Heavy metal stress

PARUL SHARMA
PHD BOTANY
HEAVY METAL
STRESS

Every year due to increase in anthropogenic activities, environmental
pollution by heavy metals is becoming an important global health
concern for both humans and plants.
Metals are natural components of all the ecosystem and some of them
as micronutrients are essential for plant growth and yield. Elevated
amounts of heavy metals contaminants resulting from urbanization and
industrialization are of serious concern. Landfill materials and their toxic
leachates enrich in heavy metal and other toxic substances affect
ground water quality and defile soils.
There are 23 heavy metals, the most common heavy metal constituents
pollutants are Cd, Al, Cr, Pb, Hg, Ni, As and Zn. Certain heavy metals
are components of our environment and soil in small quantities and are
necessary for good health but in excess they can be poisonous to
plants.
Presence of heavy metals in soil can affect physical, chemical and
biological properties of plants by affecting plant root growth, absorption
of water and nutrients and yield.

EFFECTS OF HEAVY METAL ON PLANTS
The presence of metals in the leaves can hamper plant metabolism and growth
even at very low concentrations. The oldest leaves of metal exposed plants exhibit
the highest metal content. Under metal stress, plants exhibit a thickening of root
tips and a decreased root hair density. Heavy metals decrease leaf expansion,
resulting in a more compact leaf structure and increased stomatal resistance. They
may impair leaf transpiration and Carbon dioxide fixation by decreasing leaf
conductance to carbon dioxide diffusion as a result of stomatal closure. Heavy
metals can inhibit photosynthesis of intact plants at several physiological levels:
stomata, pigment synthesis, chloroplast structure and function, and indirectly by
affecting various other metabolic pathways. Significant alteration in protein
metabolism under heavy metal stress occurs. Heavy metal exerted specific
influence on the differentiation of various tissues in the root as well as stem.
These ions seem to attack various cellular components, including cell wall and
membranes, resulting in different alterations, which ultimately lead to their
disorganization. They compete with other essential nutrients for absorption.
Insufficient supply of essential nutrients and hormones from the root adversely
influences the differentiation of tissues in stem. Heavy metals have been shown to
affect cells of cortex and pith in root and stem of plants.

CHROMIUM –
• Immobilization of chromium in vacuole of plant root cells is suggested as a main reason
for the excessive accumulation of this metal in roots .
• Cr drastically reduces seedling dry matter production and hampers the development of
stems and leaves during plant early growth stage.
• Chromium toxicity inhibits the cell division and elongation of plant roots, thus
shortening the overall length of roots. As a consequence, water and nutrient absorption
processes are severely restricted, which can lead to the decreased shoot growth.
• Cell division inhibition and imbalance of Ca2+ in cells caused by Cr, disturbing the
transport of calcium cation from plasma membrane into cytoplasm.
• Chromium is capable of creating metabolic disorders during seed germination by
disrupting the events related to the conversion of food reservoirs into energy that are
essential for the subsequent successful emergence and establishment of seedlings.
• In cowpea seeds, treated with different concentrations of Cr6+, amylase activity and
total amount of sugar were markedly decreased, resulting in depression of germination
characteristics. It is also reported that increased levels of Cr in different valence states
were associated with concomitant decline in seed germination. This can be due to
disruption in carbohydrate transfer into embryonic axis in seeds or a rise in protease
activity.
• Chromium is involved in interfering with the absorption or accumulation of a wide
range of other metals or nutrients such as Fe, Mn, Ca, Mg, K, and P in both aerial or
root parts of plants, mostly leading to their reduced cellular or tissue concentration.

ALLUMINIUM –
• The primary target of Al toxicity is roots of plants where the accumulation of
Al inflicts the inhibition of root growth in the space of minutes or hours. It can
increase the thickness of lateral roots and change their color to brown.
Reduction of root respiration and disturbances in the enzymatic regulation of
sugar phosphorylation are also caused by Al toxicity.
• Al toxicity stress negatively affects aerial parts of plants, especially as a result
of initial root damage, which hampers nutrient uptake ability of roots,
resulting in nutritional deficiency.
• Al-induced stress on shoots led to stunting of leaves, purple discoloration on
stems, leaves, and leaf veins followed by yellowing and dead leaf tips, curling
or rolling of young leaves and death of growing points or petiole.
• The reduction in stomatal aperture and decreased photosynthetic activity are
also reported to be caused by Al toxicity.
• The leaf sheaths of corn plants treated with different doses of Al had
underdeveloped epidermal and cortical cells, which was accompanied by a
decrease in the diameter of metaxylem and protoxylem in vascular bundle.
• At the ultrastructural level, alteration of chromatin configuration in nucleus
and an increase in the size and frequency of nucleolar vacuoles are ascribed to
Al stress

