Open Journal of Metal, 2013, 3, 34-41
http://dx.doi.org/10.4236/ojmetal.2013.32A1005 Published Online July 2013 (http://www.scirp.org/journal/ojmetal)
Brassinosteroids and Plant Responses to
Heavy Metal Stress. An Overview
Miriam Núñez Vázquez*, Yanelis Reyes Guerrero, Lisbel Martínez González,
Walfredo Torres de la Noval
Department of Plant Physiology and Biochemistry, National Institute of Agricultural Sciences,
San José de las Lajas, Cuba
Received June 1, 2013; revised July 4, 2013; accepted July 11, 2013
Copyright © 2013 Miriam Núñez Vázquez et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Soil contamination with heavy metals has become a world-wide problem, leading to the loss in agricultural productivity.
Plants have a remarkable ability to take up and accumulate heavy metals from their external environment and it is well
known that high levels of heavy metals affect different physiological and metabolic processes. Brassinosteroids are
considered as the sixth class of plant hormones and they are essential for plant growth and development. These com-
pounds are able of inducing abiotic stress tolerance in plants. In this paper, information about brassinosteroids and plant
responses to heavy metal stress is reviewed.
Keywords: Heavy Metal Stress; Brassinosteroids; Tolerance
1. Introduction
Soil contamination with heavy metals has become a
world-wide problem, leading to the loss in agricultural
productivity and hazardous health effects as they become
a part of the food chain [1]. However, their availability in
the soil is determined by natural processes, especially the
lithogenic and pedogenic ones, and by anthropogenic
factors [2]. Plants have a remarkable ability to take up
and accumulate heavy metals from their external envi-
ronment and it is well known that some of these metals
such as Cu, Zn, Mn, Fe are required for normal plant
growth and development at trace levels [3], since they
are structural and catalytic components of proteins and
enzymes. However, high concentrations of them and oth-
ers such as Al, Cd, Cr, Pb affect different physiological
and metabolic processes at cellular and organism levels.
Toxicity mechanisms include the blockade of func-
tional groups of important molecules, e.g. enzymes, poly-
nucleotides, transport systems for essential nutrients and
ions, displacement and/or substitution of essential ions
from cellular sites, denaturation and inactivation of en-
zymes, and disruption of cell and organellar membrane
integrity [4].
Heavy metal toxicity can elicit a variety of adaptive
responses in plants. These responses are based on mecha-
nisms that lowering metal uptake and accumulation by
plants. A common mechanism for heavy metal detoxifi-
cation is the chelation of the metal ion by a ligand. Such
ligands include organic acids, amino acids, peptides and
polypeptides. Peptide ligands include the phytochelatins
(PC), which detoxifies intracellular metals by binding
them through thiolate coordination [4].
Plant hormones such as auxins (indole-3-acetic acid,
IAA), abscisic acid (ABA) and brassinosteroids (BRs)
have been recently found to work as vital components of
stress management. Auxins have been observed to ame-
liorate the intensity of various stresses such as salinity,
drought [5], chilling [6], heat and heavy metal stress [7].
Similarly, ABA triggers plant responses to adverse envi-
ronmental stimuli [8]. Enhanced synthesis of PC contents
by exogenous ABA has been reported in Prosopis juli-
flora under Cu, Zn and Cd stress [9].
BRs are steroidal hormones which play a critical role
in a range of developmental processes and they have also
been implicated in plant responses to abiotic stress. Their
ability to improve antioxidant system by elevating the
activities and levels of enzymatic and non-enzymatic an-
tioxidants has made them a favorite tool to increase re-
sistance potential of important agricultural crops against
various abiotic stresses such as heavy metal excess
*Corresponding author.
C
opyright © 2013 SciRes. OJMetal

M. N. VÁZQUEZ ET AL. 35
[10,11].
