You are on page 1of 45

INTRODUCTION

Arsenic is ubiquitous in the environment it is the twentieth most abundant element in the Earths crust with an average concentration of approximately 3 mg kg1.Arsenic originates from anthropogenic and geochemical sources . In addition, human activities have caused an accumulation of Arsenic in soils through the production/use of Arsenic-based pesticides, manufacture of Asbased compounds, smelting of arsenic ores, mining processes and fuel utilizationThe presence of arsenic in groundwater and food poses serious health risk to people worldwide. Plants growing on such soil may biomagnify the metals in different plant parts including grains and subsequently may be transferred to the higher tropic levels. The chances of biomagnifications are larger through the staple crops, such as rice because base line level of arsenic is 10th fold higher than other cereal grains (Williams et.al 2007) . It is apparent that rice contribute significantly to human intake of arsenic in In dia (West Bengal) (Chowdhary et al 2001 ), Bangladesh (Rahman et al., 2008), and this seems true to other parts of the world, especially where arsenic level in drinking water/groundwater is on the higher side (Smith 2000, Welch et al 2000,
Tendulkar et al 2001), the main cause for arsenic exposure to the human is

rice, contributing to more than 60% of dietary arsenic exposure since rice is the major cultivated crop in As- contaminated regions of South-East Asia. (Meharg
and Rahman 2003) In large areas of Bangladesh, China, West Bengal and

Vietnam, farmers have to rely on arsenic contaminated groundwater for irrigation of crops (Hartley- Whitaker et al.,2002) In Bangladesh, nearly half of the shallow tube wells produce water containing >10 g /l arsenic,which is the current arsenic permissible limit in drinking water recommended by the World Health Organization (WHO), with the exposed population estimated at 57 million (Br. Geol. Surv. (BGS/DPHE) 2001.) The maximum permissible limit of arsenic in groundwater is 0.05 mg/l (World Health Organisation) These

ground waters, some containing more than 1 mg /l arsenic are also used extensively for irrigating rice crops in the dry season, adding more than 1000 tonnes of arsenic to the agricultural soils in Bangladesh (Ali MA,
Badruzzaman ABM, Jalil MA, Hossain MD, Ahmed MF, et al. 2003). The

existence of organic arsenic

in natural water is due to the use of organo -

arsenical pesticides .Geochemical weathering of rocks and microbial activities also contribute to arsenic mobilization in the environment, but human activity has enhanced this process and thus exacerbated the problem in some area of Bangladesh and India ground water arsenic con centration has exceeded 2000 mg /l. Poisoning has reached a massive scale in Bangladesh and West Bengal, India.. Elevated arsenic accumulation in rice has the potential to become a new disaster for the population in Southeast Asia.

. Arsenic has no known function as nutrient (Nies 1999), and may be toxic even at low concentration (in the l g /l range) to plants and animals .The accumulation of arsenic in plants not only affects the plant growth but also enters the food chain, which in turn, causes potential health risks to human beings such as skin cancer.

Rice
Rice is the seed of the monocot plant Oryza sativa. As a cereal grain, it is the

most important staple food for a large part of the world's human population, especially in East, South, Southeast Asia, the Middle East, Latin America, and the West Indies. It is the grain with the second highest worldwide production, after maize (corn). Since a large portion of maize crops are grown for purposes other than human consumption, rice is the mo st important grain with regards to human nutrition and caloric intake, providing more than one fifth of the calories consumed worldwide by the human species. Rice remains a staple food for the majority of the world's population. More than two-thirds of the world relies on the nutritional benefits of rice. Rice is naturally fat, cholesterol and sodium free. It is a complex carbohydrate containing onl y 103 calories per one-half-cup.

Of the many varieties, swamp rice, which requires flooding for two to three months during its growth, is the most important. Oryza sativa has the smallest cereal genome consisting of just 430Mb across 12 chromosomes. It is renowned for being easy to genetically modify and is a model organism for cereal biology.

Selenium
Metalloid selenium is ubiquitous in environment and its concentration in most of soil ranges from 0.01 to 2 mg/kg (Fordyce, F.M. (2005). Selenium has been widely known as a toxic element since its discovery by Berzelius in 1817; however, during the recent past, research advances have shown selenium to be an essential nutrient required to combat a number of serious deficiency disorders. Se-containing compounds have been recognised as key components of enzymes, antioxidant compounds, and hormones (Rayman, 2002). The recommended dietary allowance (RDA) of 55 g of Se per day for adults is considered essential for healthy living (National Academy of Sciences, 2004). Daily intake of less than 55 g can have serious health implications.For example, daily dietary intake of less than 25 g of selenium per day has resulted in juvenile cardiomyopathy (Keshan disease) in regions of China and arsenicosis in Bangladesh (Combs, 2001; Spallholz, & Rhaman, 2004) Dietary intake of 55200 g of selenium per day is recommended as safe and adequate to reduce the risk of bladder and prostate cancer (Combs & Lu, 2001;
Rayman, 2002; Reid et al., 2008) . In organisms that require selenium,

selenocysteine (SeCys) is an essential component in so-called selenoproteins or selenoenzymes (Rayman, M.P. 2002) 25 of which have been identified in humans (Kryukov, G.V. et al. 2003 )Selenoproteins have a redox function involved in free-radical scavenging (Lu, J. and Holmgren, A. 2009). Plants are the main source of dietary Se (Rayman, M.P. (2008). Most plant species contain less than 25 g Se g-1 dry weight and cannot tolerate high levels of Se in the environment (White et al. 2004). Yet several species of the genera Astragalus, Neptunia and Stanleya, among others, are able to hyperaccumulate Se at concentrations of up to 1015 mg Se g-1 dry weight in their shoots while growing on naturally-occurring soils containing only 210 g Se g-1 dry weight
(Virupaksha and Shrift 1965; Davis 1972, 1986). Several clinical trials

suggest high Se intake and Se supplementation may increase the risk of developing Type 2 diabetes (Bleys, et al.,2004). In contrast, a recent study has suggested that Se may be essential in reducing the risk of developing abnormal blood sugar metabolism (Akbaraly et al., 2010). SeMe-SeCys is the form of Se purported to confer the best anti carcinogenic properties, this is advantageous from a human nutrition perspective. Nevertheless, both SeMe-SeCys and SeMet confer additional health benefits over inorganic Se by either being more anti-carcinogenic or by being stored more effectively in the body
Rayman, M.P. et al. (2008)and Rayman, M.P. (2008) .Zhang et al., 1998.reported that

supplements/high Se intake are effective in reducing mammary, prostate, lung, colon and liver cancer risk. Selenium also helps in the prevention of cancer and ageing, and in the treatment of atopic dermatitis and asthma. Selenium is also required for production of thyroid ho rmone, and deficiency affects thyroid function. Health benefits conferred through Se intake are associated with the prevention of cancer, cardiomyopathy, free radical induced diseases, and protection against HIV and heavy metal poisoning (Combs & Lu, 2001) Rice (Oryza sativa L.) is the major staple crop being the source of proteins and micronutrients including selenium (Se) to nearly half of worlds population reliant upon it for sustenance. India is the second highest rice producer, producing more than one-fifth of rice worldwide (Williams et al., 2009a). Indian rice is known to be enriched in Se, but, rice cultivation in arsenic (As) contaminated areas is leading to high level of As in grains (Meharg et al.,
2004; Tuli et al., 2010) , which has emerged as an issue of huge concern.West

Bengal is the rice bowl of India and is highly arsenic (As) contaminated reaching upto 20.2 mg kg 1 in the surface horizon of pedon ( Ghosh et al.,
2006). Zavala and Duxbury (2008) reported total As concentrations in rice

grains of different countries to be 0.005 to 0.710 mg kg -168 . Geographical variation in total and inorganic As in rice grains has also been reported