COPPER –
• A plethora of research studies in clove (Syzygium aromaticum L.), in cucumber
(Cucumis sativus), and in some Eucalyptus species indicate that copper has a
propensity for the accumulation in the root tissues with little upward movement
towards shoots. Therefore, the initial characterization of Cu toxicity is the
hindrance of root elongation and growth.
• The subsequent symptoms include chlorosis, necrosis, and leaf discoloration.
• Excess Cu can become attached to the sulfhydryl groups of cell membrane or
induce lipid peroxidation, which results in the damaged membrane and the
production of free radicals in different plant organelles and parts.
• Cu at toxic levels through redox process cycling between Cu+ and Cu2+ triggers
the formation of reactive oxygen species such as singlet oxygen and hydroxyl
radical, leading to injuries to macromolecules, for example, DNA, RNA, lipids,
carbohydrates, and proteins.
• Decreased photosynthetic competence, low quantum efficiency of PSII, and
reduced cell elongation are also associated with Cu toxicity, it was shown that
toxic concentration of Cu (15 μM) depleted PSII action centers and led to
photoinhibition and disruption of its repair cycle.
• Seedling growth characteristics are shown to be adversely affected by Cu
toxicity. In spinach seeds (Spinacia oleracea L.) there is a significant negative
correlation between the root and shoot elongation with increasing Cu levels,
which was associated with a noticeable depression in seedling fresh weight.

ZINC –
• Visual signs of trouble in plants as a result of Zn toxicity are reported to
be chlorosis in young leaves and appearance of purplish-red color in
leaves, necrotic spotting between the veins in the blade of mature leaves
and inward rolling at leaf margins.
• Excess Zn2+ in cells can produce ROS and adversely influence integration
and permeability of membrane.
• Zn toxicity hampers the functionality and efficiency of photosynthetic
system in different plant species. Zn2+ reduced the content of accessory
photosynthetic pigments including Chla and Chlb by disturbing the
absorption and translocation of Fe and Mg into chloroplast.
• The elevated level of Zn2+ is reported to cause a decline in initial and
maximum Chl fluorescence, resulting in the repression of PSII activity.
• Zn in cells resulted in abnormal chromosomes, which was followed by a
sticky metaphase and premature separation of chromosomes in Bambara
groundnut (Vigna subterranean).
• Zn toxicity decreased the length of root and shoot as well as area of
leaves in tomato (Solanum lycopersicum L.).

MANGANESE –
• Mn leads to crinkled leaves, darkening of leaf veins on older foliage,
chlorosis and brown spots on aged leaves, and black specks on the stems. Mn
toxicity has been associated with a decreased CO2 assimilation but
unaffected chlorophyll (Chl) level in Citrus grandis seedlings and depleted
Chl content in pea (Pisum sativum L.) and soybean (Glycine max L.),
indicating diversity among plant species in response to Mn excess.
• In rice (Oryza sativa L.) and sunflower, Mn toxicity depressed shoot and root
growth.
NICKEL –
• Ni at excess competes with several cations, in particular, Fe2+ and Zn2+,
preventing them from being absorbed by plants, which ultimately causes
deficiency of Fe or Zn and results in chlorosis expression in plants.
• Excess nickel adversely affects germination process and seedling growth
traits of plants by hampering the activity of the enzymes such as amylase
and protease as well as disrupting the hydrolyzation of food storage in
germinating seeds.
• Ni toxicity can result in inhibited lateral root formation and development.
Moreover, the agglomeration of Ni in root apex greatly hampers mitotic cell
division in this organ, which ultimately results in growth reduction. The
induction of ROS, due to Ni toxicity is observed.