2. Brassinosteroids and Plant Responses to
Heavy Metal Stress
BRs are able to regulate the uptake of ions into the plant
cells and can be used to reduce the accumulation of
heavy metals [12], because they can reduce the metal
uptake by roots [13] and can also stimulate the synthesis
of some ligands such as the phytochelatins, which are
combined with metal ion [14,15]. They also increase the
activities of some antioxidant enzymes detoxifying the
increased production of reactive oxygen species (ROS)
generated by heavy metal stress [16-18] and so exoge-
nous applications of BRs improve the growth and meta-
bolic activity in plants under heavy metal stress.
2.1. Responses to Copper (Cu) Stress
Copper is an essential transition metal required for nor-
mal plant growth and development at trace levels [3,19].
It is an indispensable component of diverse plant meta-
bolic reactions, such as a structural element in regulatory
proteins and its participation in photosynthetic electron
transport, mitochondrial respiration, oxidative stress re-
sponse, cell wall metabolism and hormone signaling are
well established [20]. Among pollutants of agricultural
soils, Cu has become increasingly hazardous due to its
involvement in fungicides, fertilizers and pesticides [21].
However, excess of Cu metal catalyzes the formation
of ROS [22,23]. These ROS are highly toxic and oxidize
important macromolecules such as nucleic acids, proteins
and lipids, thereby disturbing cell stability and membrane
permeability [24,25]. Also, the reduced shoot and root
growth, decline in photosynthetic pigment formation
have been observed in plants under Cu stress [26].
Effects of exogenous applications of BRs (24-epi-
brassinolide, EBL) have been studied on mustard and
radish plants under copper stress. Sharma and Bhardwaj
(2007) observed an improvement in the shoot emergence
and plant biomass production under Cu stress when
mustard seeds were soaked for 8 hours in EBL (10−7,
10−9 and 10−11 M) solutions. In addition, EBL blocked Cu
metal uptake and accumulation in these plants [12].
In radish seedlings, EBL-treated seeds (10−7 M) showed
a reduced copper toxicity by stimulating the root and
shoot growth [15]. This growth response was associated
with enhanced IAA and ABA concentrations, lowered
oxidative stress (a major increase of antioxidant enzyme
activities, a major decrease of MDA and increased con-
tents of antioxidant metabolites such as proline, ascorbic
acid and total phenols) and increased phytochelatin con-
tent in the seedlings. Effects of EBL on antioxidant ca-
pacity and free radical scavenging activity of radish
seedlings were also studied [27].
2.2. Responses to Nickel (Ni) Stress
Nickel is one of the most abundant heavy metal con-
taminants of the environment due to its release from
mining and smelting practices. It is classified as an es-
sential element for plant growth [28]. However, at higher
concentrations, nickel is an important environmental
pollutant. Ni2+ ions bind to proteins and lipids such as
specific sub-sequences of histones [29] and induce oxi-
dative damage. Excess of nickel affects chlorophyll bio-
synthesis, since it affects both the synthesis of δ-ami-
nolevulinic acid and protochlorophyllide reductase com-
plex [30]. Ni2+ also replaces the Mg2+ ion of chlorophyll
pigment [31], causes the inhibition of enzymes of chlo-
rophyll biosynthesis [32] and stimulates chlorophyllase
[33], ultimately leading to a decline in the level of chlo-
rophyll pigment. Nickel also causes a significant inhibi-
tion in the activities of enzymes associated to carbon
fixation in plants [34].
EBL and 28-homobrassinolide (HBL) protect plants
under nickel stress conditions. Mustard (Brassica juncea)
plants treated with Ni2Cl and later, with a HBL foliar
spray showed no nickel toxic effect on growth, net pho-
tosynthetic rate, chlorophyll content and nitrate reductase
and carbonic anhydrase activities. Moreover, HBL treat-
ment stimulated the activities of some antioxidant en-
zymes such as peroxidases and catalases and the level of
proline [35]. Similar results were obtained in this specie
by Ali et al. (2008) using EBL foliar spray and by
Sharma et al. (2008) with HBL seed treatment for 8
hours [36,37]. EBL enhanced the level of antioxidant
system (superoxide dismutase, catalase, peroxidase and
glutathione reductase and proline), under stress condi-
tions. The influence of EBL on antioxidant system, at
least in part, increased the tolerance of mustard plants to
NiCl2 stress and thus protected the photosynthetic ma-
chinery and the plant growth.
