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Physiological and antioxidative responses of

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 Plant Gene 11 (2017) 247–254

Contents lists available at ScienceDirect

Plant Gene
journal homepage: www.elsevier.com/locate/plantgene

Physiological and antioxidative responses of medicinal plants exposed to MARK

heavy metals stress
Mahmood Malekia, Mansour Ghorbanpourb,⁎, Khalil Karimanc
a
Department of Biotechnology, Institute of Science and High Technology and Environmental Science, Graduate University of Advanced Technology, Kerman, Iran
b
Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349, Arak, Iran
c
School of Agriculture and Environment M087, The University of Western Australia, Crawley, WA 6009, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Soil heavy metal contamination is a widespread phenomenon that occurs naturally or as a result of
Heavy metals anthropogenic activities such as mining, smelting, fossil fuel combustion and agriculture. Heavy metal
Medicinal plants accumulation in agricultural soils is a major environmental constraint leading to lower crop productivity, and
ROS reduced food/feed safety. Among the non-food crops, medicinal and aromatics plants have been proposed as
Oxidative stress
alternative crops in heavy metal contaminated soils, where an environmental obstacle such as heavy metal can
Elicitor
be exploited to elicit the biosynthesis of invaluable secondary metabolites. Plants use different strategies to cope
Biochemical responses
with the heavy metals entered their cells. Tolerance to a specific heavy metal is controlled by a complex inter-
related cascade of morphological, physiological, biochemical, and genetic mechanisms. Heavy metal ions bind to
the protein groups and are able to replace specific cations in binding sites, resulting in the inactivation of
enzymes and production of reactive oxygen species (ROS), which can cause oxidative stresses such as membrane
lipid peroxidation, damage to RNA and DNA, inhibition of key enzymes, degradation of proteins and oxidation of
amino acids. The ROS include hydrogen peroxide (H2O2), singlet oxygen (½O2), superoxide anion (O2• −),
hydroxyl (HO•), alkoxyl (RO•), peroxyl (RO2•) radicals, and organic hydroperoxide (ROOH). Inside plants, ROS
can be scavenged by both enzymatic and non-enzymatic antioxidant defense systems. Among the non-enzymatic
antioxidants, phenolic compounds, ascorbic acid, tocopherol, glutathione, and carotenoids are well-known for
their role to bind/chelate heavy metals and/or scavenge the ROS generated within plant cells. Activation of
antioxidant enzymes is an intrinsic defense strategy to adjust the ROS contents of cells according to the
metabolic needs at a specific time. These antioxidant enzymes include superoxide dismutase, catalase,
glutathione peroxidase, ascorbate peroxidase, glutathione S-transferase, glutathione reductase and dehydroas-
corbate reductase. This review focuses mainly on the physiological and biochemical responses of medicinal
plants to different heavy metals stresses and the detoxification/antioxidative pathways involved, all of which
may lead to enhanced yield of secondary metabolites i.e. a desirable consequence of an undesirable
environmental factor.

1. Introduction 2010), or 5 or even higher (Lapedes, 1974; Nieboer and Richardson,

From the environmental pollution perspective, toxic elements are
categorized as (i) noncritical, (ii) toxic and relatively accessible, and
(iii) toxic, but very insoluble or very rare (Wood, 1974).
Although the term “heavy metal” has become ingrained in the
environmental literature, quite diverse definitions and uses have been
applied by academia and environmentalists (Shaw et al., 2004). Based
on encyclopedias and dictionaries of the scientific term (Shaw et al.,
2004), heavy metal may refer to all metals with a specific gravity > 4
(Anonymous, 1964; Nieboer and Richardson, 1980; Nagajyoti et al.,
1980).Thus, all the lower members (metals) of the periodic table could
be classified as heavy metal. However, the term heavy metal is
commonly used in the literature of environmental pollution but with
versatile definitions. According to Shaw et al. (2004), metal members of
the periodic table could be classified as heavy metals if they have the
following properties: (1) relatively abundant in the earth's crust, (2)
extracted and used in reasonable amounts, (3) used in places where the
public may be exposed to them, and (4) toxic to human beings. The
phytotoxicity of heavy metals varies with the taxonomical group of
plants. For flowering plants (barley, Horedum vulgare) the sequence

⁎
Corresponding author.
E-mail address: m-ghorbanpour@araku.ac.ir (M. Ghorbanpour).

http://dx.doi.org/10.1016/j.plgene.2017.04.006
Received 15 January 2017; Received in revised form 25 April 2017; Accepted 28 April 2017
Available online 02 May 2017
2352-4073/ © 2017 Elsevier B.V. All rights reserved.
M. Maleki et al.
observed is Hg > Pb > Cu > Cd > Cr > Ni > Zn, and for algae
(Chlorella vulgaris) it is Hg > Cu > Cd > Fe > Cr > Zn > Ni >
Co > Mn (Nieboer and Richardson, 1980).
Heavy metals are primarily derived from naturally occurring
geochemical materials. The heavy metal pollution may occur naturally
(surface mineralizations, volcanic outgassings, spontaneous combus-
tions, forest fires etc.) or through human activities such as mining and
agriculture (Shaw et al., 2004; Nagajyoti et al., 2010). Human inter-
ference, however, has much higher contributions towards environmen-
tal pollution than the natural occurrence (Shaw et al., 2004).
Man-made sources of metal contamination include agricultural
activities such as use of pesticide formulations containing metals, use
of contaminated sewage sludges, solid wastes or fertilizers as soil
amendments, mines, mills, metal smelters, refineries, coal- or oil-fired
electricity generating stations, municipal incinerators and automobiles
(Freedman and Hutchinson, 1981; El-Ramady et al., 2015).
