorylation occurs on threonine and serine
residues but it can also occur on tyrosine residues resulting
into many developmental and stress responses (Sasabe et
al. 2011). Alteration in cytokinesis involves the activation of
MAPKKK and proteins like mitotic kinesin. This activation
is done by cyclin dependent kinases (Sasabe et al. 2011).
MAP kinase
Downstream signaling events after sensing the ROS in
plants cells include calmodulin (the Ca binding protein), the
activation of phospholipid signaling and G‐proteins which
ultimately leads to the accretion of phosphatidic acid
and/or activation of MAPK pathways (De Pinto et al. 2012).
In this mechanism of signaling, the pathway has three ki‐
nases that are activated sequentially; MAPK kinase kinase
(MAPKK), MAPK kinase (MAPKK or MKK) and MAP kinases
(MAPK or MPK) (Mishra et al. 2006; Opdenakker et al.
2012). These then phosphorylate different cellular com‐
partments. This signaling pathway is shown to be initiated
as a result of different stresses like abiotic stress (DalCorso
et al. 2010). In reaction to a type of stress e.g. cold in plants,
MAP kinase cascade is activated that, in turn, activates
other signaling pathways, resulting into adaptation to

3. Heavy metal induced adaption in plants, Komal T et al.
Journal of Endocytobiosis and Cell Research VOL 25 | 2014 35
stress (Yang et al. 2010b). Due to this pathway, when the
concentration of stress causative agents increases, the
growth, cell division and differentiation of the plant decre‐
ases (Smekalova et al. 2013). It is proven in a study that
MPK3 and MPK6 are activated in A. thaliana in response to
short term exposure to CdCl2 at low concentration (1 µM),
following the accumulation of ROS (Liu et al. 2010). In
another study, rice MAPK cascade called Oryza sativa
MAPKK (OSMKK4 and OSMKK3) were analyzed. Rice
plants, when exposed to arsenite, showed elevated tran‐
script levels of OSMKK3 in leaves and roots in the first 30
minutes whereas the levels of OSMKK4 were raised in 3
hours (Rao et al. 2011). This cascade plays diverse roles in
signaling transferring information from sensors to respon‐
ders and controlling different processes like proliferation,
differentiation and death. Different biotic and abiotic
stresses produce different responses; these include tempe‐
rature extremes, heavy metals, salinity, high/low osmolari‐
ty, drought etc. (Taj et al. 2010; Pitzschke and Hirt 2010).
Calcium‐calmodulin system
These are known to be secondary messengers to heavy
metals as during stress conditions the concentration of
calcium greatly increases in the cell, stimulating calmodulin
proteins. This system regulates a variety of mechanisms
like regulation of genes, transport of ions across the mem‐
branes, metabolism and tolerance to different metals
(DalCorso et al. 2010). This system is mainly active in re‐
sponse to stress as seen in cold tolerance for the cold ac‐
climatization in coordination with other signaling pathways
(Yang et al. 2010b). In a study it was found that some heavy
metals such as Cd and Cu cause disturbance in the intracel‐
lular levels of Ca which causes impairment in calmodulin
signaling pathway. This regulates the Cd tolerance mecha‐
nisms. It was also found that transgenic tobacco plants over
expressing calmodulin resisted Ni and Pb toxicity efficiently
(Lindberg et al. 2012). A regulated member of this family
CRLK1 (calcium‐calmodulin receptor like kinase 1) helps in
the cold tolerance in plants even under freezing conditions
(Yang et al. 2010a).
Hormones
During heavy metal signaling, hormones have a very im‐
portant role in the regulation of development, growth of
plant along with reproduction, with key functions of the
regulation of defense mechanism against different biotic
and abiotic stresses (Pieterse et al. 2012). These are regu‐
lated in accordance with the concentration of different
metals (DalCorso et al. 2010). Recently, plant hormones
such as indole‐3‐acetic acid (IAA) also known as auxins,
brassinosteroids (BRs) and abscisic acid (ABA) have been
found to play a vital role in stress management (Vázquez et
al. 2013). Auxins seem to play an important role in the
stress regulation as seen in a case in which the exogenous
ABA induces the overproduction of phytochelatins (PCs)
under Cd, Zn and Cu stress in Prosopis juliflora (Usha et al.
