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REVIEW OF LITERATURE
The term “heavy metals” refers to any metallic element that has a relatively
high density and is toxic or poisonous even at low concentration (Lenntech, 2004).
“Heavy metals” is a general collective term, which applies to the group of metals and
metalloids with atomic density greater than 4 g cm-3, or 5 times or more, greater than
water (Nriagu and Pacyna, 1988; Hawkes, 1997). However, being a heavy metal has
little to do with density but concerns chemical properties. Heavy metals include Lead,
Cadmium, Zinc, Mercury, Arsenic, Silver, Chromium, Copper, Iron and the Platinum
group elements (Duruibe et al., 2007). Heavy metals are major pollutants that are
accumulated in the environment. Geological and anthropogenic activities are sources
of heavy metal contamination (Dembitsky, 2003).
Sources of anthropogenic metal contamination include industrial effluents,
fuel production, mining, smelting processes, military operations, utilization of
agricultural chemicals, small-scale industries (including battery production, metal
products, metal smelting and cable coating industries), brick kilns and coal
combustion (Zhen-Guo et al., 2002). One of the prominent sources contributing to
increased load of soil contamination is disposal of municipal wastage. These wastes
are either dumped on road sides or used as landfills, while sewage is used for
irrigation. These wastes, although useful as a source of nutrients, are also sources of
carcinogens and toxic metals. Other sources can include unsafe or excess application
of (sometimes banned) pesticides, fungicides and fertilizers (Zhen-Guo et al., 2002).
Additional potential sources of heavy metals include irrigation water contaminated by
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sewage and industrial effluent leading to contaminated soils and vegetables (Bridge,
2004; Jadia and Fulekar, 2009).
Uptake can be catalyzed by either channels or transporters. The application of
powerful genetic and molecular techniques has now identified a range of gene
families that are likely to be involved in transition heavy metal ion uptake into cells,
heavy metal vacuolar sequestration, heavy metal remobilization from the vacuole,
xylem loading, and unloading of heavy metals (Fig. 5). Some well-characterized
heavy metal transporter proteins are zinc-regulated transporter (ZRT), iron-regulated
transporter (IRT) like protein ZIP family, ATP-binding cassette (ABC) transporters,
the P-type metal ATPases, the natural resistance-associated macrophage protein
(NRAMP) family, multidrug resistance-associated proteins (MRP), ABC transporters
of the mitochondria (ATM), cation diffusion facilitator (CDF) family of proteins,
copper transporter (COPT) family proteins, pleiotropic drug resistance (PDR)
transporters, yellow-stripe-like (YSL) transporter and Ca2+: cation antiporter (CAX),
and so forth (Dubey, 2011; Hossain et al., 2012).
The accumulation in the soil however has become worldwide problem
leading to reduced root and shoots growth, low yield production, low nutrient
uptake and impaired homeostasis. Uptake of excess metal ions is toxic to most
plants. The bioavailability of metals from soil depends on soil factors such as
cation exchange capacity (CEC), organic matter content, the content of clay
minerals and hydrous metal oxides, pH, buffering capacity, redox potential, water
content, and temperature (Kayser et al., 2001). The toxicity of the heavy metals
within soils of high CEC is generally low, even when metal concentrations are
high (Roane and Pepper, 2000). The soil mobility and bioavailability of the
following metals usually proceed in this order: Zn > Cu > Cd > Ni (Lena and Rao,
1997; Tak et al., 2013).
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Fig. 5. Diagrammatic representation of uptake and transport of heavy metals in plants
through metal transporters (Hossain et al., 2012).
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Table 3. Worldwide emission of heavy metals from natural sources (Nagajyoti et al.,
2010).
Source
Annual emission (Kg x 105)
Global
production
Cd
Co Cu
Cr
5
Windblown dust 6-1.100
0.25
4
Volcanogenic
0.5
1.4 4
Forest wild fires 2-200
0.01
-
Vegetation
75-1,000
0.2
-
Sea salt
300-2,000
0.002
65-150
12
Hg
0.05
Mn
Ni
Pb
Zn
42.5 20
10
25
3.9 0.03
8.2
3.8
6.4
10
0.3
-
0.1
-
0.6
0.3
0.5
2.5
-
-
5
1.6
1.6
10
particles
Total
-
0.96
0.1
0.003 4
5.4 18.9 8.9 0.16
0.04 0.1
51.6 26
0.02
18.6 45.52
Table 4. Heavy metal composition of typical uncontaminated soils and agricultural
crops (Nagajyoti et al., 2010).
Heavy metals
Range in soil
Range in agricultural crops
(ppm d.wt)
(ppm d.wt)
Cd
0.01-0.7
0.2-0.8
Co
1-40
0.05-0.5
Cr
5-3,000
0.2-1.0
Cu
2-100
4-15
Fe
7,000-55,000
-
Mn
100-4,000
15-100
Mo
0.2-5
1-100
Ni
10-00
1.0
Pb
2-200
0.1-10
Zn
10-300
15-200
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Table 5. Range of heavy metal concentrations (ppm) in igneous and sedimentary
rocks (Nagajyoti et al., 2010).
Metals
Basaltic
Granite
Shales and
Black
Sand
igneous
igneous
Clays
shales
stone
As
0.2-10
0.2-13.8
-
-
0.6-9.7
Cd
0.006-.06
0.003-0.18
0.0-11
<0.3-8.4
-
Cr
40-600
2-90
30-590
26-1,000
-
Co
24-90
1-15
5-25
7-100
-
Cu
30-160
4-30
18-120
20-200
Pb
2-18
6-30
16-50
7-150
<1-31
Mo
0.9-7
1-6
-
1-300
-
Ni
45-410
2-20
20-250
10-500
-
Zn
48-240
5-140
18-180
34-1,500
2-41
Growth inhibition is a general phenomenon associated with most of the heavy
metals (Peralta et. al, 2001). The biochemical impact of metal ions on the cells is a as
a diverse as their chemical nature and the tolerance limits for heavy metal toxicity are
specific not only for species but also for each variety of crop plants (Metwally et al.,
2008). The heavy metals are frequently accumulation by agriculturally important
crops with a significant potential to impair animal and human health. Many heavy
metals are trace elements essential to plants, yet a large amount of Mn, Cu, Zn, Mo,
Co, B can have a toxic impact on a physiological process of plants (Bena and Milda,
2008). Physiological and biochemical investigation of enzymological aspects and
metal ion homeostasis (Hasan et al., 2007) helps in understanding the mechanism of
metal tolerance. The sensitivity of plants to heavy metals depends an interrelated
network of physiological and molecular mechanisms such as:
(i)
Uptake and accumulated of metals through binding to extracellular exudates
and cell well constituents
(ii)
Efflux of heavy metal from cytoplasm to extra nuclear compartments
including vacuoles
(iii)
Complexation of heavy metal ions inside the cell by various substances for
examples, organic acids, amino acids, Phytochelatins and metallotheonins
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(iv)
Accumulation of osmolytes and osmoprotectants
and induction
of
antioxidantive enzymes
(v)
Activation or modification of plant metabolism to allow adequate functioning
of metabolic pathways and rapid repair of damaged cell structures (Cho et al.,
2003; John et al., 2009; Anwesha et al., 2012).
Alteration at various stages of physiological processes result in growth
retardation, inhibition of enzymes and altered stomatal action (Barceló and
Poschenenrieder, 1990; Neelima and Reddy, 2003), reduced photosynthesis due to
low chlorophyll concentration (Somashekaraiah et al., 1992) decreased activity in
enzymes involved in CO2 fixation (Greger and Ogren, 1991). Accumulation of
phototoxic metals results in stunted growth, chlorosis and necrosis, light-mediated
rapid peroxidation and bleaching (Sandmann and Gonzales, 1989). The reduction in
plant growth during metal stress is due to low water potential compared nutrient
uptake and alteration in the activity of many key enzymes of various metabolic
pathways (Arduini et al., 1996) and disturbed microtubule organization in
meristematic cells (Eun et al., 2000). The toxic dose depends the type of ion, ion
concentration plants species, and stage of plant growth. The two mechanisms of
enzyme inhibition predominate during the process of metal uptake:
1.
Binding of the metal to sulphydryl groups, involved in the catalytic action or
structural integrity of enzymes, and
2.
Deficiency of an essential metal in metaloproteins or metal protein complexes
eventually combined with substitution of the toxic metal for the deficient
element (Babaoglu et al., 2009; Anwesha et al., 2012).
Transition metals cause oxidative injury in plant tissue and exposure of plants to
non-redox reaction metals also resulted in oxidation stress as indicated by lipid
peroxidation, H2O2 accumulation and an oxidative burst. Some metal caused a transient
depletion of GSH and an inhibition of antioxidative capacities by metabolic modelling
suggested that the reported diminution of antioxidants enzymes, especially of
glutathione reductase. Assessment of antioxidative capacities by metabolic modelling
suggested that the reported diminution of antioxidants was sufficient to cause H2O2
accumulation. The depletion of GSH is apparently a critical step in Cadmium sensitivity
since plants with improved capacities for GSH synthesis displayed higher tolerance
(Schutzendubel and Polle, 2002). Increased H2O2 levels in response to heavy metal
stress are closely linked to an improved antioxidant defense capability mediated by
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POD (Kim et al., 2010). Thus, the deleterious effects resulting from the cellular
oxidative state can be alleviated by activated enzymatic (SOD, catalase, ascorbate
peroxidase and glutathione reductase) and non enzymatic (ascorbate, glutathione)
antioxidant mechanisms (Piotrowska et al., 2010). The potential interplay between
cytosolar and mitochondrial responses to metal stress in plants (Fig. 6) in order to
obtain further insights into a broader cellular picture (Keunen et al., 2011).
Cellular enzymes and proteins contain several mercapto ligands that can
structurally chelate metals, and thereby cause these proteins to lose their functional
property. Heavy metals also generate oxidative stress that is mediated through
generation of free radicals (Seth et al., 2008). Some heavy metals directly affect
biochemical and physiological processes through inhibition of photosynthesis and
respiration leading to reduced growth (Vangronsveld and Clijsters, 1994). Therefore,
excessive heavy-metal accumulation in plants may induce toxicity by modifying
essential protein structure or replacing essential elements that is manifested by
chlorosis, growth impairment, browning of roots, and inactivation of photosystems,
among other effects (Shaw et al., 2004; Gorhe and Paszkowski, 2006; Tak et al., 2013).
Fig. 6. Schematic overview of metal-induced responses in plant cells focusing on
mitochondrial effects. Metal exposure has shown to cause mitochondrial
electron transport chain dysfunction and over-reduction, thereby increasing
mitochondrial ROS production (Keunen et al., 2011).
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2.1 Phytoremediation
Phytoremediation is an environmental cleanup strategy in which selected
plants are employed to remove the environmentally toxic contaminants (Reddy et al.,
2012). Phytoremediation is the use of plants and their associated microbes for
environment cleanup (Salt et al., 1995, 1998; Pilon-Smits, 2005). In the case of heavy
metals remediation, additional operational costs will also include harvesting, disposal
of contaminated plant mass and the accumulating plants can be incinerated and the
ashes disposed, which is much easier than excavating and disposing the contaminated
soil (Sharavanan et al., 2012).
Fig. 7. Absorbtion patterns of essential and non- essential metals in crop plants (Jadia
and Fulekar, 2009).
Phytoremediation is an efficient cleanup technology for a variety of organic
and inorganic pollutants. The organic pollutants are released into the environment
via Spills, military activities, agriculture, industry etc. Inorganic pollutants occur as
natural elements in the earth’s crust or atmosphere, and leading to toxicity.
inorganic pollutants that can be phytoremediated include plant micronutrients such
as nitrate and phosphate (Horne, 2000) plant trace elements viz., Cr, Cu, Fe, Mn,
Mo and Zn (Lytle et al., 1998), non essential elements such as Cd, Co, F, Hg, Se,
Pb, V, and W (Blaylock et al., 2000; Horne, 2000) and radioactive isotopes such as
U, Cs and Sr (Negri et al., 2000; Dushenkov et al., 2003; Pilon-Smits, 2005) as
shown in Fig. 7.
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The various strategies of phytoremediation are stated as under:

Phytostabilization also referred to as in place inactivation is primarily used for
the remediation of soil sediment, and sludges (United States Protection
Agency, 2000). It is the use of plant roots to limit contaminant mobility and
bioavailability in the soil. The experiment on phytostbilization by Jadia and
Fulekar (2008), using Sorghum to remediate soil contaminated by heavy
metals. Poplar trees are very efficient at intercepting horizontal groundwater
plumes and indirecting water flow upward because they are deep rooted and
transpires at very high rates, creating a powerful upward flow (Pilon-Smits,
2005).

Phytoextraction involves the extraction of metals by plant root and the
translocation there of to shoots. The roots and shoots are subsequently
harvested to remove the contaminants from the soil. With successive cropping
and harvesting the levels of contaminants in the soil can be reduced
(Vandenhove et al., 2001; Jadia and Fulekar, 2009). Popular species for
phytoextraction are Indian Mustard and Sunflower because of their fast growth,
high biomass, and high tolerance and accumulation of metals and other
inorganics (Salt et al., 1995; Blaylock and Haung, 2000; Pilon-Smits, 2005).

Phytovolatilization involves the use of plants to take up contaminants from the
soil, transforming them into volatile forms and transpiring them into the
atmosphere (United States Protection Agency, 2000; Jadia and Fulekar, 2009).
