The presence of metals in the leaves can hamper plant metabolism and growth even at very low concentrations. The oldest leaves of metal exposed plants exhibit the highest metal content (Ernst 1998; Mills and Scoggins 1998). Under metal stress, plants exhibit a thickening of root tips and a decreased root hair density (Punz and Sieghardt 1992). The reduction in growth and yield depends on the type of metal ions, types of growth media and species, and the stage of plant growth (Woolhouse 1983; Lata 1989a,b). The heavy metal caused inhibition in growth leads to reductions in dry matter (Pahlsson 1989), leaf area and root length (Bhattacharyya and Choudhuri 1995), and yield (Setia et al. 1987). The major effect of Cd2+ is on root growth, followed by leaf growth (Di Cagno et al. 1999). It inhibited seed germination and seedling growth in Triticum aestivum (Yadav and Yadav 1995). The plant growth is affected not only due to the toxic effect of Cd but also due to a nutritional imbalance created by Cd in the plant (Cordovilla et al. 1996; Sanita di-Toppi and Gabbrielli 1999).
The rate of photosynthesis is often regarded as a major factor regulating the crop productivity. It is the basis of crop yield and provides 90-95% of the plant dry weight, i.e., economic yield (Leuning et al. 1995; Hall and Rao 1999). Leaves are the primary site of photosynthesis and the products of their metabolism fuel the growth of all plant organs. As leaves become older, their photosyn-thetic capacity decreases (Marschner 1986; Long et al. 1996) ; Anatomical changes in leaves and structural disorganization of chloroplasts are correlated with the inhibition of photosynthesis (Terashima and Evans 1988). The leaf injuries such as chlorosis, necrosis, browning, and burning adversely affect the physiological functions especially leaf photosynthetic activity (Jafri et al.
1979). The photosynthetic efficiency depends on leaf area, chlorophyll content, and the stomatal response (Pachepsky et al. 1997). Leaves are the highly exposed organs of plants and are affected most by the environmental stresses. The rates of photosynthesis and stomatal conductance are used as early detectors of the potential stress injury to plants. The exact mechanism of the heavy metal action on photosynthesis is, however, still not clearly understood (Krupa et al. 1987; Bhardwaj and Mascarenhas 1989).
Heavy metals decrease leaf expansion, resulting in a more compact leaf structure and increased stomatal resistance (Horvath et al. 1996). They may impair leaf transpiration and CO2 fixation by decreasing leaf conductance to CO2 diffusion as a result of stomatal closure (Barcelo et al. 1988). Heavy metals in growth media can function as stressors causing physiological constraints that suppress plant vigor and inhibit plant growth. Heavy metals can inhibit photosynthesis of intact plants at several physiological levels: stomata, pigment synthesis, chloroplast structure and function, and indirectly by affecting various other metabolic pathways (Costa and Spitz 1997). Their treatments inhibit net photosynthesis in various crop plants such as corn and soybean (Bazzaz et al. 1974i ; tomato (Baszynski et al.
1980), and wheat (Setia et al. 1987). Reduction in chlorophyll content may also be due to the interference of all the metals with chlorophyll synthesis and fat metabolism, inhibiting root shoot growth, photosynthesis, nutrient uptake, leaf area, etc. (Pandey and Tripathi 2011).
Disturbances in plant water relations are widely known as one of the first effect of Cd tox-icity (Poschenrieder et al. 1989). In vivo studies have shown that leaves of plants exposed to Cd2+ accumulate high amounts of cadmium. Long-term exposure of whole plant to Cd affects chlorophyll with consequences for chloroplast development in young leaves. Cadmium may affect photosynthesis (Greger and Ogren 1991) by altering the chlorophyll content and/or sto-matal conductance (Prasad 1995).
Photosynthesis is a very sensitive indication system for toxicity (De Filippis and Pallaghy 1976; Greenfield 1942). Mercury has been shown to interfere with photosynthetic electron transfer mechanisms (Bradeen et al. 1973; Cedeno-Maldonado et al. 1972). Ultimately, mercury causes damage to photosynthetic pigments (Greenfield 1942; Puckett 1976).