2009). Steroid hormones also known as Brassino steroids
(BR), are critical in developmental and detoxification pro‐
cesses as they promote the formation of PCs which chelate
heavy metal ions accumulated in plant cells (Choudhary et
al. 2010). In another study, effects of exogenous application
of BRs were studied on raddish and mustard plants under
copper stress. It was found that BR promoted the shoot and
root growth by overcoming the copper toxicity; BRs were
responsible for antioxidant activity and increased PCs for‐
mation (Choudhary et al. 2011a). Secondary messengers
when combined together like the hormones and MAPK, can
result in effective transcription and signaling under stress
condition (Smekalova et al. 2013). Ethylene is a hormone
normally produced in age related stresses (Khan et al.
2014). Along with it, jasmonic acid and salicylic acid are
also produced. Hormone signaling pathway has positive
and negative regulators that are crucial to hormonal cross‐
talk in stress and defense mechanisms. Auxins, cytokinins,
ABA, brassinosteroids etc. adapt to changes and outcome in
antagonistic and synergistic connections that play im‐
portant roles in different abiotic stress tolerances (Peleg
and Blumwald 2011; Ha et al. 2012). In a more recent
study, various concentrations of the EBL (24‐epi‐
brassinolide); a brassinosteroid, obtained from Brassica
juncea were given to seeds of the same species prior to
sowing for 8 hours. These seeds were than exposed to Ni
stress and results showed improved growth with lower
uptake of Ni ions by the plants (Kanwar et al. 2013). The
EBL foliar spray also improved the antioxidant system by
promoting the formation of catalase, superoxide dismutase,
proline and peroxidase in bean plant under Cd toxicity
(Rady 2011). Choudhary et al. (2011b) studied the influ‐
ence of EBL on other hormones and found that under Cr
stress, EBL increases the formation of IAA to promote the
seedling growth in raddish plant. It also enhances the pro‐
duction of ABA to increase Cr tolerance (Choudhary et al.
2011b).
Formation of metal complexes
Another important strategy for heavy metal detoxification
is the heavy metal chelation by high affinity ligands. In
plants, two types of metal binding peptides are produced
which are phytochelatins (PCs) and metallothioneins
(MTs). Besides these, many other small molecules are also
used in metal chelation inside cytosol.
Phytochelatins
Phytochelatins (PCs) are a family of polypeptides, rich in
cysteine. PCs are synthesized from GSH (glutathione) and
are found in plants and fungi. The enzyme PC synthase is
activated when metal ion binds to it, which converts GSH to
PC. Ions of heavy metals such as Hg, Cu, Ni, Au, Zn and Cd
induce the biosynthesis of PCs. Cd is known to be the
strongest inducer of PCs. PC‐Cd complex is formed by the
attachment of Cd to PC through thiolic group (‐SH) of cyste‐
ine residue. These complexes are sequestered into vacuole
by ABC proteins as mentioned above. Plants are unable to
metabolize or neutralize Cd. Rather they limit Cd circula‐
tion in cytosol or transport it away through xylem and
phloem. PCs also contribute in homeostasis of Cu and Zn by
providing transitory storage for the ions. It has been found
that both heavy metal resistant and heavy metal suscepti‐
ble plants produce PCs. The process of detoxification is not
limited to metal chelation. After the chelated metal complex
is transported inside vacuole, it is stabilized there by form‐
ing complexes with organic acids or sulfides. As a part of
complex mechanism, PCs also transport metal ions.
