1

Introduction
Soil contamination with heavy metals (HMs) has now become a

major

environmental

concern

because

of

their

persistent,

nondegradative and toxic nature. HMs (density >5.0 g cm-3) such as cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), arsenic (As), copper (Cu), zinc (Zn), nickel (Ni), aluminium (Al), silver (Ag), tin (Sn) etc. are natural constituents of the earths crust and chiefly found in forms that are not available to living organisms. Some metals such as Zn, Cu, Fe, Mn and Ni act as micronutrients for plants and animals at low concentrations but become toxic at elevated concentrations (Welch, 1995), however, other metals e.g. Hg, Cd, Pb, As are of no physiological use and toxic even in traces (Holan and Volesky, 1994; Hall, 2002). Enormous quantities of HMs are continuously being released into the environment by anthropogenic activities i.e. mining, smelting of metalliferous ores, manufacturing of pesticides, paints, batteries and land application of industrial or domestic sludge and agrochemicals that contaminate urban and agricultural soils (Garbisu and Alkorta, 2003; Halim et al., 2003) and pose a significant threat to food safety and potential health risks due to their transfer from soilto-plant, which ultimately biomagnified into the food chain through ingestion of metal contaminated food (Jiang et al., 2004). Moreover, metal accumulation in the soil adversely affect the soil health particularly microbial functionalities i.e. microbial compositions, their Introduction
.

metabolic activities and symbiosis (Abou-Shanab et al., 2005), eventually leads to reduction in plant growth and crop yield (Long et al., 2002; Wang et al., 2003; Luo et al., 2005; Wani et al., 2007a, 2008). Though, both Zn and Cd are transition metals, having same ionic charge and share many similar chemical properties, but former is a micronutrient and later a non-essential and toxic metal even in trace amount. The main basis of Cd toxicity in biological systems lies in its strong affinity for thiol groups (SH) contained in cysteine residues of proteins (Wagner, 1993; Hall, 2002), its ability to bind to oxygen, nitrogen and sulphur atoms (Schutzendubel and Polle, 2002), the generation of free radicals, resulting in oxidative stress (Dixit et al., 2001). Cd, a chemical analogue of Zn and Ca (Chaney et al., 1995), also mimic the entry of these micronutrients (Hernandez et al., 1996; Sanita di Toppi and Gabbrielli, 1999) through membrane transporters and cause toxicity and micronutrient deficiency. Various conventional methods (ex-situ and in-situ) available for soil reclamation are soil excavation and dumping, containment methods (e.g., vitrification, stabilization), soil washing/flushing etc., but they all have little scope owing to high energy input, prohibitively expensive, generate secondary waste (Prasad, 2003), destroy soil structure and decrease soil productivity (Mulligan et al., 2001). Therefore, decontamination of metal polluted sites calls for energy intensive, cost-effective and environmentally sound technology to remove, sequester and detoxify the metals. Phytoremediation, employing metal accumulating plants, offers an in-situ, cost-effective Introduction
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and ecofriendly alternative to render the pollutants harmless (Salt et al., 1998), restore and maintain the biological activity of soil along with the possibility of metal bio-recovery (Baker et al., 1991, 1994). Various strategies of phytoremediation (Pulford and Watson, 2003) are phytoextraction, phytodegradation, phytostabilization,

phytovolatilisation and rhizofilteration. Out of these, phytoextraction and phytostabilization are the most effective in removing and/or inactivating toxic metals from the contaminated soil. It takes advantage of the fact that a living plant acts as a solar-driven pump which can extract and concentrate certain heavy metals from the environment (Salt et al., 1995). The criteria for metal accumulating plants are fast growing, high biomass potential, deep roots and tolerate and accumulate wide range of heavy metals in their aerial parts (Clemens, 2002). Depending plants upon the been trace element as

accumulation

capacity,

have

identified

hyperaccumulators which are able to survive on metallicolus soils without showing any metal toxicity symptoms and accumulating metals >1,000 mg kg1 dry weight for Ni, Co, Cu and Pb and above 100 and 10,000 mg kg1 for Cd and Zn, respectively, in their shoots (Baker et al., 2000). Generally, terrestrial species are preferred for phytoremediation due to their extensive root systems, ability to penetrate soil up to deeper layers, grow on low-fertility soil, extensive production of litter, release of exudates that may stimulate microbes in the rhizosphere and promote ecosystem diversity and their aboveground biomass can be harvested with subsequent resprouting Introduction
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without disturbance of the site (Stomp et al., 1994). The earliest identified terrestrial hyperaccumulating species are Thlaspi

caerulescens (Brassicaceae) which accumulated more than 3% Zn, 0.5% Pb and 0.1% Cd (Baker et al., 1991; Brown et al., 1995), Alyssum bertolonii (Brassicaceae) accumulated 1% Ni (Brooks et al., 1979), Arabidopsis halleri 157 mg kg-1 (>0.1%) of Cd (Bert et al., 2003) in their aboveground tissue but their use in the field is limited because of their slow growth and low biomass production (Ow, 1996; Ebbs et al., 1997). The accumulator plants are ideal for metal remediation as they are fast growing, producing high biomass and accumulating moderate concentration of metals coupled with

