Heavy Metal Stress

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Asiya Hameed1, Saiema Rasool2, M.M. Azooz3,4, Mohammad Anwar Hossain5, Mohammad Abass Ahanger6, Parvaiz Ahmad7 Department of Botany, Faculty of Science, Jamia Hamdard, New Delhi, India1; Department of Forest Management, Faculty of Forestry, University Putra Malaysia, Serdang, Selangor, Malaysia2; Department of Botany, Faculty of Science, South Valley University, Qena, Egypt3; Department of Biological Sciences, Faculty of Science, King Faisal University, Al-Hassa, Saudi Arabia4; Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh5; Stress Physiology Laboratory, School of Studies in Botany, Jiwaji University Gwalior, MP, India6; Department of Botany, S.P. College, Srinagar, Jammu and Kashmir, India7

CHAPTER OUTLINE 1. 2. 3. 4.

Introduction ...................................................................................................................................558 Plant Growth Response to HM Stress ............................................................................................... 558 Biochemical Response....................................................................................................................561 Generation of ROS in Cells ..............................................................................................................564 4.1 ROS Generation in Mitochondria.....................................................................................564 4.2 ROS Generation in Chloroplasts......................................................................................564 4.3 ROS Generation in Microsomes and Peroxisomes .............................................................565 4.4 Other Sources of ROS Generation in Plants .....................................................................565 5. Oxidative Damage to Biomolecules ..................................................................................................565 5.1 Oxidative Damage to DNA ..............................................................................................565 5.2 Lipid Peroxidation .........................................................................................................566 5.3 Oxidative Damage to Proteins .........................................................................................567 6. Antioxidant and HM Stress..............................................................................................................568 7. Translocation of HMs......................................................................................................................570 8. Signaling in Plants Under HM Stress ............................................................................................... 572 8.1 The Calcium-Calmodulin System ....................................................................................572 8.2 Connection with Hormone Signal Pathways......................................................................573 8.3 Role of ROS..................................................................................................................574 8.4 The MAPK Cascade .......................................................................................................575 9. Conclusions and Future Perspectives............................................................................................... 575 References ..........................................................................................................................................576

Plant Metal Interaction. http://dx.doi.org/10.1016/B978-0-12-803158-2.00024-2 Copyright © 2016 Elsevier Inc. All rights reserved.

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1. INTRODUCTION Heavy metals (HMs) are metals with a density higher than 5 g/cm3. Of 90 naturally occurring elements, there are 53 HMs, but all of them are not biologically important. Under physiological conditions based on their solubility, 17 HMs may be available for living cells and are important for organisms and ecosystem (Schu¨tzendubel and Polle, 2002). Among these metals, Fe, Mo, and Mn are important as micronutrients, whereas Zn, Ni, Cu, Co, V, W, and Cr are trace elements but at higher concentrations are toxic and the other metals like As, Hg, Ag, Sb, Cd, Pd, and U have no function and seems to be toxic to microorganisms and plants (Nazar et al., 2012). HM pollution is a not only a big problem in most parts of the world, but it is also responsible for the loss of agricultural productivity. Many industries and agricultural activities contribute to HM contamination in urban areas. HMs can directly influence growth, senescence, and energy synthesis processes because of their high reactivity. They adversely affect the absorption and transport of essential elements, thereby disturbing the metabolism and having an impact on growth and reproduction (Xu et al., 2008). More than 30 mg of lead (Pb) leads to a reduction in growth and decline in chlorophyll synthesis in leaf (Ruley et al., 2004). Sharma and Dubey (2005) have reported that Pb causes many problems like reduction in growth as well as crop production, yellowing of young leaves, reduction in absorption of essential elements such as Fe, and decline in rate of photosynthesis. When plants are subjected to HM stress production, and quenching of reactive oxygen species (ROS) were found imbalanced. ROS, such as superoxide radical ðO2  Þ and hydrogen peroxide (H2O2) and the hydroxyl radical (OH) can have detrimental effects on normal metabolism because of oxidative damage to lipids, proteins, and nucleic acids. The toxicity and tolerance to HMs vary from plant to plant, and several efforts to their tolerance mechanism at the molecular level have not been fully understood (Thapa et al., 2012). So to cope with the stress, plants need a coordination of complex physiological and biochemical processes. Gene expression, protein modifications, and changes in metabolite composition will lead to proper stress signal perception and tolerance (Urano et al., 2010).

2. PLANT GROWTH RESPONSE TO HM STRESS Growth is a complex process; therefore, HMs can affect it at several points. The toxic effect of HMs is obvious at different levels of cell structures and function of the plants. HMs use wide range of variations in the physiological and metabolic processes (Ahmad et al., 2011; Asgher et al., 2014; Khan et al., 2015). Inhibition of germination and root extension can be the result of interference with cell division or with cell elongation. It has been observed that the main influence of HMs (Hg, Cd, Co, Cu, Pb, and Zn) is the inhibition of germination, root elongation, and shoot and leaf growth (Munzuroglu and Geckil, 2002; Suzuki, 2005; Rascio et al., 2008). The elasticity of cell walls is so much reduced by it that they may break under mechanical stress (Gall et al., 2015). The inhibition of root elongation in many instances is the most sensitive parameter of HM toxicity (Schu¨tzendu¨bel et al., 2001). Because of the binding of HM cations to plant constituents, the order of toxicity usually conforms to the stability of the metal–organic complexes. In crop production, Al toxicity is one of the major growth-limiting factors in acidic mineral soils (Panda et al., 2009). The root system becomes stubby as a result of inhibition of elongation of the main axis and lateral roots because of restricted cell division. The root