(Meharg et al., 2009) . Daily intake of inorganic As (Asi) loaded rice grains

may constitute a potential As exposure pathway for humans (Meharg et al.,


2003; 2004) leading to serious health hazards such as bladder, lung, skin and

prostate cancer (Naidu et al., 2006). According to a study carried out in West Bengal (India), rice was found to contribute about 44% of total average inorganic As intake (Mondal and Polya, 2008). Arsenic to the level of 0.840 mg kg-1 has been reported in rice from Indian field sites (Norton et al., 2009). Correspondingly, Se content in Indian rice has been shown to be within the 0.005-0.233 mg kg-1 76 range ( Williams et al., 2009a). Arsenic and Se compounds can be biologically antagonistic to each other, because Se4+ and As3+ have the same electronic structure (Rosen and Liu, 2009). Thus, Se acts as a natural antidote to As by accel erating As excretion, and acting as an antioxidant component of the enzyme glutathione peroxidase, that may counteract the cancer (Spallholz et al., 2004). In contrast to As, supplemented Se intake is effective in reducing mammary, prostate, lung, colon and liver cancer risk (Duffield-Lillico et al., 2003; Rayman, 2000). Se also prevents As-induced chromosomal damage, increases biliary and urinary As excretion and reduces body As load (Sweins, 1983). Plants, more importantly rice constitute the main source of Se especially for thos consuming low protein diets (Williams et al., 2009b). Selenate is the predominant form under well oxidized soils, however selenite dominates under reduced soil conditions . Plant uptake of selenate can be mediated by sulfate transporters while silicon influx transporter (OsNIP2;1) is identified for undissociated selenite uptake at low pH in rice (Zhao et al., 2010). AsV is transported through phosphate transporters
(Tripathi et al., 2007), while AsIII through aquaporins (NIPs) in rice (Ma et al., 2008). Thus, As and Se chemistry and dynamics in

paddy fields are

complex. Zhang et al. (2006) reported the genetic difference in Se accumulation in grains of japonica rice. While, Williams et al. (2009b) concluded As accumulation has been found to limits grain Se in Bangladesh

rice. Genetic variation and the effect of different environments on grain As accumulation has also been demonstrated by (Norton et al. (2009a-b). Considering the role of Se in efflux of As from human body alongwith being antagonistic it seems worthwhile to screen out rice cultivars which accumulate least As and sufficient Se irrespective of soil consumption. For this purpose in the As content, which could eventually be useful for cultivation in arsenic prone areas, safer for huma n present study three field sites were cultivation of local rice selected in As affected areas of West Bengal, for

genotypes during Boro season (2008 and 2009). After maturation, plants were analyzed for Se and As accumulation in grains and were categorized in terms of Se and As levels, taking mean of the two Boro seasons. Further, grain As speciation of contrasting cultivars was also performed. Arsenic, selenium contained in the soil environment can enter the food chain through plant accumulation. Although selenium has been identified as a necessary element to animal life and possesses cancer chemo preventive properties from clinical trials, (Combs et al., 2001), its narrow range between deficiency and toxicity deem the uptake and accumulation of selenium worthy of extensive investigation (Brown and Arthur, 2001). In past studies, selenium has been shown to have an antagonistic affect on toxic elements in plants. Investigations over half a century ago provided evidence for a detoxifying or protective effect after toxic concentrations of selenium and arsenic were simultaneously administered to rats. More recently, the structure elucidated for the interaction of selenium and arsenic in a mammalian system was described as selenobis ( S-glutathionyl) arsinium ion [(GS) 2AsSe]
. Although the effects of selenium and arsenic have independently been studied

in various plant matrices, little research has been devoted to prov ide information on a potential selenium and arsenic interaction at the molecular level within plants.

The most common arsenic and selenium species previously found in plants and soil were AsIII, AsV , MMA, DMA, selenite (SeIV), selenate (SeVI), selenomethione (SeMet), and selenocystine (SeCys2) In general, extensive effort has been put forth to understand the metabolic pathways of contaminants such as arsenic and selenium in hyperaccumulating plants.

REVIEW OF LITERATURE
Arsenic is the chemical element that has the symbol arsenic, atomic number 33 and atomic mass 74.92. 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. Interconversion between these two arsenic species is driven by both biotic and abiotic processes and strongly influenced by the redox potential and pH. Arsenic is a notoriously poisonous metalloid with many allotropic forms, including a yellow (molecular non-metallic) and several black and grey forms (metalloids). Three metalloidal forms of arsenic, each with a different crystal structure, are found free in nature (the minerals arsenic sensu stricto and the much rarer arsenolamprite and pararsenolamprite). However, it is more commonly found as arsenide and in arsenate co mpounds, several hundred of which are known.

PHYSICAL PROPERTY
Color Phase at Room Temp. Density (g/cm3) Hardness (Mohs) Melting Point (K) Boiling Point (K) Heat of Fusion (kJ/mol) Heat of Vaporization (kJ/mol) Heat of Atomization (kJ/mol) Thermal Conductivity (J/m sec K) Electrical Conductivity (1/mohm cm) Source Gray Solid 5.778 3.5 886 --27.7 --302 50.2 30.03 Arsenopyrite enargite(misc)

Arsenic Toxicity and Implications on Human Health


The most common external manifestations to arsenic exposure include skin disorders (hyper/ hypopigmentation changes) and keratosis .which may lead to skin cancer. Exposure to arsenic is associated with cardiovascular and peripheral vascular disease, neurological disorders, diabetes mellitus and various forms of cancer (Abernathy et al., 2003)Inorganic arsenic species that are of particular concern because they are chronic human carcinogens. The Joint FAO/WHO Expert Committee on Food Additives recommends a provisional tolerable weekly intake (PTWI) of inorganic arsenic of 15 g kg1 body weight, equivalent to 130 g day1 for a 60-kg person ( World Health Organ.
1989).

Exposure to hi h levels of arseni can cause cancers of the skin, bladder, kidney, and lung, and diseases of the blood vessels of the legs and feet Diabetes, high blood pressure, and reproductive disorders. Longterm ingestion of arsenic, mainly from drinking of contaminated well water, has caused a disease called "Blackfoot" in Taiwan. Blood vessels of the leg and foot become damaged, resulting in coldness, loss of feeling and eventually gangrene in the foot.

Arseni

ndard for Drinking Water

1) USEPA-Standard for domestic water 50g As/l 006) 2)WHO-standard 50 g As/l 3)WHO-recommendation 10 g As/l 4)Banladesh/ ndia-standard 50 g As/l 5)Maximum upper limit for inorganic arsenic consumption,adult not known but estimated to be >200 g As/day.

Studies on Arsenic toxicity have shown that plant species, which are not resistant to Arsenic, suffer considerable stress upon exposure: symptoms ranging from reduction in photosynthetic rate (Stoeva et al. 2003/4) and inhibition of root growth to death (Macnair and Cumbes 1987, Paliouris and
Hutchinson 1991).

Arsenic stimulates the formation of free radicals and reactive oxygen species, resulting in oxidative stress Results in the generation of reactive oxygen species (superoxide radical, hydroxyl radical and hydrogen peroxide), which can directly damage proteins, amino acids and nucleic acids a nd can cause peroxidation of membrane lipids (Dat et al. 2000, Stoeva et al. 2003/4) Both inorganic forms of arsenic are highly toxic because they interfere with various metabolic pathways in cells such as the interaction of substrates/enzymes with the sulfhydryl groups of proteins and the replacement of phosphate in ATP for energy (Tripathi et al., 2007) .Hence, plants exposed to arsenic show symptoms including decreases in both plant growth and crop yield . Plants respond to AsV and AsIII stresses differentially by stimulating the antioxidant system and thiol metabolism respectively (Mishra et al., 2008). As (V) is rapidly reduced to As(III) in the plant tissue, sufficient cytoplasmic concentrations of As(V) will not be present to exert toxicity. Inorganic As is more toxic than the organic As, whereas As in the trivalent oxidation state is more toxic than that in the pentavalent state (Fowler 1977). Restricting the influx of As might be an important mechanism to avoid toxicity, and several metallicolous plants (plants adapted to grow preferentially in soils containing a higher than normal concentration of a particular metal), such as Holcus phosphate/AsV transport. lanatus and Cytisus striatus improve As tolerance by constitutive suppression of high -affinity

Plants exposed to arsenic substantially increase the synthesis of glutathione (GSH) and phytochelatins (PCs), the polymers of GSH (Grill, E. et al. 2006)
and Schat, H. et al. (2002) . Augmented phytochelatin (PC) synthesis has

been observed in tolerant species, such as H. Lanatus (Hartley-Whitaker, J. et


al. 2001

)and many studies point to the essential role of PCs in both

constitutive and adaptive tolerance to arsenic (Schat, H. et al. 2002) and


Hartley-Whitaker, J. et al. (2001) . Complexation of arsenic with GSH and

PCs has been demonstrated in various plants, such as Rauvolfia serpentina, H. lanatus, P. cretica, Helianthus annuus and Brassica juncea (Pickering, I.J. et al.
(2000)and Raab, A. et al. 2004) and appears to contain AsIII, exclusively.

Selenium is a chemical element with the atomic number 34, represented by the chemical symbol selenium an atomic mass of 78.96. It is a nonmetal, chemically related to sulfur and tellurium, and rarely o ccurs in its elemental state in nature. Se behaves both as an antioxidant and an anti -inflammatory agent. This is because Se in its antioxidant role, notably as GPx, can: (1) reduce H2O2, lipid and phospholipid hydroperoxides, thereby dampening the propagation of free radicals and react ive oxygen species; (2) modulate the respiratory burst, by removal of H2O2 and superoxide. Antioxidant enzymes such as peroxidases and oxyreductases that protect from oxidative stress. For example, human erythrocytes have a selenocycteine -containing glutathione peroxidase (GPx) that catalyzes glutathione-coupled reduction of and protection from hydroxyperoxides ( Wang et al., 2003). Selenium level, which is required as a micronutrient in humans and animals and has also been reported to detoxify arsenis in rats, dogs, pigs, rabbits, and humans (Alfthan et
al. 1991; Spallholz et al. 2004; Thomson 2004). Although Asian cultivars of

rice have been, in general, found to be good Se lenium accumulators (Williams


et al. 2009a), their grain trace nutrient quality decreased with increasing arsenic

content (Williams et al. 2009b).