increasing soil metal bioavailability and, hence, increasing plant uptake that are environmentally more relevant (Ginneken et al., 2007). Since phytoremediation is time consuming, it requires

acceleration by exploiting metal tolerant rhizospheric microflora, as they affect metals mobility and availability to plant through release of chelating agents, acidification and redox changes (Abou-Shanab et al., 2003, 2007), facilitating transformation to less toxic forms (Wani et al., 2008). They can also increase the metal tolerance of their host plants (Li et al., 2007) and protect against metal toxicity (Corticeiro et al., 2006). Earlier studies reported that rhizobacteria increased metal accumulation in plants (Whiting et al., 2001) and plant growth through biological N2 fixation (Zaidi et al., 2004), synthesis of phytohormone precursors and siderophores (Ahmad et al., 2008) to Introduction
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enhance nutrient availability and uptake (Cakmakci et al., 2006). In this regard, leguminous plants owe immense significance as they have symbiotic microbes associated with their roots and led to several changes in soil chemical properties i.e. pH and available N (Alvey et al., 2001) and biological properties i.e. higher microbial biomass C and N, respiration rate, ATP content and early mycorrhizal infection (Adeboye et al., 2006; Jemo et al., 2006). Apart from this, some legumes also sequester heavy metals in nodules, roots, stem, leaves and vacuoles (Georgiev et al., 1996). Therefore, such legumes, Leucaena leucocephala, Robinia pseudoacacia, Lablab purpureus, Albizia lebbeck etc., possessing dual characteristics (i.e. metal sequestration and nitrogen fixation) could be good candidates for soil decontamination (Tawfik, 2008) alongwith improvement in soil fertility (Ma et al., 2006). Sesbania drummondii, a leguminous shrub, has been reported to concentrate > 4% of Pb in shoot (Sahi et al., 2002), whereas Sesamum indicum (L.) accumulated Cr, Ni and Cd in the roots (Gupta and Sinha, 2006). The seedlings of Mung beans have also been reported to accumulate As (Van den Broeck et al., 1998), Pb (Rout et al., 2001, Singh et al., 2003) and Cr (Samantary, 2002). Recently, Dary et al. (2010) opined the usefulness of Lupinus luteus, a legume, for in situ reclamation of metal polluted soils. A range of molecular and physiological mechanisms evolve in plants for metal acquisition and homeostasis to virtually all toxic metals. One of the mechanisms of metal accumulation in plant is to synthesize metal-binding proteins and peptides, such as glutathiones Introduction
.

(GSHs, -glu-cys-gly) and phytochelatins (PCs, glucys)ngly, n= 2 to 11) that acts as a metal chelator (Pocsi et al., 2004), scavenges free radicals, and repairs damage caused by the metal induced oxidative stress intracellularly (Ferrol et al., 2009). Di Baccio et al. (2005) provided evidence of GSH synthesis and its redox status as the key factors of plant defense mechanisms in poplar exposed to Zn. Earlier studies revealed that PCs are rapidly induced in cells and tissues exposed to a range of metal cations, viz. Cd, Ni, Cu, Zn, Ag, Hg and Pb, and anions such as arsenate and selenite (Rauser, 1995; Yang and Yang, 2001; Gzyl and Gwozdz, 2005; Ramos et al., 2007; Mendoz-Cozatl et al., 2008). Molina et al. (2008) reported significant increase in total glutathione, non-protein thiols, reduced glutathione (GSH) and phytochelatins on Cd exposure to a legume, Vigna mungo. Thus, the qualitative and quantitative assessment of the GSHs and PCs production in the plant exposed to metal stress could better assist in estimating the plants efficiency for phytoremediation of metal contaminated sites. Rapid industrialization in western Uttar Pradesh (India) has exposed the soil to various industrial effluent inputs including heavy metals such as Zn, Cd, Cr, Cu, Ni etc. The industrial wastewater provides unlevied irrigation source to regional farmers during dry season, which often results in regular build up of metals in soils (Saraswat et al., 2007). To sustain these agriculture ecosystems, Leucaena leucocephala (family-Mimmosaceae), a tropical, fast

growing perennial shrub, fixes more than 500 kg Nha-1 (Aganga and Introduction
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Tshwenyane, 2003) and adapts well in a wide range of habitats including metal contaminated soils, especially in Indo-Gangetic region could be an ideal test plant for remediation of metal contaminated sites and restoration of their fertility. Keeping this in view, the present study was undertaken with the following objectives: 1. Investigation of Zn and Cd extraction potential of L.

leucocephala from electroplating and brass industry effluent fed soils and nitrogen fixation potential of the plant. 2. Evaluation of metal accumulation in different plant parts and nitrogen fixation potential of L. leucocephala along with changes in soil fertility attributes and metal concentration individually and in combination in pot culture. 3. Assessment of metal accumulation, phytochelatins production and nitrogen fixation potential of L. leucocephala in response to varying concentrations of synthetic metal solution by

monitoring of soil fertility indices in terms of micro-biochemical activities and nitrogen status in mesocosm system. 4. Verification of L. leucocephala phytoremediation efficiency in terms of metal sequestration, phytochelatins production and nitrogen fixation in response to metal stress in sand culture.

Introduction