Recently, a research was performed in which various
concentrations of the isolated EBL from Brassica juncea
leaves were given as pre-sowing treatment to the seeds of
this same species for 8 h. The pre-treated seeds were
subjected to nickel stress. Results showed that pre-treat-
ment of isolated EBL lowered the Ni ion uptake in plants
and improved growth. The amelioration of Ni toxicity
was also observed by the activities of antioxidant en-
zymes [28].
The results described above associate the BR protec-
tion to Ni stress in plants, particularly in mustard, with
the lowering of oxidative stress, because of the increase
of antioxidant enzyme activities and the proline level. It
may explain the protection to photosynthetic machinery
and the plant growth stimulation.
2.3. Responses to Cadmium (Cd) Stress
Cd is not an essential nutrient and it is one of the heavy

M. N. VÁZQUEZ ET AL.
36
metals that are known to generate toxicity even at a very
low concentration. It accumulates in plants during growth
in edible parts, thereby, endangering crop yield and their
quality. This causes a potential hazard to human and
animal health. Cd is known to cause enzyme inactivation
and damages cells by acting as antimetabolite or forms
precipitates or chelates with a number of essential me-
tabolites [38]. Cd inhibits plant growth [39], retards the
biosynthesis of chlorophyll [40], alters water balance
[41], decreases the activities of various enzymes [42], sti-
mulates stomatal closure [43] and controls photosynthe-
sis [34,44]. Cadmium stress reduces the uptake of essen-
tial mineral nutrients and also affects the activity of AT-
Pase of plasma membrane [45].
Various researchers have demonstrated that BRs re-
duce the adverse effects induced by Cd stress in plants.
Thus, Janeczko et al. (2005) found that EBL reduced the
toxic effect of Cd on photochemical processes by dimin-
ishing the damage of photochemical reaction centers and
the activity of O2 evolving centers as well as maintaining
efficient photosynthetic electron transport [46]. Probably,
the effectiveness of the protective action of EBL in-
creases in older tissue, which is more susceptible to da-
mage by Cd. Later, Anuradha and Rao (2009) reported
that EBL stimulated photosynthetic activity in radish
plants under Cd stress [11].
On the other hand, EBL foliar spray enhanced the
level of antioxidant system (superoxide dismutase, cata-
lase, peroxidase and glutathione reductase, and proline)
of bean plants under Cd stress conditions [17]. This au-
thor suggested that the elevated level of antioxidant sys-
tem, at least in part, increased the tolerance of bean
plants to CdCl2 stress, thus protected the photosynthetic
machinery and the plant growth. Similar results had been
reported in mustard [47] and chickpea [16] plants using
EBL and HBL, respectively.
The application of BRs (EBL and HBL) improved the
chlorophyll content and photosynthesis efficiency of Cd-
stressed tomato plants applied as shotgun approach [48].
Besides, BR treatment significantly increased the number
of fruits, fruit yield and lycopene and β-carotene contents
in the fruits from plants grown under Cd stress.
2.4. Responses to Other Heavy Metal Stresses
Zinc (Zn) is an essential microelement, the second most
abundant transition metal after iron (Fe) and plays a piv-
otal role in many metabolic reactions in plants [49,50].
However, high concentrations of Zn are toxic, induce
structural disorders and cause functional impairment in
plants. At organism level, Zn stress causes reduced root-
ing capacity, stunted growth, chlorosis and at cellular
level alters mitotic activity [51,52], affects the membrane
permeability, the electron transport chain, the uptake and
translocation of nutrient elements [53,54] and induces
oxidative stress by promoting the generation of ROS.
Since Zn is a non-redox metal, it cannot generate ROS
directly through Haber-Weiss reactions, over production
of ROS and occurrence of oxidative stress could be an
indirect consequence of Zn toxicity.