Metallothioneins
Like PCs, metallothioneins (MTs) are a major family of
cysteine rich, metal binding low molecular weight peptides
and are found in many organisms. Although structure of
plants MTs is different from other organisms, they interact
with heavy metals via thiolic group of their cysteine resi‐
dues. They are predicted to be involved in homeostasis and
sequestering of important heavy metal ions (Coldsbrough
2010). They also provide protection against intracellular
oxidative damage. Based on the arrangement of cysteine
residues, MTs have been divided into three classes. So
different isoforms of MTs exist and their capability to bind

4. Heavy metal induced adaption in plants, Komal T et al.
36 Journal of Endocytobiosis and Cell Research VOL 25 | 2014
and confiscate different heavy metals also varies. Hor‐
mones, cytotoxic agents and ions of the heavy metals such
as Cu, Au, Zn, Hg, Ni, Co and Cd are inducers of MTs. In a
study, three B. rapa MT genes (BrMT1‐BrMT3) were shown
to be differentially regulated in various heavy metal stress‐
es. When the seedlings were treated with Fe, the expression
of all 3 genes was variable. Upon Cu exposure, BrMT1 ex‐
pression was increased as compared to BrMT2 whereas
BrMT3 remained unchanged (Ahn et al. 2012). The tono‐
plast ABC transporter protein was also up‐regulated in
transgenic OsMT1 plants which sequestered the Cd metal
ions inside vacuole thus helping in Cd detoxification (Yang
and Chu 2011). Although animal and fungal MTs have a
clear role in heavy metal detoxification, the precise rela‐
tionship between plant MTs and heavy metals is unknown.
Amino acids, organic acids and phosphate derivatives
Organic acids including malate, citrate and oxalate have
capability to bind metals therefore they are deployed in
heavy metal tolerance mechanisms. Organic acids confer
metal tolerance in 2 ways; the external exclusion and the
internal tolerance. In former, organic acids are secreted
from roots of plants which make stable metal‐ligand com‐
plex with metal, thus hinder the metal ions from entering
and accumulating in sensitive sites in roots (Sharma and
Dietz 2009). Citrate is synthesized in plants by the citrate
synthase enzyme; it has higher affinity for metals as com‐
pared to malate and oxalate. It has been found that citrate
plays a principal role in Fe chelation. Although other heavy
metal ions like Zn, Ni, Cd and Co also have high affinity for
citrate. The amount of citrate produced depends greatly on
Ni exposure (Hassan and Aarts 2011).
Amino acids and derivatives chelate metals thus confer‐
ring plant resistance against toxic metals. Histidine is con‐
sidered the most important free amino acid in heavy metal
metabolism. Presence of putative carboxyl, amino and
imidazole groups makes histidine a versatile metal chelator
(Krämer 2010). It has been found that histidine plays a
major role in tolerance against nickel. In Ni hyperaccumula‐
tor species Alyssum and N. goesingense, the concentration of
histidine in xylem exudate is higher as compared to closely
related nonaccumulator species (McNear et al. 2010). Nico‐
tianamine (NA) is a non‐proteogenic and low molecular
weight amino acid. It is found in root and leaf cells and in
phloem also (Hassan and Aarts 2011). It is synthesized as a
result of condensation reaction of three S‐adenosyl‐L‐
methionine molecules catalyzed by NA synthase (Talke et
al. 2006). NA chelates Cu, Fe and Zn by complex formation,
which are then stored within vacuoles. In A. thaliana and
other plants, NA is involved in influx and efflux of Cu, Zn
and Fe by transporting metals from one cell to other (Klatte
et al. 2009). The membrane YSL transporter family
transport the metal‐NA complexes; such complexes are
substrates for these transporters (Gendre et al. 2007).
Phytate is a principal form of stored phosphorus in
plant cells. The molecule consists of six phosphate groups
which consent the chelation of various cations, including
Ca, Mg, Fe, Mn and K (Heumann 2002; Kumar et al. 2010).
