Download Introduction

INTRODUCTION
Environmental stress is defined as the negative impact of non-living (abiotic) or
living (biotic) factors on the organisms. When the abiotic factor is present in the
environment beyond its normal range of variation, it adversely affects the population
performance or individual physiology of the organism in a significant way i.e. stress. As
plants are especially dependent on environmental factors, therefore, abiotic stress is
particularly constraining to plants than animals. Abiotic stress is affecting the growth and
productivity of crops worldwide (Eapen and Souza, 2005). Excessive levels of metals
can result in soil quality degradation, crop yield reduction and pose significant risk to
human, animal and ecosystem health. The problem is exacerbated by their nondegradability, long-term persistence in the environment and accumulation in living
beings through food chain and biomagnification therein.
Chemically, heavy metal is defined “as the metal with a specific gravity greater
than 5” (Venugopal and Luckey, 1975). Three classes have been identified according to
their coordination properties as A, B and borderline based on their binding preferences
for oxygen, nitrogen or sulfur containing ligands. Each heavy metal belongs to either
borderline or class B (Nieboer and Richardson, 1980).
Ecologically heavy metals refer to those metals, which cause environmental
threat of any kind. The heavy metals that are of major concern are as follows: Cd, Hg,
Zn, Cu, Ni, Cr, Pb, Co, V, Ti, Fe, Mn, Ag, Sn and metalloids As and Se. Plants adapt
very differently from one another, even from a plant of the same area. When arsenic (As)
enters the food chain it becomes a potent ecological hazard which can have serious
implications. Rice (Oryza sativa L.) is regarded as a staple diet throughout the world,
especially in countries like India. Irrigation with groundwater that is contaminated with
As has led to a high As burden in rice grains and consequently increased health risk to
millions of people around the world.
1.1. Arsenic Hazards
Arsenic (As; density; 5.73 g cm-3), is ubiquitously found in various inorganic and
organic forms. Arsenic being a carcinogenic metalloid can enter into the environment by
both natural and anthropogenic activities (Abedin et al., 2002). Groundwater As
Ph.D. thesis / Richa Dave / 2013
1
Introduction
contamination and its health effects in South-East Asian countries came to limelight in
the year 1984 (Garai, 1984; Chakraborti et al., 2002). A substantial part of the GangaMeghna-Brahmaputra plain (GMB) with an area 569,749 km2 and population over 500
million was at risk. (Roychowdhury, 2008).
The amount of As that is permissible in drinking water is 10 g l-1 (Smith et al.,
2000 (WHO)). However, very high levels of As contamination of ground water and its
adverse impact on human health have been reported in many countries of the world
(Mondal et al., 2006). The magnitude of this problem is quite severe in Bangladesh
(Chowdhury et al., 2000, 2001; Smith et al., 2000; Rahman et al., 2001) followed by
West Bengal, India (Guha Mazumder et al., 1998; Mandal et al., 1998; Chakraborti et al.,
2002) and China (Sun et al., 2004; Xia and Liu, 2004). Evidence has emerged in recent
years of As contaminated groundwater in other countries in Asia like Cambodia,
Mayanmar, Pakistan, Nepal, Vietnam and the Kurdistan province of Iran (Berg et al.,
2001; Shrestha et al., 2003; Mosaferi et al., 2003). Several areas of the states of Uttar
Pradesh, Bihar, Jharkhand, West Bengal in India are As affected and thousands of people
are suffering from As toxicity and are at risk (Mondal et al., 2006).
In the 1970s the use of surface water was largely abandoned in the Bengal delta
in response to severe health effects caused by microbial pathogens. This resulted in
extensive usage of As – contaminated groundwater unawares. The high-As groundwater
is produced from shallow (<100 m) depths by domestic and irrigation wells in the Bengal
Basin aquifer system (Hoque et al, 2012). It has been reported that groundwater from
shallow tube-wells (12–33 m) contains very high amounts of As,on the other hand, the
water from deep tube wells (200–300 m) contains lesser amount of As (<50 µg l-1)
(Hossain, 2006). The subsurface mobilization of As is mainly caused by a combination
of chemical, physical and microbial factors and various theories have been propounded
to explain the mechanism of As mobilization (Hoque et al., 2012; Mondal et al., 2006).