Recently, Ramakrishna and Rao (2012) reported that
the application of EBL significantly alleviated the zinc
induced oxidative stress. EBL had a protective role on
lipid peroxidation, protein oxidation and membrane in-
tegrity in radish seedlings [55]. This BR induced lower-
ing of ROS levels, MDA and carbonyl levels could be
attributed to the increased activities of ROS scavenging
enzymes and the decreased activities of lipoxygenase
(enzyme which catalyzes the oxygenation of polyunsatu-
rated fatty acids into lipid hydroperoxides), and NADPH
oxidase (enzyme which catalyzes the formation O2− that
is converted to more stable H2O2 via complex reaction).
A similar response of Zn induced oxidative stress had
been reported in Brassica juncea plants [56].
In lower organisms like the alga Chlorella vulgaris,
Bajguz (2000) demonstrated that EBL blocked the heavy
metals accumulation in the cells. The inhibitory effect on
heavy metal accumulation was arranged in the following
order: Zn > Cd > Pb > Cu [57]. The author associated BR
protection to heavy metal stress to EBL-induced pH in-
crease in the medium, since lower pH increased the tox-
icity of heavy metals in C. vulgaris cells.
On the other hand, aluminum (Al) and chromium (Cr)
are not essential nutrients. Al toxicity is the major
growth-limiting factor for crop cultivation on acidic soil
[58] that generates oxidative stress indirectly, mediated
by its influence on membrane lipids and other peroxi-
dants such as iron [59]. Al ions are capable of binding
with lipid components of the plasma membrane [60]
causing the rigidification of plasma membrane [61] and
to DNA [62], and therefore, impairs cell division [58]. Al
is reported to inhibit the plant growth [63], mainly that of
root [64,65]. Besides this, Al also alters water relations
[41], reduces stomatal opening, decreases photosynthetic
activity and causes chlorosis and necrosis of leaves [58].
Ali et al. (2008) reported that EBL or HBL treatment
improved the response of mung bean seedlings to Al
stress [66]. This response was associated to the amelio-
rated level of antioxidant system, suggesting that, at least
in part, it was responsible for the development of resis-
tance against Al stress in these seedlings. The increase in
the degree of resistance due to the application of BRs
was reflected in the improvement of plant growth, pho-
tosynthesis and related processes, in the presence of Al.
Cr metal pollution in the biosphere has increased
sharply over the last few decades, mainly due to its an-
thropogenic release from leather, electroplating, catalytic
manufacturing, chromic acid and refractory steel indus-
tries [67]. However, in nature, Cr is usually found in tri-

M. N. VÁZQUEZ ET AL. 37
valent (Cr III) and hexavalent (Cr VI) oxidation states, of
which Cr (VI) is more phytotoxic, owing to its greater
mobility. The entry of Cr into a plant system occurs
through roots using the specialized uptake systems of
essential metal ions (Fe, S), required for normal plant
metabolism [67]. Reduced seed germination, disturbed
nutrient balance, with decreased rate of photosynthesis,
inactivation of Calvin cycle enzymes, and chloroplast
disorganization have been documented in plants under Cr
stress [67]. The oxidative stress under Cr excess is caused
by the over production of ROS.
The significant influence of EBL on the synthesis of
IAA, ABA and polyamines (PAs) of radish seedlings
under Cr (VI) metal stress was demonstrated by Choud-
hary et al. (2011). EBL could enhance the synthesis of
IAA in order to promote normal seedling growth under
Cr (VI) metal stress. On the other hand it also slightly
improved the production of ABA to increase Cr (VI)
stress tolerance. Altered synthesis of PAs observed under
the influence of EBL may be helpful in protecting the
seedlings against Cr (VI) stress by enhancing one pool of
PAs (putrescine and spermidine) and decreasing the other
pool (cadaverine) [68]. Increased levels of antioxidants
and antioxidant enzymes activities upon EBL application
with Cr (VI) metal stress also indicate its significant ef-
fect on antioxidant system of radish plants. Similarly,
reduced membrane damage, enhanced proline, photo-
synthetic pigments, sugars and radical scavenging activi-
ties also shows a major impact of EBL on radish seedling
metabolism under Cr (VI) metal stress.