Oxidative stress defense and damaged proteins repair
mechanisms
If the intracellular concentration of metal ions overcomes
the above mentioned defense mechanisms and strategies,
the plant begins to suffer oxidative stress, due to the inhibi‐
tion of metal dependent antioxidant enzyme and produc‐
tion of methylglyoxal (MG) and reactive oxygen species
(ROS) (Hossain and Fujita 2010). This includes the induc‐
tion of enzymes such as catalase (CAT) and super oxide
dismutase (SOD) and the production of non‐enzymatic free
radical scavengers. Study of literature shows many cases of
such inductions. In the leaves of Nicotiana plumbaginifolia,
APX and CAT production was induced in response to excess
Fe exposure. In the same way, CAT3 was induced in B.
juncea plants in response to Cd exposure (Minglin et al.
2005). Cd causes oxidation of CAT in pea plants therefore
reducing its activity, so the plant responds by upregulating
the transcription of CAT gene (Romero‐Puertas et al. 2007).
SOD activity increases in response to prolonged activity of
metals. Wheat leaves respond to excess Cd by increased
SOD levels (Lin et al. 2007).
Glutathione (GSH)
The production of ROS is also stumbled upon by the activa‐
tion of ascorbic acid glutathione scavenging system. GSH is
a low molecular weight, non‐enzymatic antioxidant. It is
one of the major redox buffers and antioxidant found abun‐
dantly in all plant cell compartments (Hossain et al. 2010;
Yadav 2010). GSH plays a role in control of (hydrogen per‐
oxide) H2O2 levels; up‐regulation of GSH levels is of crucial
importance because it induces the defensive strategies
against ROS and MG through different pathways, which
includes the activation and expression of enzymes associat‐
ed with GSH and argininosuccinate (AsA) (Hossain et al.
2012a). GSH plays a key role in various environmental
stresses, metal tolerance and metal chelation because it
acts as an ROS scavenger and a substrate for PC biosynthe‐
sis (Krämer 2010). GSH shields proteins against denatura‐
tion triggered by oxidation under stress conditions. It is
also involved indirectly, in protecting membranes by main‐
taining the reduced state of zeaxanthin and α‐tocopherol
(Hossain and Fujita 2011). GSH (Glu‐Cys‐Gly) is the major
intracellular antioxidant inside the cell and is the precursor
of PCs; it also forms complexes with heavy metals such as
Cd (Wójcik and Tukiendorf 2011). Nevertheless, the major
role of GSH as antioxidant depends on its intracellular
concentration and it varies substantially under Cd toxicity.
It has been observed in B. juncea and B. campestris that Cd
concentration increases the GSH concentration in response
to increasing Cd (Anjum et al. 2008). In Phaseolus vulagaris
and Pisum sativum, Cd treatment induces ascorbate peroxi‐
dase (APX) (Romero‐Puertas et al. 2007). An elevation in
GSH concentration in the leaves, roots and stems under
various metal stresses (Hg, Cd and Pb) was reported by
Huang et al. (Huang et al. 2010). In Holcus lanatus, an As
tolerant species, GSH levels were increased significantly as
induced by As, as compared to species which were sensitive
to As (Verbruggen et al. 2009).
It has been demonstrated in recent studies that nitric
oxide (NO) influences the GSH synthesis, as revealed in a
study with Medicago truncatula in which increased NO in
roots increased the GSH and glutathione (GS) gene expres‐
sion (Xu et al. 2011). S‐nitroso glutathione (GSNO) is
formed, during the interaction of GSH with NO, that may
perhaps interconnect the reactive nitrogen and ROS based
signaling pathways (Xiong et al. 2010).