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becomes stunted and brittle and apices become swollen and damaged (Panda et al., 2009). Al causes extensive root injury, especially in root cap region, and hampers the mineral and water uptake (Panda et al., 2009). The severity of inhibition of root growth is a suitable indicator of genotypical differences in Al toxicity (reviewed by Rout et al., 2001). HMs induce the toxicity by binding with the organic compounds called complexes. These in turn lose their capacity to function and eventually lead to death of the affected cells. HMs interfere with the cell division and thereby reduce the growth of both root and shoot meristem. Inhibition of mobilization of nitrogen and phosphorous during seedling growth have been observed in spinach on exposure to mercury (Hg) (Gothberg et al., 2004). Similarly, Ashger et al. (2014) have observed reduced sulfur assimilation because of cadmium stress in Brassica spp. Toxicity of Hg is more pronounced at lower concentrations than others. On exposure to HMs, root and shoot growth declines and could be positively correlated with low nitrogen associated with reduced nitrate reductase (NR) activity or low pigment content (reviewed in Nazar et al., 2012). Seedlings contaminated with HMs are likely to have stunted and poorly developed root system that inhibits the potential to absorb nutrients and moisture from soil and thereby hamper further growth. Significant reduction in number and leaf area indicate an interference of HMs on plants and can be attributed to the reduced photosynthetic activity and low nitrogen content (Faizan et al., 2011). The declining leaf area indicates not only retarded growth, but also a morphological adaptation to stressed conditions. This could also be attributed to the effect of Cd treatment on the leaf area that modifies the relative transpiration surface as found in Phaseolus coccineus exposed to Cu stress. Cd, Al, and Hg are highly toxic and inhibit the uptake of water and nutrients in peas and are closely coincided with the extensive accumulation especially in roots. This blocks the entry or binding of essential ions like Ca, Mg, and K to ion-carriers (Wang and Greger, 2004). John et al. (2009) also reported the decline in shoot and root length in Brassica juncea subjected to Cd and Pd stress. Decline in growth in mustard plants because of higher levels of Cd and Pb is also reported by Ahmad et al. (2012), and this decline is attributed to less nutrient uptake and lowering of water potential. Arsenic (As) also affects root growth more severely than shoot growth possibly because of the retention of As in the shoots in higher amount than in the stem (Stoeva et al., 2005). The inhibiting effects of HMs such as Cd and Hg on morphological parameters have been reported in Abelmoschus esculentus. The phytotoxic symptoms in response to HMs may be due to a range of interactions at the cellular level. Excess HMs may stimulate the formation of free radicals that damage the cellular functioning of the plant (Ahmad et al., 2011, 2012). HMs resulted in the decreased dry matter and seed yield, lowered protein content in seeds, indicating an adverse effects exerted by HMs. Cd has been found to be the most toxic metal for crop yield and caused the most severe reduction in the dry weight of shoot, root, and seed yield (Ahmad et al., 2011, 2012; Ashger et al., 2014). Several studies have also demonstrated a relatively higher phytotoxicity of Cd and Co than that of Mn. The reduction in the fresh and dry weight of shoot and root because of HM stress has been reported by many workers (Ahmad et al., 2011; Qui et al., 2013, 2014). Amendment of soil with HMs at concentrations higher than the normal levels resulted in a conspicuous decrease of root and shoot biomass expressed in terms of dry weight (Ghani, 2010). It has earlier been reported that increasing Co supply resulted in decreased root biomass indicating an alteration of physiology and metabolism of plants. Biomass loss (fresh weight) under metal treatment has also been reported by many workers (Ahmad et al., 2011, 2012; Mostofa and Fujita, 2013; Mostofa et al., 2014).

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The majority of the responses in relation to environmental stress in plants are linked with growth, differentiation, and physiological aspects such as photosynthesis, ion uptake, and transport. Under Cd stress, plants show a number of symptoms like chlorosis, leaf rolling, and stunting. In certain species, this resulted in structural disorders and thereby growth restrictions (Djebali et al., 2010; Jin et al., 2008). Cadmium stress leads to decrease in shoot dry weight that is concomitant with a significant reduction in both length and diameter of the internodes as well as in leaf area and thickness, but it is dependent on tissue age. Cd that reached the leaves resulted in further physiological and structural damages. Leaf growth was inhibited and blade thickness was diminished because of the reduced enlargement of mesophyll cells resulting in increasing tissue dry weight/fresh weight ratio, especially in young leaves. Under Cd stress, stomatal conductance declined sharply, suggesting that stomatal functionality may be compromised (Perfus-Barbeoch et al., 2002). Decreased stomatal conductance and photosynthetic rate have been reported by Ahmad et al. (2011) and Asgher et al. (2014). Changes in plant water relations with a decline in transpiration rate have been observed in other Cd-exposed species (Ahmad et al., 2011) and were ascribed to decreased leaf blade expansion. Exposure of aquatic plant Eladea canadensis to HM was found to inhibit cell division, but induced a significant enlargement of only one of the two layers constituting the leaf blade. This suggests that the changes in leaf cell enlargement caused by Cd may actually be due to specific morphogenetic effects rather than to impaired water balance (Rascio et al., 2008). Cu or Cd applied alone or in combination caused significant reduction in root diameter, width, and thickness of the leaf midrib (Kasim, 2006). The diameter of xylem vessel, parenchyma cell area, pith, cortex of root, dimensions of stem, vascular bundles, number of xylem arms in root, frequency of stomata on abaxial leaf surface, and reduction in grain yield have also been observed because of HM stress. The increase in the number of xylem tissues from an increased rate of transpiration and thickening of the cell wall with reduction in number of cells in the cortical region of the roots because of accumulation of HMs was also reported by Rauser and Ackerley (1987). Extensive inhibitions in the number and diameter of xylem vessels have been reported in many plant species. This could be due to inhibition in the water flow transportation to shoots in plants thus contributing the imbalance in the water transport. Cd causes damages in the leaves and thus renders loss in transpiration, leading to decreases in stomatal conductance, which is concomitant with a decreased leaf blade expansion. Arsenic (As) stress resulted in loss of root hairs and damage to epidermal cells and the cortex with those cells losing their shape and showing signs of shriveling and disintegration. Incomplete differentiation of stele and formation of pith is also reported in As-treated plants. As causes permanent damage to the meristem but root border cells accumulates high levels of As and limiting its movement in to the root. The major responses elicited by stem to Cd and Hg are disintegration of cells, inhibition, or alteration of stem area (Parmar et al., 2013). ROS attacks various cellular components, including cell wall and membranes resulting into different alterations, which ultimately lead to their disorganization as has also been reported (Janicka-Russak et al., 2008; Ahmad et al., 2011, 2012, 2015; Tran and Popova, 2013). The alteration in stem structure could be correlated with structural changes vis-a-vis dysfunctioning of root system following HM treatments. An insufficient supply of essential nutrients and hormones from the root adversely influences the differentiation of tissues in stem (Aloni, 2010). The stem diameter decrease may be the result of interference with cell division or cell elongation.