Of these, the important theories are the pyrite oxidation and oxy-hydroxide reduction
(Hossain, 2006) and the arsenic dissolution and relaease in the deltaic region have been
modelled upon the contribution of microbes, organic matter as well as palaeosol
formation (Hoque et al., 2012). Flooding induces reducing (anaerobic) conditions in soils
(Reynolds et al., 1999) hence As(V) is reduced to As(III) and adsorved As(V) is released
as As(III). Alluvial and deltaic environments are mainly characterized by reducing
Ph.D. thesis / Richa Dave / 2013
2
Introduction
conditions which cause high As release in groundwater (Smedley and Kinniburgh, 2002).
The
toxicity
order
of
arsenicals
is
as
follows:
inorganic
As(III)>organic
As(III)>inorganic As(V)> organic As(V) (Mondal et al., 2006).
When As enters the food chain, it causes wide spread distribution throughout the
plant and animal kingdoms. Both long and short term exposures have been found to be
hazardous and can lead to skin, bladder, lung and prostate cancers, cardiovascular
diseases, diabetes, anemia as well as reproductive, developmental, immunological and
neurological effects. (Cullen and Reimer 1989; Mandal and Suzuki, 2002; da Silva et al.,
2005; Roychowdhury, 2008).
1.2 Arsenic contamination of rice
Figure 1.1: Arsenic uptake and translocation in rice from flooded soils (Based on
Zhao et al., 2010 & Carey et al, 2010).
Arsenic contamination in plant-based foods is an important source of Asi
(Brammer and Ravenscroft, 2009). Rice is specifically a problem regarding the entry of
As into the food chain, owing to a combination of anaerobic growing conditions and
specific plant physiological characteristics (Booth, 2009). It is also the dietary staple for
Ph.D. thesis / Richa Dave / 2013
3
Introduction
half the world’s population. Intake of Asi from eating rice can be substantial; it is the
dominant source for populations based on a rice diet and not exposed to high
concentrations of As in drinking water (Zhao et al., 2010). Even for populations exposed
to elevated Asi in drinking water, such as As-affected areas in South Asia, Asi intake
from rice is significant, accounting for ∼50% (Mondal and Polya, 2008). There is an
urgent need to understand how plants assimilate and metabolize As in order to develop
mitigation strategies against this widespread contamination in the food chain (Zhao et al.,
2010).
The global average concentration of As in soil is about 5 mg kg−1.
Uncontaminated soils typically contain <10 mg kg−1 total As, but the concentration can
reach hundreds or thousands of mg kg−1 in contaminated environments (Zhao et al.,
2010; Hoque et al., 2012).
The bioavailability of As to plants is governed by edaphic properties,
environmental conditions and modification of the soil in the rhizosphere; these factors
interact to influence As speciation in the soil. Arsenic has four oxidation states: −3, 0,
+3, and +5, the last two being the most common in the terrestrial environment. Arsenate
[As(V)] is the predominant species in aerobic soils, whereas arsenite [As(III)]
predominates in anaerobic environments such as submerged soils (Zhao et al., 2010).
1.3. Arsenic uptake and translocation within the plant
Arsenate (As(V)) is the main As species in aerobic soils. It has a strong affinity
for iron oxides/hydroxides in soil; thus the concentrations of arsenate in soil solutions are
usually low. Wenzel et al. (2002) reported ≤ 53 nM arsenate in the soil solutions from a
range of uncontaminated and moderately contaminated soils and up to 2.3 µM in a
highly contaminated soil. Arsenate is an analogue of phosphate and is known to be taken
up by plants via the high affinity phosphate transport systems (Asher and Reay, 1979;
Meharg and Macnair, 1992a, Abedin et al., 2002a).