Glutathione S‐transferases (GST)s
GSTs belong to superfamily of multifunctional phase II
metabolic isoenzymes which are best known for their abil‐
ity to detoxify xenobiotics. GSTs basically catalyze the con‐
jugation of reduced form of GSH with various compounds
to form derivatives that can be sequestered in the vacuole
or secreted from the cell. In addition they also defend
against oxidants and abiotic induced oxidative stress
(Hossain et al. 2012b). An increased GST activity was seen

5. Heavy metal induced adaption in plants, Komal T et al.
Journal of Endocytobiosis and Cell Research VOL 25 | 2014 37
in barley (Hordeum vulgare) when subjected to Cu, Hg, Co,
Cd, Pb and Zn (Valentovičová et al. 2009). In the same way,
another study with rice seedlings showed increased activity
of GST in response to Cd stress (50 µM Cd, 7 days) (Hu et al.
2009). In the callus of the onion, the GST activity increased
substantially in response to Cd stress (1 mM CdCl2)
(Rohman et al. 2010). Time and dose affect the GST levels in
plants so it is likely that in case of severe toxicity, more
effective responses like GST are activated, when basal anti‐
oxidant mechanisms are used up and deplete (Hossain et al.
2010). In a study by Dixit et al. (2011) it was observed that
tobacco plants in which GST gene expression are upregu‐
lated show less lipid peroxidation due to less Cd accumula‐
tion than wild type plants indicating better Cd tolerance.
Glutathione Peroxidase (GPX)
GPXs belong to family of enzymes with peroxidase activity.
They make up important part of the plants, antioxidant
network in different cellular organelles. Their primary
activity is the catalyzation of the reduction process of free
H2O2 to water, ROOHs (organic hydroperoxidases) and lipid
hydroperoxidases to alcohol in the presence of GSH and
other reducing agents (Foyer and Noctor 2011). The mam‐
malian glutathione peroxidases possess higher affinity to
lipid peroxidases than H2O2, and they are homologous to
most of the plants GPX genes. A. thaliana exposed to Cd
stress (11 and 10 µM for 7 days) showed an increase in GPX
activity, indicating protection by GPX against lipid peroxi‐
dases (Semane et al. 2007). Additionally it was also found
that Cu, Hg, and Ni induce and increase the activity of GPX
whereas Co, Zn and Pb exposure has no substantial effect
on its activity (Valentovičová et al. 2009). It was reported
that GPX activity was induced under As stress in other
plants (Gupta et al. 2009).
Dehydroascorbate reductase (DHAR)
DHAR is an important enzyme required in the AsA‐GSH
(ascorbate‐glutathione) reaction in higher plants. The
ascorbic acid is oxidized to form dehydroascorbic acid
(DHA) through spontaneous disproportion. DHAR then
reduces DHA to AsA using GSH (Chen et al. 2003; Yang et al.
2009). It was observed that enzyme activity of DHA reduc‐
tase increased in two barley genotypes when exposed to Cd
stress for 1‐25 days (Chen et al. 2010). In the same way, the
DHA reductase activity amplified in wheat leaves and roots
when exposed to different concentrations of Cd (Paradiso
et al. 2008). Under Ni stress, its activity increased in rice
roots and shoots, showing that Ni maintains the elevated
levels of AsA by activating the AsA regenerating system
(Maheshwari and Dubey 2009). Increase in DHAR activity
was seen in two tomato cultivars; “Josefina” and “Kosaco”
when imperiled to boron stress, in addition to reduction in
AsA content, which indicated that the enzymatic activity
increased to reduce the oxidative stress (Cervilla et al.
2007). In transgenic A. thaliana plants, in which genes for
CAT and GST were overexpressed; when exposed to Cd
stress, a considerable increase in DHA reductase activity
was seen, however, it dropped in non‐transgenic plants
(Zhao et al. 2009).
