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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