Arsenite (As(III)) is the dominant As species in reducing environments such as
flooded paddy soils (Marin et al., 1993; Xu et al., 2007). Thermodynamically, reduction
of arsenate to arsenite can occur quite readily at intermediate redox potentials. Flooding
of paddy soils leads to mobilization of arsenite into the soil solution and enhanced As
bioavailability to rice plants (Xu et al., 2007). The arsenite concentration in soil solutions
Ph.D. thesis / Richa Dave / 2013
4
Introduction
from flooded paddy soils typically varies from 0.01 to 3 µM; these concentrations are
generally higher than those of arsenate found in uncontaminated aerobic soils. As(III)
and undissociated methylated As species are transported through the nodulin 26-like
intrinsic (NIP) aquaporin channels (Zhao et al., 2010; Mosa et al., 2012; Ma et al, 2008).
Uptake of both As(V) and As(III) is an active process, described by two additive
hyperbolic functions over high affinity (low concentration) and low affinity (high
concentration) ranges respectively (Abedin et al., 2002a). Uptake of organic species of
As, DMA and MMA is also known to take place though at a lower rate than inorganic
ones (Meharg, 2004). After entering into roots through high-affinity phosphate
transporters, arsenate [As(V)] is readily reduced to arsenite [As(III)] (Zhao et al., 2010).
In rice, it has been shown that As(III) is exported subsequently to the xylem by the
silicon efflux transporter Lsi2, resulting in root-to-shoot transport of As (Ma et al, 2008).
Another transporter Lsi6, belonging to aquaporin family and localized in the shoot
epidermis may not be responsible for As(III) retranslocation in the grain, therefore, the
grain loading of As in rice may be through phloem mass flow (Carey et al., 2010) (Fig.
1.1.).
1.4. Arsenic induced phytotoxicity
Arsenic is a non-essential metalloid and does not play any role in biological
system and thus its mere presence may initiate disturbances in the proper functioning of
the cell (Patra et al., 2004). Though As is a metalloid it possesses various metallic
properties and causes toxicity to plants in a manner similar to other heavy metals. Based
on their chemical and physical properties heavy metals cause toxicity in three different
ways: (a) enhanced production of reactive oxygen species (ROS) such as superoxide
radicals (O2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), (b) blocking of
essential functional groups in biomolecules and (c) displacement of essential metal ions
from biomolecules (Garg and Singla, 2011). ROS production is a normal phenomenon
occurring during electron transport through electron transport chains in chloroplast and
mitochondria and other compartments also like glyoxysomes and peroxisomes (Gill and
Tuteja, 2010). Heavy metals stimulate generation of ROS, either by direct electron
transfer or as a consequence of metal mediated inhibition of metabolic reactions (Elstner,
1991). There is significant evidence that exposure to inorganic As species results in the
Ph.D. thesis / Richa Dave / 2013
5
Introduction
induced generation of ROS (Hartley-Whitaker et al., 2001a; Srivastava et al., 2005,
Singh et al., 2006). This probably occurs through the conversion of As(V) to As(III), a
process which readily occurs in plants (Meharg and Hartley-Whitaker, 2002). The
transfer of light energy or misdirected electrons to oxygen generates singlet oxygen (1O2)
and O2. Thereafter, in a chain reaction of single electron transfer, other ROS are
produced (Gill and Tuteja, 2010).
A serious imbalance in any cell compartment between the production and
dismutation of ROS leads to oxidative stress resulting in dramatic physiological changes
(Foyer and Noctor, 2003). This manifests itself as oxidative damage to membranes and
macromolecules such as RNA, DNA and proteins.
ROS attack membrane lipids particularly unsaturated fatty acids, such as linolenic
acid. These react with the methylene group between two double bonds in
polyunsaturated fatty acids, followed by rearrangement of double bond and thus forming
conjugated dienes. Hydrogen abstraction followed by oxygenation produces lipid
peroxides, which accelerate further damage by initiating radical chain reactions (Gill and
Tuteja, 2010). Peroxidized fatty acids are unstable and undergo reductive cleavage by
reduced metals such as Fe2+ by Fenton-type reaction. Lipid peroxidation finally leads to
formation of a number of degradation products particularly aldehydes, such as
malondialdehyde (MDA) (Garg and Singla, 2011; Gill and Tuteja, 2010).