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3. BIOCHEMICAL RESPONSE The biochemical basis of HM toxicity is not always clear. Cd, Cu, and Hg affect sulfhydryl groups in proteins and inactivate them. For a radioactive metal such as copper, an excess supply may result in uncontrolled redox reactions, giving rise to the formation of toxic free radicals. This may lead to lipid peroxidation and membrane leakage (Qadir et al., 2004; Ahmad et al., 2011, 2012). Other HMs may inactivate major enzymes by replacing the activating cation. For example, Zn may replace the Mg in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), reducing the activity of this enzyme and hence the photosynthetic capacity. Like Zn, Cd also affects photosynthesis. Fluorescence measurements indicate that the Calvin cycle is the major process affected and that this subsequently leads to a “downregulation” of photosystem II (PSII) (Nagajyoti et al., 2010). Cd affects the mineral composition even in Cd-resistant species. It reduces the concentration of Mn, Cu, and chlorophyll in the leaves, even at a concentration in solution that has no effect on biomass production (Li et al., 2008). Various environmental stresses decrease the chlorophyll pigments and have been reported in many plants (Ahmad et al., 2011, 2012; Tomar et al., 2015; Ahanger et al., 2015). Loss in chlorophyll pigment can lead to damage in the photosynthetic apparatus. Net photosynthesis is also sensitive to Cd, which directly affects chlorophyll biosynthesis (Li et al., 2008; Ahmad et al., 2011, 2012; Asgher et al., 2014) and proper development of chloroplasts (Djebali et al., 2010; Jin et al., 2008). Pisum sativum and Zea mays showed an impaired photosynthesis because of lower PSII activity under metal stress conditions (Hattab et al., 2009; Wang et al., 2009). HM stress shows negative influence on the photosynthetic attributes and the enzymes like Rubisco (Asgher et al., 2014). The photosynthetic apparatus reveals a sensitive parameter against HM stress. The changes in this mechanism have found to be dose-dependent and vary from one plant to other. Cd can be accumulated at excessive levels in plants, which in turn adversely affects plant growth and different biochemical and physiological processes (John et al., 2009; Irfan et al., 2014; Ahmad et al., 2011, 2012, 2015). Cu plays a central part in physiological processes in plants, including photosynthesis related plastocyanin and membrane structure, respiration, and necessary cofactors for oxidative enzymes. Not all elements are essential for plant growth but some play a key role at low levels because the HMs stimulate the metabolism and hence growth. The chlorophyll analysis proved that there is a significant decrease in pigment contents because of the higher Cu exposure (Chu et al., 2006; Tanyolac et al., 2006; Singh et al., 2007). A remarkable decrease in the chlorophyll has been found because of elevated HM concentrations that lead to many consequences such as inhibition of enzyme activity, replacement of central metal Mg with HMs, decrease in source of essential metals involved in chlorophyll synthesis such as Fe and Zn (Mostofa and Fujita, 2013; Mostofa et al., 2014), and retardation of metabolic pathways. Certain metals are very important for the normal functioning of the cell; for example, Zn serves an activator of various enzymes and encodes DNA. Likewise, Mn is cofactor and also part of the oxygen-evolving complex in chloroplast. Molybdenum is an axial component of regulation of nitrogen. Nickel is an essential component of some enzymes and has been reported in some beans and tea plants. When these metals are in excess, they inhibit a large number of plant enzymes such as Calvin cycle and chlorophyll biosynthesis and consequently decrease the photosynthetic activity. Despite this, Ni alters the plant water relations and increases the defense mechanism by activating the antioxidative enzymes in plants. Displacement of essential metal ions inhibits the enzyme activity during chlorophyll biosynthesis (Shakya et al., 2008).