ALLUMINIUM TOXICITY IN SOIL -
Al is known as an inhibitory element for the growth of plants, especially in acidic
soils with pH values as low as 5 or 5.5 where the most phytotoxic form of
aluminum (Al3+) is prevalent. The most easily recognized symptom of Al toxicity is
the inhibition of root growth, and this has become a widely accepted measure of
Al stress in plants.

The root apex (root cap, meristem, and elongation zone) accumulates more Al and
attracts greater physical damage than the mature root tissues. Indeed, only the apical
2 to 3 mm of maize roots (root cap and meristem) need be exposed to Al for growth to
be inhibited. When Al is selectively applied to the elongation zone or to all of the root
except the apex, growth is unaffected. A number of changes to the ultrastructure of
cap cells in maize roots after a 2-h treatment with Al suggested that Al might inhibit
root growth indirectly, via a signal-response pathway involving the root cap, hormones,
and secondary messengers.

Inhibition of Ca Uptake by AI-
Transport of Ca into cells is energetically passive and is probably mediated by
membrane-spanning channels. Many polyvalent cations (e.g. La3+, Ga3+, and
Gd3+) inhibit Ca transport, and the ability of Al to reduce Ca uptake and
translocation in plants is well documented.
Three interactions occurs:
(a) inhibition of Ca uptake
(b) displacement of Ca from the apoplast
(c) disruption of Ca homeostasis in the cytoplasm.
Al inhibited Ca uptake in Al-sensitive wheat lines. Studies established a
correlation between Al toxicity and the inhibition of Ca uptake that met all the
criteria for a primary cause of toxicity: the effect is measurable within minutes,
it involves the root apex, and it is consistent with the long-term symptoms of Ca
deficiency.
A large proportion of the total Ca in root tissue resides in the apoplast, where it
is required for membrane stability and normal cell development. Al can displace
apoplasmic Ca by competing for ligand or by reducing the negative potential
difference on the membrane surface.

ALLUMINIUM TOLERANCE –
1. By exclusion -
Several plant species are known to secrete organic acids from their
roots in response to Al3+ . Citrate, oxalate and malate are some of
the commonly released organic acid anions that can form sufficiently
strong complexes with Al3+ to protect plant roots. Increased Al3+
resistance correlates with greater rates of organic acid exudation.
Al3+ interacts with the cell, perhaps via a receptor protein (R) on the
plasma membrane, to activate the transcription of genes that
encode proteins involved in the metabolism of organic acids and
their transport across the plasma membrane. Organic acid anions
form a stable complex with Al, thereby keeping Al3+ outside of root
cells. Although many types of organic acids are found in root cells,
only a few of these are specifically secreted in response to Al3+
exposure. This suggests that specific transport systems for organic
acid anions exist on the plasma membrane. In wheat and maize, this
transport system has been identified as an anion channel. Anion
channels are membrane-bound transport proteins that allow the
passive flow of anions down their electrochemical gradient. Patch-
clamp studies on protoplasts prepared from wheat roots showed that
Al3+ activates an anion channel in the plasma membrane that is
permeable to malate and chloride.

2. Aluminum tolerance by internal accumulation –
It is well known that some Al-tolerant plant species can accumulate high
concentrations of Al without showing symptoms of Al toxicity. Remarkably, Al
stimulates the growth of Melastoma malabathricum, a tropical rainforest
species known to accumulate Al. Buckwheat leaves accumulate over 400
mg/kg dry weight when grown on acid soils. Hydrangea plants can accumulate
high concentrations of Al (>3000 mg/kg dry weight) in leaves over several
months of growth. Recent evidence has shown that these Al-accumulator
species detoxify internal Al by forming Al–organic acid complexes. Complexes
of Al citrate (1:1) in Hydrangea leaves and Al oxalate (1:3) in buckwheat have
been identified by Al nuclear magnetic resonance. A current hypothesis is that
chelation of Al by organic acids effectively reduces the activity of Al3+ in the
cytosol, preventing the formation of complexes of Al and essential cellular
components.