Arsenic and Selenium compounds can be biologically antagonistic to each other, because Se 4+ and As3+ have the same electronic structure. Thus, Selenium acts as a natural antidote to arsenic by accelerating arsenic excretion, sequestering arsenic by complexation and an antioxidant component of the enzyme glutathione peroxidase that may counteract the cancer . Antagonistic effects or mutual detoxication between arsenic and selenium have been reported in humans and other animals (Levander, 1977; Moxon, 1938; Zeng,
2001)

Mechanisms of arsenic uptake, translocation and detoxification in plants


Arsenate with dissociation constants (pKa) of 2.2, 6.97, and 11.5, most arsenic acid (H3AsO4) is dissociated as the oxyanions H 2AsO4or HAsO4 -under normal pH conditions (pH 48), and they are the chemical analogs of corresponding phosphate ions. Arsenate is taken up by plant roots via the phosphate transporters. In contrast to arsenate, arsenous acid (H 3AsO3, pKa = 9.2, 12.1, and13.4) is mostly undissociated at normal pH conditions (>94%undissociated at pH <8.0). Therefore, plant roots take up arsenite mainly as the neutral molecule As(OH)3. As in microorganisms and mammalian tissues (Bhattacharjee H, Rosen BP. 2007 .) MMA and DMA have lower dissociation constants than arsenite (pKa = 4.2 and 6.1, respectively). This explains the sensitivity of their uptake to the external pH increasing as the pH of the medium decreased. Small amounts of organic As (monomethylarsonic acid, MMA; and dimethylarsinic acid, DMA) can also be taken up through unknown transporters. Long distance transport of As from root-to-shoot takes place in the form AsV and AsIII. Plants assimilate sulfate (SO 4) to form cysteine (Cys) for the synthesis of glutathione (GSH) in two ATP-dependent

steps: in the first, rate-limiting step, g-glutamylcysteine (gEC) is synthesized by g-glutamylcysteine synthetase (g-ECS) using cysteine and g-glutamic acid (gGlu) as substrates; and in the second step, GSH is synthesized by glutathione synthetase (GS), using glycine (Gly) as a substrate. In response to arsenic, plants induce synthesis of phytochelatins (PCn), the polymers of GSH, through the enzyme phytochelatin synthase (PCS). PCn can be transported from root -toshoot and vice versa. Before detoxification, AsV is reduced to AsIII by arsenate reductase (AR) using GSH as a reductant. Phytochelatins and GSH coordinate with AsIII to form a variety of complexes. These complexes can be sequestered in the vacuole by ABC-type transporters, although direct evidence is lacking. In addition, large amounts of unbound AsIII are found in vacuoles but whether

these

are

transported

as

free AsIII

is

not

know.

Arsenic detoxification(PC)
A very general strategy of heavy metal/metalloid detoxification mechanism employed by plants is the production of PCs. The thiol (SH) -rich metal-binding peptides form AsSH complexes to detoxify Arsenic toxicity (Jocelyn 1972).
(Schmoger et al. 2000) suggesting that PCs play an important role in

decreasing the toxicity of arsenic in terrestrial plants. Both As(III) and As(V) trigger the formation of PCs in plants (Grill et al. 2006) In contrast to As(III), however, As(V) has no affinity for thiol groups, and it is therefore unlikely that

PCs would complex As(V). Hence, it is suggested that once inside the cytoplasm, As(V) is partially reduced to As(III) (which can then form complexes with PCs) (Jocelyn 1972). It can therefore be speculated that the reduction of As(V) to As(III) plays an important role in the detoxification of arsenic in higher plants (Quaghebeur and Rengel 2005). Arsenic a consequence of its pervasiveness, nearly every organism, from E. coli to humans, has mechanisms for arsenic detoxification, most of which involve transport systems that catalyze extrusion from the cytosol (Bhattacharjee and
Rosen, 2007).

Different forms of selenium found in soils influence its mobility, uptake, and metabolism by plants. The major influences on uptake a re soil pH and salinity. Some soil anions, such as phosphate, increase plant selenium uptake because increased soil-solution anion concentrations compete with selenium anions for adsorption sites. Other anions, such as chloride or sulfate, actually enhance or inhibit uptake by affecting plant metabolism. Inorganic selenides and elemental selenium are mostly insoluble except under conditions of low pH in moist, reducing environments. In these conditions organic selenides may also be found as selenium amino acids, such as selenoglutathione. Selenate and selenite compete with other anions, such as phosphate, sulfate, oxalate, and molybdate for adsorption sites.

Uptake of Selenium in plants


Selenate is the predominant form of Se in alkali ne and well-oxidized soils ( pH > 15), whereas in well-drained mineral soils with pH from acidic to neutral (pH < 15), Se exists predominantly as selenite (Elrashidi, M.A. et al. (1987) Under strongly reduced soil conditions ( pH < 7.5), selenide becomes the dominant form. (Elrashidi, M.A. et al. 1987) .Plant uptake of selenate can be mediated by sulfate transporters owing to the chemical similarity between selenate and

sulphate (Anderson, J. (1993) Translocation of Se from root to shoot depends on which Se species is supplied to the plant. In plants fed with selenate, Se is readily translocated to the shoot, and selenate is the predominant species in the xylem sap (Li, H.F. et al. (2008) By contrast, in selenite-treated plants, most of the Se stays in the roots, with little selenite being detected in the xylem sap (Li, H.F. et al. (2008). Selenite taken up by roots is readily converted to other forms, including selenomethionine (SeMet) and selenomethionine Se-oxide hydrate (SeOMet), but mostly into unidentified and water -insoluble forms( Li,
H.F. et al. (2008) Thus, in general, Se translocation from root to shoot is lower

in plants fed with selenite than in those fed with selenate order: SeO42- > H2Se02 > SeO22- > Se.

Arvy, M.P.

et.al.(1993) Selenium uptake resulting in reduced plant growth following the

ANTIOXIDANT SYSTEM IN PLANTS


The primary response of plants is the generation of reactive oxygen species (ROS) upon exposure to high levels of heavy metals. Various metals either generate ROS directly through Haber-Weiss reactions or overproduction of ROS and occurrence of oxidative stress in plants could be the indirect consequence of heavy metal toxicity (Wojtaszek, 1997; Mithofer et al.,
2004). The indirect mechanisms include their interaction with the antioxidant

system (Srivastava et al., 2004), disrupting the electron transport chain and disturbing the metabolism of essential elements .One of the most deleterious effects induced by heavy metals exposure in plants is lipid peroxidation, which can directly cause biomembrane deterioration. Malondialdehyde (MDA), one of the decomposition products of polyunsaturated fatty acids of membrane is regarded as a reliable indicator of oxidative stress .The ROS produced as a result of oxidative stress causes a variety of harmful effects in plant cells, such as inhibition of photosynthetic activity, inhibition of ATP production, lipid peroxidation, and DNA damage. Plants have evolved a variety of mechanisms

to counteract the effects of ROS in cellular compartments. These mechanisms involve a variety of molecular antioxidants such as nonprotein thiol (NP -SH), cysteine, glutathione (GSH), ascorbic acid, proline and antioxidant enzymes such as SOD, APX, GPX, CAT, and GR. GSH plays a key role in protecting membranes to damage by free radicals by trapping them in aqueous phase and as a part of ascorbate glutathione cycle (Mishra et al., 2006). . However, plants have developed a very potential mechanism to combat with such adverse environmental heavy metal toxicity problems . Plants produce low molecular weight Thiols that show high affinity for toxic metals .The most important/critical low molecular weight biological thiols are glutathione (GSH) and cysteine. GSH is a sulfur containing tri-peptide thiol with the formula glutamatecysteine- glycine.

1) GLUTATHIONE AND GLUTHIONE REDUCTASE


Glutathione, glutamyl cysteinyl glycine (GSH) is the major low molecular weight thiol compound in most plants .Glutathione exists in two form reduced glutathione (GSH) and oxidized glutathion e (GSSG). The reduction potential of glutathione depends on the intracellular GSH/GSSG ratio. Change in the redox ratio of glutathione mainly depends on the pH, total GSH concentration, GSH biosynthesis and GSH catabolism. Several studies have indicated th at exposure of plants to high level of heavy metals induces ROS, either directly or indirectly by influencing metabolic processes. GSH participate in the control of H 2O2 level of plant cells. Change in the ratio of its reduced (GSH) to oxidized (GSSG) form during degradation of H 2O2 is important in certain redox signaling pathways. It has been suggested that the GSH/GSSG ratio, an indicative of the cellular redox balance, may be involved in ROS perception. Reduced glutathione (GSH) acts as an antioxidant and involve directly in the reduction of most ROS generated during stress. Glutathione acts as disulphide reductant to protect thiol groups on enzymes, regenerate ascorbat e and react with singlet

oxygen and hydroxyl radicals.It acts as a protein disulphide reductant, which detoxifies herbicides by conjugation, either spontaneously or by the activity of one of a number of glutathione - S-transferases (GST), and regulates gene expression in response to environmental stress and pathogen attac k. It also participates in the regeneration of ascorbate from dehydro -ascorbate via the enzyme dehydro-ascorbate reductase. In such reactions GSH is oxidized to glutathione disulphide (GSSG). GSH is regenerated by glutathione reductase (GR) in a NADPH-dependent reaction.
Dehydro-ascorbate + 2 GSH=Ascorbate + GSSG in presence of dehydro ascorbate reductase(DHAR). GSSG + NADPH= 2 GSH + NADP in presence of glutathine reductase(GR) .