Heavy metals are grouped into essential and nonessential micro-
nutrients for normal plant growth. The essential micronutrients such as
Co, Fe, Mn, Mo, Ni, Zn, and Cu are required for normal plant growth
and take part in redox reactions, electron transfer, and other important
metabolic processes. Nonessential metals such as Pb, Cd, As, Cr, and Hg
are potentially highly toxic for plants (Sebastiani et al., 2004; Rai et al.,
2004).
People from different cultural backgrounds all around the world,
generally assume that chemical drugs are efficient enough to cure
diseases. However, medicine has been progressing at a rapid pace, and
majority of chemical drugs have been demonstrated to cause side
effects, so people have opted to use the medicinal herbs with minimal
side effects (Bahmani et al., 2014; Bahmani et al., 2015; Saki et al.,
2014). Nowadays, people have become more interested in using
medicinal plants to treat diseases due to lower health risks. According
to the World Health Organization (WHO) report, about 80% of people
tend to use the native medicinal herbs to treat their diseases as the first
line of primary health care (Sahito et al., 2003). Medicinal plants can be
consumed in the form of fruits, vegetables, extracts, or drugs for disease
treatment and preventive healthcare (Sahito et al., 2003).
Accumulation of heavy metals in the environment has been an
ongoing process over the past decades, mainly caused by human
interferences in the nature. Medicinal plants growing in their natural
environment are consequently exposed to different types of heavy metal
contaminations. In this review, we explore the physiological and
biochemical responses of various medicinal plants to different heavy
metals and focus on the antioxidative pathways and secondary meta-
bolites involved.
2. Heavy metals uptake
Heavy metals in soil may move towards the root surface either
through diffusion along with the water or by ion exchange between clay
particles and the root surface. Release of metals from soil particles can
be facilitated by root/microbial exudates and activities within the
rhizosphere.
Movement of the elements into roots occurs either by passive
diffusion mediated bythe cell membrane, or via the more common
processes of active transfer against concentration and/or electrochemi-
cal potential gradients (Clarkson and Luttge 1989; Fergusson, 1990;
Stroinski, 1999). The active uptake has been adapted by plants for
absorption of essential trace metals but, simultaneously, the other
available elements are also taken up (Silver, 1983). Metals can move
inside the cell along a concentration gradient through cation channels
located within the cell membrane (Kochian, 1995). Foliar uptake is
another route of entry of metals into plant cells that could happen
through stomata, leaf cuticle, or both. The entry of metals into plant
cells through the foliage is of particular significance from the pollution
point of view because of the possible aerosol deposit from the atmo-
sphere (Kabata-Pendias and Pendias 1984; Lodenius, 1990; Misra and
248
Plant Gene 11 (2017) 247–254
Table 1
Noncritical and toxic metals (Wood, 1974).
Noncritical Toxic but rare Toxic and accessible
Na, K, Mg, Ca, Fe, Li, Ti, Hf, Zr, W, Nb, Ta, Be, Co, Ni, Cu, Zn, Sn, As, Se, Te,
Sr and Al Re, Ga, La, Os, Rh, Ir, Pd, Ag, Cd, Pt, Au, Hg, Tl, Pb,
Ru, Ba Sb, Bi
Mani, 1991; Suszcynsky and Shann, 1995).
3. Effects of heavy metals on medicinal plants growth and
development
Many reports have shown the accumulation of heavy metals in
different parts of medicinal plants (Table 1) (Jiang et al., 2001, 2006;
Chan, 2003; Haider et al., 2004; Rai et al., 2004, 2005; Rai and
Mehrotra, 2008; Kováčik et al., 2006; Srivastava et al., 2006; Khan
et al., 2007; Krejpcio et al., 2007; Mishra et al., 2007; Pandey et al.,
2007; El-Rjoob et al., 2008; Jonnalagadda et al., 2008). The growth and
development of medicinal plants can be adversely affected by high
concentrations of heavy metals (Jiang et al., 2001; Rai and Mehrotra,
2008). Increase of chromium (Cr) concentration in the environment can
cause a significant decrease in plant biomass, root and shoot length, and
contents of proteins, sugars, chlorophyll and carotenoids in Phyllanthu-
samarus (Rai and Mehrotra, 2008). Similar outcomes were previously
obtained in P. amarus plants exposed to Cd while the starch content
increased (Rai et al., 2005). A significant reduction in plant growth and
activity of photosystem II with a concomitant increase in proline levels
were observed in Indian mustard (Brassica juncea) exposed to Cd2 + in a
concentration-dependent manner (Sharmila et al., 2016). Exposure to
Se and Hg completely arrested the shoot development of Portulaca
oleracea at all the concentrations applied. However, concentration-
dependent changes were observed in the development of roots, ranging
from their complete inhibition to variation in their initiation time,
number and length (Thangavel et al., 1999). In another study, Mono-
choriahastata plants exposed to Cd showed several visual toxicity
symptoms such as withering, chlorosis, and falling of leaves, which
were more severe at 15 mg L− 1 of Cd (Baruah et al., 2017). The Cd
quantification by ICP-OES showed that the Cd concentrations were
significantly higher in root tissues than those in the shoot and found to
be in the following order: root > stem > leaf. Seedlings of Allium
sativum L. exposed to 10− 3–10− 2 M Cd exhibited a substantial growth
reduction, but did not develop chlorosis (Jiang et al., 2001). Prolonged
exposure or application of higher metal (Cd and Cu) concentrations to
the roots of 3-week-old Arabidopsis thaliana seedlings resulted in a
growth reduction, combined with visible signs of chlorosis (Cuypers
et al., 2011). Some medicinal plants may get acclimatized to the heavy
metal stress and gradually resume normal growth depending on the
heavy metal species and concentration. When two-months-old seedlings
of Catharanthus roseus grown in soil were transferred to the soil
containing high levels of CdCl2 and PbCl2, senescence of lower leaves
and extensive chlorosis were noticed after four days. However, the
affected plants gradually acclimatized and after 20 days, the chloro-
phyll content was almost comparable to that of plants grown under
normal conditions. In case of CdCl2 treatment, a stunted growth with
reduced leaf area, reduced biomass and sterility were recorded after six
months, while PbCl2-treatedplants showed normal growth and flower-
ing. The total alkaloid content was also found to be decreased in roots
of CdCl2-treated plants whereas no change was observed in case of
PbCl2 (Pandey et al., 2007) (Table 2).