Inside the cell, toxicity of As(V) and As(III) varies due to the differences in their
chemical properties and affinities for the reactive groups. Because As(V) is a phosphate
analogue, it competes with phosphate inside the cell for binding sites, for example by
replacing phosphate in ATP it forms unstable ADP-As that leads to the disruption of
energy flows in cells (Meharg, 1994). However, Bertolero et al. (1987) has pointed out
that because As(V) is rapidly reduced to As(III) in plant tissue, As(V) will not normally
have high enough cytoplasmic concentrations to exert toxicity. Arsenite exerts high
toxicity to plants by reacting with sulfhydryl groups (–SH) of enzymes and cellular
proteins, leading to inhibition of cellular function and death (Ullrich-Eberius et al.,
1989). Arsenic may also affect the protein synthesizing machinery of the cells by their
effect on enzymes of nitrogen metabolism (Jain and Gadre, 1997; Schmidt et al., 2005).
Though, a recent study published in the Science journal by Wolfe-simon et al
(2011) sparked a debate in the world’s scientific community over the issue of As. In the
Ph.D. thesis / Richa Dave / 2013
6
Introduction
study a stain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate
as a nutrient when phosphate is limiting and to specifically incorporate As into its DNA
in place of phosphorus. This debate lead to further studies by Reaves et al. (2012) and
Erb et al. (2012) challenging the discovery and claiming that as opposed to previous
study, GFAJ-1 is an arsenate-resistant, but still a phosphate-dependent bacterium.
1.5. Role of amino acids in stress tolerance
Amino acids are the building blocks of proteins among which, histidine, proline,
cysteine and glycine along with other amino acids are known to be induced significantly
upon heavy metal exposure (Dwivedi et al., 2010). Metal induced production of ROS
may also modify amino acids leading to their loss. Amino acids can be peroxidized by
free radicals turning them into second toxic messengers in cells and tissues consequently,
even resulting in the oxidation and depletion of vital antioxidants in vivo (Gebicki &
Gebicki, 1993). Reactive oxygen (ROS)-mediated oxidation of proteins, free amino acids
and proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side
chains, nitration of aromaticamino acid residues, nitrosylation of sulfhydryl groups,
sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino
groups, and to conversion of some amino acid residues to carbonyl derivatives.
Oxidation can lead also to cleavage of the polypeptide chain and to formation of crosslinked protein aggregates. Furthermore, functional groups of proteins can react with
oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives
(glycation/ glycoxidation) to produce inactive derivatives (Stadtman and Levine, 2003).
On the other hand, histidine, proline, cysteine and glycine along with other amino
acids are known to be induced significantly upon heavy metal exposure (Davies et al.,
1987; Dwivedi et al., 2010). Proline and cysteine are the two most important amino
acids involved in stress tolerance but not much is known about their free to bound ratios.
Proline has been reported to accumulate in tissues/organs of plants subjected to various
abiotic stresses including heavy metal toxicity and appears to be a preferred organic
osmoticum for many plants (Mishra and Dubey, 2006). Amino acids or protein content,
along with other mineral nutrients in the food crops, will affect a great portion of the
world population, especially in developing countries where rice grain is the main source
Ph.D. thesis / Richa Dave / 2013
7
Introduction
of protein. Thus, quantification of various amino acids in response to different
concentrations of As seems imperative in rice (Dwivedi et al., 2010).
As
As
As
As
As
As
As
As
Figure 1.2: Amino acids induced during arsenic stress. (Based on Sharma & Dietz,
2006 & Dwivedi et al, 2010).
Upon exposure to metals, plants often synthesize a set of diverse metabolites that
accumulate to concentrations in the millimolar range, particularly specific amino acids,
such as proline and histidine, peptides such as glutathione and phytochelatins (PC), and
the amines spermine, spermidine, putrescine, nicotianamine, and mugineic acids. Thus,
nitrogen metabolism is central to the response of plants to heavy metals (Sharma and
Dietz, 2006).
The scheme presented in Figure 1.2 displays all stress responsive amino acids and
the metabolic link. Except for PC with metal dependent activation of enzyme activity,
nicotianamine, and mugineic acid synthesis, the responses may not or not in each case be
the primary plant reactions to heavy metals. However, from the data available, it has
become clear that changes in the contents of these metabolites bear functional
significance in the context of metal stress tolerance (Sharma and Dietz, 2006).