Arsenite has a high affinity to the sulfhydryl (SH) groups of peptides, such as glutathione (GSH) and phytochelatins (PCs; polymers of GSH), whereas As(V), in its inorganic form, does not bind to thiol ligands (Raab et al. 2007). A variety of complexes of As(III) with GSH and PCs have been detected in plants
(Raab et al. 2005, 2007), which are supposedly sequestered inside the vacuole (Bleeker et al. 2006)

2) Superoxide dismutase(SOD):
Superoxide dismutase, the family
2 to

of

metalo-enzymes

catalyses

the

disproportionation of superoxide

molecular oxygen and H 2O2 (Scandalios,

J. G., Plant Physiol., 1993) Superoxide dismutase removes superoxide and

hence decreases the risk of hydroxyl radical formation from superoxide via the metal-catalysed HaberWeiss-type reaction. Three isozymes of SOD, namely Mn-SOD, Cu/Zn-SOD and Fe-SOD have been reported in various plant species. Mn-SOD is predominantly found in mitochondria( Bowler, C.et al 1989) and peroxisomes (Sandalio, L. M. et al 1987).Cu/Zn-SOD initially supposed to be located in the cytosolic fraction(Hernandez, J. A.et al 1999) has lately also

been reported from chloroplastic and mitochondrial fractions(Hamilton, III, E.


W. and Heckathorn, S. A. 2001). Similarly, Fe-SOD though predominantly

detected in chloroplasts, has also been reported from cytosolic, mitochondrial


(Salin, M. L., 1988) and peroxisomal fractions.

3)Ascorbic acid and Ascorbate peroxide.


Ascorbate is present in chloroplasts, cytosol, vacuole and apoplastic space of leaf cells in high concentrations (Polle, A., Chakrabarti, K., Schurmann, W.
and Rennenberg, H. 1990) . It is perhaps the most important antioxidant in

plants, with a fundamental role in the removal of hydrogen peroxide (Foyer, C.


H. Alscher, R. G. and Hess, 1993) Oxidation of ascorbate occurs in two

sequential steps, first producing mono-dehydroascorbate, and if not rapidly re reduced to ascorbate, the mono-dehydro-ascorbate disproportionates to ascorbate and dehydro -ascorbate Ascorbate peroxidase activity has m ainly been reported from chloroplast and cytosol (Chen, G. X. and Asada, K., Plant
1989) However some recent studies have also reported its occurrence in

mitochondria as

well .In the chloroplasts, SOD and ascorbate peroxidase

enzymes exist in both soluble and thylakoid-bound forms. Superoxide generated at the membrane surface can thus be trapped and converted immediately to H2O2 to be scavenged by the membranebound ascorbate peroxidise (
Neubauer, C. and Schreiber, U., Z. Naturforsch. C, 1989).Two enzymes are

involved in the regeneration of reduced ascorbate, namely mono -dehydroascorbate reductase which uses NAD(P)H directly to recycle ascorbate and dehydro-ascorbate reductase.

4 Mono-dehydro-ascorbate + 2H2O= 4 Ascorbate + O2 in presence of light.

Objectives
1. To study the response of selenium on growth parameters viz. root length, shoot length and weight in contrasting rice varieties under As exposure. 2. To st u d y t he g ro wt h a n d photosynthetic response i n ri c e c ul t i va r s u nde r di ffe re n t c on c e nt ra t i on of As su pp l e me nt e d wi t h Se . 3. In t e ra c t i ve e f fe c t o f Se on a nt i oxi da n t s sy s t e m o f ri c e c ul t i va r s u nde r d i f fe re n t c o nc e n t r a t i o n o f As . 4. To me a su re t he t h i ol s vi z . c y st e i ne , NP SH, GSH a nd GS SG c o nt e nt du ri n g As st re s s su ppl e me n t e d wi t h Se . 5. To study the effect of selenium on arsenic uptake and translocation in rice genotypes.

MATERIALS AND METHODS


Two c o mmo n l y c u l t i va t e d r i c e c ul t i va r s BRG- 1 2 a n d Got ra b ho g we re p ro c ure d fr o m R. R. S. , Chi n su ra h se l e c t e d f o r t h e st ud y ba s e d o n p re vi o u s sc re e n i n g. Se e ds o f ri c e we re s oa ke d i n mi l l i Q wa t e r f or 24 ho u rs a n d t he n t ra n s fe rre d o n P e t ri di sh e s fo r ge r mi na t i on. Thre e da y s ol d se e dl i n gs we re t ra n sf e r re d t o t h e t ra y c o n t a i ni n g 3 l i t He wi t t n u t ri e nt s ol ut i on. The e x p e ri me n t s we r e c a r ri e d o u t i n c ont ro l l e d c ond i t i o n s by p r o vi d i n g 1 4 h l i ght a nd 1 0 h da r k pe r i od (2 6 0 - 3 5 0 m mol m - 2 s - 1 ). The t e mpe ra t u re i s ke p t a t 25 o C du r i n g t he da y a nd 20 o C d ur i n g t he ni gh t , t he re l a t i ve hu mi d i t y i s 7 0% t ha t ma y be ma i n t a i n e d b y hu mi di fi er. Aft e r 7 da y s gr o wt h, t h e sol ut i on o f t ra y wa s c ha n ge d. 7 da y s ol d se e d l i n g wa s t h e n e xp o s e d t o di ff e re nt c o nc e n t r a t i o n (0, 25 M ) o f a r se n i c ( As II I) su p pl e me n t e d wi t h Se [ IV] ( 25

M ) t r e a t me n t p re p a re d b y di l u t i n g s t o c k s ol ut i on of a r se na t e (p re pa re d b y t he s a l t Na 2 H As O 4 ) a nd se l e ni t e ( Na 2 Se O 3 ) wi t h He wi t t n ut ri e nt s ol ut i o n. Af t e r 7 da y s t re a t me nt , pl a nt s we re ha r v e st e d, wa sh e d wi t h do ub l e d i s t i l l e d wa t e r, b l o t t e d a n d u se d fo r t h e s t u d y o f va ri ou s p a ra me t e r s.

a . P r e p a r a ti o n o f r i c e s e e d l i n g s :

Se e d s we r e st e ri l i z e d i n 0 . 1% Hg Cl 2 s ol ut i on f or 30-4 5 se c o nd fo l l o we d by wa s hi n g wi t h de i o ni z e d wa t e r. The se e d s ge r mi n a t e d i n h yd ro po ni c so l ut i o n . Th e c o mp o s i t i o n of n u t ri e n t so l u t i on p ro v i d e d by H e wi t t (1 9 6 6 ) us e d fo r t he gro wt h o f r i c e c ul t i va r s.

Fig. Germinated Seedlings of both variety BRG -12 & Gotrabhog

Plants of rice in Hydroponic condition

The composition of Hewitt nutrient medium used is as follows (Hewitt,


1966)

CHEMICALS

CONCENTRATION

NH4NO3 K2SO4 CaCl2 MgSO4 KNO3 KH2PO4 EDTA

4.4 mM 2 mM 4 mM 1.5 mM 1.4 mM 1.3 mM 50 QM

FeSO4.7H2O H3BO4 ZnSO4 MnSO4 Na2MoO4 CoSO4.7H2O CuSO4

50 QM 10 QM 0.001 mM 0.005 mM 0.000 mM 0.2 QM 1 QM

b . St oc k sol ut i o n o f a r se n i c (1 0 00 Q M ) wa s p re pa re d b y

di s so l vi n g 0. 3 1 2 g i n 1 l i t o f He wi t t s ol u t i on u si n g Na 2 HAs O 4 ( ICN) .
c . St oc k so l ut i o n of se l e ni u m (1 00 0 M ) wa s p re pa re d by

di s so l vi n g 0. 1 7 2 g i n 1 l i t o f He wi t t s ol u t i on u si n g Na 2 Se O 3 .
2. Gr o wt h p a r a me te r s

Pl a nt

b i o ma s s

wa s

me a s ure d

on

f re sh

we i ght

b a si s.