Poplar is also most used species for phytovolatilization because of its high
transpiration rate (Pilon-Smits, 2005).

Rhizofiltration is primarily used to remediate extracted ground water, surface
water and waste water with low contaminant concentrations (Ensley, 2000).
Rhizofiltration can be used for Pb, Cd, Cu, Ni, Zn, and Cr, which are primarily
retained within the roots (United States Protection Agency, 2000).

Phytodegradation of trichloroethylene and atrazine poplar has been the most
popular and efficient species so far, owing to its high transpiration rate and
capacity to degrade and volatilize these pollutants (Schnoor et al., 1997; PilonSmits, 2005) as shown in Fig. 8.
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f
Fig. 8A. Models representation phytoremediation technology used for remediating
polluted water, soil, or air. The possible fates of pollutants during
phytoremediation (a-f). The pollutant represented by red circles can be
stabilized or degraded in the rhizosphere, sequestered inside the plant
tissue, or volatilized (Pilon-Smits, 2005).
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Fig.8B. Molecular mechanisms proposed to be involved in transition metal
accumulation by plants. (a) Metal ions are mobilized by secretion of
chelators and by acidification of the rhizosphere. (b) Uptake of hydrated
metal ions or metal-chelate complexes is mediated by variousuptake
systems residing in the plasma membrane. Inside the cell, metals are
chelated and excessmetal is sequestered by transport into the vacuole. (c)
From the roots, transition metals aretransported to the shoot via the xylem.
Presumably, the larger portion reaches the xylem via the root symplast.
Apoplastic passage might occur at the root tip. Inside the xylem, metals are
present as hydrated ions or as metal-chelate complexes. (d) After reaching
the apoplast of the leaf, metals are differentially captured by different leaf
cell types and move cell-to-cell through plasmodesmata.Storage appears to
occur preferentially in trichomes. (e) Uptake into the leaf cells again is
catalysedby various transporters [not depicted in (f)]. Intracellular
distribution of essential transition metals (¼ trafficking) is mediated by
specific metallochaperones and transporters localized in endomembranes
(please note that these processes function in every cell). Abbreviations and
symbols: CW cell wall, M metal, filled circles chelators, filled ovals
transporters, bean-shapedstructures metallochaperones (Modified according
to Clemens et al., 2002, Babula et al., 2012).
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2.2 Plants Uptake Metals
Bioavailability of metals is the primary factor responsible for the uptake of
metals. In soils, metals exist as a variety of chemical forms in a dynamic equilibrium
governed by the physical, chemical and biological processes of the soil.
Bioavailability of soil pollutants, a primary basis of remediation efficacy, refers to a
fraction of the total pollutant mass in the soil and sediment available to plants. Uptake
of metals by plants involves root interception of metal ions, entry of metal ions into
roots and their translocation to the shoot through mass flow and diffusion. Plants have
evolved highly specific mechanisms to take up, translocate, and store these nutrients.
For example, metal movement across biological membranes is mediated by proteins
with transport functions. In addition, sensitive mechanisms maintain intracellular
concentration of metal ions within the physiological range. In general, the uptake
mechanism is selective and plants preferentially acquired some ions over others. Ion
uptake selectivity depends upon the structure and properties of membrane
transporters. These characteristics allow transporters to recognize, bind and mediate
the trans-membrane transport of specific ions. For example, some transporters
mediate the transport of divalent cations, but do not recognize mono-or trivalent ions
(Wani et al., 2012).
2.3 Biochemical Response Evaluation of C3 and C4 Plant Systems Exposed to
Heavy Metal Stress Under Elevated CO2 and Temperature
The plants viz., C3 and C4 exposed to heavy metals stress under elevated
atmospheric CO2 and at elevated temperature follow the same trend as in normal
conditions however; there are significant alterations in biochemistry of photosynthesis
of both types of plant systems. The effect of heavy metals on the enzymes those
catalyses the photosynthetic reactions e.g. PEPC, PPDK (pyruvate phosphoenol
dikinase), NADP-ME (NADP dependent malic enzyme) (Doubnerova and Ryslava,
2011). PEPC enzyme that catalyze the reaction of bicarbonate and phosphoenol
pyruvate (PEP) to yield 4 C acid compound oxaloacetic acid (OAA) need divalent
metal ions such as Mg+2 and Mn+2 for activation. These metal ions along with other
such as Fe and Zn are cofactors and may be replaced by the heavy metals that inhibit
the activity of enzyme resulting into less CO2 supply at the rubisco site thereby
reducing the net productivity.
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Despite having toxic manifestations of heavy metals the abundant PEPC
favors the production of OAA in C4 plants, which is further converted into acidic
malate by the action of NAD dependent malate dehydrogenase enzyme (NADMDH
EC 1.1.1.37). This NAD-MDH enzyme also act as detoxifying agent as it also
catalyze the formation of stable compounds of malate and metals such as aluminum
(Ma and Furukawa, 2003). C3 plants however; are devoid of such defense
mechanisms as PEPC is not the primary carbon dioxide fixing enzymes and face the
oxidative stress caused by heavy metals. Since photosynthetic efficiency depends
largely on the activity of rubisco (Sage, 2004), elevated atmospheric CO2 increases
the photorespiration in C3 plants as a result of oxygenase activity of rubisco
increasing the oxidative conversion of metals present in the cell. The oxidative
conversion of metals in C3 plant is also favored by the higher atmospheric
temperature (Du and Fang, 1982). The oxidation of metals in living organisms is thiol
(-SH) containing compound mediated with the hydrogen peroxide (H2O2) catalase
complex. Catalase is present in peroxisomes of plant cells. In C3 plants high
photorespiration rates as a result of elevated concentration of CO2, peroxisomes are
large and numerous while in the C4 plants the peroxisomes of mesophyll cells are
small and fewer in number (Tolbert, 1971). The milieu of interior cells of C4 plants is
highly reductive, thus it prevents the oxidative conversion of metals (Srivastava et al.,
2012) as shown in Fig. 9.
2.4 Cadmium
Cd is a highly toxic trace element and has been ranked No. 7 among the top 20
toxins (Abraham et al., 2013). Cadmium belongs to the group of heavy metals and is
highly toxic to plants and animals (Alloway, 1991). Cd a naturally occurring non
nutrient heavy metal, is one of the most important heavy metal pollutants due to high
toxicity to living organisms (Han et al., 2007; Zhang et al., 2010) and released to the
environment as a result of anthropogenic activities (Wagner, 1993; Djebali et al.,
2010). Major anthropogenic sources of Cd are Cd-containing phosphate fertilizers,
sewage and industrial emissions (Adriano, 1986). Mining and smelting industries also
release substantial amounts of Cd into the environment (Nriagu and Pacyna, 1988;
Anuradha and Seeta Ram, 2009).
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Fig. 9. Photosynthesis diagram of C3 and C4 plant showing the role of PEPC enzyme
in heavy metal response whereas Phosphoenolpyruvate carboxylase (PEPC)
enzyme acts as metal detoxifying agent (Srivastava et al., 2012).
The presence of Cd in soil leads to crop yield losses and human health hazards
(Seregin and Ivanov, 2001). Cadmium is non-essential to biota and is highly mobile
and available to plants. It is toxic to humans at lower concentrations than some other
toxic elements in plants, and its effects on humans are cumulative (Singh and
McLaughlin, 1999). Cd interferes with many intercellular functions mainly by
formation of complex with side group of organic compounds resulting in inhibition of
essential activities. Since Cd has a half-life of approximately 20 years in the human
body, there are concerns that consumption of foods containing relatively high levels
of Cd may lead to chronic toxicity (FAO/WHO, 1993). The World Health
Organization has set a maximum provisional tolerable intake limit for an adult at 6070 μg Cd per day (WHO, 1972) and the Codex Alimentarius Commission of
FAO/WHO proposed a limit on the concentration of Cd in cereal grains and oil seeds
traded on the international markets.
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2.5 Natural Occurrence
Cadmium is rarely found in a pure state. It is present in various types of rocks
and soils and in water, as well as in coal and petroleum. Cadmium is widely
distributed in the earth’s crust at an average concentration of about 0.1 mg kg-1 and is
commonly found in association with zinc. However, higher levels of Cd are present in
sedimentary rocks: marine phosphates often contain about 15 mg Cd kg-1. Cadmium
enters the environment mainly through the ground, because it is found in manures and
pesticides. It is released into the environment by power stations, heating systems,
metal working industries or urban traffic. It is widely used in electroplating, pigments,
plastic stabilizes and nickel- cadmium batteries (Sanita di Toppi and Gabbrieli, 1999).
2.6 Visual Symptoms of Cadmium
Cd impairs negative effects on the plant growth and development (Benavides
et al., 2005). It is recognized as an extremely significant pollutant element due to its
high toxicity and large solubility in water (Pinto et al., 2004). Cd induces chlorosis,
necrosis, vein reddening, and root and shoot growth retardation, besides reduced
nutrient uptake (Sanita di Toppi and Gabbrielli, 1999; Mohamed et al., 2012; Irfan et
al., 2013). Cd is not an essential nutrient for plants, and it can accumulate at higher
levels in aerial organs (Wang et al., 2007; Zou et al., 2008), inducing phytotoxicity
manifested in leaf roll, chlorosis, growth reduction, and eventually death (Benavides
et al., 2005; Zou et al., 2008, 2012). The most common effect of Cd toxicity in plants
is stunted growth, leaf chlorosis and alteration in the activity of many key enzymes of
various metabolic pathways (Arduini et al., 1996). The reduction in plant growth
during the stress is due to low water potential, hampered nutrient uptake and
secondary stress such as oxidative stress. The visible symptoms of Cadmium toxicity
in plants are described as chlorosis, leaf rolling, stunting of growth, necrosis and red
organ coloration. Chlorosis in Cd stress may appear due to induced iron deficiency
(Haghiri, 1973), phosphorus deficiency or reduced manganese transport (Goldbold
and Hutterman, 1985) The inhibition of root Fe (III) reductase induced by Cadmium
led to Fe (III) deficiency, and seriously affects the photosynthesis (Alkantara et al.,
1994).
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2.7 Cadmium in Soil
Soil pollution by heavy metals has become a critical environmental concern
due to its potential adverse ecological effects. Among the heavy metals, Cd is of
special concern due to its relatively high mobility in soils and potential toxicity to
biota at low concentrations (Das et al., 1997; An, 2004). Cd in soils is derived from
both natural and anthropogenic sources. Natural sources include underlying bedrock
or transported parent material such as glacial till and alluvium. The major natural
sources for mobilisations of Cd from the earth’s crust are volcanoes and weathering of
rocks. Within the biosphere the Cd is translocated by different processes. Naturally a
very large amount of Cd is released into the environment, about 25,000 t a year. The
main anthropogenic input of Cd to soils occurs by industrial waste from processes
such as electroplating, manufacturing of plastics, mining, paint pigments, alloy
preparation, and batteries that contain Cd, composts, or fertilizers. Even domestic
sewage sludge, which originated from the strictest control sources, contains Cd and
adds it to pollution. From the sewage systems, Cd enters rivers and streams and
therefore contaminates other places or accumulates in the sludge. The addition of Cd
in metal rich sewage sludge may also result in contamination of ground water (Moradi
et al., 2005). The average natural abundance of Cd in the earth’s crust has most often
been reported from 0.1-0.5 ppm, but much higher and much lower values have also
been cited depending on a large number of factors. Igneous and metamorphic rocks
tend to show lower values, from 0.02 to 0.2 ppm, whereas sedimentary rocks have
much higher values, from 0.1 to 25 ppm. Fossil fuels contain 0.5-1.5 ppm Cd, but
phosphate fertilizers contain from 10 to 200 ppm Cd (Cook and Morrow, 1995). Soils
can be polluted by Cd from a variety of sources such as phosphate fertilizer and
sewage sludge. The Cd concentration in soil solution of uncontaminated soils usually
ranges from 0.04 to 0.32 μM Cd while mildly polluted soils contain 0.32-1 μM Cd
(Wagner, 1993; Tran and Popova, 2013).
The concentrations of Cadmium in the surface soil ranges between 0.1 and 0.4
mg kg-1 (Page et al., 1981). The median Cadmium concentration in non- volcanic
soils ranges from 0.01-1 mg kg-1, but in volcanic soil levels of up to 4.5 mg kg-1 Cd
have been found (Korte, 1983). Sources of Cadmium in soil include ashes from fossil
fuel combustion, waste from cement manufacture and the disposal of municipal refuse
and sewage sludge. Wastes from non- ferrous metal production, manufacture of
Cadmium-containing articles, as well as the ash residues from refuse incineration
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contain elevated Cadmium levels and are responsible for contamination of the ground
water. The anthropogenic emission of Cd have been estimated to be 3000 tons year-1
(Sanita di Toppi et al., 1999), however, this amount is negligible compare to Cd
originating from anthropogenic sources (Durcekova et al., 2007). Highly polluted
soils containing over 100 mg Kg-1 Cd are reported in china, France and some other
countries (Goncalves et al., 2009). The concentrations of Cadmium in Lithuanian
soils are not as high, but there exists another problem :in the field close to the
highways, the previous main risk factor lead has now been replaced by Cadmium
(Antanaitis et al., 2007; Januskaitiene, 2012).