The relative toxicity of heavy metals may depend on the soil
property, plant species, age of the plant and heavy metals species
(Shaw, 1995a, 1995b; Shaw and Rout, 2002). Phaseolus aureus seeds
germinated over Hoagland's solution containing Hg or Cd showed
significant inhibition of root elongation, and the inhibition was more

Table 2
Effects of heavy metals on medicinal plants growth and metabolism.
Plant species
Allium sativum L.
Catharanthus roseus L.
Bowiea volubilis,
Eucomis autumnalis,
Merwilla natalensis
Portulaca oleracea
Phyllanthus amarus
Phyllanthus amarus
Ocimum tenuiflorum
Bacopa monnieri
249
Pimpinella anisum L. (anise),
Carum carvi L. (caraway),
and Foeniculum vulgare L.
(fennel)
Hypericum perforatum
Matricaria chamomilla
Thalictrum rugosum
Vaccinium corymbosum
Brassica juncea L.
Monochoria hastata
Mentha crispa
Ocimum basilicum L. and
Mentha spicata L.
Withania somnifera L.
M. Maleki et al.
Heavy metal Concentration Effects References
Cd 10− 2–10− 3 M Growth reduction Jiang et al., 2001
CdCl2 50–500 μM Inhibition of seed germination, reduction of α-amylase and protease activity in treated seeds, in Pandey et al. (2007)
PbCl2 case of CdCl2 treatment, a stunted growth, reduced biomass and sterility were recorded
Cu, Zn, Cd, Pb, 1–2 mg L− 1 Inhibition of germination, reduction of shoot and root growth Street et al. (2007)
Hg
Se, 0.1–5 ppm Reduction of shoot and root growth Thangavel et al. (1999)
Hg
Cd 20, 50 and 100 ppm Decrease in fresh and dry weight, length of root and shoot, contents of protein, chlorophyll, Rai et al. (2005)
carotenoids and sugar
Cr Decrease in fresh and dry weight, length of root and shoot, contents of protein, chlorophyll, Rai and Mehrotra (2008)
carotenoids and sugar
Cr 10–100 μM Reductions in photosynthetic pigments, protein, cysteine, ascorbic acid and non-protein thiol Rai et al. (2004)
contents
Cd 10–200 μM Reductions in total chlorophyll, protein content. Singh et al. (2006)
Induction in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and
guiacol peroxidase (GPX)
Cd, Cu, Pb, Zn Stimulation of seed germination by Cd and Pb at 6 and 100 mgL− 1, respectively. Jeliazkova and Craker
Reduction of seed germination and root growth by Cu and Zn (2003)
Ni 25 and 50 mM Decrease in the concentration of hypericinand pseudohypericin Murch et al. (2003)
Cd 60 and 120 μM Significant decline in chlorophyll and water content in the leaves Kováčik et al. (2006)
Cu 200 to 500 μM Stimulatory effect on the production of berberine Kim et al. (1991)
Cd 50 and 100 μM Increased phenolic compounds Manquian-Cerda et al.
(2016)
Se, Cd Se mitigates the negative effects of Cd stress in mustard plants through the regulation of Ahmad et al. (2016)
osmoprotectants, antioxidant enzymes, and secondary metabolites
Cd 5–15 mg L− 1 Visual toxicity symptoms such as withering, chlorosis, and falling of leaves Baruah et al. (2017)
Pb 900, 1800, 3600, 7200, and 9000 mg kg− 1 Increasing the production of essential oils Sá et al. (2015)
Pb, Cu and Cd Pb(500, 600, 750, 900 mg kg− 1), Cu (270, 300, Increasing the essential oils quantitative in O. basilicum. However, no significant change in Kunwar et al. (2015)
500,700 mg kg− 1) and Cd (6, 10, 20, 30 mg kg− 1) essential oil composition was observed for M. spicata grown in metal amended soils
Cd 5–300 μM N, P, and K deficiency at higher doses of Cd that also caused stunting of growth, chlorosis, and Mishra et al. (2014)
necrosis
Plant Gene 11 (2017) 247–254
M. Maleki et al.
evident in the seeds germinated and grown over Hg-containing nutrient
solution (EC = 3,μM) than those germinated and grown over Cd-
containing nutrient solution (EC = 13.2,μM). When five-day-old seed-
lings were exposed to various concentrations of Hg or Cd, all the
seedlings died within 24 h of exposure to Cd at a concentration as low
as 30 μM, but not even a single death was recorded in the seedlings
exposed to Hg at concentrations as high as 200 μM. Nevertheless, the
observed lethality of Cd for the fully grown seedlings was found to be
only species-specific (Shaw and Rout, 2002).