Co nt r ol a n d t re a t e d pl a nt s we re h a r ve s t e d, wa she d wi t h d ou b l e di st i l l e d wa t e r, bl o t t e d ge nt l y t o re mo ve e x c e s s wa t e r a n d we i g he d. Roo t a n d sh oo t l e n g t h we re me a su re d b y me t ri c sc a l e .
3 . B i oc h e mi c a l a n a l y s i s (i ) P h o t o s y n t h e ti c p i g me n t s e s ti ma t i o n

Phot o syn t he t i c p i gme n t s of t re a t e d a nd u n t re a t ed p l a n t s (10 0 mg) we re e x t ra c t e d i n 4 ml o f 8 0% c h i l l e d a c e t one ( v / v ) a n d c e nt ri fu g e d a t 10 , 0 00 g f o r 10 mi n a t 4 C. Th e a b so rba nc e o f

t he e x t ra c t wa s t a k e n a t 4 80, 5 1 0, 64 5 a n d 66 3 n m a s p e r pr o c e d ure o f A r n o n (1 9 4 9 ) . Ch l o r op h yl l c on t e n t s we re c a l c ul a t e d a c c o rd i n g t he f ol l o wi n g fo r mul a : To t a l Chl o ro ph y l l ( mg g - 1 f w) = [20.2 (A645) + 8.02 (A663)] x V d x 1000 x W


[12.7 (A663)  2.63 (A645)] x V

Chl o r oph yl l a ( mg g - 1 f w) =
d x 1000 x W

Chl o r oph yl l b ( mg g - 1 f w) =

[22.9 (A645)  4.68 (A663)] x V

d x 1000 x W

Ca ro t e n oi d c on t e n t i n t h i s e xt ra c t wa s c a l c ul a t e d by t he fo r mul a gi ve n b y D u x b u r y a n d Y e nt s c h (1 9 5 6 ).

[7.6 (A480) 1.49 (A510)] x V

Ca r ot e no i d ( mg g

-1

f w) =
d x 1000 x W

whe re , A 4 8 0 , A 5 1 0 , A 6 4 5 , A 6 6 3 = a b s or b a nc e a t t he s e wa ve l e n gt h s V = vo l u me o f fi na l e x t ra c t ( ml ) W = we i ght o f p l a n t sa mp l e ( g ) d = wi d t h o f t he c u v e t t e ( 1 c m)
(i i ) P r o te i n me a s u r e me n t

Prot e i n c o nt e nt wi l l be r e c or de d a s pe r L o wr y e t a l
(1 9 5 1 ).

Ab o ut 2 00 mg of t re a t e d a n d u nt re a t e d p l a n t s wi l l be c ru s he d i n 5 ml 10 % TC A a nd c e nt ri fu ge d a t 1 0, 00 0 g f or 1 0 mi n a t 4 C. Supe r n a t a n t i s t he n de c a n t e d a n d pe l l e t s wi l l be wa s h e d t wi c e wi t h 1 N Na OH. Th e pe l l e t s wi l l be a ga i n c e nt ri fu g e d i n 5 ml 1 N Na OH a nd fi n a l sup e r na t a n t wi l l b e c ol l e c t e d. Re a ge n t A (1 ml ) a nd r e a ge n t B (1 ml ) wi l l be a dd e d t o re a ge nt C t o ma ke 10 0 ml . Fi v e ml o f a bo ve so l ut i o n wi l l be a dde d t o fi na l sup e r n a t a n t ( 0 . 5 m l ) a nd ke p t f o r 1 0 - 15 mi n a t 30 r C. Re a ge nt D (0 . 5 ml ) wi l l be fi n a l l y a dde d a nd t ho ro u gh l y mi x e d. Af t e r 30 mi n t he a b s or ba nc e wi l l be re c o r de d a t 7 00 n m. Bo vi ne se r u m a l bu mi n ( SRL) wi l l be u se d a s a st a nda rd. Pr ot e i n c ont e nt wi l l b e e xp re s se d a s mg g - 1 f w.

(i i i ) Cy s te i ne c o n t e n t

As t re a t e d a n d un t r e a t e d r i c e l e a ve s/ ro ot s (3 0 0 mg F W) wi l l be e xt ra c t e d i n 5 % c hi l l e d pe rc h l o ri c a c i d ( HCl O4 ), c e n t r i fu ge d a t 1 0, 00 0 f or 1 0 mi n a t 4 0 C; c y s t e i ne c on t e n t wi l l b e me a su re d i n sup e r n a t a n t u s i n g a c i d ni nh y d ri n re a ge n t ( G a i t o n d e 1 9 5 7 ) .
F or p r e p a r a ti o n o f e v e r y 1 0 ml o f n i n h y d r i n r e a g e n t : 2 5 0 mg

of n i n h y dr i n wi l l be di s so l ve d i n 6 ml g l a c i a l a c e t i c a c i d a n d 4 ml HCl . Re a c t i on mi x t u re (3 ml ) c ont a i ne d 1 ml each of

su pe rn a t a n t , gl a c i a l a c e t i c a c i d & n i n h yd ri n r e a ge nt . M i xt u re wi l l be he a t e d fo r 15 mi n a t 95 0 C & t he n ra pi d l y ke pt a t r oo m

t e mpe r a t u re pre p a r e d

&

a b s o r ba n c e kn o wn

wi l l conc.

be of

me a s ure d c y st e i ne

at

56 0

n m.

Cy st e i ne c o n t e n t wi l l be c a l c ul a t e d f ro m t he st a nd a rd c u r ve using ( L - c y st e i n e hyd roc h l o r i de , si g ma ) & i s e x pr e s se d a s n mo l g - 1 F W. (i v ) N o n p r o te i n t h i o l ( N P S H ) e s ti ma ti o n Le ve l o f NP - SH i n c on t r ol & me t a l l o i d e xp os e d p l a n t s wi l l be me a su re d f ol l o wi n g t he me t h od o f El l ma n ( 1 9 5 9 ) . P l a n t ma t e ri a l (7 0 0 mg) wi l l b e h o mo ge ni z e d i n 3 ml o f 6. 6 7 % c h i l l e d 5 - su l f o sa l yc i l i c a c i d. Af t e r c e nt r i f u ga t i o n a t 1 30 00 x g fo r 10 mi n a t 4 0 C. NP - SH c on t e n t wi l l b e me a s ure d i n t he s up e rn a t a nt by d i l ut i n g it


5

to
mM

ten

t i me s

wi t h El l ma n
mM DTNB

reagent in 120

(1 : 9 )
mM

(c o n t a i n i n g

EDTA,

0.6

P o ta s s i u m P h o s p h a te B u ffe r , pH - 7 . 5 ) . Mi x t u re wi l l be l e ft fo r

15 mi n fo r c ol or de v e l o p me n t & a bs or ba n c e wi l l be re c o r de d a t 412 n m. St a n da r d c u r ve wi l l b e ma de u si n g kn o wn c o nc e n t r a t i on o f GSH ( L - gl ut a t h i on e re duc e d, s i gm a ) f o r c a l c ul a t i n g t he NP- SH va l u e . Th e NP- SH c ont e nt i s e x pre s se d a s mo l g- 1 F W.


(v ) T o ta l g l u t a t h i o n e d e te r mi n a t i o n

Fo r t ot a l g l u t a t hi one ( GS H+ G S SG ), f r oz e n l e a ve s t i s su e wi l l be ho mo ge n i z e d i n 0. 1 M s o d i u m ph o sp ha t e 0 . 0 05 M EDTA b u f fe r (p H 8. 0) a n d 25 % me t a p ho sp h o ri c a c i d ( u se d a s a pro t e i n pre c i p i t a nt ). De t e r mi n a t i on of total gl u t a t hi o ne wi l l be pe r fo r me d f l uo re me t r i c a l l y by t h e me t h od of H i s si n a n d H i l f


(1 9 7 6 ).

(vi) E s ti ma ti o n o f me l o n d i e l d e h y d e c o n te n t

Li p i d pe r o x i da t i o n wi l l be de t e r mi n e d b y e s t i ma t i o n o f t he M D A c o n t e n t f ol l o wi n g H e a t h a n d P a c k e r (1 9 6 8 ) wi t h sl i ght mod i fi c a t i on. Pl a nt ma t e r i a l (2 00 mg) wi l l be ho mo ge n i z e d i n 3 ml o f 0 . 1% TC A. Th e h o mo ge na t e wi l l be c e nt ri fu g e d a t 10, 00 0 g f o r 5 mi n. Fo r e ve ry 1 ml of a l i quo t , 4 ml o f 2 0 % TCA containing 0 . 5% t hi oba rb i t u ri c acid wi l l be a d de d . M i xt u re wi l l be he a t e d a t 9 5 C fo r 3 0 mi n a n d t he n c oo l e d qui c kl y on i c e ba t h. Af t e r c e n t r i f u ga t i o n o f t he mi xt ure a t 10, 00 0 g f o r 1 5 mi n, t he a b so rb a nc e o f t he s up e rna t a n t wi l l b e t a ke n a t 5 32 a nd 6 00 n m. Co r re c t i on of n on - sp e c i fi c t u rb i d i t y wi l l be ma de by su b t ra c t i n g t he a bs or ba n c e va l u e t a ke n a t 6 0 0 n m f r o m t he a b s or ba nc e a t 5 32 n m. The l e ve l o f l i p i d

pe r o xi da t i o n wi l l be e xp re s se d a s Q mol of M D A fo r me d pe r gra m o f fre s h we i g ht u s i n g t he e xt i nc t i o n c oe ff i c i e nt o f 15 5 mM - 1 c m - 1 .