The agricultural application of phosphate fertilizers is greatly responsible for
the direct input of cadmium to arable soils. The cadmium content for phosphate
fertilizers varies widely and depends on the origin of the rock phosphate. Long-term
continuous application of phosphate fertilizers has been shown to cause increased soil
cadmium concentrations. The application of municipal sewage sludge to agricultural
soils as a fertilizer can also be a significant source of cadmium. Polluted soils can
contain cadmium levels of up to 57 mg kg-1 (dry weight) resulting from sludge
applied to soil and up to 160 mg kg-1 in the vicinity of metal-processing industry. The
increasing amount of metals in soil is a growing agronomic problem limiting crop
productivity over the world (Durcekova et al., 2007). Thus, increased Cd in the soil
leads to increased Cd in crop plants (Zarcinas et al., 1999; Al-Faiyz et al., 2007). The
successive addition of Cd in soils results decrease in the yield of different plant parts,
i.e. root, stem, leaf and seed of different species. Cadmium being toxic to plant, its
increased concentration in soil reduce growth and impairs metabolism (Foy et al.,
1978; Clijsters and Van Assche, 1985). The background cadmium concentration in
agricultural soils remains less than 1mg kg-1 (Sharma and Pushpendra, 2012).
Cd total content and its plant availability are affected by chemical and physical
properties of soil. Organic matter content, content of clay particles and sequioxides,
and soil pH are main parameters affecting adsorption of this element in soils
(Adriano, 2001). Low molecular weight organic acids present in the rhizoshere soil
influenced the solubilisation of particulate bound cd into soil and its subsequent
accumulation by plant (Pavlikova et al., 2007).
Cadmium contamination in arable
soils and surface water has become severe due to improper management of waste and
application of chemicals containing Cd especially in developing countries (Helal et
al., 1999; Muhling and Lauchli, 2003). In arid and semi arid region, it is likely that
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bio-solids, which contain heavy metals such as Cd, are used on saline soils in order to
improve soil quality (Weggler-Beaton et al., 2000; Muhling and Lauchli, 2003; Zong
et al., 2007).
2.8 Cadmium Uptake and Transport in Plant
Cadmium uptake in plants has been studied extensively. Cd is present in soils
mainly as a free ion or as soluble complexes or adsorbed at ion exchange sites of the
inorganic soil constituents. About 50% of Cd accumulated in Thlaspi caerulescens, a
Cd hyperaccumulator species is believed to be taken up from the soil solution. Cd can
be easily transported within plants (Epstein and Bloom, 2005) in the form of metalloorganic complexes. The mechanisms of uptake, translocation and deposition depend
upon the bio-availability of soil, pH, temperature, redox potential and concentration of
other elements (Fig. 10). Cd can easily penetrate the root system of xylem through the
apoplastic and/or symplastic pathway and reach tissues of aerial parts of the plants
(Salt and Rauser, 1995; Yang et al., 1998; Irfan et al., 2013).
Subsequent to the metal uptake into the root system, three processes govern
movement of the metal into the xylem, sequestration of metal inside the root cells,
symplastic import into the stele and release into the xylem (Clemens et al., 2002). The
membrane potential which is negative on the inside of plasma membrane and might
exceed 200 mV in the root epidermis cells provides a strong driving force for the
uptake of cations through secondary transporters. The transport of cadmium in plant
is closely related to the plant metabolism (Kabata-Pendias and Pendias, 2001).
Cadmium easily penetrates the roots through the cortical tissues and is translocated to
the above ground tissues (Yang et al., 1998). It is easily taken up and accumulated by
plants therefore its concentration in plant tissues can excess Cd concentration in soil
in several crop species including barley (Durcekova et al., 2007). Cd is rapidly taken
up by roots and can be loaded into xylem for its transport into leaves (RodriguezSerrano et al., 2009). Cd ions compete with elements such as K, Ca, Mg, Fe, Mn, Cu,
Zn and Ni for uptake through the same transmembrane carrier (Sanita di Toppi and
Gabbrielli, 1999). The uptake and translocation of mineral nutrients such as Fe, Zn,
Cu and Mn under Cd stress have been reported in crops such as soyabean (Cataldo et
al., 1983; Drazic et al., 2004), rice (Rubio et al., 1994; Liu et al., 2003a), and barley
(Zong et al., 2007).
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REVIEW OF LITERATURE
Cadmium enters the roots, it can reach the xylem through an apoplsatic and/or
simplastic pathway (Salt et al., 1995a) complexed with several legends, such as
organic acids and phytochelatins (Salt et al., 1995b). Normally, cadmium ions are
retained in roots and only a small fraction of this is transported to the shoots (Cataldo
et al., 1983). At the cell wall, a part of Cd is bound to the carboxy-groups of mucilage
uronic acids. This fraction cannot be transported to the shoots, therefore, cannot be
removed by shoot biomass harvesting. Thus, it is possible for plant exhibiting
significant metal accumulation into roots to express a limited capacity for
phytoextraction (Lasat, 2000).
Cadmium influx across the plasma membrane in roots occurs via
concentration- dependant process exhibiting saturable kinetics (Cataldo et al., 1983;
Costa and Morel, 1994). Cd is taken up metabolically in low and non- metabolically
in high supply. Some external factors such as light intensity, temperature, etc. may
influence mainly the metabolic way. Evidences show that Cd can also enter the
cytosol by voltage- gated Ca channels (Blaylock and Huang, 2000). Because of their
similar properties, it was suggested that Cd and Zn may be taken up and translocated
within the plant via similar pathways. Generally, the gradient of Cd concentrations in
the plant declines in the order root> leaves> seeds (Hart et al., 1998), but there are
some differences among the species and genotypes regarding Cd allocation in the
shoot. For example, Cd transport to the shoots represents 2% in soybean (Cataldo et
al., 1983), 10-20% in sugar beet (Greger and Lindberg, 1986).
2.9 Cadmium Accumulation by Plants
Cd is known to compete with several essential metal ions. It is reported that
plants accumulate a variety of cations under iron deficiency including Cd (Wuana and
Okieimen, 2011; Irfan et al., 2013). Although not essential for plant growth, Cd is
readily taken up and accumulated in plants at variable amounts (Koopmans et al.,
2008). The accumulation of Cd in roots and shoots depends on binding to
extracellular matrix, complexing inside the cell and on the transport efficiency of the
plants (Djebali et al., 2010). In general, plant accumulation of a given metal is
function of uptake capacity and intracellular binding sites. Generally the content of
cadmium in plant tissue decreases in order: root> stem> leaves>fruits> seeds. It has
been hypothesized that cadmium accumulation in developing fruits could occur via
phloem mediated transport.
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REVIEW OF LITERATURE
Fig. 10. Cadmium uptake and processing in plant (Irfan et al., 2013)
The shoot and root Cd content in cucumber (Cucumis sativus), Wheat
(Triticum aestivum), oat (Avena sativa), and tomato (Lycopersicon esculentum) grown
in nutrient solution with 100µm Cd increased 5 to 10 times it the metal concentration
increases 10 times. Greger and Lindberg (1986) reported a 4 to 10 fold increase in the
Cd content in the roots of the sugar beet (Beta vulgaris L.) when the Cd concentration
was raised from 5 to 10 µM. They also reported that the Cd content of the shoots was
only 10% to 20% of that of the roots.
Plants often accumulate large quantities of Cd without toxicity symptoms
(Lehoezky et al., 1998), which enters into food chain that may endanger human
health. Its high mobility in soil-water plant system does help easy Cd entry into food
chain. Thus using cultivars those have low metal accumulation potential could
provide an option for farmers to cope with such a risk and to reduce the influx of
pollutants into food chain. Since, cultivars of the same crop species differ widely in
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REVIEW OF LITERATURE
their response to heavy metal stress, the most appropriate option is to select one
having the genetic architecture to accumulate low Cd in edible parts be selected (Liu
et al., 2003, 2007; Yu et al., 2006). Genetic differences among plant types for Cd
absorption and accumulation is grains have been observed recently (Liu et al., 2007;
Niaz et al., 2010).
The plant cell avoids Cd accumulation in the cytosol by compartmentalizing
Cd in sub cellular compartments, although this distribution is not clearly established
yet. In maize plants, Cd may be mainly associated with cell walls and vacuoles of
bean roots (Lozane-Rodriguez et al., 1997). The accumulation of Cd in crop plants
has become a major threat to agriculture and human health. Considerable Cd
accumulation in edible parts of crops, including rice, occurs in these areas (Arao and
Ae, 2003; Arao et al., 2003; Ishikawa et al., 2005; Ippei Ogawa et al., 2009).
2.10 Role of Cadmium in Photosynthetic Pigments
Photosynthesis is also sensitive to Cd imposition, chlorophyll being one of the
targets (Somashekaraiah et al., 1992) as well as the enzymes involved in CO2 fixation
(Greger and Ogren, 1991; De-Filppis and Ziegler, 1993; Sandalio et al., 2001; Tantrey
and Agnihotri, 2010). Cd produces alterations in the functionality of membranes by
inducing lipid peroxidation (Fodar et al., 1995) and disturbances in chloroplast
metabolism by inhibiting chlorophyll biosynthesis and reducing the activity of
enzymes involved in CO2 fixation (De-Filppis and Ziegler, 1993; Romero-Puertas et
al., 2002).
In plants, Cd is particularly damaging to the photosynthetic apparatus.
Although inhibition of Rubisco activity in the Calvin cycle is considered as a primary
plant response to Cd stress (Siedlecka et al., 1997), photosystem I and II are also
affected (Krupa et al., 1993) and levels of total chlorophyll and carotenoid can be
lowered. In addition, Cd has been demonstrated to increase non photochemical
quenching in Brassica napus.
Cd damages the photosynthetic apparatus, lower chlorophyll content
and
inhibits the stomatal opening (Barcelo and Poschenrieder, 1990). Cadmium induces
profound changes in the physiology of plants. Despite its very low relative
concentration in chloroplasts, it seriously blocks the activity of photosyntehtic
processes at different routes. Cd decreases photosynthetic rate due to reduced
chlorophyll content and the enzymatic activity involved in CO2 fixation (Greger and
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REVIEW OF LITERATURE
Ogren, 1991; Moussa et al., 2010). The primary targets of Cd toxicity are also PS II
and an enzymatic phase of photosynthesis particularly Ribulose-1,5-bisphosphate
carboxylase / oxygenase (Krantev et al., 2008; Popova et al., 2008). However, in
other researches no direct Cd effects on the photosystem are found in Brassica juncea
(Haag-Kerwer et al., 1999) or Arabidopsis thaliana (Perfus-Barbeoch et al., 2002;
Januskaitiene, 2010).
Plants exposed to various concentrations of Cd: supplied as CdCl2, exhibit
reduced photosynthesis and transpiration depending on the concentration of Cd
solution and the effect generally become more pronounced with time. High Metal
concentration, 100mm Cd, reduced photosynthesis and chlorophylls (50-60%) as well
as Rubisco content, 35% and activity, 70% (Pietrini et al., 2003). It was formed that
Cd+2 impeded CO2 fixation without affecting the rates of electron transport of PS I or
PS II or the rate of dark respiration (Greger and Ogren, 1991). However, the high
sensitivity of photosynthesis to Cd could not be due to some photochemical events as
there were some contradictory results of fluorescence analyses. Yet photosynthesis is
also sensitive to Cd, which directly affects chlorophyll biosynthesis (Ekmekei et al.,
2008; Li et al., 2008) and proper development of chloroplasts (Djebali et al., 2005,
2010; Jin et al., 2008).
The effect of Cadmium on the photosynthetic process is a subject of intensive
investigations. The Attention of researchers is mainly focused on the possible sites of
toxic cadmium effect the pigment apparatus (Stiborva et al., 1988), electron transport
(Greger and Ogren, 1991).
The effect of Cd on chloroplast ultrastructure and the
integrity of the membrane system and leads to increased plastoglobule number,
changing the lipid composition and the ratios of the main structural components of
thylakoid membranes. At the same time, no one way data exist for the functional
activity of the photo synthetic apparatus in plants, subjects to cadmium stress.
Inhibition was mostly established, but in some cases stimulation of CO2 fixation was
also detected in plants treated in vivo with cadmium concentrations (Sheoran et al.,
1990; Gregar and Ogren, 1991).
The presence of stimulating effect on the integral photosynthetic process was
explained by the concentrating cadmium effect upon-the photosynthetic apparatus,
due to a stronger inhibition of plant growth as compared to that of the photosynthetic
parameters in young bean plants. There are different opinions about the cadmium
effect on membrane bound photochemical reactions and their relative part in the
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REVIEW OF LITERATURE
integral process of inhibition. According to Baszynski et al. (1980), Sheoran et al.
(1990) Cd, when applied in vivo, inhibits the electron transport of photosystem 2 (PS
II) in tomatoes, clover, alfalfa and pea plants. Greger et al. (1991) suggest that Cd
does not affect the functional activity of either PS II or PS I and suppose that its
inhibitory effect on CO2 fixation is main connected with photo-phosphorylating and
bio chemical reactions. The inhibition of photosynthesis by Cd is well documented. A
Marked decrease of photosynthesis rate (PN) for different plant species under
exposure to Cd stress has been demonstrated (Buszynski and Zurek, 2007; Kupper et
al., 2007; Krantev et al., 2008).