The dependence of toxicity of heavy metals on the age of plants has
also been documented in other studies. Seedlings of Phaseolus coccineus
were exposed to Cuat the stationary growth stage of the primary leaves,
and a significant decrease in Chla/b and Chl/Car ratios occurred, but
not when they were exposed at the intensive growth stage of the
primary leaves (Maksymiec et al., 1995, Maksymeic and Baszynski,
1996a, 1996b).
Heavy metal uptake by medicinal plants may interfere with N, P and
K uptake and cause deficiency of these macronutrients. Studies by
Mishra et al. (2014) demonstrated that supplemental exposure of the
medicinal plant Withania somnifera, to Cd caused NPK deficiency at
higher doses of Cd along with stunting of growth, chlorosis, and
necrosis. Total photosynthetic pigment contents enhanced at 50 μM
(1.75-folds higher than control) and reduced by 0.13-folds at 300 μM in
comparison with control. Total chlorophyll (chl a + chl b), chl a, chl b,
and carotenoids decreased by 0.1- to 0.2-folds, respectively, in compar-
ison with control at the toxic Cd concentrations (200 to 300 μM).
Some heavy metals can diminish the toxic effects of another heavy
metal. For example, the mustard (Brassica juncea) plants exposed to
elevated levels of Cd exhibited reduced biomass, pigment content, and
relative water content (RWC) (Ahmad et al., 2016). However, supple-
mentation of selenium (Se) neutralized the negative effects of Cd and
increased biomass, pigment content, and RWC. Osmolyte (proline and
glycine betaine) and sugar content increased under Cd stress and a
further increase was observed with addition of Se. Moreover, Cd
decreased the protein content and supplementation of Se increased it
to appreciable levels.
In addition to the visible toxicity symptoms, damage to fine cellular
structures is the consequence of metal-induced ROS accumulation. The
chlorophyll content of Potamogeton crispus declined with increasing
Pb2 + concentration (from 5 to 50 mg L− 1). Simultaneously, high
concentrations of Pb2 + aggravated the ultrastructural damage to the
leaf cells including swelling of chloroplasts, disruption and disappear-
ance of chloroplast envelopes; swelling of mitochondrial cristae,
deformation and vacuolation of mitochondria; condensation of chro-
matin, dispersion of nucleoli, and disruption of nuclear membrane (Hu
et al., 2007).
Heavy metals could prevent seed germination of medicinal plants
depending on the heavy metal species and concentration. Germination
of Catharanthus roseus seeds (imbibed in GA3 and KNO3 for 24 h)
treated by different concentrations of CdCl2 and PbCl2 was inhibited by
both heavy metals. Both α-amylase and protease activity reduced
substantially in seeds received increasing concentrations of CdCl2 and
PbCl2 (Pandey et al., 2007). Street et al. (2007) showed that Cu and Zn
at 1 mg L− 1 significantly (p < 0.05) reduced the germination percen-
tage of Eucomis autumnalis. Low concentrations (1–2 mg L− 1) of Cu and
Zn negatively affected the root development of the three species Bowiea
volubilis, E. autumnalis and Merwilla natalensis. Also, the Hg concentra-
tions of 0.5 and 1 mg L− 1significantly decreased the germination
percentage of B. volubilis and E. autumnalis, respectively. Cadmium
and Hg at 2 mg L− 1 showed a detrimental effect on the root growth of
B. volubilis. All these heavy metals at 0.5 mg L− 1 significantly decreased
the shoot length of M. natalensis. Jeliazkova and Craker (2003)
investigated the effect of heavy metals (Cd, Cu, Pb, and Zn) on seed
germination and root growth in Pimpinella anisum L. (anise), Carum
carvi L. (caraway), and Foeniculum vulgare L. (fennel). Results showed
that Cd at 6 mg L− 1stimulated seed germination and root growth of
250
Plant Gene 11 (2017) 247–254
caraway by approximately 20% as compared with the control. How-
ever, seed germination and root growth of anise plants reduced at 6 and
10 mg L− 1 of Cd and also at 100 and 500 mg L− 1of Pb. Germination of
caraway seeds, however, was stimulated at 100 mg L− 1of Pb. Copper
and Zn significantly reduced the seed germination and root growth in
all the three plant species. It, therefore, means that the effects of heavy
metals on seed germination and root growth of medicinal plants may
depend on plant/heavy metal species.
4. Effects of heavy metals on secondary metabolism of medicinal
plants
Heavy metals have the potential to elicit the production of
secondary metabolites in medicinal plants, but the effects depend on
the plant species as well as heavy metals type and concentration. It
means that in-depth research needs to be performed to figure out the
appropriate heavy metal species and concentration for a given medic-
inal plant. Medicinal plants that tolerate heavy metal stress may keep
their active components unchanged. The therapeutically active com-
pounds phyllanthin and hypophyllanthin enhanced in
Phyllanthusamarus plants due to the abiotic stress caused by certain
levels of Cd (Rai et al., 2005). Rai and Mehrotra (2008) showed that the
concentration of phyllanthin and hypophyllanthin increased up to
certain levels of Cr. Rai et al. (2004) studied the effects of Cr on
Ocimum tenuiflorum. The Cr-treated plants yielded more eugenol (a
major component of Ocimum essential oil) in comparison to the control
(14.46, 24.61, 16.80, 3.83% more eugenol in plants received 10, 20, 50
and 100 μM of Cr, respectively). The production of phenolic compounds
and antioxidant response of Vaccinium corymbosum L. were investigated
in plantlets exposed to Cd2 + at 50 and 100 μM for 7, 14 and 21 days
(Manquian-Cerda et al., 2016). Treatment by Cd2 + increased the
content of malondialdehyde (MDA), with the highest increase at
14 days. Abundance of chlorogenic acid, the main phenolic compound
found in blueberry plantlets, increased with the addition of Cd2 + to the
growth medium.