3 . E s ti ma t i o n o f a n t i o x i d a n t e n z y me s E n zy me e x t r a c ti o n

0. 5 gm of fre s h ri c e t i s s ue wi l l be h o mo ge n i z e d i n a n e xt ra c t i o n b u ff e r c o n t a i n i n g 5 0 m mol / L Tr i s - HC L bu ffe r ( p H 7. 0) 0. 1 m mol / L EDTA 1 m mol / L P M SF a nd 0. 3 g g - 1 F W P VP. Th e h o mo ge na t ge wi l l be f i l t e re d t h ro u gh fou r f ol d s o f mu sl i n a nd c e n t r i f u ge d a t 15, 0 0 0 x g f o r 3 0 mi n i n So r va l l RC5 C re fr i ge ra t e d c e n t r i f u ge . The c l e a r supe r n a t a nt wi l l be

use d fo r t he c ru de e n z y me a c t i vi t y fo r a s sa y o f c a t a l a se , pe r o xi da se a nd gl u t a t h i o n e r e du c t a se .

(i ) A s sa y o f c a ta l a se

For t he me a s u re me nt o f t he C A T ( E C -1 . 1 1 - 1 . 6 ) a c t i vi t y , e xt ra c t i o n wi l l be d o ne i n t h e b uf fe r c on t a i ni n g 50 mM Tr i s HCl (p H - 7 ), 0. 1 mM EDTA, 1 mM P M SF & 0. 3 g g- 1 o f f w P VP. The c a t a l a se a c t i vi t y wi l l be a s s a ye d b y t he me t hod o f Ae b i


(1 9 7 4 ). The a s sa y sy s t e m c o mp r i se d o f 5 0 m mol / L s o di u m

pho s p ha t e b u f fe r ( p H 7. 0 ), 20 m mol / L H 2 O 2 a nd a sui t a b l e a l i q uo t of e n z y me i n t h e fi na l vo l u me o f 3 ml . de c re a se i n t he a bs o rb a nc e wi l l be t a ke n a t 2 4 0n m .

(i i ) A s s a y o f A sc o r b a te P e r o x i d a se

The a c t i vi t y of A P X ( E C 1 . 1 1 . 1 . 1 1 ) wi l l b e me a su re d a c c or d i n g t o t he me t h od of N a k a n o a n d A s a d a ( 1 9 8 1 ) by e s t i ma t i n g c oe f fi c i e nt t he 2. 8 ra t e mM - 1 of a sc or ba t e The 3 o x i d a t i on ml (e xt i nc t i o n mi x t u r e c m - 1 ). re a c t i o n

c ont a i n e d 50 mM pho s pha t e bu f fe r (p H 7. 0 ), 0. 1 mM H 2 O 2 , 0. 5 mM sod i u m a sc o rb a t e , 0. 1 mM EDT A a n d a s u i t a b l e a l i qu ot of e nz y me e xt ra c t . The c ha n g e i n a b so rb a nc e wi l l be mo ni t o re d a t 290 n m a nd e nz y me a c t i vi t y wi l l be e xp re s se d a s un i t s mg - 1 pro t e i n ( 1 u n i t = Q mo l e s o f a s c o rb a t e o x i d i z e d mi n - 1 g - 1 f w).

i i i . A s s a y o f G u a i a c ol P e r o x i d a s e
G P X ( E C 1 . 1 1 . 1 . 7 ) a c t i vi t y wi l l be a s sa y e d a c c o rd i n g t o

t he me t h od o f H e me d a a n d K l e i n (1 9 9 0 ). A 10 0 ml of re a c t i o n mi x t u re wi l l be pre pa re d b y a ddi n g 10 ml of 1% gua i a c o l ( v/ v), 10 ml o f 0. 3 % H 2 O 2 a nd 8 0 ml o f 50 mM pho s p ha t e bu f fe r (p H 6. 6). En z y me e x t ra c t (7 5 l ) wi l l be a dde d t o re a c t i on mi xt ur e i n a fi na l vol u me o f 3 ml . Th e i nc re a s e i n a b so rb a nc e d u e t o oxi da t i o n of gua i a c o l (e x t i nc t i o n c oe f fi c i e n t 26. 6 mM - 1 c m - 1 ) wi l l be mo ni t o re d a t 47 0 n m. Enz y me a c t i vi t y wi l l be e x pre s se d a s u ni t s mg - 1 pr o t e i n (1 u ni t = Q mo l e s o f g ua i a c o l o xi d i z e d mi n - 1 g - 1 f w).
(i v ) A s s a y o f g l u ta t h i o n e r e d u c ta se

For t he e s t i ma t i o n o f t he G R ( E C - 1 . 6 , 4 . 2 ) a c t i vi t y p l a n t ma t e ri a l wi l l be e x t ra c t e d i n 0. 1 M Po t a s si u m p ho sp ha t e b uf fe r (p H - 7 . 5) c on t a i ni n g 0. 5 mM EDT A. The a c t ivi t y o f gl ut a t h i on e re d u c t a se wi l l b e a s sa ye d by t he m e t ho d o f S mi t h e t. a l . , (1 9 8 8 ). The re a c t i on mi x t u re c o nt a i n e d 1 ml o f 0. 2 M p ot a s si u m pho s p ha t e b u ffe r (p H - 7. 5 ) c o n t a i n i n g 1 mM EDTA, 0. 5 ml o f 3 mM 5, 5 - di s t h i ob i s 2- n i t ro be n z oi c a c i d ( DTNB) i n 0. 01 M pho s p ha t e b u f fe r (p H - 7. 5 ), 0. 25 ml H 2 O, 0. 1 ml 2 mM N ADP H, 0. 05 ml e nz y me e xt ra c t & 0. 1 ml 2 0 mM GS SG. The c o mp o ne nt s wi l l b e a d de d i n t h e or de r a s a bo ve di re c t l y t o a c u ve t t e & t he re a c t i o n wi l l b e st a rt e d by t he a ddi t i on of GS SG. Th e i n c re a se in a b so r ba nc e wi l l be mon i t or e d f or 5 mi n a t 41 2 n m. Th e ra t e o f e nz y me ac t i vi t y wi l l be e x p re s se d a s mol e s o f GSS G re du c e d pe r mi n pe r gm f w.

( v ) A s sa y o f s u p e r o x i d e d i s m u t a s e (S O D ) The a c t i vi t y o f s u p e r o x i d e d i s m u ta se ( E C - 1 . 1 5 -1 . 1 ) wi l l be a s sa ye d by a d op t i ng the me t hod o f B e a u c h a mp


and F r i d o v i c h ( 1 9 7 1 ) , b y me a su ri n g i t s a b i l i t y t o i nh i b i t t h e

pho t oc he mi c a l re duc t i on o f Ni t ro bl ue Te t r a z o l i u m ( NBT). The 3 ml re a c t i o n mi x t u re c on t a i n 4 0 mM ph o sp ha t e bu ffe r (p H7. 8), 13 mM me t h i o n i n e , 7 5 m NBT, 2 m Ri b of l a vi ne, 0 . 1 mM EDT A & a s ui t a b l e a l i qu o t o f e nz y me e xt ra c t . Ri bo f l a vi ne wi l l be a dde d a t t h e e n d & t e s t t u b e s wi l l b e sh a ken a n d pl a c e d 30 c m b e l o w l i ght s ou rc e c on s i st i n g o f 15 W fl uo re sc e n t l a mp s. Re a c t i on wi l l be st a r t e d by s wi t c h i n g o n t he l i ght & 30 mi n, t he re a c t i o n wi l l be st o ppe d by s wi t c hi n g o f t he l i gh t wi t h n o e nz y me se r ve d a s c on t r o l . The a b s or ba nc e of t he s o l u t i o n wi l l be t a ke n a t 5 6 0 n m. Ac t i vi t y of SOD wi l l be me a su re d by su b st ra c t i n g NBT re d u c t i on i n l i ght wi t h p r ot e i n f ro m NBT re d u c t i o n i n l i ght wi t ho ut p ro t e i n. One u ni t o f a c t i vi t y i s t h e a mo un t of p r o t e i n re q u i re d t o i nh i b i t 5 0 % i n i t i a l re d uc t i on of NBT und e r l i ght .

5 . M e t a l e s ti ma ti o n

Fo r e st i ma t i o n o f t ot a l As a n d Se , dri e d p l a n t sa mp l e s we re po wd e r e d a nd d i ge s t e d i n 3 mL H NO3 a t 1 20 0 C fo r 6 h ( Smi t h e t a l . , 20 0 8 ). The l e ve l of As a nd Se wa s qua n t i e d by Ind uc t i ve l y Cou pl e d Pl a s- ma M a s s Spe c t ro met e r ( ICP - M S, Agi l e n t 75 00 c e ).