Carotenoids are the secondary light absorbing pigments called the accessory pigments. They provide essential photoprotective mechanisms, blocking the formation of ROS (Young and Britton 1990). Carotenoid is less affected (Clijsters and Van Assche 1985) or is generally increased by the heavy metal exposure (Foyer and Harbinson 1994; Ralph and Burchertt 1998). In the green alga Chlorella vulgaris, the enzyme protochloro-phyllide reductase was found to be inhibited in the presence of sublethal concentration of Hg, which resulted in the reduction of chlorophyll biosynthesis and accumulation of protochloro-phyll (De Filippis and Pallaghy 1976).
Proteins are the most abundant molecules in the cell, making up more of the dry weight. They are found in all cellular components forming the basis of the cell structure and function. Each kind of protein is specialized for its biological function. They operate as enzymes, transporting and regulatory proteins and also serve as structure and storage of compounds. Significant alteration in protein metabolism under heavy metal stress has been reported by a number of workers. Some of them observed an increase in protein synthesis (Shah and Dubey 1997) while others observed a decrease (Costa and Spitz 1997). In majority of plant cells, proteins are synthesized in cytoplas-mic compartments. Most proteins have a lifetime less than that of cell and therefore are degraded and, if necessary, resynthesized (Nozaki 1986; Nwokolo and Smartt 1996).
Plants appear to contain a diversity of metal binding metallothioneins (MTs) with the potential to perform distinct roles in the metabolism of different metal ions. The change in the biochemical characterization for metal tolerance involved the de novo synthesis of metal binding proteins.
Lue-kim and Rauser (1986) reported an induction of Cd-binding protein from crude extracts of roots of tomato with an apparent molecular weight 31,000 Da in high ionic strength and 21,500 Da at low ionic strength. Increase in soluble protein was also reported by Vogeli-Lange and Wagner (1990) in tobacco leaves on Cd exposure at a concentration of 20 mm. Lozano-Rodriguez et al. (1997) observed an increase in the soluble protein content of pea root and shoot upon treatment of Cd at a concentration of 0.05 mM, whereas the same had no effect on maize. Ali et al. (1998) have observed an increase in the protein content of Bacopa monniera plant-lets on exposure to Cd stress. Hirt et al. (1989) reported stimulation of the protein and RNA synthesis in suspension cells of Nicotiana tabacum on exposure to Cd stress at a concentration of 100 mM. They observed that the increase in protein content was probably due to the synthesis of new proteins to detoxify the intracellular Cd by binding with the same and rendering the internal concentration of free Cd low enough to minimize the toxic effect and allow stimulation of RNA synthesis.
Gil et al. (1995( reported that total soluble protein as well as Rubisco decreased with time at Cd concentrations of 15 and 30 mg/L. Rubisco constitutes more than 50% of the leaf soluble protein and is the key enzyme in photosynthesis (Woolhouse 1974); hence, any decline in leaf soluble protein including Rubisco will have an adverse impact on Rubisco activity and ultimately on photosynthesis. Kevreson et al. (1998) observed a decrease in total soluble protein and Rubisco activity in sugar beet plant with decreasing leaf water status and generation of ROS.
Once NO3 is absorbed by root, it can be assimilated in the root itself, transported to the shoot or stored in vacuoles (Srivasankar and oaks 1996). Assimilation of nitrate reaction takes place in the cytoplasm of cells in both roots and shoots. The uptake and assimilation of nitrate are regulated mainly by an enzyme, nitrate reductase (NR), which plays a rate-limiting role in plant metabolism (Galvan et al. 1992; Khan 1996). This complex enzyme is substrate inducible and predominantly utilizes NADH as a co-factor (Selvaraj et al. 1995). Nitrite (NO-2) is very toxic to plants, but because the activity of nitrite reductase (NIR) is normally higher than that of NR, normally nitrite does not accumulate and is rapidly converted into NH4+.
NR was found to be most sensitive cytosolic enzyme, while peroxidase was the most resistant (Ernst 1998). In leaves, NR is activated by photosynthesis, reaching the activation state of 60-80%. In the dark, or after stomatal closure, leaf NR is inactivated down to 20-40% of its maximum activity (Ahmad and Abdin 1999; Kaiser et al. 1999). NR is very sensitive to heavy metal stress and any change in this enzyme affects nitrogen assimilation pathways and thus growth (Hemalatha et al. 1997; Hall and Rao 1999). Reduced NR activity has been reported in many heavy metal treated plants such as Glycine max, Zea mays, and Pisum sativum (Chugh et al. 1992). Percent inhibition in NR activity by toxic metals increased with increasing metal concentrations (Vyas and Puranik 1993). NR shows a considerable variation in response to metal ions, which are species and cultivar specific (Bharti and Singh 1993; Dabas and Singh 1995).