Medicinal plants may lose their capability to synthesize active
components under high concentrations of heavy metals. Seedlings of
St. John's wort plants grown in a sterile, controlled environment
supplemented with 25 or 50 mM of Ni completely lost their capacity
to produce or accumulate hyperforin and demonstrated a 15–20-fold
decrease in the concentration of hypericin and pseudohypericin (Murch
et al., 2003).
Active components of certain medicinal plants that are tolerant to
the heavy metal stress could remain unchanged. After 10 days exposure
of 4-week-old chamomile (Matricaria chamomilla L.) plants to low
(3 μM) and high (60 and 120 μM) Cd concentrations, dry mass
accumulation and N content were not significantly altered under any
levels of Cd. However, there was a significant decline in the chlorophyll
and water content of leaves. Among the coumarin-related compounds,
herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-β-
d-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased sig-
nificantly at all the Cd levels tested. Cadmium had no significant effect
on umbelliferone, a stress-related metabolite of chamomile. Lipid
peroxidation was not also affected by even 120 μM Cd. Cadmium
accumulation was approximately seven- (60 μM Cd treatment) to
eleven- (120 μM Cd treatment) fold higher in roots than leaves. At
high concentrations, Cd stimulated K leakage from roots, while it could
stimulate K uptake at the lowest concentration (Kováčik et al., 2006).
Cell suspension cultures of medicinal plants supplemented with
heavy metals may also alter their active components. Stimulatory effect
of CuSO4 on the production of berberine was found in cell suspension
cultures of Thalictrum rugosum (Kim et al., 1991). Maximum enhance-
ment in berberine production was achieved by adding CuSO4at
concentrations between 200 and 500 μM. In addition to stimulating
secondary metabolites production, addition of CuSO4 permeabilized
cells, releasing a large amount of berberine into the medium.
M. Maleki et al.
The chemical composition of essential oils could also be affected by
high concentrations of heavy metals. Sá et al. (2015) investigated the
effect of different concentrations (900, 1800, 3600, 7200, and
9000 mg kg− 1) of Pb on the yield and chemical composition of
essential oil and phytoaccumulation of garden mint (Mentha crispa
L.). Length of root and aerial parts was not significantly affected by Pb
treatments. However, the leaf number, budding, and green mass were
greatly influenced by the presence of Pb in soil, showing a remarkable
resistance. A high concentration of Pb significantly enhanced the
essential oil yield, which was approximately 10 times higher than that
of the control. The chemical composition of M. crispa essential oil was
also affected by high Pb concentrations, and its major component
(carvone) concentration varied from 39.3% for plants grown in non-
contaminated soil (control) to 90% for all cultivations in Pb-contami-
nated soils.
In a greenhouse study, saplings of O. basilicum L. and M. spicata L.
were planted in pots containing soils treated with different concentra-
tions of Pb, Cu and Cd (Kunwar et al., 2015). The essential oil
composition of aerial parts was analyzed by GC and GC–MS. There
were significant quantitative variations in the essential oil of O.
basilicum grown in all heavy metal amended soils. In case of M. spicata,
however, no significant changes were observed in essential oil compo-
sition for plants treated with heavy metals. This is a typical example
where various plant species have differential responses to metals,
highlighting the necessity to choose the right medicinal plant when
dealing with a specific metal contamination in soil. A schematic model
of signaling pathways in heavy metal-induced enhancement of second-
ary metabolite production in plant cell is shown in Fig. 1.
Fig. 1. A schematic diagram illustrating the signaling pathways involved in heavy metal-
induced enhancement of secondary metabolite production. Reactive oxygen species
(ROS) generated from heavy metal-induced oxidative stress oxidize the polyunsaturated
fatty acids (PUFA) to polyunsaturated fatty acids hydroperoxide (PUFA-OOH), which are
subsequently converted to oxylipins, leading to the expression of genes involved in
biosynthesis and accumulation of secondary metabolites in plant cells. Other signaling
pathways associated with heavy metal exposure include production of ethylene and
jasmonic acid through the precursor 12-oxo-phytodienoic acid (OPDA), which are also
induced under stress- and pathogenesis-related component genes.
251
Plant Gene 11 (2017) 247–254
5. ROS production and scavenging
Heavy metals can induce oxidative stress by generating free radicals
and toxic oxygen species (Singh et al., 2006). These oxygen species
react with lipids, proteins, pigments and nucleic acids and cause lipid
peroxidation, membrane damage and inactivation of enzymes, thus
affecting the cell performance and viability. The deleterious effects
resulting from the cellular oxidative state may be alleviated by
enzymatic and non-enzymatic antioxidant pathways of the plant and
may vary at various cellular and sub cellular levels in different plants
(Singh et al., 2006). Plants use a diverse array of enzymes such as
superoxide dismutase (SOD), ascorbate peroxidase (APX), guiacol
peroxidase (GPX), catalase (CAT) as well as low molecular weight
antioxidants like cysteine, non-protein thiol and ascorbic acid to
scavenge different ROS types, thereby protecting potential cell injury
and tissue dysfunction (Halliwell, 1987; Singh et al., 2006).
Heavy metals stress can cause reduction of molecular oxygen and
produce intermediate products such as superoxide radicals (O2•-),
hydroxyl radicals (OH•-) and hydrogen peroxide (H2O2), which are
potentially more toxic and reactive than O2.