CADMIUM TOXICITY IN SOIL -
The release of Cd into the environment constitutes a significant pollution
problem. The release of Cd from anthropogenic activities is estimated to be
about 4,000 to 13,000 tons per year, with major contributions from mining
activities, and burning of fossil fuels. Treated sewage sludge (“biosolids”) and
phosphate fertilizers are important sources of Cd contamination in agricultural
soils.
Cd uptake by plants has great impact and relevance not only to plants but also to
the ecosystem, in which plants form an integral component. The ability of some
plant species to uptake and hyperaccumulate Cd in edible parts increases the risk
of Cd assimilation by animal consumers through trophic transfer.
Some phosphatic fertilizers and phosphorites contain high concentrations of Cd
and are considered as the potential cause of increasing Cd contamination in rice.
The accumulation of Cd in plants may cause several physiological, biochemical
and structural changes. Cadmium accumulation alters mineral nutrients uptake,
inhibits stomatal opening by interacting with the water balance of plant, disturbs
the Calvin cycle enzymes, photosynthesis, carbohydrate metabolism, changes the
antioxidant metabolism, and lowers the crop productivity.

As Cd is unable to participate directly in biological redox reactions, it
induces oxidative stress via different indirect mechanisms. Once inside
the plant, Cd stimulates the activity of NADPH oxidases, resulting in
extracellular superoxide, H2O2 accumulation and lipid peroxidation and
oxidative burst.
In soil-plant relationship, Cd may influence physiological processes and
biochemical mechanisms primarily by affecting concentration and
functions of mineral nutrients. Cadmium has been shown to interact with
the availability of nutrient elements and also some of these nutrients
have protective role against the toxic effects of Cd stress.

CADMIUM ACCUMULATION IN PLANTS –
Cadmium accumulation by higher plants can occur through foliar or root uptake.
However, the primary point of entry for Cd into plants is through the roots. The
degree to which higher plants are able to take up Cd depends on its
concentration in the soil and its bioavailability. Cadmium bioavailablity in soils is
modulated by the presence of organic matter, pH, redox potential, temperature,
light intensity, cation exchange capacity and concentrations of other elements.
In particular, Cd ions seem to compete with other micro and macronutrients such
as calcium and zinc for the same transmembrane carriers, which might lead to
plant nutrient deficiencies. Cadmium uptake also appears to be decreased in the
presence of dissolved organic matter because ligands on the organic matter
effectively bind Cd ions.
The main route for uptake of cadmium across the plasma membrane is the large
negative electrochemical potential produced as a result of the membrane H +
translocating adenosine triphosphatases (ATPases).
During their transport through the plant, metals become bound to cell walls,
which can explain why normally Cd 2+ ions are mainly retained in the roots, and
only small amounts are translocated to the shoots. But once loaded in the xylem
sap, Cd is translocated to the aerial parts of plants through the transpiration
stream, where they might be present as a divalent ion or complexed by several
ligands, such as amino acids, organic acids and/or phytochelatins.

The partitioning of Cd to different plant organs plays important role in toxicity of
Cd to plants. The amount of Cd that accumulates in plant is limited by several
factors including -
1) Cd bioavailability within the rhizosphere
2) Rates of Cd transport into roots via either the apoplastic or symplastic
pathways
3) The proportion of Cd fixed within roots as a Cd- phytochelatin complex and
accumulated within the vacuole
4) Rates of xylem loading and translocation of Cd
Rhizosphere is an important environmental interface connecting plant roots and
soil. The influence of root exudates on Cd bioavailability and toxicity is a
consequence of change in the rhizosphere pH, redox potential and the number and
activity of rhizospheric microbes, and the capacity for chelating with Cd ions.
The roots of some plants, such as wheat and buckwheat, excrete organic acid such
as oxalic acid, malic acid and citric acid that can chelate with Cd to prevent its
entrance into roots. In addition, the combination of organic phosphate acids and
Cd ions would produce cadmium phosphate complexes unavailable to plants.