It inhibits the synthesis of chlorophylls (Azevedo et al., 2005) and their stable
binding to proteins (Horvath et al., 1996), thereby decreasing the accumulation of
pigment - lipoprotein complexes, particularly photosystem (PS) I, Cd interacted with
the protein subunits of light harvesting complex II. In vitro studies with isolated
Chloroplasts showed that PS II is extremely sensitive to Cd (Chugh and Sawhney,
1999). The maximum quantum efficiency of PS II as shown by ratio of variable
fluorescence to maximum fluorescence (Fv/Fm) decreased under Cd stress, indicating
inhibition of electron transfer reactions in PS II (Sigfridsson et al., 2004; Azevedo et
al., 2005; Kupper et al., 2007). The target sites of Cd action have been suggested at
both the donor and the acceptor sides of PS II (Sigfridsson et al., 2004). The
degradation of Chloroplast structure caused by Cd stress are often observed in plants,
which may be associated with a significant accumulation of Reactive oxygen species
(ROS) in plants resulting from Cd induced oxidative stress (Schutzendubel et al.,
2001; Romero-Puertas et al., 2004). However, in vitro studies indicate that CO2
fixation is inhibited by Cd without any significant effect on photochemical reactions
in isolated protoplasts and chloroplasts, which implies that Cd limits PN by affecting
the dark phase of the photosynthetic process. It has been suggested that the site of Cd
action locate either at the carboxylation step and/or the regenerated phase of Calvin
Cycle. Disturbances in the activities of Rubisco and other enzymes of the dark phase
of photosynthesis were observed in cd stressed plants (Malik et al., 1992, Burzynski
and Zurek, 2007).
Maize (Zea mays L.) seedlings were grown in nutrients solution culture
containing 0. 5 and 20 m Cd and the effects on various aspects of photosynthesis
were investigated after 24, 48, 96 and 168 h of Cd treatments. Photosynthetic rate
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REVIEW OF LITERATURE
(PN) decreased after 48 h of 20 m Cd and 96 h of 5 M Cd additions respectively.
Chl a and total chl content in leaves declined under 48 h of Cd exposure. Chl b
content decreased on extending the period of Cd exposure to 96 h. The maximum
quantum efficiency and potential photosynthetic capacity of PS II, indicated by Fv/Fm
and Fv/Fo respectively, were depressed after 96 h on set of Cd exposure (Wang et al.,
2009).
The photosynthetic apparatus appears to be especially sensitive to cadmium
negative effect. The primary targets of cadmium impact are PS II and the enzymatic
phase
of
photosynthesis,
particularly
ribulose-1,5-bisphosphate
carboxylase/
oxygenase (Krantev et al., 2008; Popova et al., 2008) whereas in other research no
direct cadmium effects on the photosystem have been found in either Brassica juncea
(Haag-Kerwer et al., 1999) or Arabidopsis thaliana (Perfus-Barbeoch et al., 2002).
Chlorophyll fluorescence is considered as very useful technique to reveal the extent of
damaging modifications in acclimated or stressed plant leaves (Krantev et al., 2008).
Among the chlorophyll fluorescence parameters, the maximum photo chemical
efficiency of PS II in dark- adapted leaves (Fv/Fm) is recognized as a good indicator
for photo-inhibitory or photo-oxidative effects on photosystem II.
Cd is an effective inhibitor of photosynthesis (Chugh and Sawhney, 1999;
Vassilev et al., 2005; Mohamed et al., 2012). A linear relationship between
photosynthesis and inhibition of transpiration was observed in clover, lucerne, and
soybean that suggest Cd inhibited stomatal opening (Barcelo and Poschenrieder,
1990). Cd damages the photosynthetic apparatus, in particular the light harvesting
complex II and photosystems I and II (Krupa et al., 1993; Siedlecka et al., 1997). Cd
also causes alteration in leaf gas exchange (Costa and Morel, 1994; Lopez-Climent et
al., 2011), stomatal closure in higher plants (Poschenrieder et al., 1989) and an overall
inhibition of photosynthesis (Ekmekci et al., 2008; Mohamed et al., 2012). In A.
thaliana Cd altered the activity of photosynthetic apparatus (Mohamed et al., 2012)
while decreased the potential quantum yield of PSII (Maksymiec et al., 2007).
Similarly, the synthesis and level of pigments are decreased in other plant species
under the influence of Cd (Ekmekci et al., 2008; Mohamed et al., 2012; Irfan et al.,
2013).
In many species, such as oilseed rape (Brassica napus) (Baryla et al., 2001),
sunflower (Di Cagno et al., 2001), Thlaspi caerulescens (Kupper et al., 2007), maize,
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REVIEW OF LITERATURE
pea, barley (Popova et al., 2008), mungbean (Wahid et al., 2008), and wheat (Moussa
and El-Gamal, 2010), the evidence showed that photosynthesis was inhibited after
both long-term and short-term Cd exposure. A large number of studies have
demonstrated that the primary sites of action of Cd are photosynthetic pigments,
especially the biosynthesis of chlorophyll (Baszynski et al., 1980) and carotenoids
(Prasad, 1995). According to Baryla et al. (2001), the observed chlorosis in oilseed
rape was not due to a direct interaction of Cd with the chlorophyll biosynthesis
pathway and most probably it was caused by decreasing of chloroplast density. The
Cd induced decrease in pigment content was more powerful at the leaf surface
(stomatal guard cells) than it was in the mesophyll. In addition, the change of cell
size, and the reducing of stomata density in the epidermis in Cd-treated leaves were
observed. Thus, Cd might interfere directly with chloroplast replication and cell
division in the leaf.
Cd ions are known to affect the structure and function of chloroplasts in many
plant species such as Triticum aestivum, Beta vulgaris (Greger and Ogren, 1991),
Vigna radiata (Keshan and Mukherji, 1992), Spinacea oleracea (Sersen and Kralova,
2001), and Phaseolus vulgaris (Padmaja et al., 1990). The main target of the
influence of Cd are key enzymes of CO2 fixation: ribulose-1,5-bisphosphate
carboxylase (RuBPCase) and phosphoenolpyruvate carboxylase (PEPCase). It has
been shown that Cd ions lower the activity of RuBPCase and damage its structure by
substituting for Mg ions, which are important cofactors of carboxylation reactions and
also Cd can shift RuBPCase activity towards oxygenation reactions (Siedlecka et al.,
1998). Stiborova (1988), Malik et al. (1992) demonstrated that Cd caused an
irreversible dissociation of the large and small subunits of RuBPCase, thus leading to
total inhibition of the enzyme. In addition to the negative effects of Cd on the
photosynthetic carboxylation reactions PSII electron transport and especially oxygen
evolving complex were found to be very sensitive to the effect of Cd (Clijsters and
Assche, 1985). Mechanism of inhibition of Cd, it is generally accepted that the wateroxidising complex (OEC) of PS2 is affected by Cd by replacing the Ca2+ in Ca/Mn
clusters constituting the oxygen-evolving centres (Sigfridsson et al., 2004) or by some
modifications in the Qb-binding site (Geiken et al., 1998). Cd also produces
alterations in the functionality of membranes by inducing changes in their lipid and
fatty acid composition (Popova et al., 2009; Tran and Popova, 2013).
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2.11 Effect of Cd on Mineral Nutrition
It has been reported that uptake, transport, and subsequent distribution of
nutrient elements by the plants can be affected by the presence of Cd ions. In general,
Cd has been shown to interfere with the uptake, transport, and use of several elements
(Ca, Mg, P, and K) and water by plants (Das et al., 1997). In sugar beet, deficiency of
Fe in roots induced by Cd was observed (Chang et al., 2003).
Cd also reduced the absorption of nitrate and its transport from roots to shoots,
by inhibiting nitrate reductase activity in the shoots (Hernandez et al., 1996).
Appreciable inhibition of the nitrate reductase activity was also found in plants of
Silene cucubalus (Mathys, 1975). Nitrogen fixation and primary ammonia
assimilation decreased in nodules of soybean plants during Cd treatments (Karina et
al., 2003). The observation of Cd-treated soybean seedlings showed that there was an
increase in laccase activity (laccases are responsible for lignin biosynthesis), during
the early stage of Cd treatment, whereby Cd induced the lignin synthesis in early
stage of root growth and as a result might cause inhibition of root elongation. How Cd
inhibits the uptake of other elements is not yet completely clear. In maize, Cd
treatment induced an inhibition of H+ATPase in root cells. Many studies revealed that
H+ATPase is an integral protein associated with the plasma membrane and is located
preferentially at the epidermal and cortical cell layers of roots. H+ATPase functions as
an ion transporter across the plasma lemma and this is dependent on the
electrochemical gradient generated by the plasma membrane H+ATPase. Thus, Cd
which causes a decrease in activity of H+ATPase, might inhibit absorption of some
essential elements (Astolfi et al., 2005).
In addition, data on poplar (Populus jaquemontiana var. glauca) showed that
Cd can inhibit mineral nutrition by competition between this metal and other metal
ions (Solti et al., 2011). It is known that Cd might inhibit the chelating process of Fe
and the loading of Fe into the xylem. Thus, the metals that are transported in the
xylem, like as occurred with Fe were influenced by Cd. The mechanism was like the
influence of Cd on Ca in competition for Ca transporters. The alkaline earth metals
except Mg. It should be mentioned that several plant nutrients have many direct as
well as indirect effects on Cd availability and toxicity. Direct effects include
decreased Cd solubility in soil by favoring precipitation and adsorption (Matusik et
al., 2008), competition between Cd and plant nutrients for the same membrane
transporters (Zhao et al., 2005), and Cd sequestration in the vegetative parts to avoid
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REVIEW OF LITERATURE
its accumulation in the grain/edible parts (Hall, 2002). Indirect effects include dilution
of Cd concentration by increasing plant biomass and alleviation of physiological
stress (Tran and Popova, 2013).
2.12 Role of Cadmium in ROS
Cadmium is a non-redox metal, but it leads to the formation of reactive
oxygen species (ROS) such as superoxide radicals (O2-•), singlet oxygen (1O2),
hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) (Munoz et al., 2008; Verma et
al., 2008; Zou et al., 2012). Recently the cellular production of reactive oxygen
species (ROS) in leaves from pea plants under Cd stress has been reported (RomeroPuertas et al., 2004). ROS were detected in epidermal transfer and mesophyll cells,
with plasma membrane being the main source of ROS, although mitochondria and
peroxisomes were also involved (Romero-Puertas et al., 2004). Concerning the
mechanisms of ROS production, Cd does not participate in Fenton-type reactions
(Stoch and Bagchi, 1995) but can indirectly favour the production of different ROS
such as hydrogen peroxidase (H2O2), superoxidase (O2-) and hydroxyl radical (.OH),
by unknown mechanisms, giving rise to an oxidative burst (Olmas et al., 2003;
Romero Puertas et al., 2004; Garnier et al., 2006; Rodrigues-Sarrano et al., 2009).
The enzymes superoxidase dismutase (SOD), catalase (CAT), and peroxidase
(POx) are involved in the detoxification of O2- (SOD) and H2O2 (CAT, POx), thereby
preventing the formation of (.OH) radicals. Ascorbate peroxidase and glutathione
reductase as well as glutathione are important components of the ascorbate
glutathione cycle responsible for the removal of H2O2 in different cellular
compartments (Jimenez et al., 1997; Noctor et al., 1998; Rodrigues-Sarrano et al.,
2009). A common consequence of most abiotic and biotic stresses that they at some
stages of stress exposure is an increased production of reactive oxygen species.
Reactive oxygen species may lead to the unspecific oxidation of proteins and
membrane lipids or may cause DNA injury. Both Cd induced generation of ROS
(Olmos et al., 2003; Cho and Seo, 2005) or Cd caused inhibition of enzymatic
(Sandalio et al., 2001) or non enzymatic antioxidants (Rellan-Avarez et al., 2006)
have been reported. The crucial role of ROS regulation in the cell at the presence of
elevated concentration of toxic metals is supported also by observation that some
heavy metals hyper accumulator plants have increased antioxidant apparatus
compared to non-accumulators (Iannelli et al., 2002; Durcekova et al., 2007).
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Generally, heavy metals cause oxidative damage to plants, either directly or
indirectly through reactive oxygen species (ROS) formation. Certain heavy metals
such as copper and iron can be toxic through their participation in redox cycles like
Fenton and/or Haber-Weiss reactions. In contrast, Cd is a non-redox metal unable to
perform single electron transfer reactions, and does not produce ROS such as the
superoxide anion (O2•-), singlet oxygen (1O2), hydrogen peroxide (H2O2), and
hydroxyl radical (OH•), but generates oxidative stress by interfering with the
antioxidant defence system (Benavides et al., 2005; Cho and Seo, 2005; Gratao et al.,
2005). Cd inhibits the photoactivation of photosystem 2 (PSII) by inhibiting electron
transfer. Thus, Cd could lead to the generation of ROS indirectly by production of a
disturbance in the chloroplasts. In addition, other reports suggested that Cd may
stimulate the production of ROS in the mitochondrial electron transfer chain (Heyno
et al., 2008). Treatment of pea and rice plants with Cd stimulates the plasma–
membrane-bound NADPH oxidase in peroxisomes and thus generates ROS. In
Medicago sativa exposure to Cd for 6-24 h caused a rapid accumulation of peroxides
and depletion of glutathione (GSH) and homoglutathione (hGSH), and led to redox
imbalance. The Cd-induced cell death in bright yellow-2 (BY-2) tobacco cells was
preceded by NADPH-oxidase-dependent accumulation of H2O2 followed by cellular
O2 and fatty acid hydroperoxide accumulation (Gill and Tuteja, 2010). The
manifestations of ROS damages in plants involve lipid peroxidation, protein
peroxidation, and DNA damage (Tran and Popova, 2013).