Superoxide radicals can reduce metal ions (Mn +) of cells such as
3+
Fe and Cu2 + (Heldt, 2005):
2⋅O−2 + 2H+ → O2 + H2 O2
Superoxide dismutase can catalyze the conversion of superoxide
radicals into H2O2 and O2 (Heldt, 2005):
2⋅O−2 + 2H+ → O2 + H2 O2
Metal ions are reduced in reaction with H2O2 and form hydroxyl
radicals (Heldt, 2005):
H2 O2 + M(n−1)+ → OH− + ⋅OH + M n+
Hydroxyl radicals are very aggressive and can destroy enzymes and
lipids through oxidation. The plant cell has no protective enzymes
against •OH. Therefore, it is necessary for cells to prevent reduction of
metal ions via elimination of superoxide radicals using SOD. Hydrogen
peroxide has a damaging effect on many enzymes and can be eliminated
by CAT and APX (Heldt, 2005). Catalase is a key enzyme that catalyzes
the decomposition of hydrogen peroxide to water and oxygen, and
protects plant cells from the oxidative damage (Chelikani et al., 2004).
Ascorbate peroxidase is another important enzyme for eliminating of
H2O2. During this process, ascorbate, an important plant antioxidant, is
oxidized and converted to radical monodehydroascorbate, which is
spontaneously reduced to the ascorbate through the reduced ferredox-
in. In another way, two monodehydroascorbate molecules can be
converted to ascorbate and dehydroascorbate. Also, dehydroascorbate
could be reduced to ascorbate by glutathione (GSH) in chemical
reactions catalyzed by dehydroascorbatereductase. Glutathione is also
an important antioxidant in plant cells composed of three amino acids,
glutamate, cysteine, and glycine. The GSH oxidation leads to formation
of a disulfide (GSSG) bond between the cysteine residues of two
glutathione molecules. Reduction of GSSG is catalyzed by the enzyme
glutathione reductase, mediated by NADPH as the reducing agent
(Heldt, 2005).
Production of H2O2 is a common plant response to metal exposure.
Cuypers et al. (2011) showed a strong elevation of the H2O2 content in
both roots and leaves of Cd-exposed A. thaliana seedlings, but only in
leaves of the 5 M Cu-exposed plants.
Plasma membranes are considered as a primary target for heavy
metal stress in both roots and leaves (Cuypers et al., 2011). The extent
of lipid peroxidation can differ based on the plant species, tissues types
and the heavy metal involved. In A. thaliana seedlings, exposure to Cu
increased lipid peroxidation in roots stronger than the Cd exposure
(Cuypers et al., 2011). Malondialdehyde is the product of lipid
peroxidation under stress and is regarded as a marker for oxidative
stress. The increased MDA content indicated severe oxidative stress in
M. Maleki et al.
ashwagandha shoots by stimulating excessive ROS generation that
damaged membranes (Mishra et al., 2014). The MDA content increased
in Withania somnifera under Cd stress (Mishra et al., 2014).The MDA
content has also been shown to be increased following treatment of
Catharanthus roseus by both CdCl2 and PbCl2 (Pandey et al., 2007).
Oxylipins are biologically active signaling molecules derived from
oxygenated polyunsaturated fatty acids and are involved in regulation
of gene expression under abiotic stress conditions (Cuypers et al.,
2011). Lipoxygenases (LOXes) are the primary enzymes for biosynthe-
sisofoxylipins. In roots of A. thaliana seedlings, the cytosolic LOX1 was
strongly induced by both Cd and Cu treatments, whereas the LOX2 gene
expression reduced in Cu-exposed plants and also after exposure to 5 M
Cd. Both LOX mRNAs increased in leaves under heavy metal treat-
ments, but the response was stronger for LOX2 (Cuypers et al., 2011).
This shows that the gene expression pattern (here, LOXes) can differ
based on the plant tissue and heavy metal type.
Various nonenzymatic agents accumulate in plants upon heavy
metal exposure, which are involved in heavy metal detoxification and
ROS-scavenging pathways (Mishra et al., 2014). Mishra et al. (2014)
showed that nonenzymatic antioxidants like phenolics, AsA, and
nonproteinthiol (NPSH) have profound role in Cd detoxification at all
concentrations of Cd. Further, other nonenzymatic antioxidants such as
proline, tocopherol, flavonoid, GSH, total ascorbate, and DHA were
involved in Cd detoxification up to certain concentrations, i.e.,
150–200 μM (except proline). Ascorbate (AsA) and DHA are the most
abundant low-molecular weight antioxidants playing a vital role in
defense against metal-induced oxidative stress caused by enhanced
levels of ROS (Mishra et al., 2014).
The O2•− molecules released by NADPH oxidases is an important
source of ROS production at the plasma membrane, a process that is
differentially affected by the heavy metals supplied (Cuypers et al.
(2011). Expression levels of NADPH oxidases (RBOHC/F) genes depend
on the plant organs and heavy metal used. Inhibition of the expression
of NADPH oxidases (RBOHC/F) genes was observed in roots of Cu-
exposed A. thaliana seedlings. Cadmium exposure on the other hand,
resulted in a significant induction in expression of RBOHD gene. In
leaves, the highest induction caused by Cd exposure was found for the
RBOHC gene, and expression of the RBOHF gene was also strongly
induced. Copper exposure significantly increased the abundance of
RBOHD gene transcripts (Cuypers et al., 2011). The outcomes showed
that the expression of NADPH oxidase is dependent on plant tissues,
heavy metals and types of gene transcript.