REFERENCES
1. Ali, et al.,2003 Fate of arsenic in the environment. Bhaka: Buet -UNU International Symposium, International Training Network Centre, Bangladesh University of Engineering and Technology, United Nations University, Tokyo, P. 1-6. 2. Anderson, J. (1993) Selenium interactions in sulfur metabolism. In Sulfur Nutrition and Assimilation in Higher Plants: Regulatory Agricultural and Environmental Aspects (De Kok, I.S. et al., eds), pp. 4960, SPB Academic. 3. Arvy, M.P. (1993) Selenate and selenite uptake and translocation in bean plants (Phaseolus vulgaris). J. Exp. Bot. 44, 10831087 4. Alfthan GAA, Arvilommi H, Huttunen JK (1991) Selenium metabolism and platelet glutathione peroxidase activity in healthy finnish men: effects of selenium yeast, selenite, and selenate. Am J Clin Nutr 53:120 125 5. Abernathy CO, Thomas DJ, Calderon RL. Health effects and risk assessment of arsenic. J Nutr 2003;133:1536S8S

6.

Akbaraly et al., 2010,Bleys, et.al., 2004) A global survey of effects of genotype and environment on selenium concentration in lentils ( Lens culinaris L.): Implications for nutritional fortification strategies Food Chemistry Volume 125, Issue 1, 1 March 2011, Pages 72-76

7.Arnon, D I (1949). Copper enzymes in isolated chloroplast, polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15.

7.

Br.Geol. Surv.

Surv.

(BGS/DPHE)

2001.

Arsenic

Contamination

of

Groundwater in Bangladesh. Vol. 1: Summary. Keyworth, UK: Br. Geol. 8. Bowler, C., Alliotte, T., De Loose, M., Van Montgu, V. and Inze, D., EMBO J., 1989, 8, 3138 9. Brown KM, Pickard K, Nicol F, Beckett GJ, Duthie GG, Arthur JR. Effects of organic and inorganic selenium supplement ation on selenoenzyme activity in blood lymphocytes, granulocytes, platelets and erythrocytes. Clin Sci 2000; 98: 59399 10. Bhattacharjee H, Rosen BP. 2007. Arsenic metabolism in prokaryotic and eukaryotic microbes. In Molecular Microbiology of Heavy Metals , ed. DH Nies, S Silver, pp. 371406. 11. Beauchamp, C and Fridovich, I ( 1971 ). Superoxide dismutase: improved assays andan assay applicable to acrylamide gels. Anal, Biochem. 44:
276-286 and Arsenic Technol. 41:2930-2936.

12. Combs & Lu, 2001; Gailer, Madden, Burke, Denton, & Aposhian, 2000; Moghadaszadeh & Beggs, 2006; Rayman, 2002 A global survey of effects of genotype and environment on selenium concentration in lentils (Lens culinaris L.): Implications for nutritional fortification strategiesFood Chemistry, Volume 125, Issue 1, 1 March 2011, Pages 7276

13. Chowdhary, et al .,Uptake Kinetics of Arsenic Species in Rice Plants Plant Physiology., march 2001 vol. 128 no. 3 1120-1128 14. Clark LC, Combs Jr GF, Turnbill BW, et al,2001 Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: a randomized controlled trial. JAMA 1996; 276: 195763. 15. Chen, G. X. and Asada, K., Plant Cell Physiol., 1989, 30, 987998 16. Dwivedi S, Tripathi RD, Srivastava S, Singh R, Kumar A, Tripathi P, Dave R, Rai UN, Chakrabarty D, Trivedi PK, Tuli R, Adhikari B, Bag MK, Arsenic affects mineral nutrients in grains of various Indian rice (Oryza sativa L.) genotypes grown under arsenic-contaminated soils of West Bengal. Protoplasma 2010b;245:113 -24. 17. Duffield-Lillico AJ, Slate EH, Reid ME, Turnbull BW, Wilkins PA, Combs Jr GF, Park HK, Gross EG, Graham GF, Stratton MS, Marshall JR, Clark LC, Selenium supplementation and secondary preventi on of nonmelanoma skin cancer in a randomized trial. J Natl Cancer Inst 2003;95:1477-81. 18. Duxbury, AC, Yentsch, CS (1956) Plankton pigment monograph. J. Mar.
Res. 15:93-101.

19. Dat, J., S. Vandenabeele, E. Iranova, M. Van Montagu, D. Inze, and F. Van Breusegem, 2000: Dual action of the active oxygen species during plant stress responses. Cell. Mol. Life Sci. 57, 779795. 20. Elrashidi, M.A. et al. (1987) Chemical-equilibria of seleniumin soils a theoretical development. Soil Sci. 144, 141 152 21. Fordyce, F.M. (2005) Selenium deficiency and toxicity in the environment. In Essentials of Medical Geology (Selinus, O. et al., eds), pp. 373415, Elsevier Rayman, M.P. (2002) The argument for increasing selenium intake. Proc. Nutr. Soc. 61, 203 215

22. Foyer, C. H. and Lelandais, M., in Photosynthetic Responses to the Environment (ed. Yamamoto, H. Y.), Am. Soc. Plant Physiologists, Rockville, MD, 1993, vol. 8, pp. 88 101. 23. Fowler, B. A., 1977: Toxicology of environmental arsenic. In: R. A Goyer, and M. A. Mehlan, eds. Toxicology of Trace Elements, 79122, Wiley, New York. 24. Gailer 2000 Chemical Form of Selenium in Naturally Selenium -Rich Lentils (Lens culinaris L.) from Saskatchewan J. Agric. Food Chem., 2007, 55 (18), pp 7337734 Publication Date (Web): August 9, 2007. 25. Grill, E., Mishra, S., Srivastava, S., Tripathi, R.D., 2006. Role of phytochelatins in phytoremediation of heavy metals. In: Singh, S.N., Tripathi, R.D. (Eds.), Environmental Bioremediation Technologies. Springer, Heidelberg, pp. 101145 26. Ghosh AK, Sarkar D, Bhattacharyya P, Maurya UK, Nayaka DC. Mineralogical study of some arsenic contaminated soils of West Bengal. India Geoderma 2006;136:300-320. 27. Hartley-Whitaker J. 2002 et.al Arsenic uptake and metabolism in arsenic resistant and nonresistantplant species. New Phytol. 154:294 28. Hewitt, EJ (1966) Sand and water culture methods used in the study of plants nutrition, 2 nd edn. 29. Hernandez, J. A., Campillo, A., Jimenez, A., Alarcon, J. J. And Sevilla, F., New Phytol., 1999, 141, 241251. 30. Hamilton, III, E. W. and Heckathorn, S. A., Plant Physiol., 2001,Rio, L. A. et al., Plant Physiol., 1998, 116, 1195 1200. 31. Jocelyn, P. C., 1972: Biochemistry of the SH Group. Academic Press, London. 32. Kryukov, G.V. et al. (2003) Characterization of mammalian selenoproteomes. Science 300, 1439 1443

33. Lu, J. and Holmgren, A. (2009) Selenoproteins. J. Biol. Chem 284, 723727. 34. Levander OA. Metabolic interrelationships between arsenic and selenium. Environ Health Perspect 1977;19:159 64. 35. Li, H.F. et al. (2008) Selenium uptake, translocation and speciation in wheat supplied with selenate or selenite. New Phytol. 178, 92 102 36. Moxon AL. The effect of arsenic on the toxicity of seleniferous grains. Science 1938;88:81. 37. Meharg AA. Arsenic in rice-understanding a new disaster for South-East Asia. (2003)Trends Plant Sci 2004;9:415720 38. Meharg AA, Rahman M. Arsenic contamination of Bangladesh paddy soils: Implications for rice contribution to arsenic consumption. Environ Sci Technol 2003;37:229-34. 39. Meharg AA.2004 Baseline soil variation is a major factor in arsenic accumulation in Bengal delta paddy rice. Environ Sci Technol 2009;43:172427 40. Mondal SP, Polya DA. Rice is a major exposure route for arsenic in Chakdaha block, Nadia district, West Bengal, India: A probabilistic risk assessment. Appl Geoche 2008; 23:2987 -2998 41. Meharg, A.A.1994, Macnair, M.R., 1992. Suppression of the highaffinity phosphateuptake Mishra, S., Srivastava, S., Tripathi, R. D., & Trivedi, P. K. (2008). Thiol metabolism and antioxidant system complement each other during arsenate detoxification in Ceratophyllum demersum L. Aquatic Toxicology, 86, 205215

42. Mosaferi, M, Yunesion, M, Mesdaghinia, A., Naidu, A, Nasseri, S, Mahvi, AH. (2003). Arsenic occurrence in drinking water of I.R. of Iran : the case of Kurdistan Province.