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HMs have been found to decrease chlorophyll a, chlorophyll b, and total chlorophylls in various plant species (Ahmad et al., 2011, 2012). The inhibit various enzymes that affect the metabolic processes responsible for chlorophyll biosynthesis (Pourraut et al., 2011). The decrease in the chlorophyll content of the cells may be either because of reduced synthesis or of accelerated degradation of light-harvesting pigments. HMs affect both light and dark reactions of photosynthesis, inhibiting the photosynthetic electron transport chain and PSII being the most sensitive target (Gajewska and Skłodowska, 2008). Mercury ions complexed with organic compounds also cause deterioration of 33 KDa polypeptide of PII submembrane. Decreased photosynthetic pigments are also because of the prolonged duration of HM exposure. There could also be a consequence of cadmium-induced change in Mg and Fe content in leaf (Benavides et al., 2005). Significant reduction in chlorophyll content may also be due to strong oxidation of the photochemical apparatus (Mishra and Dubey, 2006) and a reduction in chloroplast size and density. Photosynthesis of woody plants shows a decreasing trend under HMs before the symptoms become visible (Huang et al., 2010). These effects are directly or indirectly related to biochemical and molecular changes in response to HMs (Semane et al., 2010). Lipids and proteins provide an important indicator of oxidative stress as they rapidly get damaged under environmental stress. This decrease may be due to increased proteolytic activity (reference), various membrane modifications by protein denaturation, or interaction with thiol group proteins (Anjum et al., 2015). HMs are also responsible for inhibiting uptake of Mg2þ and Kþ, decreasing synthesis, and increasing protein degradation and prevention of Rubisco activity (Jiang et al., 2009). Decrease in protein content because of HM–induced oxidation of proteins mediated by H2O2 to increase proteolytic activity and have been proposed as an index of oxidative stress (Romer-Puertas et al., 2002). It could be due the interference of metals with sulfur containing amino acids and protein or it is caused either by reduced de novo synthesis or by increased decomposition of protein to amino acids (Anjum et al., 2015). Reports pertaining to reduced protein content because of metal stress are well-documented (Zhang et al., 2005; John et al., 2009; Sethy and Ghosh, 2013; Tiwari et al., 2013). Presence of HMs caused a decrease in the nitrate content indicating the least accumulation of nitrate in the leaves and might be due to reduced nitrate transport from root to shoot (Chaffei et al., 2004). Cd toxicity seems to reduce the mobilization of endogenous nitrate because it interferes with the metal Mo, which acts as the key enzyme to regulate the nitrate (Khudsar et al., 2001). Decrease in NR activity might be related to the reduction of nitrate concentration in plant tissue as a result of HM stress. It plays a central part in the maintenance of the whole plant system and any interference with it decreases enzyme activity and hence changes its conformation and stability by binding with eSeSe and eSH groups of protein and eCOOH and eNH2 group of enzymes. Cdinduced supraoptimal generation of ROS could interfere with the active state of NR, rendering it inactive. The NR activity exhibited a progressive decline in response to increasing dose of Cd (Irfan et al., 2014). The inhibition in NR activity is suggested to be from a reduced supply of nicotinamide adenine dinucleotide (NADH), disorganization of chloroplast, decreased nitrate supply to the site of enzyme synthesis, water stress created by metals, and direct effects on protein synthesis because it has a strong affinity with the thiol group of enzymes. Gill et al. (2012) reported a differential response of leaf N and NR activity in response to Cd stress garden cress (Lepidium sativum L.). NR activity and N content in the leaves were reduced under higher Cd treatments, which resulted in disruption of the coordination between carbon, S, and N metabolism and thus poor plant growth and susceptibility to Cd stress. Unaltered activity of NR complemented the carbon and S metabolism and antioxidant machinery for possible tolerance to Cd toxicity at a lower Cd level.

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Nitrate reduction in vivo depends upon the supply of NADH as source of electrons and the inhibition of NR activity may be due to decreased rate of photosynthetic supply, respiration, and NADH availability. Decline in NR activity because of stress might also be due to the reduced uptake of nitrogen or adverse effect of NR enzyme activity itself (Iqbal et al., 2015). Nitrate and NR in response to As and Al have been observed in rice because of reduced assimilation of nitrogen (Sharma and Dubey, 2005). Sugar acts as a regulatory molecule to control seed and embryo development. Vinod et al. (2012) reported that HM modified the carbohydrate accumulation in wheat leaves and its level was significantly reduced in leaf tissues in response to Cu and Zn stress. Similar decrease in sugar content was also reported by Singh et al. (2007). Manivasagaperumal et al. (2011) reported that sugar and starch content showed a decreasing trend with progressive increase in Zn content in cluster Bean. The observed decline in total sugar with respect to the high level of Zn may be due to its role on the enzymatic reactions related to the cycles of carbohydrate catabolism. Recently, the importance of glucose as a direct and central signal molecule in modulating plant development has been recognized; for example, glucose acts as a key regulator in many vital processes such as germination; root, stem, and shoot growth; photosynthesis; carbon and nitrogen metabolism; flowering; and senescence (Rolland et al., 2002). Additionally, sugars such as glucose have been identified to be involved in these physiological processes through the regulation of transcription factors (Li et al., 2009). On the other hand, as an important component of cell wall polysaccharides, glucose also plays important roles in cell wall formation (Moore et al., 2003). The cell wall is a pivotal site for Cd storage in plants (LozanoRodrı´guez et al., 1997; Carrier et al., 2003), and up to 70–90% of the total Cd is accumulated in the cell walls in Athyrium yokoscense (Nishizono et al., 1987). Recently, Shi et al. (2015) showed that exogenous glucose alleviated Cd toxicity is mediated through increasing Cd fixation in the root cell wall and sequestration into the vacuoles. Proline (Pro) acts as an indicator under HM stress and plays an important role in the osmoprotection, reconstruction of chlorophyll, protection of enzyme from denaturation, tolerance to stress by osmoregulation, and stabilization of protein synthesis and also acts as a source of energy to restore the growth. Proline enhancement is also because of de novo synthesis or decreased degradation of Pro (Liang et al., 2013). Proline accumulation is quite pronounced under HM stress (Ahmad et al., 2011, 2012) and may contribute to osmotic adjustment, enzyme protection, and stabilizing the structure of organelles and macromolecules. Ni inactivates the proteins by suppressing the activity of ribonuclease and protease (Maheshwari and Dubey, 2011). In response to Cd stress, Pro content was found to increase in B. juncea, Triticum aestivum, and Vigna radiata (Dhir et al., 2004), but Pro accumulation decreased on exposure to Cd2þ in hydrophytes (Ceratophyllum, Wolffia, and Hydrilla). Similarly, increasing Pro content in response to Cd2þ in sunflower was also reported (Zengin and Munzuroglu, 2006). Increased level of Pro was observed in Brassica oleracea in the presence of Cd and Hg as reported by Theriappan et al. (2011). In many plant species, elevated levels of Pro were correlated with metal tolerance and metal-induced Pro accumulation seems to be very common in a wide variety of plants. In this case, Pro acts as an osmoprotectant and prevented metal-induced osmotic stress. Although Pro and betaine do not take part directly in metal chelation, they stabilize native state of proteins by regulating their water and hence these osmoprotectants help in maintaining the conformational characteristics and integrity of proteins. Recently studies in plants have demonstrated the capacity of Pro to detoxify ROS. In many plant studies, exogenous application of Pro and glycine betaine (GB) showed enhanced tolerance against metal-induced oxidative stress that was due to the