1.6. Arsenic tolerance through antioxidant system in plants
Ph.D. thesis / Richa Dave / 2013
8
Introduction
In order to avoid oxidative damage, plants have evolved a complex antioxidant
defense system including enzymes and molecules (Dat et al., 2000). The most prominent
antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT) and a large
number of peroxidases (POX) including guaiacol peroxidase (GPX) and ascorbate
peroxidase (APX), and glutathione reductase (GR). The major ROS-scavenging
pathways of plants include SOD, found in almost all cellular compartments, the water–
water cycle in chloroplasts, the ascorbate–glutathione cycle in chloroplasts, cytosol,
mitochondria, apoplast and peroxisomes, and CAT in peroxisomes (Gratão et al., 2005).
The most important function of water-water cycle is the reduction of O2

to
H2O2 at the site of its generation by SOD. SOD is thus considered as first defense in
the antioxidant pathway against ROS as it dimutates O 2 , which are the precursor of
other ROS. This dismutation, known as Mehler reaction, occurs at a very fast rate
(Polle and Rennenberg, 1994). SOD, by its action, influences the concentration of O 2
and H2O2, the two Haber-Weiss reaction substrates.
The main pathway in which H2O2 is reduced to water is ascorbate-glutathione
cycle, also referred to as Asada-Foyer-Halliwell pathway that uses ascorbate (AsA)
and glutathione (GSH) as reducing substrates. In this cycle, APX uses two molecules
of AsA to reduce H2O2 to water, with the concomitant
generation of two molecules of monodehydroascorbate
(MDHA). APX family consists of at least five different
isoforms (Asada, 1992) including thylakoid, soluble
stromal, cytosolic, peroxisomal and apoplastic isoenzymes
(Gratão et al., 2005). MDHA is a radical with a short
lifetime that, if not rapidly reduced, disproportionates to
AsA and dehydroascorbate (DHA). Within the cell MDHA
can be directly reduced to ascorbate. The electron donor for
MDHA reduction may be b-type cytochrome, reduced ferredoxin or NAD(P)H+H+.
The latter reaction is catalyzed by monodehydroascorbate reductase (MDHAR).
Despite the possibility of enzymic and nonenzymic regeneration of ascorbate directly
from MDHA, rapid disproportionation of MDHA radical means that some DHA is
always produced when ascorbate is oxidized in leaves and other tissues. DHA is reduced
to ascorbate by the action of dehydroascorbate reductase (DHAR) using GSH as the
Ph.D. thesis / Richa Dave / 2013
9
Introduction
reductant. This reaction generates oxidized glutathione (GSSG), which is in turn rereduced to GSH by NAD(P)H+H+, a reaction catalyzed by GR, which is a flavoprotein.
Ascorbate and GSH are not consumed in this pathway but participate in a cyclic transfer
of reducing equivalents, involving four enzymes, which permit the reduction of H2O2 to
H2O using electron derived from NAD(P)H+H+.
SOD-Cu(II) + O2 
SOD-Cu(I) + O2
SOD-Cu(I) + O2 
SOD-Cu(II) + H2O2
CAT
2 H2O2
2 H2O + O2
GPX
H2O2 + RH2
2 H2O + R
Peroxides can also be metabolised directly by peroxidases, such as GPX, present
throughout the cell and CAT in the peroxisomes (Bowler et al., 1992; Azevedo et al.,
1998; Polidoros and Scandalios, 1999). The ping-pong mechanism of GPX is the same as
APX, but GPX prefers aromatic electron donors such as guaiacol and pyrogallol.
Peroxidases also fulfill various other roles and their cellular distribution (cell walls,
endoplasmic reticulum, Golgi apparatus and vacuoles), is presumably connected with
different physiological functions (Gaspar et al., 1991). The protective action of CAT is
limited because of its discrete localization in the peroxisomes, its relatively poor affinity
for its substrate and its sensitivity to light-induced inactivation (Feierabend and Engel,
1986; Feierabend et al., 1992). CAT, unlike peroxidases, does not require any substrate
for the conversion of H2O2 to water and molecular oxygen
The equilibrium of SOD, APX and CAT is essential in order to determine the
steady-state level of O2 and H2O2. Compensatory mechanisms are induced if the
balance is altered. For instance, when CAT activity is reduced in plants, other ROS
scavenging enzymes, such as APX and GPX are up-regulated (Vandenabeele et al.,
2004).