Amino acid catabolism in plants is generally concerned with the production of metabolites for other biosynthetic pathway. They serve as precursor of many kinds of small molecules such as glutathione and proline that have important and diverse biological roles. Increase in amino acid pool was observed by heavy metal stress in maize germinating seed (Nagoor 1999).
Proline is an imino acid with aliphatic side chain, but differs from other members of the set of 20 amino acids in that its side chain is bonded to both the nitrogen and the a-carbon atoms. The resulting cyclic structure markedly influences protein architecture. Usually, glutamate is the precursor of proline. Proline a total free amino-acid accumulated in plants when they experience moisture stress conditions and decrease on release of stress (Pandey and Tripathi 2011). It has been shown to play an important role in ameliorating such conditions as drought, salinity, and heavy metal stress (Andrade et al. 1995). It has been used as a single parameter to measure physiological dryness (Lutts et al. 1999). It oxidizes in turgid tissues rapidly and also gets affected with the duration of stress conditions (Jager and Meyer 1977) . Cadmium has a strong and positive relation with proline accumulation. A number of workers reported an increase in proline content under Cd stress (Wu et al. 1995; Nagoor 1999). Wu et al. (1995) studied the impact of Cu2+ and Cd2+ on intracellular proline level in four species of algae and reported that proline accumulation is the general response of algal cells to Cd stress.
It may be argued that proline acts as a sink for nitrogenous compounds resulting from the degradation of proteins and protects cell metabolism from the harmful nitrogenous compounds (Aspinall and Paleg 1981; Yancey et al. 1982). It is often considered to be involved in stress resistance mechanisms by acting as an osmoprotectant thereby facilitating osmoregulation, protection of enzymes, stabilization of the protein synthesis machinery, and regulation of cytosolic acidity, etc (Alia and Saradhi 1991).
Heavy metal exerted specific influence on the differentiation of various tissues in the root as well as stem. Elevated concentrations of these metals induced drastic anatomical changes. Different heavy metals of supraoptimal concentrations have been shown to inhibit various metabolic processes as in plants resulting in their reduced growth and development (Bala and Setia 1990; Davies 1991). There is paucity of information on the differentiation of tissues in plants in response to heavy metal toxicity.
Differential anatomical changes in the root and stem of Solanum melongena have been observed (Mehindirata et al. 1999). The major responses elicited by roots to cadmium treatments appear to be caused by accumulation of these metals in the tissues. These ions seem to attack various cellular components, including cell wall and membranes, resulting in different alterations, which ultimately lead to their disorganizations, as has also been reported by Setia and Bala (1994) . Accumulation of Ni and Cd occur in root tissues in toxic amounts in a number of plant species (Woolhouse 1983). The development of large intercellular spaces in root core following heavy metal treatments resembles those resulting from exposure of roots to anaerobiosis due to water logging. The formation of large intercellular spaces (aerenchyma) is an adaptive response to anaerobiosis (Erdmann et al. 1986). With the Cd concentration, root area increases; this might be due to expansion of cells and formation of air spaces in cortical regions. Insufficient supply of essential nutrients and hormones from the root adversely influences the differentiation of tissues in stem (Davies 1991; Setia and Bala 1994). Heavy metals have been shown to affect cells of cortex and pith in root and stem of plants.
A major factor limiting metal uptake into roots is slow transport from soil particles to root surfaces (Nye and Tinker 1977; Barber 1984). With the possible exception of volatile mercury for all other metals, this transport takes place in soil solution. In soil, metal solubility is restricted due to absorption to soil particles. Some of the soil binding sites are not restricted due to adsorption to soil particles. Some of the soil binding-sites are not particularly selective, e.g., they bind Cd as strong as Ca. Non specific binding occurs at clay-cation exchange sites and carboxylic groups associated with soil organic matter. Other sites are more selective and bind Cd stronger than Ca, e.g., most clay particles are covered with a thin layer of hydrous Fe, Mn, and Al oxides. These selective sites maintain the Cd activity in the soil solution at low levels (Chaney 1988).
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