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upregulation of antioxidant defense system and glyoxalase systems (Hossain et al., 2010; Kumchai et al., 2013; Irfan et al., 2014). In response to Cd stress in mung beans, inhibition or insufficient activation of antioxidant enzymes and glyoxalase pathway enzymes were observed. Importantly Pro or betaine pretreated seedlings modulated the activities of antioxidative and glyoxalase pathway enzymes and rendered the plant more tolerant to Cd-induced oxidative stress (Hossain et al., 2010). Recently, Irfan et al. (2014) found a good correlation between Pro accumulation and Cd stress tolerance in B. juncea. In another study, Ahmad and Gupta (2013) reported that stimulation of Pro accumulation was found to be associated with enhanced antioxidant defense system and regulation in expression of phytochelatin (PC) synthase (PCS), metallothionine-2, glutathione reductase (GR), and glutathione synthetase genes under As stresses that rendered the plants more tolerant to As stress. In vitro grown cabbage (B. oleracea var. capitata) showed enhanced tolerance to molybdenum (Mo) toxicity when the seedlings were supplemented with exogenous Pro, and it was observed that supplementation of 80 mM Pro to the Mo-treated medium could help 50% seedlings to overcome the toxicity (Kumchai et al., 2013). Addition of Pro in Mo-stressed seedlings upregulated the antioxidant enzyme (superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione S-transferase (GST), and catalase (CAT)) activities that provided metal chelation and enhanced tolerance. One of the important roles of Pro is the enhancement of endogenous glutathione (GSH) level from which PCs are synthesized, where the metal binds to the constitutively expressed enzyme PCS, thereby activating it to catalyze the conversion of GSH to PCs (Zenk, 1996).

4. GENERATION OF ROS IN CELLS Oxygen is an important constituent for the sustainability of all the organisms. The reduction of O2 to H2O provides the energy to the organism, but when it is incompletely reduced, ROS is generated, which is highly reactive and oxidizes biological molecules. Chloroplasts, mitochondria, and peroxisomes are the main sites for ROS production. ROS can be produced from both endogenous and exogenous substances. Potential endogenous sources include mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell activation.

4.1 ROS GENERATION IN MITOCHONDRIA Mitochondria are also potential sites for ROS production primarily generating O2  and H2O2 because of the overreduction of electron transport chain that harbors electrons with sufficient free energy to directly reduce O2. Direct reduction of O2 to O2  takes place in the flavoprotein region of NADH dehydrogenase segment of the respiratory chain. During mitochondrial electron transport, the oxygen radical is markedly enhanced in the presence of antimycin A, which blocks electron flow after ubiquinone. This results in the accumulation of reduced ubiquinone that may undergo autooxidation, resulting in the production of O2  (Mittler, 2002; Gill and Tuteja, 2010). Several observations reveal ubiquinone as a major H2O2-generating location of the mitochondrial electron transport chain in vitro and it would appear that O2  is a major precursor of H2O2.

4.2 ROS GENERATION IN CHLOROPLASTS Photosynthesizing plants are especially at the risk of oxidative damage, because of their oxygenic conditions and the abundance of the photosensitizers and polyunsaturated fatty acids (PUFAs) in the

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chloroplast envelope. In photosynthetic electron transport, the photoreduction of O2 to O2  takes place and is called the Mehler reaction. Although photoreduction of O2 is an important alternative sink for the consumption of excess energy because it is always associated with the generation of toxic ROS. The photoproduction of ROS rate is enhanced under the conditions in which photon intensity is in excess of that required for the CO2 assimilation. Under the conditions of photon excess the relaxation systems suppresses the photoproduction of ROS in chloroplast, such as photorespiration, the cyclic electron flows through either PSI or PSII, and the downregulation of PSII quantum yield as regulated by the xanthophylls cycle and the photon gradient across thylakoid membrane (Asada, 1999).

4.3 ROS GENERATION IN MICROSOMES AND PEROXISOMES In plants, O2  is known to be produced during nicotinamide adenine dinucleotide phosphate-oxidase (NADPH)-dependent microsomal electron transport. In the presence of NADPH or related reduced compounds, a chain reaction starts that provides the basis for the H2O2 producing NADH-oxidase activity of peroxidases (Mittler, 2002). H2O2 production is distinguished from that by the phagocyte-type NADPH-oxidase by (1) different Km values for oxygen, (2) different requirements for NADH and NADPH, and (3) different sensitivities of the two enzymes to inhibitors such as cyanide, azide, and diphenyleneiodonium. Based on these differences, rapid H2O2 production in some plant species triggered by pathogen attack and elicitor treatment has been attributed to the NAD(P) H-oxidase activity of apoplastic peroxidases. In addition to its NAD(P)H-oxidase activity, horseradish peroxidase can reduce H2O2 to OH (Chen et al., 2014). During the infection of barley by Erysiphe graminis, a germin-like oxalate oxidase was induced and is another alternative source of ROS (Dumas et al., 1995). Allan and Fluhr (1997) reported that addition of fungal elicitor gives rise to two distinct ROS-producing mechanisms in epidermal cells of tobacco. The two sources of ROS production are (1) NADPH oxidase and/or a xanthine oxidase and (2) peroxidase and/or amine oxidase.