43. Ma JF, Yamaji N, Mitani N, Xu X-Y, Su Y-H, McGrath SP, Zhao F-J (2008) Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc Natl Acad Sci 105:9931 9935 44. Meharg AA, Rahman M. Arsenic contamination of Bangladesh paddy soils: Implications for rice contribution to arsenic consumption. Environ Sci Technol 2003;37:229-34. 45. Macnair, M. R., and Q. Cumbes, 1987: Evidence that arsenic tolerance in Holcus lanatus L. is caused by an altered phosphate uptake system. New Phytol. 107, 387394 46. Neubauer, C. and Schreiber, U., Z. Naturforsch. C, 1989, 44, 26227 47. Norton GJ, Islam MR, Deacon CM, Zhao FJ, Stroud JL, Mcgrath SP, Islam S, Jahiruddin M, Feldmann J, Price AH, Meharg AA (2009) Identification of low inorganic and total grain arsenic rice cultivars from Bangladesh. Environ Sci Technol 43:6070 6075 48. Quaghebeur, M., and Z. Rengel, 2005: Arsenic speciation governs arsenic uptake and transport in terrestrial plants. Microchim. Acta 151, 141 152. 49. Paliouris, G., and T. C. Hutchinson, 1991: Arsenic, cobalt and nickel tolerances in two populations of Silene vulgaris (Moench) Garcke from Ontario, Canada. New Phytol. 117, 449459. 50. Polle, A., Chakrabarti, K., Schurmann, W. and Rennenberg, H., Plant Physiol., 1990, 56,72-89. 51. Pickering IJ, Prince RC, George MJ, Smith RD, George GN, Salt DE. 2000. Reduction and coordination of arsenic in Indian mustard. Plant Physiol. 122:117177 52. Raab A,Williams PN, Meharg A, Feldmann J. 2007. Uptake and translocation of inorganic and methylated arsenic species by plants. Environ. Chem. 4:197 203 53. Raab, A., Ferreira, K., Meharg, A.A., & Feldmann, J. (2007). Can arsenic-phytochelatin complex formation be used as an indicator for

toxicity in Helianthus annuus? Journal of Experimental Botany, 58, 13331338. 54. Williams PN, Prince AH, Raab A, Hossain SA, Feldmann J, Meharg AA (2005) Variation in arsenic speciation and concentration in paddy rice related to dietary exposure. Environ Sci Technol 30:234 -278. 55. Raab A, Feldmann J, Meharg AA. 2004. The nature of arsenicphytochelatin complexes in Holcus lanatus and Pteris cretica. Plant Physiol. 134:111322 56. Rayman, 2002 A global survey of effects of genotype and environment on selenium concentration in lentils (Lens culinaris L.): Implications for nutritional fortification strategiesFood Chemistry, Volume 125, Issue 1, 1 March 2011, Pages 72-76 57. Reid, M.E. et al. (2008) The nutritional prevention of cancer: 400 mc per day selenium treatment. Nutr. Cancer 60, 155 163 58. Rayman, M.P. (2008) Food-chain selenium and human health: emphasis on intake. Br. J. Nutr. 100, 254268.

59. Rosen BP, Liu L. Transport pathways for arsenic and selenium: A minireview. Environ Int 2009;35:512-5. 60. Scandalios, J. G., Plant Physiol., 1993, 101, 712. 61. Sandalio, L. M., Palma, J. M. and del Rio, L. A., Plant Sci., 1987, 51, 1 8 62. Schat H, Llugany M, Vooijs R, Hartley-Whitaker J, Bleeker PM. 2002. The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non -hyperaccumulator metallophytes. J. Exp. Bot. 53:238192 63. Spallholz JE, Mallory BL, Rahman MM (2004) Environmental hypothesis: is poor dietary selenium intake an underlying factor for

arsenicosis and cancer in Bengladesh and West Bengal. India Sci Total Environ 323:2132 64. Schmoger, M. E. V., M. Oven, and E. Grill, 2000: Detoxification of arsenic by phytochelatins in plants. Plant Physiol. 122, 793 867 65. Spallholz JE, Mallory BL, Rahman MM. Environmental hypothesis: is poor dietary selenium intake an underlying factor for arsenicosis and cancer in Bengladesh and West Bengal, India. Sci Total Environ 2004;323:21-32. 66. Sweins A. Protective effect of selenium against arsenic -induced chromosomal damage in 1983;98:249-52. 67. Stoeva, N., M. Berova, and Z. Zlatev, 2005: Effect of arsenic on some physiological parameters in bean plants. Biol. Plant. 49, 293 296. 68. Srivastava, S., Tripathi, R.D., Dwivedi, U.N., 2004. Synthesis stress in Cuscuta reflexa Physiology 161, 665674 69. Smith, AH, Lingas, EO, Rahman, M. (2000). Contamination of drinking water by arsenic in Bangladesh: a public health emergency. Bulletin of the World Health Organization 78: 1093-103. 70. Spallholz JE, Mallory BL, Rahman MM (2004) Environmental hypothesis: is poor dietary selenium intake an underlying factor for arsenicosis and cancer in Bengladesh and West Bengal. India Sci Total Environ 323:2132 71. Thomson CD (2004) Assessment of requirements for selenium and adequacy of selenium status: a review. Eur J Clin Nutr 58:391 72. Tendukar, N, Bhattacharya, P, Mukherjee, AB. (2001). Preliminary assessment of arsenic contamination in groundwater in Nepal. In: Abstracts, international conference on arsenic in the Asia-Pacific region: of phytochelatins and modulation of antioxidants in response to cadmium an angiospermic parasite. Journal of Plant cultured human lymphocytes. Hereditas

managing arsenic for our future. Adelaide, Australia: CSIRO- Land and Water. P 103-5 73. De Koe, T., and N. M. Tripathi RD, Srivastava S, Mishra S, Singh N,Tuli R, et al. 2007. Arsenic hazards: strategies for toleranc and remediation by plants. Trends Biotechnol. 25:15865 74. Tripathi RD, Srivastava S, Mishra S, Singh N, Tuli R, Gupta DK, Mathuis FJM (2007) Arsenic hazards; strategies for tolerance and remediation by plants. Trends Biotechnol 25:158 165 75. Virupaksha and Shrift 1965; Davis 1972, 1986 Spatial Imaging, Speciation, and Quantification of Selenium in the Hyperaccumulator Plants Astragalus bisulcatus and Stanleya pinnata Plant Physiology 142:124-134 American Society of Plant Biologists. 76. Williams PN, Lombi E, Sun G-X, Scheckel K, Zhu YG, Feng X, Zhu J, Carey AM, Adomako E, Lawgali Y, Deacon C, Meharg AA. Selenium characterization in the global rice supply chain. Environ Sci Technol 2009a;43:602430. 77. Welch, AH, Westjohn, DB, Helsel, DR. and Wanty, RB. (2000) Arsenic in ground water of the United States: occurrence and geochemistry. Groundwater 38, 589-604 78. White, P.J. et al. (2004) Interactions between selenium and sulphur nutrition in Arabidopsis thaliana. J. Exp. Bot. 55, 1927 1937 79. Wojtaszek, P., 1997. Oxidative burst: an early plant response to pathogen infection. Biochemical Journal 322, 681 692 80. Williams PN, Islam S, Islam R, Jahiruddin M, Adomako E, Soliaman ARM, Rahman GKMM LuY, Deacon C, Zhu Y-G, Meharg AA (2009a) Arsenic limits trace mineral nutrition (selenium, zinc and nickel) in Bangladesh rice grain. Environ Sci Technol 43:8430 8440. 2006

81. Williams PN, Islam S, Islam R, Jahiruddin M, Adomako E, Soliaman ARM, Rahman G KMM, Lu Y, Deacon C, Meharg AA. Arsenic limits trace mineral nutrition (selenium, zinc and nickel) in Bangladesh rice grain. Environ Sci Technol 2009b;43:8430 8436. 82. Williams PN, Islam S, Islam R, Jahiruddin M, Adomako E, Soliaman ARM, Rahman GKMM LuY, Deacon C, Zhu Y-G, Meharg AA (2009a) Arsenic limits trace mineral nutrition (selenium, zinc and nickel) in Bangladesh rice grain. Environ Sci Technol 43:8430 8440. 83. Zhang LH, Shi WM, Wang XC (2006b) Difference in selenium accumulation in shoots of two rice cultivars. Pedosphare 16:646 653 84. Zavala YJ, Duxbury JM, Arsenic in rice: I. Estimating normal levels of total arsenic in rice grain. Environ Sci Technol 2008;42:3856 -60. oil 202:3339 85. Zhang XK, Zhang FS, Mao DR (1998) Effect of iron plaque outside roots on nutrients uptake by rice (Oryza sativa L.): zinc uptake by Fe -deficient rice. Plant Soil 202:3339 86. Zeng H. Arsenic suppresses necrosis induced by selenite in human leukemia HL-60 cells. Biol Trace Elem Res 2001;83:115. 87. Zhang LH, Shi WM, Wang XC (2006a) Difference in selenite absorption between high-and low-selenium rice cultivars and its mechanism. Plant Soil 282:183193