Glutathione reductase (GR)
GR belongs to an enzyme family which catalyzes the reduc‐
tion of GSSG to GSG using NADPH. Heavy metals in particu‐
lar Cd reduce the GSH/GSSG ratio and activate antioxidant
enzymes such as GR and SOD. The cysthiol group of re‐
duced glutathione (GSH) is oxidized and glutathione reduc‐
tase (GR) catalyzes the reverse reaction by using NADPH,
thus acting as a defense mechanism against Cd‐generated
oxidative stress (Yannarelli et al. 2007). GR helps cell to
resist toxicity caused by RO metabolites; it maintains the
reduced form of GSH and ascorbate in cell which sequen‐
tially retains the cellular redox state heavy metal stress
(Hossain et al. 2011). Nouairi et al. (2009) reported in their
study that glutathione reductase activity in B. napus in‐
creased significantly at lower concentrations of Cd ions and
then dropped when concentrations were raised after 15
days of treatment. While, in B. juncea leaves the GR enzyme
activity levels were unaffected under variable concentra‐
tions of Cd ions. In a study on two cultivars of mung beans,
the GR activity was increased in Cd tolerant and also in Cd
sensitive genotype, in response to Cd stress (Anjum et al.
2011). Whereas, in another study it was observed that the
GR activity decreases considerably when subjected to Cd
stress on seedlings of mung bean (Hossain et al. 2010),
indicating that the GR activity is greatly influenced by the
difference in genotype. In another study it was reported
that GR enzymatic activity in B. napus roots decreased
while in leaves the glutathione reductase buildup was com‐
paratively high when exposed to Cu (Russo et al. 2008).
Upregulation of GR contributes in maintenance of
GSH/GSSG ratio and higher GSH levels under heavy metal
stress which are in turn, used by many enzymes that de‐
pend on GSH involved in ROS and MG metabolism (Hossain
et al. 2012a; Hossain et al. 2010).
Heat shock proteins (HSPs)
Heat shock proteins are signaling molecules released in
metal induced as well as other forms of abiotic stresses.
HSPs are found in all types of cells and are expressed not
only in response to elevated temperatures but in other
stresses also (Dubey 2011). They protect and repair pro‐
teins and act as molecular chaperons to ensure correct
folding. The induction of HSPs by several heavy metal ions
(Al, Cu, Hg, Cd and Zn) has been reported (Dubey 2011).
Gene expression under heavy metal stress
Plants’ adaptation strategies are controlled by genetically
determined and well organized signaling system. To eluci‐
date the plants, heavy metal response strategies,
knowledge of these genes is necessary. The Cd and Hg
treated alfalfa seedlings were studied for the expression of
GSH pathway. The expression of GS, GR1 (cytosolic), GR2
(plastidic), GPX and PCs were studied and analyzed in RT‐
PCR. The specific concentration (30 µM) of Cd has no effect
on the expression of these genes; but same amount of Hg
clearly affected the buildup of some transcripts. Hg caused
accumulation of GR1 and GR2 gene transcripts to the peak
after the metal supply (Ortega‐Villasante et al. 2007). Simi‐
larly the expression of GSH metabolic pathway genes (GST,
GS, GR and γ‐ECS) were studied in Camellia sinensis under
Cd stress. In this plant Cd exposure caused oxidative stress
and upregulation of these genes except GST. Same genes
were studied in A. thaliana with Cd or Cu stress. The syn‐
thesis of GSH, GS and γ‐ECS genes was enhanced (Yadav
and Mohanpuria 2009). In another study it was demon‐
strated that when rice roots were subjected to Cd accumu‐
lation, GST and APX genes were overexpressed (Lee et al.
2010). Lycopersicon esculentum was studied for heavy
metal stress tolerance genes under As or Cr stress. It was
found that HSP 90‐1 and GR‐1 transcripts accumulated in
response to both stresses (Goupil et al. 2009). GST and GR
gene expression increases in Al tolerant soybean on the
exposure to 10 µM Al (Duressa et al. 2010). The glyoxalase I
gene was found to be overexpressed in wheat seedlings in
response to Zn (ZnCl2 10‐20 µM for 24 hours) stress (Lin et
al. 2010).