4.4 OTHER SOURCES OF ROS GENERATION IN PLANTS Other important sources of ROS in plants that have received little attention are detoxification reactions catalyzed by cytochrome P450 in cytoplasm and endoplasmic reticulum. ROS are also generated at plasma membrane level or extracellularly in apoplast in plants. pH-dependent cell wall peroxidases, germin-like oxalate oxidases, and amine oxidases have been proposed as a source of H2O2 in apoplast of plant cells. pH-dependent cell wall peroxidases are activated by alkaline pH that, in the presence of a reductant, produces H2O2. Alkalization of apoplast upon elicitor recognition precedes the oxidative burst and production of H2O2 by a pH-dependent cell wall peroxidase has been proposed as an alternative way of ROS production during biotic stress (Mittler, 2002; Ahmad et al., 2010).

5. OXIDATIVE DAMAGE TO BIOMOLECULES 5.1 OXIDATIVE DAMAGE TO DNA Plant genome is very stable, but its exposure to biotic and abiotic stress might damage DNA. The higher concentration of ROS can be important mediators of damage to cell structures, nucleic acids, lipids, and proteins (Tuteja et al., 2009). The OH is known to react with all components of the DNA molecule, damaging both the purine and pyrimidine bases and also the deoxyribose backbone.

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ROS-induced DNA damage involves single or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose modifications, and DNA cross-links. DNA damage can result either in arrest or induction of transcription, induction of signal transduction pathways, replication errors, cell membrane destruction and damage to photosynthetic proteins and genomic instability (Tuteja et al., 2009). Mitochondrial DNA (mtDNA) and cytoplasmic DNA (ctDNA) are close to major sites of ROS production and high rates of modification and equally high repair rates have been reported in mammalian mitochondria. No comparable information is available for plant cells. However, the presence of multiple copies of mtDNA and ctDNA enables the cell to select against negative mutations. 8-hydroxyguanine (8-OH-G) is the most commonly observed modification in DNA and was first reported in human urine. This oxidized DNA product is important because it is both relatively easily formed and is mutagenic. It is a good biomarker of oxidative stress of an organism. It has been noted that 8-OH-G undergoes keto-enol tautomerism and therefore, 8-OH-G is often called 8-oxoguanine or 8-oxo-G; however, 8-oxo-G and 8-OH-G are equivalent. The major type of DNA damage caused by exposure to ultraviolet B is the formation of dimers between adjacent pyrimidines (i.e., ultraviolet photoproducts consist primarily of cyclobutane pyrimidine dimmers and 6-4PPs dimers). Several mechanisms are available for repairing DNA damage both in the nucleus and in the mitochondria. These include direct reversal of the damage, replacement of the base, and replacement of the whole nucleotide (Tuteja et al., 2009). Specific and general repair mechanisms that can repair DNA base modifications have also been observed. The efficiency of repair mechanisms may be enhanced following exposure to ROS because expression of many DNA repair enzymes is up regulated following oxidative stress.

5.2 LIPID PEROXIDATION Metal-induced generation of ROS results in the attack of not only DNA in the cell nucleus, but also other cellular components involving PUFA residues of phospholipids, which are extremely sensitive to oxidation (Abd-Allah et al., 2015). PUFAs linoleic acid (18:2) and linolenic acid (18:3) are particularly susceptible to attack to 1O2 and HO,, giving rise to complex mixtures of lipid hydroperoxides (Ahmad et al., 2010). The peroxidation of lipids is considered the most damaging process known to occur in every living organism. Membrane damage is sometimes taken as a single parameter to determine the level of lipid destruction (i.e., lipid peroxidation). Now, it has been recognized that during lipid peroxidation, products are formed from polyunsaturated precursors that include small hydrocarbon fragments such as ketones, malondialdehyde (MDA), etc. and compounds related to them. Some of these compounds react with thiobarbituric acid to form colored products called thiobarbituric acid reactive substances that can be measured by monitoring their absorption at around 530 nm. Extensive PUFA peroxidation decreases the fluidity of the membrane, increases leakiness, and causes secondary damage to membrane proteins. Several aldehydes (e.g., 4-hydroxy-2-nonenal (HNE) and MDA), as well as hydroxyl and keto fatty acids, are formed as a result of PUFA peroxidation. The aldehyde breakdown products can form conjugates with DNA and proteins. Aldehydes formed in the mitochondria may be involved in causing cytoplasmic male sterility in maize because a restorer gene in this species encodes a mitochondrial aldehyde dehydrogenase (Miller et al., 2010). In plant cells, some of the PUFA oxidation products function as secondary messengers either directly or after enzymatic modification (Mittler, 2002; Miller et al., 2010). The overall process of lipid peroxidation consists of three stages: initiation, propagation, and termination (Gill and Tuteja, 2010).