Glutathione, ascorbate, carotenoid, flavonoid, tocopherol, various amino acids,
phenols, and other thiols are amongst important non-enzymic antioxidants (Larson,
Ph.D. thesis / Richa Dave / 2013
10
Introduction
1988). Carotenoids play important role in chloroplasts and are important terminator of
radical chain reactions (Krinsky et al., 1994).
The general scheme for chlorophyll-sensitized production of singlet oxygen and
subsequent quenching by carotenoids can be summarized as follows:
In the presence of oxygen, a new chain carrying
Chl + hυ
1
Chl* (singlet)
peroxy radical species of β-carotene is formed:
1
Chl*
3
Chl* (triplet)
β-Carotene + ROO
3
Chl* + O2
Chl1 + 1O2
1
O2 + Car
3
Singlet oxygen
3
Car* + O2
β-Carotene
Car*
β-Car + O2
β-Car
β-Car-OO
β-carotene radical (β-Car•) can be removed
through interaction with another peroxyl radical.
Car + Heat
β-Car + ROO
Inactive products
1.7. Thiolic metabolism
After accumulation of metal/metalloid by the plant two important strategies
mainly operate i.e. chelation via different ligands and subsequent compartmentalization
to vacuole and dismutation of induced ROS as primary and secondary detoxification
strategies, respectively. Following uptake, arsenate is reduced efficiently to arsenite in
plant cells, and that most plants have a high capacity for arsenate reduction.
Cysteine (Cys) and GSH constitute major thiols of the cell, which play roles not
only in relieving the oxidative stress but also in metalloid primary detoxification.
Cysteine is regarded as the terminal metabolite of sulfur assimilation and is the pivotal
sulfur-containing compound for production of a variety of metabolites containing
reduced sulfur, including GSH and phytochelatins (PCs) (Leustek et al., 2000; Saito,
2000). The inorganic sulfur in the environment, sulfate ion in the soil and SO2 in the air
are fixed into the cysteine by sulfur assimilation pathway in plants (Saito, 2000). Sulfur
is taken up by the plant exclusively in the form of sulfate and translocated unmetabolized throughout the plant, which is subsequently reduced to cysteine by the
pathway.
GSH is a tripeptide, γ-Glu-Cys-Gly, that exists interchangeably with oxidized
form (GSSG). Certain plants contain tripeptide homologue of GSH in which the carboxy
terminal Gly is replaced by other amino acids. These are γ-Glu-Cys-β-Ala
Ph.D. thesis / Richa Dave / 2013
11
Introduction
(homoglutathione), γ-Glu-Cys-Ser (hydroxymethylglutathione) and γ-Glu-Cys-Glu. Two
ATP-dependent steps catalyzed by γ-glutamylcyesteine synthetase (γECS) and
glutathione synthetase (GS) lead to sequential formation of γ-glutamyl cysteine (γEC)
and GSH.
In the plants, the physiological significance of GSH is divided into two
categories, sulfur metabolism and defense (Grill et al., 2006). GSH is the predominant
cellular non-protein thiol (NP-SH) and regulates sulfur uptake at root level. It is used by
the GSH-S-transferases (GSTs) in the detoxification of peroxides generated due to
metalloid induced oxidative damage and xenobiotics and is a precursor of the PCs, which
are crucial in controlling the cellular heavy metal concentration. GSH acts as an
antioxidant and redox buffer (Moons, 2003).
Besides, GSH is required as a reductant in the enzymatic reduction of As(V) to
As(III) by the AR enzyme in plants (Duan et al., 2005). The reduction of As(V) to
As(III) can also be brought about nonenzymatically by GSH (Delnomdedieu et al.,
1994). GSH can also directly quench ROS species, such as singlet oxygen and OH
(Xiang and Oliver, 1998). Studies have shown that GSH accumulates in response to
increase in ROS generation or is constitutively higher in plants adapted to exacting
conditions. Increased rate of GSH accumulation provoked by H2O2 generation may be
accompanied by the changes in the rate of sulfate uptake (Rausch and Wachter, 2005).