Cd can also negatively interfere with important plant processes such as water
transport, oxidative phosphorylation in mitochondria, photosynthesis and chlorophyll
content (Djebali et al., 2005). Cd is capable of including oxidative, stress which in
turn can result in a variety of antioxidant responses (Tames et al., 2008). Some of the
damage reported is related to leaf structure disorganisation, reduced intercellular air
spaces, drastic structural thylakoid alterations in the chloroplasts (Djebali et al.,
2005), Stomatal closure, softening of cell wall thickening and decrease content and
efficiency of Rubisco activity. Cd produces disturbances in the plant antioxidant
defences, producing an oxidative stress (Romero-Puertas et al., 2002, Rodriguez
Serrano et al., 2006, 2009). As consequences tissues injured by oxidative stress
generally
contain
increased
concentrations
of
carbonylated
proteins
and
malondialdehyde and show an increased production of ethylene. Cadmium can cause
oxidative stress by inducing the generation of reactive oxygen species such as H2O2,
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O2- radicals and .OH radicals, as well as disturbances in the antioxidative systems for
the detoxification of ROS. Cd is a non-redox metal unable to perform single electron
transfer reactions and does not participate directly in production of ROS such as the
superoxide anion (O2-), single oxygen (O2), hydrogen peroxide (H2O2) and hydroxyl
radical (OH.), but generates oxidative stress by interfering with the antioxidant
defense system (Gratao et al., 2005; Gonsalves et al., 2007).
Chloroplasts, the photosynthetic organs, are highly sensitive to cadmium
toxicity induced damage (Sandalio et al., 2001). Cadmium could lead to be
regeneration of ROS indirectly by production of disturbance in the chloroplasts. ROS
are also produced by the reaction of chloroplast O2 and electrons that escape from the
photosynthetic electron transfer system under normal circumstances. Cd inhibits the
photoactivation of photosystem II by inhibiting the electron transfer (Sigfridsson et
al., 2004). Other reports suggest that cadmium may also stimulate the product of ROS
in the mitochondrial electron transfer chain (Heyno et al., 2008). Cd toxicity induces
the oxidative stress by disturbing the balance between ROS generation and their
removal. Heavy metals, including cadmium are associated with oxidative stress, plant
damage and changes of metabolism such as nutrient uptake, contents of pigments,
protein, chlorophyll synthesis, the profile concentration or activity of isozymes and
enzymes of stress metabolism (Clemens, 2006; Ghani and Wahid, 2007; Zhang et al.,
2010). Several reports have proved that cadmium weakens the antioxidative
protection by depleting the glutathione pool, interacting with essential thiol groups in
proteins, Zn displacement, altered calcium and sulfohydryle homeostasis, or via
generation of reactive oxygen species by redox cycling quinones (Stohs and Bagchi,
1995; Dinaker et al., 2009).
2.13 Metabolic Process
Cd interferes with plant metabolic processes, causing root growth retardation,
suberization, damage to internal and external root structures, decreased root hydraulic
water conductivity, interference with nutrient absorption and translocation leading to
nutrient imbalance, decreased chlorophyll content, interference with enzymatic
activities related to photosynthesis and a decrease in stomatal opening and
conductance (Prasad, 1995; Benavides et al., 2005; Zhang et al., 2010). Cd is one of
the most toxic metals for plants and animals affecting many of metabolism. A number
of detrimental effects of Cd on metabolism have also been reported, such as decreased
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nutrient uptake (Ghnaya et al., 2007), changes in nitrogen metabolism (Wang et al.,
2007), changes of water balance and inhibition of stomatal opening (Sandalio et al.,
2001; Djebali et al., 2010).
In plants, exposure to Cd causes reductions in biomass production and
nutritional quality, and inhibition of photosynthesis, stomatal, conductance,
transpiration rate (Shi and Cai, 2008), and other metabolic activities (Januskaitiene,
2010). These toxic effects have been associated with alterations in fundamental
metabolic processes of higher plants such as photosynthesis (Krupa et al., 1993) or
cell respiration (Greger et al., 1991; Hernandez and David, 1997). The uptake,
distribution and effects of Cd on different metabolic processes seem to be controlled
by several plant factors such as species and/or variety (Page et al., 1972) water and
nutritional status (Barcelo and Poschenrieder, 1990; Oliveira, 1994).
2.14 Antioxidant Enzymes
A variety of proteins function as scavengers of superoxide and hydrogen
peroxide. These include, among others, superoxide dismutase (SOD), catalase (CAT),
ascorbate
peroxidase
(APx),
monodehydroascorbate
reductase
(MDHAR),
dehydroascorbate reductase (DHAR), peroxidases (POD), and glutathione reductase
(GR), and non-enzymatic scavengers, including, but not limited to, glutathione
(GSH), ascorbic acid (ASA), carotenoids, and tocopherols. SOD, GR, APx, POD, and
CAT showed variations in their activities that depend on the Cd concentration and
plant species used. Increased activity of SOD has been detected in many Cd treated
plants, such as pea (Sandalio et al., 2001), wheat (Milone et al., 2003), and bean
(Cardinaels et al., 1984).
Decline in the enzymatic activity of CAT and SOD has been associated with
Cd toxicity in Phaseolus vulgaris (Chaoui et al., 1997), Phaseolus aureus (Shaw,
1995), H. annuus (Gallego et al., 1996), and Pisum sativum (Sandalio et al., 2001).
Variable activity of CAT has been observed under Cd stress. Yilmaz and Parlak
(2011) reported that the observed high tolerance of Groenlandia densa to Cd stress
was partially due to high activity of CAT. Its activity increased in rice, mustard,
wheat, chickpea, and black bean (Vigna unguiculata subsp. cylindrica) roots and
declined in soybean, Phragmites australis, Capsicum annuum and Arabidopsis under
Cd stress (Gill and Tuteja, 2010). APx and GPx are scavengers of H2O2 in ROS
detoxification. An increase in leaf APx activity under Cd stress has been reported in
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Ceratophyllum demersum, mustard, wheat, and black bean. An increase in GPx
activity in Cd-exposed plants was reported in wheat, Arabidopsis and Ceratophyllum
demersum.
It was found that an initial increase in GPx activity in spruce needles subjected
to Cd stress, and subsequent Cd treatments caused a decline in the activity (Gill and
Tuteja, 2010). A decrease in POD activity caused by Cd was reported in mustard
(Brassica juncea) (Markovska et al., 2009). An increase in GR activity was found in
cotton, Arabidopsis, blackgram, wheat, and mustard upon Cd treatment (Markovska et
al., 2009; Gill and Tuteja, 2010). Cd stress increases the activity of POD in radish (ElBeltagi et al., 2010) and causes no significant change in the leaves of pea plants. An
increase in ASH content during Cd exposure was found in barley. In contrast, a
decrease in ASH in the roots and nodules of soybean under Cd stress was also
observed. Cd also decreased the ASH content in cucumber chloroplasts and in the
leaves of Arabidopsis and pea, whereas it remained unaffected in Populus canescens
roots (Gill and Tuteja, 2010). An increase in GSH levels, which resulted in enhanced
antioxidant activity against Cd toxicity, has been found in the leaves and chloroplasts
of Phragmites australis Trin. (Cav.) ex Steudel. Increased concentration of GSH has
been observed with increasing Cd concentration in pea, Sedum alfredii, and black
bean.
A decrease in GSH, which could weaken the antioxidative response and
defensive strength against Cd stress in the more sensitive genotypes, was also
found in pea (Metwally et al., 2005). The role of proline as an antioxidant was
reported in tobacco cells exposed to Cd stress. Islam et al. (2009) reported that
tobacco cells exposed to Cd treatment accumulated high levels of proline and by
this way they can alleviate the inhibitory effect of Cd on cell growth (Tran and
Popova, 2013).
Proline accumulation in cd2+ stress has been demonstrated in shoots
of Brassica juncea, Triticum aestivum and Vigna radiata by Dhir et al. (2004) and
Zengin and Munzuroglu (2006) in sunflower. Cd produced an enhancement of lipid
peroxidation in Phaseolus vulgaris (Chaoui et al., 1997), Helianthus annuus (Gallego
et al., 1996), and Pisum sativum (Lozano-Rodriguez et al., 1997; Tran and Popova,
2013).
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2.15 Protein Content
Heat-shock proteins (HSPs) are presently known as proteins (Tran and
Popova, 2013). Palma et al. (2002) reported a decrease in protein content in Brassica
juncea under Cd stress due to enhanced protein degradation process as a result of
increased protease activity. The reduced protein content under Cd Stress could have
resulted from induced lipid peroxidation and fragmentation of proteins due to toxic
effects of reactive oxygen species. In B. juncea, John et al. (2009) observed a
decrease of 24.9 and 87.3% in the protein content respectively at 450 and 900 µM Cd.
In Albizia procera, a decrease in crude protein content with increasing cadmium
concentration up to 10 ppm.
Cadmium stress causes the de-naturation of proteins. Stress proteins restore
the native conformation of proteins denaturated by cadmium or to decompose them.
In Cd treated maize plants, Reddy and Prasad (1993) observed the synthesis of 70
KDa phosphoprotien, heat shock protein (HSC) 70. The accumulation of mRNA of
an 18 KDa heat shock protein gene, 51 kDa soluble protein was found. This protein
was designated as a Cd stress-associated protein. It was generated mainly in the root
tissue of treated and control seedlings and located below the plasma membrane and
outer periphery of the tonoplast (Mittra et al., 2008). In poplar (Populus tremula L.)
exposed to Cd for a short term or a longer term treatment, it was found that stressrelated proteins, like HSPs, proteinases, and pathogenesis-related proteins, increased
in abundance in leaves. Lee et al. (2010) reported that Cd affected the synthesis of 36
proteins in rice. In roots, the synthesis of 16 proteins was increased, while the
synthesis of 1 protein was reduced. In leaves, the synthesis of 16 proteins was upregulated, while the synthesis of 3 proteins was down-regulated. Treatment of tomato
plants with a low Cd concentration (10 μM) induced changes in 36 polypeptides,
while higher Cd concentration (100 μM) induced changes in 41 polypeptides
(Rodríguez-Celma et al., 2010). In 3-week-old Arabidopsis thaliana seedlings
exposed to 10 μM Cd, it was found that among 730 determined proteins 21 were upregulated in response to Cd (Tran and Popova, 2013).
2.16 Chromium
Chromium (Cr) is one of the most toxic heavy metals found 7th abundantly in
the earth’s crust (Panda and Choudhary, 2005; Nematshahi et al., 2012), which
attenuates the environment. Although toxic, it has nutritive importance too (Kabata49
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Pendias and Pendias, 2001). Despite these factors, in contrast to other toxic metals
like cadmium, lead, mercury and aluminum (Shanker et al., 2005). The metal was first
discovered in the Siberian red ore (crocoites) in 1798 by the French scientist
Vauquelin. As a matter of fact, the name chromium is derived from the Greek word
“χρώμα” (chroma-color) due to that propriety of the element (Santos and Rodriguez,
2012).
It is a transition element located in the group VI-B of the periodic table with
a ground-state electronic configuration of Cr 3d54s1 (Shanker et al., 2005).
Chromium can exist in several chemical forms, displaying oxidation numbers from
0 to VI. There are mainly two stable oxidation states of chromium (viz., Cr 6+ and
Cr3+), Cr6+ is considered to be more toxic than Cr 3+ (Panda and Patra, 2000, Ahmad
et al., 2011). The intermediate states of +4 and +5 are metastable and rarely
encountered (Zayed and Terry, 2003; Maria, 2012). Cr (III) is the predominant form
in most minerals and is favoured by reducing and strongly acidic conditions, while,
Cr (VI) occurs under oxidizing and alkaline conditions (Oze et al., 2004; Dermatas
et al., 2012). Cr(VI) is conceived as the most toxic form of Cr, highly soluble in
water, which usually occurs amalgamated with oxygen as chromate (CrO4 2−) or
dichromate (Cr2O72−) oxyanion (Shanker et al., 2005). Cr salts are used in many
industrial process products such as leather tanning, electroplating, steel production,
metal finishing, catalyst application, pigment manufacturing, and metal corrosion
inhibitors (Zayed and Terry, 2003; Nath et al., 2005; Venkateswaran et al., 2007).
2.17 Occurrence of Chromium
The provenance of Cr in environment is both natural and anthropogenic.
Chromium is a naturally occurring elements found in rocks, animals, plants, soil and
in volcanic dust and gases. Whereas chromium (VI) is generally produced by
industrial processes (Lakshmi and Sundaramoorthy, 2010). Therefore, they are
present in the effluents of industries and in municipal sewage (Nath et al., 2005;
Venkateswaran et al., 2007). Chromium is a naturally occurring element abundant in
rocks, animals, plants, soil, and in volcanic dust and gases. Cr is released from a wide
spectrum of anthropogenic activities such as smelting of metallic ores, industrial
fabrication, and commercial applications of metal into the environment, which
ultimately have significant antagonist biological and ecological effects (Kotas and
Stasicka, 2000). Other sources of anthropogenic contamination include the
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augmentation of manure, sewage, sludge, fertilizers, and pesticides to soil. Normal
range of Cr is from 10 to 50 mg/ kg depending on the parental material (Pandey and
Pandey, 2008; Hayat et al., 2012). The most important sources of Cr (III) are fugitive
emissions from road dust and industrial cooling towers; also, Cr (VI) compounds are
still used in the manufacture of pigments, in metal-finishing and chromium-plating, in
stainless steel production, in hide tanning, as corrosion inhibitors, and in wood
preservation.