Earlier, Arora et al. (2010) had reported the effect of
EBL treatment to regulate the diminution of Cr metal
toxicity in mustard plants [69].
Besides, the interaction of EBL with lead (Pb) on the
growth of algae and ion accumulation in Chlorella vul-
garis cells was studied by Bajguz (2000) [57] while the
effect of BRs on oxidative stress generated by manga-
nese stress in maize leaves was reported by Wang et al.
(2009) [70].
3. Concluding Remarks
BRs reduced the heavy metal toxicity in plants. Firstly,
this reduction was associated with lesser ion uptake and
accumulation and with the increasing activity of ATPase,
an enzyme responsible for acid secretion and changes in
membrane level [57]. The proton pump generates an H+
electrochemical gradient and provides a driving force for
the rapid ion fluxes required for the uptake of various
nutrients such as K+, Cl–, 3 NO

, amino acids and sucrose
across the plasma membrane [71]. The regulation of
H+-ATPase activity [72] not only allows nutrient uptake
in plant cells but also controls water fluxes [73]. In addi-
tion to this, proton secretion induced by BRs has been
reported too. This proton secretion was accompanied by
an early hyperpolarization of the plasma membrane, in-
dicating that proton pumps could be targets of BRs [74].
On the other hand, the lesser ion accumulation induced
by BRs may be explained by the ability of these com-
pounds to increase some ligands which form chelates
with metal ion as, for example, phytochelatins [14]. Fur-
ther, BRs have also influence on electrical properties of
membranes and transport of ions by altering their per-
meability and structure, stability and activity of mem-
brane enzymes.
All the above results confirmed that phytotoxicity
from heavy metals is closely related to oxidative stress
and so to the production of ROS in plants. An imbalance
between ROS production and ROS scavenging leads to
oxidative burst. ROS can react with nucleic acids, pro-
teins and lipids and causes membrane damage and en-
zyme inactivation resulting in inhibition of plant growth
[75]. In plants, removal of ROS and cellular homeostasis
are governed by antioxidative enzymes such as superox-
ide dismutase (SOD), peroxidase (POD) and catalase
(CAT) and the enzymes of ascorbate-glutathione cycle
and various non-enzymatic antioxidants such as carote-
noids, α-tocopherol, proline, phenols, ascorbate and glu-
tathione via scavenging and neutralization [76,77].
The elevation in the activities of antioxidant enzymes
by BRs is well documented and it is known that it is a
gene regulated phenomenon. Cao et al. (2005) demon-
strated on the basis of molecular, physiological and ge-
netic approaches the elevation in antioxidant enzymes
was the consequence of enhanced expression of det 2
gene, which enhanced the resistance to oxidative stress in
Arabidopsis [78]. Similarly, Xia et al. (2009) reported
that BRs-induced stress tolerance is associated with in-
creased expression of genes encoding antioxidant en-
zymes such as SOD, CAT, POD, GR and APX in leaves
of cucumber [79]. Various reports have also shown that
the application of BRs modified antioxidant enzymes
activities under other abiotic stresses such as water defi-
cit [80], salinity [81], high temperature [82] and chilling
[83].
Plant protection induced by BRs under heavy metal
stress has been related to interactions of these steroidal
hormones with other plant hormones such as IAA and
ABA. Recently, Choudhary et al. (2012) reviewed the
benefits of brassinosteroid crosstalk and they concluded
that the versatile role of BRs may be attributed to multi-
layer interactions with other plant growth regulators af-
fecting the post-transcriptional fate of the target response
[84], although they pointed out that the mechanisms be-
hind the pleiotropic action of BRs and the execution of
BR-induced responses still remain poorly understood at
this time.
So, the focus of future research should be to elucidate
the mechanism by which BRs confer tolerance to heavy

M. N. VÁZQUEZ ET AL.
38
metal stress at cellular and molecular levels.
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