Feedback inhibition of γECS by GSH has often been considered as fundamental control
over synthesis of GSH (Alscher, 1989). Another mechanism, through which tissue GSH
content might be modified, is altered de novo synthesis of γ-ECS and/or GS (Noctor and
Foyer, 1998). Degradation of GSH through the enzymes called as γ-glutamyl
transpeptidases (γGTs) may also affect its cellular concentrations.
GSTs constitute an important class of enzymes related to GSH metabolism that
catalyze the conjugation of the GSH to a variety of hydrophobic, electrophilic and
usually cytotoxic substrates. The ‘original’ functions of plant GSTs are poorly
understood. GSTs catalyze alternative isomerization or peroxidase reactions and are
involved in tyrosine metabolism or hydroxyperoxide detoxification, respectively. GSTs
may also function in cellular redox homeostasis, act as stress signaling proteins or
regulate apoptosis (Marrs, 1996; Moons, 2003).
Ph.D. thesis / Richa Dave / 2013
12
Introduction
In response to heavy metals (Cd, Zn etc.) and metalloids (As) plant synthesize
sulphur rich peptides. In plants, As is reported to significantly induce the synthesis of
Phytochelatins (PCs) (Schat et al., 2002). Many studies conclude the essential role of
PCs for both normal and constitutive tolerance to As (Hartley-Whitaker et al., 2001b;
Schat et al., 2002; Li et al., 2004). Arabidopsis cad1 mutant deficient in PCs are sensitive
to As (Ha et al., 1999) and in addition, inhibition of PC synthesis by BSO almost
completely abolished the tolerance to As(V) in both nonmetallicolous and metallicolous
populations of Holcus lanatus (Hartley-Whitaker et al., 2001b).
PCs are enzymatically-synthesized cysteine rich polypeptides mediating the high
affinity binding and promoting vacuolar sequestration of heavy metals. PCs were first
idendified and characterized in fission yeast Schizosaccharomyces pombe and were
termed as cadystins (Murasugi et al., 1981). Grill et al. (1985) discovered the presence of
metal binding peptides in plant system (Rauvolffia serpentina cell culture) and named
them phytochelatins. These are synthesized by the action of enzyme, γ-glutamylcysteinyl
dipeptidyl transpeptidase, trivially called as phytochelatin synthase (PCS) using GSH or
PCs as substrate. The general structural formula for PCs has been given as (-Glu-Cys)nGly, where n ranges 2-11. Other families have also been detected based on having
different C-terminal residues. The main classes include: Canonical PCs, [γ-Glu-Cys]nGly with C-terminal Glycine, homo-PC, [iso-(PC)-β-alanine], Hydroxymethyl-PC, [iso(PC)-Serine], Iso-PC, [iso-(PC)-Glu and iso-(PC)-Gln], desGluPCs (Cys-[γ-Glu-Cys]nGly) and Des-Gly PC (Zenk, 1996; Sarry et al., 2006). Synthesis of PCs is induced by
the entry of a variety of metal/metalloid ions (Ag+, As5+, Au+, Bi3+, Cd2+, Cu2+, Hg2+,
Ni2+, Pb2+, Sb3+, Se4+, Sn2+, Te4+, W6+, Zn2+, Fe2+, Ga3+, In3+, Pd2+; Grill et al., 1987;
Maitani et al., 1996) into the cell. PCs are found in almost all higher plants, algae,
bryophytes, pteridophytes and gymnosperms (Gekeler et al., 1988; 1989, Mehra and
Tripathi, 2000). These have also been detected in several fungi including S. pombe, S.
cerevisiae, Candida glabrata, Mucor racemosus and Articulospora tetracladia (Grill et
al., 1986a; Mehra et al., 1988; Mehra and Tripathi, 2000; Miersch et al., 2001) and even
in a nematode, Caenorhabditis elegans (Clemens et al., 2001; Vatamaniuk et al., 2001).