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Once formed, peroxyl radicals can be rearranged via a cyclization reaction to endoperoxides (precursors of MDA), with the final product of the peroxidation process being MDA. Peroxidation of lipids is an autocatalytic process that is terminated for example by the recombination of radicals (R, þ R, / nonradical product) or depletion of the substrate. MDA can react with DNA bases guanine (G), adenine (A), and cytosine (C) to form adducts M1G, M1A, and M1C, respectively. M1G adducts were found to range in tissue at levels ranging from below the limit of detection to as high as 1.2 adducts per 106 nucleosides (which corresponds to approximately 6000 adducts per cell). Site-specific experiments confirmed that M1G is mutagenic in Escherichia coli, inducing transversions to thiamine (T) and transitions to A (Fink et al., 1997). The mutation frequencies are comparable with those reported for 8-oxo-G in similar systems. M1G is repaired by both bacterial and mammalian nucleotide excision repair pathways and is also repaired in E. coli by mismatch repair. Plants exposed to HM stress exhibited an increase in lipid peroxidation because of the generation of free radicals. This was related to blockage of electron flow in PSII by metal ions that lead to the formation of excited chlorophyll that in turn causes the production of free radicals. Treatment with Cd significantly increased the accumulation of lipid peroxides in P. sativum (Metwally et al., 2005), Arabidopsis (Guo et al., 2008), B. juncea (Ahmad et al., 2011; Asgher et al., 2014), and Bechmeria nivea (Liu et al., 2007).

5.3 OXIDATIVE DAMAGE TO PROTEINS Protein oxidation is defined here as a covalent modification of a protein induced by ROS or byproducts of oxidative stress. Most types of protein oxidations are essentially irreversible, whereas a few involving sulfur-containing amino acids are reversible. Protein oxidation is widespread and often used as a diagnostic marker for oxidative stress. The amino acids cysteine (Cys) and methionine (Met) are very reactive, especially with 1O2 and HO,. ROS causes the oxidation of thiol to disulfide (R1eSeSeR2, cysteine) and is a very important metabolic redox regulation mechanism. Cysteine sulfhydryl groups can be generated from the disulfides by endogenous cellular reducing agents, such as GSH and protein disulfide isomerase, which reverse the formation of non-native disulfide bridges. Chloroplasts, mitochondria, and other cellular compartments of the cell contain large number of potential Trx-regulated proteins that reduces cysteine. The further oxidation of cysteine via cysteine sulfenic acid (ReSOH) to cysteine sulfinic acid (ReSO2H) is also enzymatically reversible and probably involved in signaling pathways. Another reversible modification is oxidation of methionine to methionine sulfoxide. In chloroplasts, the small heat shock protein is inactivated by methionine sulfoxidation, but revitalized by reduction catalyzed by the enzyme peptide methionine sulfoxide reductase using Trx as the reductant. Met has been suggested to act as an endogenous antioxidant. Carbonylation is the most commonly occurring oxidative protein modification and is irreversible. Protein carbonylation may occur because of direct oxidation of amino acid side chains (e.g., Pro and arginine to g-glutamyl semialdehyde, lysine to amino adipic semialdehyde, threonine to amino ketobutyrate). It can also be induced through the interaction of proteins with oxidative byproducts of other molecules, such as lipid peroxidation derivatives such as HNE and MDA. Protein carbonylation can also be observed in glycation reactions, in which lysine can be nonenzymatically derivatized with oxidized sugars.

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In stressed tissues, a significant proportion of all proteins are carbonylated. Plant protein analysis by Nguyen and Donaldson (2005) gave an estimation of 4 nmol carbonyl groups per milligram of protein. Cadmium (Cd)-treated pea plants raised the carbonylation level from 4 to 5.6 nmol/mg protein. The carbonylation level increases from 6.9 to 16.3 nmol/mg of peroxisomal protein as a result of Cd treatment of the intact plant; this can be the result of the higher local ROS concentration in the peroxisomes (Romero-Puertas et al., 2002). It has been reported that protein carbonylation increased during vegetative phase and decreased during reproductive phase in Arabidopsis to limit the transfer of oxidatively damaged components to the offspring. Protein carbonylation is higher in mitochondria than in chloroplasts and peroxisomes. This indicates that the mitochondria are more susceptible to oxidative damage and/or the removal of modified proteins is less efficient in the mitochondria. Several carbonylated proteins have been identified in the mitochondrial matrix. In dry Arabidopsis seeds, most of the carbonylation was found in the storage proteins, but carbonylation of a number of other proteins increased strongly during seed germination. The oxidized proteins derived from several cellular compartments including the cytosol, chloroplasts, and mitochondria (Ahmad et al., 2010; Gill and Tuteja, 2010). Oxidative modification of proteins can have multiple adverse effects on the cellular physiology. For example, oxidative modification of enzymes has been shown to inhibit their activities. Enzymes that have metals on or near the active sites are particularly sensitive to metal catalyzed oxidation. Depending on the extent of oxidative damage, such as percentage of molecules that are modified and how long the modification has remained in the system, the affronts can have mild to severe effects on cellular functions.