Peroxidase activity and MDA content peaks were observed in
Potamogeton crispus with an increase in Pb2 + concentration (from 5
to 50 mg L− 1), whereas SOD and CAT activities decreased firstly and
then rose (Hu et al., 2007). Rai et al. (2004) studied the effects of Cr on
O. tenuiflorum. Chromium induced lipid peroxidation coupled with K
leakage. Toxic effects of Cron O. tenuiflorum were reflected by the
reductions in photosynthetic pigments, protein, cysteine, ascorbic acid
and non-protein thiol contents. However, leaves had enhanced proline
content in Cr-treated plants. Hyperactivity of SOD, GPX and CAT
indicated that antioxidant enzymes played an essential role in protect-
ing plants from Cr toxicity. Singh et al. (2006) studied the effects of
different Cd concentrations on B. monnieri, and observed that toxic
concentrations of Cd caused oxidative damage as evidenced by
increased lipid peroxidation and reduced contents of chlorophyll and
protein. However, B. monnieri was able to combat metal induced
oxidative injury through activation of various enzymatic and non-
enzymatic antioxidants. The impact of short-term (2 days) and long-
term (4 days) applications of Cd (40 μg/g soil) on spinach plants was
investigated in sulphur (S)-sufficient (300 μM SO42 −) and S-deficient
(30 μM SO42 −) soils (Bagheri et al., 2016). Compared with the control
(+ S and − Cd), the oxidative stress was increased by S deficiency (− S
and − Cd), Cd stress (+S and + Cd) and their combination (− S and
+ Cd). The trend of stress severity was as follows: (S deficiency) < (Cd
stress) < (S deficiency and + Cd stress). The maximum decline in
252
Plant Gene 11 (2017) 247–254
ascorbate (ASA), dehydroascorbate (DHA), total ascorbate (ASA + D-
HA, hereafter TA), glutathione (GSH) and total glutathione (GSH
+ GSSG, hereafter TG) was observed under S deficiency. However,
the TA and total TG contents increased significantly, besides a marginal
increase in their reduced and oxidized forms, when Cd was applied in
the presence of sufficient S. The S supply also enhanced the activity of
SOD, APX, glutathione reductase (GR) and CAT under Cd stress,
whereas their activity was diminished by S deficiency and Cd stress
under S deficiency conditions. Contents of soluble proteins and photo-
synthetic pigments declined under both S and Cd stresses, where the
highest decline belonged to S deficiency and dual stress treatments,
respectively. Plant fresh and dry weights, although adversely affected
by every stress applied, declined most under the dual stress conditions.
It may be concluded that an optimal level of S is required for better
response of SOD, APX, GR and CAT activity under Cd stress.
Cadmium stress has also been shown to induce stress-related
responses and defense pathways in roots of hydroponically grown
Medicago truncatula seedlings (Rahoui et al., 2017). The Cd stress
(100 μM) initially increased the ROS levels, enhanced antioxidative
(total SOD, CAT, and PRX) and ascorbate–glutathione-related metabo-
lism enzymes (APX and MDAR) in all the genotypes tested except A17
and TN1.11. In agreement with peroxidase enhancement, the soluble
phenolic compounds were also accumulated under Cd treatment.
However, the lignification event was restricted to the recently created
protoxylem elements established in the root tip area, usually constitut-
ing the elongation zone. In the absence of necrotic reactions, develop-
mental changes such as lignin deposition, increase in cellulose and
pectin contents, intercellular meatus, along with condensed and
deformed hairs were detected in roots of Cd-treated plants.
Rajpoot et al. (2016) studied the response of rice (Oryza sativa L.)
seedlings raised for 8 days in hydroponics in Yoshida nutrient medium
containing 200 μM NiSO4. The negative effects caused by the Ni stress
included a decline in height, reduced biomass, loss of plasma membrane
integrity in roots, increased levels of O2−, H2O2 and %OH, enhanced
lipid peroxidation, and a decline in photosynthetic pigments. Moreover,
the levels of antioxidative enzymes SOD, CAT, and GPX increased and
alterations in their isoenzyme profile patterns were also observed under
Ni treatment.
Mustard (Brassica juncea) plants exposed to elevated levels of Cd
exhibited increased production of H2O2 and lipid peroxidation, electro-
lyte leakage, along with enhanced activities of antioxidant enzymes
such as SOD, APX and GSH (Ahmad et al., 2016). Supplementation of Se
decreased accumulation of H2O2 and lipid peroxidation, and increased
the activities of antioxidant enzymes to greater levels, and regulated Cd
accumulation in roots and shoots. Ascorbic acid and flavonoids
decreased with elevated concentrations of Cd; however, tocopherol
and total phenols increased with the same concentrations of Cd.
Selenium application maintained AsA and flavonoid content, and
further increases in tocopherol and total phenols were observed.
Overall, the results confirmed that exogenous application of Se can
mitigate the negative effects of Cd stress in mustard plants via
regulation of osmoprotectants, antioxidant enzymes, and secondary
metabolites.
ROS accumulation in plants has been shown to be linked with
damages to DNA, proteins and lipids (Lopez et al., 2006). Lin et al.,
2007 investigated the response of Vicia faba plants exposed to 5 and
10 mg L− 1 Cd for 4 d by monitoring the distribution of Cd in plant, the
effects on the cell lipids, antioxidative enzymes and DNA damages in
leaves. The Cd concentration in plants increased in Cd-treated plants.