Step (i):
γEC-Gly + PCS
Step (ii): γEC-PCS + (γ-EC)n-Gly
Ph.D. thesis / Richa Dave / 2013
γEC-PCS + Gly
(γEC)n+1-Gly + PCS
13
Introduction
Besides detoxification of toxic metal/metalloid ions, other roles for PCs have also
been suggested, such as homeostasis of essential metal ions such as Cu and Zn, transport
of metal from root to shoot (Gong et al., 2003), sulfur (Steffens, 1990) and GSH
metabolism (Beck et al., 2003). PCS may be activated by heavy metals (Grill et al.,
2006) or metal-GSH complexes (Vatamaniuk et al., 2000). Kinetic analysis of PCS
catalyzed reaction indicated that synthesis of PCs consists of two distinct steps; iformation of γEC concomitant with the cleavage of glycine from GSH, ii- transfer of
γEC unit from the enzyme to acceptor molecule i.e. GSH or oligomeric PC peptides
(PCn).
PCS genes were identified in A. thaliana, S. pombe and Triticum aestivum
(Vatamaniuk et al., 1999; Ha et al., 1999) for the first time and thereafter the gene has
been identified in other plants, prokaryote and the model nematode, C. elegans (Clemens
et al., 2001; Oven et al., 2002a; Heiss et al., 2003; Tsuji et al., 2005; Dong et al., 2005;
Loscos et al., 2006, Colón-Ramos et al., 2007). PCS gene is thought to be constitutively
expressed in plants (Grill et al., 1989; Howden et al., 1995; Chen et al., 1997) and there
is self-regulation of its activity by heavy metals (Zenk, 1996; Cobbett, 2000). However,
after characterization of PCS gene in other systems, it has been demonstrated that PCS
activity may be regulated at various levels (Ha et al., 1999; Lee and Korban, 2002; Heiss
et al., 2003).
Overexpression studies targeting various enzymes of the cysteine, GSH and PC
biosynthetic pathway and those involved in As detoxification have been carried out and
have given significant results (Tripathi et al., 2007). The gene products relevant to As
tolerance include those involved in uptake and transport of As(V) and As(III), reduction
of As(V) to As(III), the synthesis of metalloid binding peptides, and membrane
transporters involved in vacuolar As sequestration and As extrusion. Understanding the
mechanistic details of these processes will help develop high biomass plants suitable for
hyperaccumulation. From the present knowledge of various aspects of PCS and PCs,
there is also a possibility of their use for detoxification of xenobiotics and as bioindicator
and biosensor of metal pollution (Grill et al., 2006).
Ph.D. thesis / Richa Dave / 2013
14
Introduction
High Affinity Pi transporte
NIP subfamily of aquapori
Si/AsIII efflux carrier (Lsi2
AtABCC1 and AtABCC2 va
transporters
Figure 1.3: Arsenic tolerance strategies in rice plant
AR, arsenate reductase; PCS, phytochelatin synthase; arsM, As(III)-S-adenosylmethionine
methyltransferase.
arsenate (High affinity Pi) transporter; plasma membrane aquaporin channel
Lsi1; unidentified arsenite efflux transporter; As-thiol transporter (AtABCC1 &
AtABCC2); tonoplast aquaporin channel for As(III) transporter to vacuole; arsenite efflux carrier
Lsi2.(Based on: Zhu and Rosen 2009; Tripathi et al., 2007, 2012; Song et al, 2010).
Ph.D. thesis / Richa Dave / 2013
15
Introduction
OBJECTIVES OF THE WORK
The main objective of this study was to identify tolerant varieties and their metabolic
specifications which make them tolerant or suitable for cultivation in As contaminated
sites. The work entailed the study of effects of As on plant metabolism, uptake and
toxicity along-with the alleviation of the deleterious effects of As through various
antioxidative mechanisms and thiol metabolism in rice (O. sativa) plants.
The following are the objectives of the study:
 Screening and characterization of As tolerant and sensitive varieties.
 Arsenic uptake, As speciation in rice.
 Oxidative stress and antioxidant responses during As stress in rice.
 Amino acid response to As stress in rice plant.
 Response of thiol metabolism including phytochelatins during As stress.
 Phytochelatin synthase activity as modulated by As in rice plant.
Ph.D. thesis / Richa Dave / 2013
16