2.18 Visual Symptoms of Chromium
Symptoms of Chromium toxicity were expressed differently in different
plants. Cr phytotoxicity includes inhibition of seed germination or of early seedling
development, reduction of root growth, leaf chlorosis and depressed biomass (Sharma
et al., 1995; Hamid et al., 2012; Nematshahi et al., 2012). Soane and Saunder (1959)
and Rout et al. (1997) found the toxicity symptoms in corn and mung bean due to the
application of Chromium in excess by which the plants were severely stunted, and the
leaves had a tendency to roll around the shoot; the leaves were narrow and purple
green with an intense purple colour on the lower two inches of the lower blade. In
tobacco, no specific toxic symptoms were marked; but shoot development was
depressed and consequently no inflorescence developed. Hunter and Vergnano (1953)
reported that oat plants affected by chromium toxicity were stunted with poorly
developed roots and small necrotic lesions. They also reported that plants receiving
5.0 ppm Chromium in the nutrient solution were usually normal, but showed signs of
mild chlorosis of the leaves at 10 ppm chromium; the plants were stunted and most of
the leaves showed chlorosis and necrotic lesions at 25 and 50 ppm chromium. Roots
showed normal growth at 5 and 10 ppm chromium but poor root developed at higher
concentrations. Hewitt (1953) reported Cr toxicity in maize plants. Chromium toxicity
caused wilting of the tops of soybean plants at 5 ppm Cr only after two days of
treatment (Turner and Rust, 1971). Barcelo et aI. (1985) reported that visual
symptoms of toxicity in bush bean (Phaseolus vulgaris cv. Contender) occurred at 2.5
and 5ppm of Cr treatment. The plants showed chlorosis in trifoliated leaves as in case
of iron deficiency. Samantaray et al. (1996b) observed severe chlorosis and stunted
growth of rice (Oryza sativa cv. Pathara) grown on chromite minewaste containing a
high percentage of chromium. Corradi et al. (1993) recorded suppression of lateral
shoots with a diminishing trend with the increase in the dose of chromium. Lateral
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root production was completely inhibited at 10 ppm Cr (VI). At the time of harvest,
older leaves of these plants treated with 5 ppm Cr showed interveinal chlorosis with
the development of interveinal necrotic areas. A high dose of Cr (30 or 60 ppm) added
to the plants resulted in the death of plants within three days of treatment in
hydroponics as well as pot culture experiments. Samantaray et al. (1996a) also
reported severe chlorosis and necrosis of leaf in E. colona at high concentrations of Cr
in solution culture (Samantaray et al., 1998).
2.19 Chromium in Soils
Cr as one of several heavy metals that cause serious environmental
contamination in soil, sediments and ground water, is a matter of serious problem
(Hamid et al., 2012). The common chromium mineral is chromite, and the chromium
content of acid igneous and sedimentary rocks ranges from 5-120 ppm (KabataPendias and Pendias, 1992). These metals are deposited in different soil profiles
leading to long-term metal contamination (Lakshmi and Sundaramoorthy, 2010).
Chromium is an average concentration of 100 ppm, ranging in soil between 1 mg/kg
and 3000 mg/kg; in sea water from 5 μg/L to 800 μg/L and in rivers and lakes
between 26 μg/L and 5.2 mg/L. Normally, Cr is mined from chromate but native
deposits are not unheard off. One of the most interesting characteristics of this metal
is its hardness and high resistance to corrosion and discoloration. The importance of
these proprieties resulted among others in the usage of this metal in the development
of stainless (Santos and Rodriguez, 2012). Generally, the parent material determines
the levels of chromium in soil, and typical soil chromium contents range from 20-65
ppm (Kabata-Pendias and Pendias, 1984). However, Langard (1980) has identified the
elevated levels of chromium with anthropogenic contamination mainly through
industrial processes.
Alloway and Ayres (1997) have reported an average concentration of 100 ppm
for chromium in earth’s crust, and natural chromium content in surface soils has been
estimated to range from 5-1100 ppm, with a mean value of 60 ppm (Ward, 1995).
Furthermore, Kabata-Pendias and Pendias (1992) have reported the mean chromium
content for world sandy soils to be 47 ppm. Discarded manufactured products and
coal ashes constitute the major sources of anthropogenic chromium inputs into soils,
and the total worldwide inputs of chromium into soils is estimated to be 898 x 103
tons per year (Nriagu, 1990). Values representing the maximum allowable limits
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(MAL) of chromium contents in soils for many countries have also been documented
by Kabata-Pendias (1995); Great Britain (50 ppm), Canada (75ppm), Austria and
Poland (100 ppm), as well as 200 ppm for Germany. Chromium of environmental
concern is the waste chromium, which has the characteristic spinel structure,
FeO.Cr2O3, whose trivalent chromium can be converted to hexavalent chromium after
an alkaline roasting process (Bartlett and James, 1996), leaving them as residuals that
become waste forms of chromium in soil-water system. Generally, very little
chromium is leached from soil due its presence as an insoluble Cr2O3. xH2O
(Fishbein, 1981). However, the mobility and toxicity of chromium depend on its
oxidation state; trivalent chromium is relatively immobile, often bound to both
organic and inorganic ligands in soils, more stable and extremely less toxic than
hexavalent chromium (Ross et al., 1981), which is a peculiar industrial pollutant.
Furthermore, the biological reduction of toxic and more mobile hexavalent chromium
to trivalent chromium by organic matter occurs in soils (Barlett and Kimble, 1976),
and this is responsible for the low chromium availability to plants. The major
processes by which the trivalent chromium is transported from soil include aerial and
surface water transport through aerosol formation and runoff respectively.
Both Cr (III) and Cr (VI) differ in terms of mobility, bioavailability and
toxicity. Cr (III), on the other hand, is less toxic, less mobile, and is mainly found
coalesced to organic matter in soil and aquatic environment (Becquer et al., 2003).
Since trivalent and hexavalent Chromium concentrations in soil can range from 0.1 to
250 ppm Cr, and in certain areas, soil content may be as high as 400 ppm Cr (Langard
and Norseth, 1979). Overall, most soils have been shown to contain on average 50
ppm Cr (Hartel, 1986). Chromium in ambient air originates primarily from industrial
sources (i.e., steel manufacturing and cement production) and the combustion of fossil
fuels. The content in coal and crude oil varies from 1 to 100 mg Cr/L and from 0.005
to 0.7mg Cr/L, respectively (Revathi et al., 2011). A number of soil processes and
environmental factors may affect the form and biomobilization potential of chromium
(Order, 2002). In soils, Cr is present mostly as insoluble chromium hydroxide
aqueous solution [Cr (OH)3 aq] or as Cr (III) adsorbed to soil components (Maria,
2012).
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2.20 Chromium Uptake, Translocation in Plant
The first interaction Cr has with a plant is during its uptake process. Cr is a
toxic, nonessential element to plants; hence, they do not possess specific mechanisms
for its uptake. Therefore, the uptake of this heavy metal is through carriers used for
the uptake of essential metals for plant metabolism. The toxic effects of Cr are
primarily dependent on the metal speciation, which determines its uptake,
translocation and accumulation. The pathway of Cr (VI) transport is an active
mechanism involving carriers of essential anions such as sulfate (Cervantes et al.,
2001). Fe, S and P are known also to compete with Cr for carrier binding (Wallace et
al., 1976). Independent uptake mechanisms for Cr (VI) and Cr (III) have been
reported in barley. The use of metabolic inhibitors diminished Cr (VI) uptake
whereas it did not affect Cr (III) uptake, indicating that Cr (VI) uptake depends on
metabolic energy and Cr (III) does not (Skeffington et al., 1976). In contrast, an
active uptake of both Cr species, slightly higher for Cr (III) than for Cr (VI), was
found in the same crop (Ramachandran et al., 1980). Golovatyj et al. (1999) have
shown that Cr distribution in crops had a stable character which did not depend on
soil properties and concentration of this element; the maximum quantity of element
contaminant was always contained in roots and a minimum in the vegetative and
reproductive organs (Shanker et al., 2005). Vegetables grown at tannery
contaminated sites could take up and accumulate chromium (Cr) at concentrations
that are toxic (Nigussie et al., 2012).
Chromium uptake and translocation by plant cells were very low and chromium
concentration associated with the root was greater than that in the leaf which in turn
was greater than that in the fruit (Ramachandran et al., 1980). Similar results were
also reported on the uptake of chromium (VI) over chromium (III) in soils (Cary et
al., 1977a,b; Lahouti and Peterson, 1979). Often plants growing on low chromium
soils may appear to have lower concentrations of chromium as compared to the same
plants grown on high chromium soil which may be due to contamination (Cary and
Kubota, 1990). Addition of up to 1% chromium as Cr (OH)3 to soils increased the
chromium concentration in alfalfa and buckwheat (Cary et al., 1977b). Many workers
noted that the lower chromium concentrations were found in the fruit with increased
levels in the stem and the highest concentration in the leaf (Desmet et al., 1975a, Cary
et al., 1977a,b; Samantaray et al., 1998). Sequestration of most of the Cr in the
vacuoles of the root cells to render it non-toxic could be the reason for poor
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translocation of Cr to shoot, which may be a natural toxicity response to the plant
(Hayat et al., 2012) as shown in Fig. 11-13.
Fig. 11. The overview of chromium resources and uptake responses in plants (Hayat
et al., 2012)
The uptake and distribution mechanisms of Cr in vegetative and reproductive
parts of the plant are not yet fully understood. As Cr is a non-essential element to
plants, they do not possess specific mechanisms for its uptake. Therefore, carriers used
from uptake of heavy metals are used for its uptake. The mechanism of Cr uptake and
translocation in plants differs with lapse of time. Both active and passive transports
seem to be involved in the uptake mechanisms; former prevails at lower concentration,
whereas latter becomes more significant at toxic levels (when membrane selectivity
has been lost). The impact of Cr contamination in the physiology of plants depends on
the metal speciation which is responsible for its mobilization, subsequent uptake,
translocation, and accumulation resulting in toxicity of the plant system. The pathway
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of Cr (VI) transport is an active mechanism involving carriers of essential ions such as
sulfate (Cervantes et al., 2001). Fe, S, and P are known also to compete with Cr for
carrier binding (Wallace et a1., 1976). The root plasma membrane is the first
functional structure to come in contact with toxic metals and is thought to play a
critical role in plant tolerance. The use of metabolic inhibitors diminished Cr (VI)
uptake, whereas it did not affect Cr (III) uptake indicating that Cr (VI) uptake depends
on metabolic energy and that of Cr (III) does not. Uptake of Cr (III) seems to be
passive while that of Cr (VI) seems to be active (Barcelo and Poshenrieder, 1997). The
uptake of Cr (VI) is mediated by the sulfate carrier with lower affinity (Skeffington et
al., 1976), Cr (III) tightly binds to carboxyl group of amino acids in proteins forming
binuclear complexes (Schlosser, 1991). It has been reported that after uptake of Cr
(IV), it is immediately reduced to Cr (III) in the cells (Myttenaere and Mousny, 1974).
Cr (III) is located in the cytosol inside the cell (Yamamoto et al., 1981). The
persistence of Cr in soil can lead to increase in the uptake by the plants and the
persistence is due to low mobility and recalcitrant nature of the metal.
Srivastava et al. (1999a,b) suggested that in Lycopercicum esculentum
carboxylic acid and amino acids present in the root are involved in the enhanced
uptake of chromium in the roots. Cr mainly moves in the xylem of the plants. Cr
distribution in crops had a stable character which does not depend upon soil properties
and the concentration of this element. The maximum quantity of element contaminant
was always contained in roots and the minimum in the vegetative and reproductive
organs (Zayed et al., 1998; Pulford et al., 2001). As the metal pollutants are nondegradable and are readily taken up by plants (Lakshmi and Sundaramoorthy, 2010).
Both forms, Cr (III) and Cr (VI), may be taken up by plants. Uptake of Cr (III) is
considered passive, while that of Cr (VI) is considered to be active (Liu et al., 2008;
Ahmad et al., 2011).
2.21 Chromium Accumulation and Absorption by Plants
In 7 out of 10 crops analyzed, more Cr accumulated when plants were grown
with Cr (VI) than with Cr (III) (Zayed et al., 1998). Skeffington et al. (1976) from
radioactive tracer studies using 51Cr reported that Cr mainly moved in the xylem of
the plants. In bean, only 0.1% of the Cr accumulated was found in the seeds as against
98% in the roots (Huffman and Allaway, 1973a). The reason of the high accumulation
in roots of the plants could be because Cr is immobilized in the vacuoles of the root
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cells, thus rendering it less toxic, which may be a natural toxicity response of the plant
(Shanker et al., 2004a). Since both Cr (VI) and Cr (III) must cross the endodermis via
symplast, the Cr (VI) in cells is probably readily reduced to Cr (III) which is retained
in the root cortex cells under low concentration of Cr (VI) which in part explains the
lower toxicity of Cr (III). Although higher vascular plants do not contain Cr (VI)
reducing enzymes, they have been widely reported in bacteria and fungi (Cervantes et
al., 2001; Shanker et al., 2005).