The enhanced levels of lipid peroxidation in leaves and H2O2 concen-
tration in root tissues were attributed to the oxidative stress caused by
Cd. Damage to DNA was also detected by neutral/neutral, alkaline/
neutral and alkaline/alkaline Comet assay. Raised levels of DNA
damage occurred with reference to the oxidative stress in leaves,
proposing that the oxidative stress induced by Cd accumulation in
plants contributed to the DNA damage observed. Therefore, damage to
M. Maleki et al.
DNA was possibly an important mechanism of Cd-phytotoxicity in Vicia
faba plants.
Kumar et al., 2013 studied the response of the Talinum triangulare
(Jacq.) Willd. Plants exposed to 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM of
Pb for 7 days, by focusing on the root cellular biochemistry, DNA
damage, structural, and elemental analyses. The results revealed that
Pb stress increased the ROS production, lipid peroxidation, protein
oxidation, cell death, and DNA damage and reduced the protein content
in a dose-dependent manner.
The heavy metal-induced ROS can activate signal transduction
cascade through the mitogen-activated protein kinase (MAPK) path-
ways (Panda and Achary, 2014; Jonak et al., 2004; Lin et al., 2005; Liu
et al., 2010). The MAPK cascade consisting of the MAPKKK-MAPKK-
MAPK module is considered as one of the major routes that mediates
transduction of extra cellular stimuli to the intracellular responses
(Panda and Achary, 2014). MAPKKK activates MAPKK through phos-
phorylation and MAPKK can phosphorylate MAPK. The MAPKs, extra-
cellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal
kinase (JNK) and p38 MAPK transmit extracellular signals into the
nucleus, and have been shown to participate in a broad range of cellular
functions such as cell growth, differentiation and apoptosis. Studies
have shown the role of MAPK in heavy metal stress. Treatment with Cd,
inorganic Hg or tributyltin can activate ERK, JNK and p38 MAPK
(Matsuoka and Igisu, 2002). The MAPK cascade also plays important
role in regulation of genes and activates transcription factors (TFs),
enzymes and proteins (Luo et al., 2016; Yang et al., 2003). Yeh et al.
(2004) found that OsMAPK2 expressed in rice suspension-cultured cells
in response to 100–400 μM Cd. Exposure of alfalfa (Medicago sativa)
seedlings to excess Cu or Cd ions activated four distinct MAPKs: SIMK,
MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK
activation revealed that SIMK, MMK2, MMK3, and SAMK were
activated very rapidly by Cu ions, while Cd ions induced delayed
MAPK activation (Jonak et al., 2004). Exposure to 10 μM CdCl2 for 8 h
caused a prolonged activation of Erk kinase and accumulation of c-fos
mRNA (Ding and Templeton, 2000). The c-Jun kinase/stress-activated
protein kinase (SAPK) was also activated by Cd2 +. The studies
mentioned above indicate that specific cascades and enzymes could
be targeted by different heavy metals.
6. Conclusions and future perspectives
Medicinal plants can be exposed to different kinds of abiotic stresses
in their natural environment such as cold, drought, salinity, and heavy
metal toxicity. Occurrence of heavy metals in biosphere is due to both
natural phenomena and anthropogenic activities related to industriali-
zation and urbanization. Large quantities of heavy metals are found in
the soil and aquatic ecosystems, while a relatively smaller proportion is
present in the atmosphere as particulate or vapors. Heavy metals
toxicity influences the performance of individual plants, plant commu-
nities, and consequently affects functionality of the whole ecosystem. In
soil, the heavy metals accumulation is of a great concern as they can be
taken up and assimilated by agricultural and medicinal plants that are
consumed by humans as food or medicine. The uptake of heavy metals
triggers a set of complex physiological and biochemical changes
followed by alterations in plant primary and secondary metabolism.
In general, seed germination, growth and development of medicinal
plants can be adversely affected by high concentrations of heavy
metals. The relative phytotoxicity of heavy metals, however, depends
on the soil properties, plant species, age of the plant, and heavy metals
species and concentration.
Heavy metals also affect the production of secondary metabolites,
which is of high significance in the context of medicinal plants. Many
studies have confirmed the positive effects of heavy metals on produc-
tion of valuable secondary metabolites in medicinal plants. However,
achievement of the desirable effect on secondary metabolism may
depend on the medicinal plant species and species/concentration of the
253
Plant Gene 11 (2017) 247–254
heavy metals. This means that in-depth research needs to be performed
to figure out the appropriate heavy metal species and optimal concen-
tration for a given medicinal plant and/or metabolite.
The ROS generated in medicinal plant cells under heavy metal stress
is an initial signal of stress, followed by MAPK cascades and activation
of many TFs, and enzymes such as antioxidant enzymes and proteins. In
general, the secondary metabolism may be upregulated through the
enzymatic/nonenzymatic pathways that are activated to scavenge the
ROS generated during the metal stress. Therefore, when dealing with
metal-contaminated soils that are not suitable for crop production, the
viable option could be the cultivation of appropriate medicinal plants
for production of valuable secondary metabolites such as phenolics,
essential oils, AsA, nonproteinthiol, tocopherol, flavonoid, etc. Finally,
certain heavy metals can mitigate the adverse effects of other metals,
which need to be further considered when managing heavy metal
stress.
Our knowledge is quite limited on the foliar uptake of heavy metals,
biochemical pathways involved, and the effects on biosynthesis of
different secondary metabolites in medicinal plants, which is important
for managing airborne heavy metal pollution. Furthermore, the role of
enzymatic/non-enzymatic antioxidants and carrier genes in detoxifica-
tion of heavy metals is not fully elucidated for different types of
medicinal plants, which needs to be explored for both root and foliar
routes of heavy metals uptake.
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