Heavy metal stress causes a reduction in molecular oxygen and releases highly reactive intermediate products such as hydrogen peroxide (H2O2), superoxide radicals and hydroxyl radicals known as reactive oxygen species.

1. Heavy Metal Stress
Afsa Fayaz Bhat

2. • 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
define 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.

4. 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

5. 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

6. 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

7. COPPER
1. 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.
2. The subsequent symptoms include chlorosis, necrosis, and leaf discoloration.
3. 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.
4. 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.
5. 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.
6. 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.

8. 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.).

9. 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.

10. 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

11. 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.

12. • 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
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

13. • 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

14. 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.

15. 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
Al. Buckwheat leaves accumulate over 400 mg/kg dry weight when grown
acid soils. Hydrangea plants can accumulate high concentrations of Al
mg/kg dry weight) in leaves over several months of growth. Recent evidence
has shown that these Al-accumulator species detoxify internal Al by
Al–organic acid complexes. Complexes of Al citrate (1:1) in Hydrangea
and Al oxalate (1:3) in buckwheat have been identified by Al nuclear
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.

17. CADMIUM TOXICITY IN SOIL
1. 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.
2. 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.
3. 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.

18. 2. 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

19. 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.

20. • 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.

21. 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.

22. • 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.

24. 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.

25. • 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
with a capacity to hyperaccumulate Zn, Cd and Ni. T. caerulescens plants have been found
contain more than 100 mg/ Kg Cd frequently, and more than 1,000 mg/Kg Cd
with very large variations between sites and populations. It was able to accumulate
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.

27. 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.

28. Genes and functions contributing to
Cd detoxification in Plants.

29. 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.

30. • 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

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