6. Heavy metal induced adaption in plants, Komal T et al.
38 Journal of Endocytobiosis and Cell Research VOL 25 | 2014
Soil remediation
Besides all adaptation strategies and repair mechanisms
adopted by plants to cope with high concentrations of toxic
metals, there are measures, which can be taken to boost
plants, defense systems. These measures include soil reme‐
diation technologies i.e. solidification/stabilization, excava‐
tion, soil washing, thermal treatment, soil‐vapor extraction
and bioremediation (Hao et al. 2011). All these technologies
can be used to treat soil metal toxicity but bioremediation
is the most effective, environment friendly and economic
option (Ali et al. 2013b). Bioremediation techniques (phy‐
toremediation) require less technological intervention and
are more suited for developing countries. Phytoremedia‐
tion can be used as a promising clean up technology for
contaminated soils. Calcareous soil containing excess cad‐
mium and zinc can be phytoextracted or phytostabilized
using poplar plant (Populous alba L.) (Hu et al. 2013).
Likewise extremely acidic tailings can be phytostabilized
during compost assisted process using plant species like
mesquite, buffalo grass and catclaw acacia (Solis‐
Dominguez et al. 2012). Solanum nigrum L. can be used for
the hyperaccumulation of zinc and cadmium along with the
phytostabilization of nickel, where metallothioneins play
significant role in plant nickel homeostasis (Ferraz et al.
2012). Same is the case with tree species where whole trees
can be used to serve the purpose like Peltophorum ptero‐
carpum, A. mangium, L. leucocephala,P. macrocarpus, E.
camaldulensis and L. floribunda, where A. mangium when
used along with organic fertilizers gives excellent results
(Meeinkuirt et al. 2012). In addition, byproducts of biosolid
compost, leonardite, sugarbeet lime etc. are effectively used
for phytostabilization of trace elements in semi‐arid envi‐
ronments (Perez‐de‐Mora et al. 2011).
Phytoremediation processes can be improved further
by microbial assisted phytoremediation where A. capillaris
compost plant growth promoting bacteria is used for tailing
dams in their phytostabilization (Nicoara et al. 2014). An‐
other example includes plant growth promoting bacteria
with Vicia faba that were used to phytostabilize moderate
copper contaminated soils where Enterobacter cloacae,
Rhizobium leguminosarum bv. Viciae and Pseudomonas sp.2,
examined with co‐inoculation. This results in effective
copper migration from the soil (Fatnassi et al. 2013). Ura‐
nium and other radioactive elements contamination is an
important issue that should be solved immediately as not
only it pollutes the environment but also it has long half‐
life. In an experiment, uranium has been successfully rhi‐
zofiltred (type of phytoremediation) using Hellianthus
annus (sunflower) and Phaseolus vulgaris helping to eradi‐
cate radioactive toxins (Lee and Yang 2010).
Other strategies that can also be adopted for soil metal
remediation include phytodegradation, phytoextraction,
phytotransformation, phytostimulation and phytovolati‐
lization (Park et al. 2011). All these filtration processes can
be best applied by following approach of Haslmayr, which
includes assessment of risk, site investigation remediation
strategy, realization of measures, monitoring and reuse
(Haslmayr et al. 2014).
Conclusion
Different human activities with the increase in human
population have resulted in increase of some toxic com‐
pounds in the soils not present naturally as their constitu‐
ents. This has resulted in various health problems and
risks. This process results in accumulation of heavy metals
in the food chain incorporating not only plants but also
animals. The plants have certain defense mechanisms to
shield themselves and to respond to stimulus to minimize
the health risks. Different anti‐oxidant systems including
enzymatic or non‐enzymatic, symptoms elucidation and
mechanisms of tolerance vary from plant to plant. This
reviewed literature explains different mechanisms of plant
defenses against various toxic metals.
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