It is of great interest to determine the absorption and distribution pattern of
metals in different vegetable parts, especially in the edible parts, due to the increasing
toxicity problems caused by metals in the soil-plant system (Baxter et al., 1983) so as
to estimate the basal load that a crop can stand without exceeding permissible limits
recommended for consumption. The absorption and accumulation of chromium in
different plants were reported in Citrus sinensis (0.2 to 0.3 ppm), pear (Pyrus
commuhis) (0.03 to 0.85 ppm), Triticum spp. (10.2 to 14.8 ppm) and corn (Zea mays)
(0.22 to 0.74 ppm) by using different types of culture systems (Liebig et al., 1942;
Saint-Rat, 1948; Vergnano, 1959; Shimp et al., 1957). In oat (Arena sativa) grown in
serpentine soil, the accumulation of chromium varied between 3 to 11 ppm (Soane
and Saunder, 1959). They also reported that the accumulation of chromium in leaves
varied between 4 and 14 ppm and in roots from 13 to 175 ppm in tobacco (Nicotina
tobacum) grown in serpentine soil.
Mung bean plants grown in chromite mine wastes accumulated 62.29 to 70.73
ppm chromium (Samantaray and Das, 1997). Chromium accumulated mainly in the
roots and was poorly transported to shoots (Moral et al., 1994; Samantaray and Das,
1997) possibly due to spatial localization in a specific sub cellular compartment in the
root cells (Barcelo et al., 1985). The level of chromium in plants grown in solution
culture containing chromium ranged from 3.8-10.53 (ng Cr/g dry weight) in tomato,
12-31 (ngCr/g dry weight) in potato, 19-42 (ng Cr/g dry weight) in wheat and 18-30
(ng Cr/g dry weight) in bean (Huffman and Allaway, 1973). The dynamics of metals
in the absorption as well as the accumulation in various plant parts have been studied
in soilless cultures (Moral et al., 1996). Hexavalent chromium was found to be
preferentially absorbed by the roots (Ishihara et al., 1968). Subsequently, Moral et al.
(1996) found that trivalent chromium was also absorbed by the roots; less is
accumulated and transported to aerial parts. Hunter and Vergnano (1953) reported that
the chromium accumulation varied between 0.04-3.9 ppm in the oat plants grown in
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Fig. 12. Hypothetical model of chromium transport and toxicity in plant roots
(Shanker et al., 2005).
Fig. 13. Schematic representation of translocation of Cr and Ni from root to leaf
(Kumar and Maiti, 2013).
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solution culture. Accumulation and distribution of chromium were observed in trees,
shrubs and herb naturally growing on chromite mine wastes containing high
percentage of chromium (Samantaray et al., 1998a,b). Cary and Kubata (1990)
explained the effects of chromium concentration in soil on chromium accumulation in
plants (Samantaray et al., 1998). This higher Cr (III) uptake has been reported to be
detrimental to crops. The effect of Cr (III) on Vigna radiate germination and seedlings
revealed that high Cr concentration had a significant reduction in shoot and root
length. The Chromium has also been found to reduce water status and mineral
nutrition of bean plant (Azmat and Khanum, 2005).
An important reason for enhanced accumulation of chromium in the root may
be due the presence of organic acids in the root exudates which form complexes with
chromium, thereby making them available for the uptake by root. Chromium
accumulation was comparatively lower in Vigna radiata, therefore present in small
quantities in the shoot whereas maximum accumulation was shown by Vigna
unguiculata (Hayat et al., 2012). Shanker et al. (2005) suggested that generally in
plants chromium translocation from root to shoot is very slow. In both species of
Vigna, chromium accumulation was very high in the roots compared to the stem
(Hayat et al., 2012). In bean, only 0.1% of the Cr accumulated was found in the seeds
as against 98% in the roots (Huffman and Allaway, 1973). The reason for the higher
accumulation in roots of the plants could be because Cr is immobilized in the
vacuoles of the root cells, thus rendering it less toxic, which may be a natural toxicity
response of the plant (Shanker et al., 2004). Since both Cr (VI) and Cr (III) must cross
the endodermis via symplast, the Cr (VI) in cells is probably readily reduced to Cr
(III), which is retained in the root cortex cells under low concentration of Cr (VI)
which in part explains the lower toxicity of Cr (III). Although higher vascular plants
do not contain Cr (VI) reducing enzymes, they have been widely reported in bacteria
and fungi (Cervantes et al., 2001). These metals are mostly absorbed by plants easily
and prove toxic to plants that can be observed as growth retardation (Arun et al.,
2005). Plants may absorb toxicant trace metals either directly from the atmosphere
through leaves (or) roots from soil and water (Lakshmi and Sundaramoorthy, 2010).
2.22 Chromium Assimilation by Plants
Chromium is a common contaminant of surface waters and ground waters
because of its occurrence in nature, as well as anthropic sources (Babula et al., 2008).
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Cr (III) and Cr (VI), being the most stable are also the important in terms of
environmental contamination. Very few studies have attempted to elucidate the
transport mechanisms of Cr in plants, but factors like oxidative Cr state or its
concentration in substrate play important roles (Babula et al., 2008). Due to its higher
solubility and thus, bioavailability, Cr (VI) is more toxic at lower concentrations than
Cr (III), which tend to form stable complexes in soils (Lopez-Luna et al., 2009). Also,
the pathway of Cr (VI) transport is thought to be an active mechanism involving
carriers of essential anions such as sulfate (Cervantes et al., 2001). Fe, S and P are
known also to compete with Cr for carrier binding (Wallace et al., 1976). Also Cr
absorption and translocation have been show to be modified by soil pH, organic
matter content and chelating agents, among others. However some plants (such as
soybean and garlic) have the capacity to reduce Cr (VI) to unstable intermediate like
Cr (V) and Cr (IV), or eventually to the more stable form, Cr (III); this represents the
detoxification pathway of Cr (VI) (Babula et al., 2008). As this mechanism of
detoxification is performed readily in the roots and as Cr is immobilized in the
vacuoles of the root cells, the amount of Cr translocated to the aerial portion of the
plants is very little (Shanker et al., 2005).
2.23 Role of Chromium in Photosynthesis
Like other metals, Cr can affect photosynthesis severally and in many different
steps, which can ultimately translate in loss of productivity and death. Shanker et al.
(2005), Cr(VI) can easily cross biological membranes and has high oxidizing
capacity, generating reactive oxygen species (ROS) which might induce oxidative
stress (Pandey et al., 2009). ROS are generated in normal metabolic processes like
respiration and photosynthesis, being chloroplasts one of the main sites of reactive
oxygen production and detoxification (Mittler, 2002). However, because the
chloroplast has high amounts and complex systems of membranes rich in
polyunsaturated fatty acids, this organelle might also be a target for peroxidation
(Hattab et al., 2009b) and one of the ways by which photosynthesis is affected. A
common parameter affected by Cr is the amount of photosynthetic pigments, which
tends to decrease when plants or algae are exposed to high doses of this metal
(Rodriguez et al., 2011; Subrahmanyam, 2008; Vernay et al., 2007). Juarez et al.
(2008) using algae, demonstrated that, ROS caused structural damage to the pigmentprotein complexes located in the thylakoid membrane (e.g. the destabilization and
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degradation of the proteins of the peripheral part of antenna complex), followed by
the pheophytinization of the chlorophylls (substitution of Mg2+ by H+ ions), and
destruction of the thylakoid’s membranes. It has also been demonstrated that Cr
affects, and might even inhibit, pigment biosynthesis, among others, by degrading δaminolaevulinic acid dehydratase (Vajpayee et al., 1999), an essential enzyme in
chlorophyll biosynthesis. Vernay et al. (2007) also presented evidence that this metal
probably competed with Fe and Mg for assimilation and transport to the leaves and
therefore affected different steps of pigment biosynthesis. Several hypotheses
explaining these results have been proposed, e.g., structural alterations in the
pigment–protein complexes of PSII or impairment in energy transfer from antennae to
reaction centers (like a diversion of electrons from the electron-donating side of PS I
to Cr (VI) are the most endorsed (Shanker et al., 2005). Recently, Henriques (2010)
implied that Cr (VI) might not be directly responsible for the damage to the
chloroplast, as the valence state of Cr depends of the local pH and redox values. For
instance, in irradiated chloroplasts, the previously mentioned conditions would favor
the less toxic Cr (III) form over the highly toxic Cr (VI). Appenroth et al. (2000)
demonstrated that Cr damaged the water oxidizing centers (WOC) associated to PSII
and Henriques (2010) proposed that this could be explained by the reduction of the Ca
and Mn availability, caused by Cr, which are fundamental in the structure and
functioning of the water oxidizing centers.
Besides the photochemical process, Cr is also known to cause distress in the
biochemical aspects of photosynthesis. Vernay et al. (2007, 2008) discussed that
despite that loss of biomass and wilting were common symptoms of Cr exposure;
little was known about Cr effect on water status and gas exchange. Subrahmanyam
(2008) also commented that it was unclear if Cr-induced inhibition of the
photosynthetic process was also due (among others previously mentioned factors) to
Cr-induced interference with the Calvin cycle’s enzymes. In those reports, the authors
proved that Cr consistently affected parameters like E (transpiration rate), gs (stomatal
conductance), A (photosynthetic rate) and Ci (substomatal CO2 concentration). One of
the main conclusions of those articles was that even though the decrease in gs seemed
to be responsible for the variation in water regulation status, the increase in
substomatal CO2 concentration induced by Cr accumulation clears stomatal
conductance as the responsible for the decrease in photosynthetic rate.
Subrahmanyam (2008) and by Vernay et al. (2007) that the reduction in
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photosynthetic rate might lay in the functional status of the Calvin cycle enzymes.
Unfortunately, the availability of data regarding Cr putative effects on the enzymes of
the Calvin cycle is far less than what exists for other parameters.
The recent works of Dhir et al. (2009), Bah et al. (2010) provided one of the
first insights to Cr-induced effects at the Calvin cycle enzymes. Dhir et al. (2009)
found a significant decrease in ribulose-bisphosphate carboxylase oxygenase
(RuBisCO) activity induced by exposure to wastewaters (rich in Cr) from an
electroplating unit and suggested that this results could be explained by a substitution
of Mg2+ in the active site of RuBisCO subunits by metal ions; decline in RuBisCO
content as a result of oxidative damage a shift in the enzymes activity from
carboxilation to oxygenation. On the other hand, Bah et al. (2010) performed a
proteomic analysis of Typha angustifolia’s leaves exposed to metals and found that
exposure to Cr induced the expression of ATP synthase, RuBisCO small subunit and
coproporphyrinogen III oxidase. A protective mechanism against metal toxicity at the
photosynthetic level, which might be responsible for the metal tolerance displayed by
T. angustifolia. Furthermore, the increased expression of ATP synthase was indicative
of the high energetic requirements needed to cope with metal toxicity (Santos and
Rodriguez, 2012).
2.24 Interaction of Chromium with Different Heavy Metals
The interaction of metals and other microelements are very important for plant
growth and development nbut principally it depends on the availability of the metals.
However, availability in soil depends on several soil conditions, such as pH or redox
potentials (Bartlett and Kimble, 1976). Hunter and Vergnano (1952) reported the
association of nickel, cobalt, copper, zinc, manganese and molybdenum with high
concentrations of the element in the leaf tissue of oat plants, but this was not always
so with chromium and aluminium. Toxic effects of nickel, chromium, copper and
molybdenum were associated with the reduced nitrogen content of the plants and
nickel, cobalt, chromium, zinc and manganese increased the concentration of
phosphorus. Therefore, tile solubility of metals can be increased or decreased
depending on the presence of other elements in the soil-plant system. Moral et al.
(1996) established another important point for the interaction between chromium and
iron and copper upon absorption which could be associated with the chemical
properties of these metals, as the charge (Cr+3 and Fe+3), effective ionic and metal
radius (Cr and Cu). In the same way, the contents of zinc can be diminished with Cr
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treatments. They also observed antagonistic and synergistic effects of different
elements and metals in stem, branch and leaf where a competitive interaction of
chromium and copper was noted. However, a synergistic interaction between B-Cr
was found in root, nevertheless, in aerial parts, an antagonistic effect was observed.
Simultaneously, in fruits no effect of chromium treatments were noted on Fe, Cu and
Zn contents, but, boron concentration increased in the presence of chromium. Turner
and Rust (1971) found that 0.l ppm of chromium in nutrient culture resulted in the
decrease in concentration and contents of Ca, K, R Fe and Mn in shoots and Mg, P, Fe
and Mn in roots as compared to the untreated soyabean plants. They also observed
that in soil culture, chromium treatment significantly increased concentrations of Ca,
K, Mg, R Bo and Cu in shoots of soybean. Chromium interferred with the ability of
the plants to obtain these elements from soil.