CADMIUM TOXICITY IN PLANTS –
• Cadmium being a divalent cation may compete with Ca, Mg or iron (Fe) in
their transport across membranes. It is taken up by plants via cation transport
systems normally involved in the uptake of essential elements, such as
members of ZIP and NRAMP families or Ca channels and transporters. Cadmium
entry through the Ca channel in the leaves disturbs the plant-water
relationship, causing stomatal closure in many plants, leading to lower
transpiration rate, and inhibition of photosynthesis through an adverse effect
on chlorophyll metabolism. This subsequently leads to growth inhibition and
imbalance in the nutrient level.
• Cd-induced inhibition of photosynthesis has also been attributed to an
inhibition of the activity of key enzymes of the Calvin cycle and the
photosynthetic electron transport chain in rice, and inhibition occurred at the
uptake level or in translocation of nutrients.
• Cadmium may interfere with the nutrient uptake by altering the plasma
membrane permeability, leakage of nutrients through plasma membrane and
affect the element-transport processes across the membrane.
• An excess Cd supply increased macronutrient and decreased micronutrient
concentrations.
• A high percentage of polymorphism in rice DNA is reported following exposure
to Cd. This might be mainly related to DNA breaks.

• Cadmium alters the conformation of proteins, for example enzymes, transporters or
regulator proteins, due to its strong affinity as ligand to sulfhydryl and carboxylic
groups.
• Different transporters are involved in the translocation of nutrients into the aerial
part of the plant at different levels, and Cd can inhibit these transporters. Toxic heavy
metals compete with the transport systems operating for micronutrient uptake, and
this occurs by using the same transmembrane carriers used for the uptake of Ca2+,
Fe2+, Mg2+, Cu2+ and Zn2+ ions.
• Another important toxicity mechanism is due to the chemical similarity between Cd2+
and functionally active ions situated in active sites of enzymes and signaling
components. Thus, Cd2+ ions can interfere with homeostatic pathways for essential
metal ions and the displacement of divalent cations, such as Zn and Fe, from proteins
could cause the release of “free” ions, which might trigger oxidative injuries.

CADMIUM TOLERANCE MECHANISM –
1. Metal Binding Ligands -
Plants, like all living organisms, have evolved a suite of mechanisms that control and
respond to the uptake and accumulation of both essential and non-essential metals.
These mechanisms include the chelation and sequestration of metals by particular ligands
and, in some cases, the subsequent compartmentalization of the ligand– metal complex
in vacuoles.
The vacuole of plant cells plays an important role in the homeostasis of the cell. The
vacuolar membrane, named tonoplast, functions as an effective and selective metal
diffusion barrier. Vacuolar compartmentalization prevents the free circulation of Cd ions
in the cytosol and forces them into a limited area. Several studies have shown that the
vacuole is the site of accumulation of a number of metals including Cd. One example is
the accumulation of Cd and phytochelatins (PCs) in the vacuole involving an ATP-binding
cassette (ABC) transporter (Hall 2002 ).
Among the metal-binding ligands in plant cells, the PCs and metallothioneins (MTs) are
the best characterized. MTs are cysteine-rich polypeptides encoded by a family of genes
whereas PCs are a family of enzymatically synthesized cysteine rich peptides.
In plants, PC–Cd complexes are sequestered in the vacuole. In mesophyll protoplasts
derived from tobacco plants exposed to Cd, almost all of both the Cd and PCs
accumulated were confined to the vacuole.

2. Plant Metal Accumulation –
Although Cd is not an essential or beneficial element for plants (with the exception
mentioned before), they generally exhibit measurable Cd concentrations, particularly in
roots, but also in leaves, most probably as a result of inadvertent uptake and
translocation.
T. caerulescens , of the Brassicaceae family is one of the best known hyperaccumulator
plants with a capacity to hyperaccumulate Zn, Cd and Ni. T. caerulescens plants have
been found to contain more than 100 mg/ Kg Cd frequently, and more than 1,000 mg/Kg
Cd occasionally, with very large variations between sites and populations. It was able to
accumulate >10,000 mg/kg Cd in the shoots without showing any symptoms of
phytotoxicity.
The hyperaccumulation trait has been hypothesized to perform several ecological
functions in hyperaccumulator plants: (1) metal tolerance/ disposal; (2) interference
with other plants (elemental allelopathy); (3) drought resistance and (4) defence against
some herbivores and pathogens.

ROLE OF PHYTOCHELATINS -
Phytochelatins, as a pathway for metal homeostasis and detoxification, have
been identified in a wide range of living organisms from yeast and fungi to
many different species of animals. In plants, PCs are found to be part of the
defensive act not only against metal-related stresses but also in response to
other stressors such as excess heat, salt, UV-B, and herbicide . PCs are
reported to be used as biomarkers for the early detection of HM stress in
plants.
PCs HAVE THE GENERAL STRUCTURE -
Early analyses demonstrated PCs consisted of only the three amino acids: Glu, Cys and
Gly with the Glu, and Cys residues linked through a g-carboxylamide bond. PCs form a
family of structures with increasing repetitions of the g-Glu-Cys dipeptide followed by a
terminal Gly; (g-Glu-Cys)n-Gly, where n has been reported as being as high as 11, but is
generally in the range of 2 to 5. PCs have been identified in a wide variety of plant
species and in some microorganisms. They are structurally related to glutathione (GSH;
g-Glu-Cys-Gly) and were presumed to be the products of a biosynthetic pathway. In
addition, a number of structural variants, for example, (g-Glu-Cys)n-bAla, (g-Glu-Cys)n-
Ser, and (g-Glu-Cys)n-Glu, have been identified in some plant species.

Genes and functions contributing to
Cd detoxification in Plants.

PCs ARE SYNTHESIZED FROM GSH –
Numerous physiological, biochemical, and genetic studies have confirmed that GSH is the
substrate for PC biosynthesis. The induction of PCs in the presence of Cd coincided with a
transient decrease in levels of GSH. Furthermore, the exposure of either cell cultures or
intact plants to an inhibitor of GSH biosynthesis, buthionine sulfoximine, conferred
increased sensitivity to Cd with a corresponding inhibition of PC biosynthesis. This could
be reversed by the addition of GSH to the growth medium. By far the most detailed
characterization of the pathway of PC biosynthesis has come from studies in the fission
yeast (Schizosaccharomyces pombe), and in Arabidopsis. Genetic studies have confirmed
GSH deficient mutants of the fission yeast and Arabidopsis are also PC deficient and
hypersensitive to Cd.
ROLE OF PCs-
The precipitous induction of PCs occurs inside cells as result of the varying levels of
multiple types of HMs where PCs via sulfhydryl and carboxyl groups can attach to some
HM cations and anions such as Cd, Cu, Ag, Zn, Pb, Ni, and Ar. However, Cd2+ ions are
found to be the most effective stimulator of PCs synthesis. Cytosol is the place where PCs
are manufactured and actively shipped from there in the form of metal-phytochelatin
complexes of high molecular weight to vacuole as their final destination by Mg ATP-
dependent carrier or ATP-binding cassette (ABC) transporter.

In Solanum nigrum L. the production of PCs was enhanced in roots when the plant was
exposed to 200 μmol·L−1 Cu, which resulted in the immobilization of Cu excess in the root
and its preclusion from moving toward the shoot.
The stimulation of different As-PC complexes in roots of some rice cultivars subjected to
the elevated levels of arsenic reduced the transport of As from soil or root to the aerial
parts and grains. These strategies can be effective in terms of preventing toxic metals
from reaching the consumable parts of crops.
A prolonged exposure of Brassica juncea to Cd resulted in 3-fold higher accumulation of
PCs in leaves than roots.
Treating maize plants with Cd for a longer period of time led to decreased PCs action in
roots and increased level of phytochelatin synthase in leaves. The feedback regulation
process or substrate reduction may be accountable for this phenomenon.
Phytochelatins along with antioxidative enzymes can form a synergistic defensive regime
in plants under HM stress which, in turn, can strengthen plant’s resistance against metal
intoxication. The increased enzymatic biosynthesis of PCs coupled with the heightened
activity of antioxidative system in Brassica chinensis L. led to an effective detoxification
of Cd.
In transgenic tobacco plants, artificial synthesis of phytochelatin gene enhanced
their resistance to varying levels of cadmium. The transgenic Arabidopsis plants
were much better HM accumulators than wild type Arabidopsis as a result of
expressing synthetic phytochelatins (ECs). The overexpression of arsenic-
phytochelatin synthase 1 (AsPCS1) and yeast cadmium factor 1 (YCF1) (isolated from
garlic and baking yeast) in Arabidopsis thaliana resulted in an increased tolerance to
Cd and As and also enhanced its ability to accumulate the metals to a greater
extent.

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