The inhibitory effects of chromium on plant growth are thought to be the
result of specific interaction between chromium and phosphorus (Moral et al., 1995)
or Fe (Canon, 1960) in plant nutrition. Turner and Rust (1971) concluded that
chromium in hexavalent form specifically interfered with the uptake of Fe or P by
plants. It appeared that a broad range of elements was affected by treating soybean
plants with chromium soil. There was no significant effect of chromium treatment on
Ca, Zn, Bo or Cu concentrations but the total contents of calcium and zinc were
depressed with the addition of high concentration of chromium. The interaction
mechanism with organic acids might play an important role in the inhibitory and
stimulatory effects of chromium on the translocation of different mineral nutrients
(Barcelo et al., 1985). Interaction with Zn, Ca and Fe has been reported, but effects
varied widely in different species and cultivars (Turner and Rust, 1971; Foroughi et
al., 1976; Skeffington et al., 1976). The translocation of phosphorus in bush bean
grown on soil with pH 6.0 was inhibited by the addition of hexavalent chromium
(Turner and Rust, 1971; Barcelo et al., 1985). In nutrient culture, Turner and Rust
(1971) reported that the concentrations and total contents of Ca, K, P, Fe and Mn in
shoots and Mg, R, Fe and Mn in roots showed apparently non-significant increases
due to chromium treatment at 0.05 ppm and significant decreases at all treatments
>0.1 ppm. They also stated that there were no significant effects of chromium
treatment on Na and Zn concentrations but the total Zn content tended to decrease
with chromium treatment at high concentrations. Riedel (1985) reported that the
uptake of Cr (VI) was approximately linear with time, proportional to external Cr (VI)
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concentration and inversly proportional to external sulphate ion concentration in
diatom (Thalassiosira pseudodana).
The concentration of Cr (VI) that restricted growth also inhibited sulphate
uptake. Skeffington et al. (1976) examined the uptake of chromate by barley seedlings
and found that it was competitively inhibited by sulphate and concluded that chromate
entered the roots through the sulphate uptake system. Several workers reported
chromate to be an inhibitor of sulphate uptake by plants. It usually inhibited sulphate
uptake though only when the ratio of sulphate to chromate ranged from 1:10 to 1:1
(Smith, 1976; Coughlan, 1977; Deane and Brien, 1981). Turner and Rust (1971)
found Cr (VI) to interfere with Co, K, Mg, R Fe and Mn uptake in nutrient cultures
and Co, K, Mg, R, B and Cu in soil culture. Cary et al. (1977a) described a Cr and Fe
interaction in plants and postulated a similar or related process of translocation for the
two elements. Soane and Saunder (1959) felt that Cr (III) and Fe (III) acted
analogously causing the acute phosphate (P) deficiency symptoms which they
observed in sand culture experiments with oats. Hewitt (1953), and Anderson and
Nilsson (1973) found Fe chlorosis to be the primary symptom of high chromium
presence.
Cunnigham et al. (1975) correlated increasing Cr content in sludge with
decreasing concentrations of Cu, Zn, Ni, Cd and Mn in plant tissues. They attributed
this phenomenon to a possible blockage of plant absorption sites. Grove and Ellis
(1980) reported the changes in the soil chemistry of Fe and Mn resulting from addition of chromium compounds to soil. They also reported the formation of a mixed
hydrous oxide of Fe (III) and Cr (III). The precipitated compound was apparently
quite stable at low pH. Sludge addition raised the pH to quite high levels on all soil
and decreased the solubility of Fe (Samantaray et al., 1998).
2.25 Effect of Chromium on ROS Generation
Heavy metal usually from ROS either indirectly or through involvement in a
redox reaction. Cr was thought to be a non redox metal that could not participate in
Fenton reactions,however other studies have shown that Cr can indeed participate in
Fenton reactions, proving its redox character (Shi and Dalal, 1989). Cr reactivity can
be considered from its interaction with glutathion, NADH and H2O2, forming OHradicals in cell free systems (Shi and Dalal, 1989; Aiyar et al., 1991). Production of
H2O2, OH- and O2- under Cr stress has been demonstrated in many plants, generating
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oxidative stress leading to demage of DNA, proteins and pigments as well as initiating
lipid peroxidation (Bagchi et al., 2000; Panda et al., 2003; Panda, 2003; Panda and
Chaudhury, 2005). The source of chromium and subsequent imposition of oxidative
stress is summarized as shown in Fig. 14.
Cr at both toxic and mild concentrations can inhibit uncoupled electron
transport (Dixit et al., 2001), indicating the electron transport chain to be a common
site of Cr binding in plants. Inhibition of electron transport by Cr may be a
consequence of redox change in the Cu and Fe carriers,where Cr may Br transferred
by cytochrome in the mitochondria to reduce it or the reduced heme group of
cytochrome may act as a site for Cr binding ,blocking electron transport (Dixit et al.,
2001). The severe inhibition of cytochrome oxidase activity may be due to the binding
of Cr to complex IV where Cr may also bind to cytochrome a3 (Dixit et al., 2001).
Another alternative mechanism is the generation of O2- radicals in the mitochondria
(Scandalios, 1993; Vranova et al., 2002). In pea plants, the treatment of Cr at different
concentrations showed that O2- is generated in the cytochrome b region (complex III)
of root mitochondria. The O2- generation at this specific site was high under Cr (Dixit
et al., 2001; Panda and Chaudhury, 2005). The Cr induced inhibition transport is
represented in Fig. 15.
2.26 Role of Chromium in Mineral Nutrition
Chromium, due to its structural resemblance with some essential elements, can
affect mineral nutrition of plants in a byzantine way. Cr-induced changes in ion
concentrations were associated with a significant reduction in plant biomass. It is
suggested that Cr stress interferes with the functions of mineral nutrients in rice
plants, thus causing a serious inhibition of plant growth (Adriano, 1986). The toxic
effects of Cr may particularly be linked to the interactions of Cr with essential nutrient
elements due to its changeable valences (Turner and Rust, 1971). The negative effect
of Cr on the uptake of nutrients, such as Zn, Fe, Ca, Mg, Mn, and Cu (Turner and
Rust, 1971; Moral et al., 1995; Chatterjee and Chatterjee, 2000; Gardea-Torresdey et
al., 2004; Zeng et al., 2010a). Chromium accumulation and tolerance varied
genotypes within a crop (Zeng et al., 2008a).
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Fig. 14. Involvement of chromium in inhibition of electron transport system in plants
(Panda and Chaudhury, 2005).
Fig. 15. Involvement of Chromium in inhibition of electron transport system in plants
(Panda and Chaudhury, 2005).
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It is important to obtain additional information on the possible mechanism of
Cr tolerance by studying Cr stress on the sub cellular distribution and chemical form
of mineral nutrients among the genotypes. The results show that Cr concentration was
significantly and negatively correlated with the dry weight of all plant organs. In rice
roots, Cr concentration had a significantly positive correlation with Mg concentration,
but a significantly negative correlation with Ca, Zn, and Fe concentrations. In rice
stems, significant and positive correlations between Cr and Ca/Mg concentrations, but
negative correlations between Cr and Zn/Fe concentrations were found. In rice leaves,
there was a significant and positive correlation between Cr and Ca concentration,
negative correlation between Cr and Mg/Zn concentrations, and no relationship with
Fe concentration (Zeng et al., 2010b). Furthermore, Cr showed a significant and
negative correlation with the accumulation of Ca, Mg, Zn, and Fe. Cr stress may
significantly inhibit the growth of rice plants by interfering with uptake, translocation,
and accumulation of nutrients. The relationships between Cr and Zn, Fe, Ca, or Mg in
sub cellular distribution varied with plant organs and sub cellular fractions. Cr and
other four nutrients were found in roots and stems; however, there was no significant
correlation between Cr and the other four nutrient elements in the mitochondrial
fraction of rice leaves. In the supernatant soluble fractions of each rice organ
(root/shoot), Cr showed a significant correlation with Ca and Mg, but no correlation
with Zn and Fe. Both, Ca and Mg acquired as divalent cations play an important role
in plant growth and development (Zeng et al., 2010a).
Generally, Ca2+ is required as a component of plant cell wall and cell
membranes, acting as a counter cation to balance organic anions in plant vacuoles,
and is an intracellular secondary messenger of plant cells (Marschner, 1995; White
and Broadley, 2003). Magnesium is required to stabilize membranes (Cowan, 1995),
maintain energy and nucleic acid metabolism (Shaul, 2002), and activate many
enzymes (Smith et al., 1995). Zinc is an essential component of some key enzymes,
such as oxidoreductases, transferases, and hydrolases, while Fe is a cofactor of
many ubiquitous proteins participating in crucial metabolic pathways in all living
organisms (Briat et al., 1995). Therefore, it is necessary to determine the effect of
Cr stress on the sub cellular distribution and chemical forms of these elements. It
has been shown that the sub cellular distribution of Ca and Mg in all rice plant
organs was significantly affected by Cr stress. In roots, the Ca concentration in cell
wall decreased, whereas soluble fractions increased under high Cr level. It may be
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suggested that more Ca2+ was transported into the vacuole of root cells in order to
regulate ionic concentration and osmotic homeostasis when the plants were exposed
to Cr stress. On the contrary, in stem, Ca concentration increased in the cell wall but
decreased in dramatic reduction of Mg concentration in all sub cellular fractions of
rice roots and an increase in all sub cellular fractions except for mitochondria of rice
leaves. In stem, the Mg concentration in cell wall, chloroplast, and mitochondrial
fractions increased, and in nucleus and soluble fraction, it reduced at high Cr level.
It may be assumed that when plants are exposed to Cr stress, Mg uptake is inhibited
and more Mg2+ will be transported to the leaves from roots in order to maintain the
normal function of leaves (Zeng et al., 2010a). However, it is indicated that the
effect of Cr stress on higher plants differs greatly from that on thallophytes (Horcsik
and Balogh, 2002). Inorganic Zn is the predominant chemical form of this element
in rice. Root Zn concentration in the inorganic form was markedly reduced under Cr
stress. It may be assumed that the quantity of Zn translocated from roots to shoots
will recede in plant, exposed to Cr stress, as the main form is inorganic Zn, whereas
Fe concentration in the forms of pectinates/protein-integrated metal and oxalic salt
was greatly increased under Cr stress. In stem, the concentration of Zn and Fe in the
forms of inorganic metal and water-soluble metal chelated with organic acid
increased under Cr stress. This unambiguous effect of stress on the concentration
and Fe/Zn forms may be attributed to the activated root exudation (Zeng et al.,
2008b).
Exudates, especially organic acids such as citric or malic acid, may have
stimulated the uptake and transport of metal ions. However, more supporting
evidences are required to substantiate this assumption. Moreover, it is well
documented that genotypic differences exist in plant heavy metal tolerance in terms
of mineral uptake and enzyme activity (Wu et al., 2003). It is reported that Ca, Zn,
and Mg can alleviate some stresses on plants (Wu and Zhang, 2002; Tobe et al.,
2003; Hassan et al., 2005; Kashem and Kawai, 2007). It may be concluded that Cr
stress affects the distribution and chemical forms of Ca, Mg, Fe, and Zn ions in
plant organs and cells, resulting in imbalance between inorganic nutrients. The
extent of these nutrient imbalances was related to genotype differences in Cr
accumulation and soluble fractions, at high Cr level (Zeng et al., 2010a). Moreover,
a significant decrease of Ca concentration in the chloroplast and nucleus fractions
was observed in both stem and leaves, thus probably causing damage to chloroplast
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and nucleus structure. Cr stress also caused a metabolic pool to channelize the
production of new biochemically related metabolites which may confer resistance or
tolerance to Cr stress (e. g., phytochelatins and histidine) (Schmoger, 2001; Hayat et
al., 2012).
2.27 Antioxidant Enzyme
Plants need to effectively eliminate the toxic reactive oxygen species (ROS)
which are generated as a result of environmental stresses. Plants have developed a
complex antioxidant system which scavenges these ROS particles thereby providing
protective shield against the oxidative attack (Vranova et al., 2002). The antioxidant
response of plants to metal-induced oxidative stress is variable and depends on type of
plant and metal involved. Unlike other heavy metals such as Cd, Zn and Fe,
information on antioxidative response against Cr is little. Among the two plants H.
annuus and Solanum nigrum, the overall antioxidant property of S. nigrum is found to
be comparatively higher than H. annuus (Vijayalakshmi et al., 2010). The increase in
antioxidant enzymes activity observed might have been in direct response to the
generation of superoxide radical by Cr-induced blockage of the electron transport
chain in the mitochondria. The increase noticed due to Cr (VI) indicated that its
augmentation assumable generates more singlet oxygen than Cr (III).
The decrease in the activity of the enzyme as the concentration of the external
Cr increased is definitely because of the inhibitory effect of Cr ions on the enzyme
system itself. In pea plants, ROS generation (superoxide, hydrogen peroxide, and
hydroxyl radical) was enhanced in the chloroplasts isolated from plants exposed to Cr
stress (Pandey et al., 2009b). The antioxidative enzymes catalase (CAT), guaiacol
peroxidase (GPx), glutathione reductase (GR), ascorbate peroxidase (APx) and
superoxide dismutase (SOD) have been thoroughly studied for plants like rice, wheat,
and pea, etc. Activity of CAT in response to Cr has been studied in many crop plants
like rice, wheat, green gram, Brassica juncea (Panda and Patra, 2000b; Panda et al.,
2002; Panda and Khan, 2003; Pandey et al., 2005). The response of CAT to Cr varied
from plant to plant. In rice, Cr either induced CAT activity or suppressed it. Activity
of superoxide dismutase increased at lower Cr level while it decreased at higher Cr
level. Ascorbate peroxidase was found to be most sensitive to Cr stress.
Monodehydroascorbate reductase activity was higher at lower concentrations of Cr
but decreased at higher concentration. Activities of dehydroascorbate reductase and
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glutathione reductase (GR) in the isolated chloroplasts increased when plants were
exposed to Cr. Ascorbate and glutathione (GSH) pools decreased which was more
evident in the GSH pool as the duration of Cr treatment increased (Pandey et al.,
2009b; Hayat et al., 2012).
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