6. ANTIOXIDANT AND HM STRESS HM generates the ROS including H2O2 leading to oxidative stress in plants (Hossain et al., 2012; Mostofa et al., 2014; Shahid et al., 2014; Asgher et al., 2014). To cope up with free radicals, plants have developed a strong defense mechanism called antioxidant defense systems composed of both enzymatic and nonenzymatic components. Enzymes such as CAT, APX, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), GST, GR, peroxiredoxin, and nonenzymatic compounds such as ascorbate (AsA), GSH, a-tocopherol, and flavonoids are constantly alert to regulating the concentration of ROS, including H2O2 (Asada, 1999; Miller et al., 2010; Kapoor et al., 2015). The delicate balance between ROS production and scavenging that allows this duality in function to exist in plants is thought to be organized by a large network of genes that tightly regulates ROS production and scavenging (Petrov and Van Breusegem, 2012). HMs have been found to decrease CAT activities and increase APX in B. juncea (Ahmad et al., 2011). Varying responses to Cd-induced oxidative stress are probably related both to the levels of Cd supplied and to the concentration of thiolic groups already present or induced by Cd treatment. Thiols possess strong antioxidative properties and they are consequently able to counteract oxidative stress (Mostofa and Fujita, 2013; Mostofa et al., 2014; Shahid et al., 2014). The AsA-GSH cycle is essential in HM–induced plants to combat oxidative damage (Mostofa and Fujita, 2013; Mostofa et al., 2014). The increase in APX and GR activities in metal stresses plants maintains AsA and GSH turnover and activation of the H2O2 scavenging AsA-GSH cycle. CAT often decreases following exposure to HM concentration (Hossain et al., 2010; Ahmad et al., 2011). Differential responses of SOD showed increases in both leaves and roots. Cd-induced increase in SOD

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have been found in B. juncea (Ahmad et al., 2011) and Morus alba (Ahmad et al., 2012). Increase in SOD activity may be attributed to increased production of ROS or increased expression of gene encoding SOD. Reduction of SOD confers to inactivation of enzyme by H2O2 produced in different cellular compartments. SOD is a key enzyme in scavenging ROS by converting oxygen radicals to hydrogen peroxide in chloroplast, mitochondria, and cytosol. Increase in SOD activity shows high ROS accumulation that in turn is removed by antioxidative enzymes by inhibiting oxygen radical accumulation. Efficient functioning of SOD blocks O2 driven cell damage by converting it to H2O2, which is then reduced to water and molecular oxygen by action of enzymes (i.e., APX and CAT) working at different locations in the cell (Mittler, 2002; Foyer and Noctor, 2005). Increased APX under Hg stress confers the role in detoxification of H2O2 into water using AsA as an electron donor, resulting in the formation of dehydroascorbate. GR is also associated in this pathway. Enhancement in the activities of SOD, APX, and GR under HM stress (Cd, Zn, Cu, Pb, and Fe) may be considered as circumstantial evidence for tolerance mechanism developed by this plant. Increased GR activity could be explained by transcriptional or translational modifications to keep surplus GR level to protect against HM stress (Romero-Puertas et al., 2002). The susceptibility to oxidative stress is a function of the overbalance between the factors that increase oxidant generation and those substances that exhibit antioxidant capability (Ahmad et al., 2010, 2011). GR catalyses the reduction of glutathione disulfide (GSSG) to GSH at the expanse of NADPH and plays an important role in the maintenance of GSH pool. Increase in GPX occurs as a result of de novo protein synthesis or the activation enzymes that persists in plant cells to combat the ROS effects. CAT plays an effective role in protecting the mechanisms occurring in mitochondria and peroxisomes by scavenging the free radicals, especially hydrogen peroxide, during photorespiration as well as under stress conditions. Pb has been shown to inhibit the CAT activities. This decrease in CAT led to an increase in lipid peroxidation as a result of decrease in H2O2 detoxifications. Decrease in CAT is also from modification and inactivation of CAT enzymes and also because of binding of nonessential elements. SOD, APX, and CAT are adapted to environmental stress and are called a plant-protective enzymatic system. These enzyme activities, except for CAT, increase under HM stress and have been reported in A. esculentus. Despite SOD, APX, and CAT, HMs also affect the activities of other enzymes such as amylase, alcohol dehydrogenase, and NR. Most of the effects are inhibitory. Enzymatic mechanisms involved in metabolic pathways represent the major defense strategy to combat against ROS production. SOD catalyses the dismutation of superoxide molecules into H2O2 and O2. H2O2 is subsequently converted to water by peroxidases and CAT and in the way the enzyme activity is balanced. Extensive studies have been carried out on ROS activities of enzymes regarding the defense system on plants against HM stress. Among the water-soluble antioxidants found in plants, AsA is one of the most abundant and serves as a major redox buffer, cofactor for enzymes, regulator of cell division and growth, and signaling molecule (Gallie, 2013). Different type of ROS (1O2, O2  , and OH) can be neutralized by AsA or it can act as a cofactor of APX. Under HM stress conditions, plant cells must maintain higher levels of AsA to counter the adverse effects of oxidative stress. Differential regulation of AsA levels was observed in response to Cd stress. Decrease in AsA content and AsA redox state observed in response to Cd stress was observed in rapeseed, rice, and Arabidopsis (Hsu and Kao, 2007; Smeets et al., 2009; Chao et al., 2010; Hasanuzzaman et al., 2012; Keunen et al., 2013). Liu et al. (2007) showed that AsA contents increased in B. nivea plants under low-level Cd stress, whereas the contents decreased under

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high-level Cd stress. These findings also demonstrated that higher endogenous AsA is essential to detoxify ROS directly by proper functioning of APX. Importantly, exogenous pretreatment with AsA or its precursor can modulate the gene expression related with HM stress tolerance (Zhao et al., 2005; Paradiso et al., 2008; Chao et al., 2010). GSH is one of the most powerful antioxidant involved in both signal transduction and defense against ROS and methylglyoxal (MG) (Singla-Pareek et al., 2006; Jin et al., 2008; Hossain et al., 2012). ROS-induced oxidation of protein is inhibited by GHS. GSH also indirectly played roles in protecting membranes by maintaining a-tocopherol and zeaxanthin in reduced states. The broad range of functions of GSH in modulating HM stress tolerance is well-documented (Hossain et al., 2012) and as a first-line defense response against Cd stress, plants synthesize GSH and PCs to form Cd-SH complexes and to diminish free Cd ions in the cytoplasm (Guo et al., 2008; Mishra and Dubey, 2006; Khan et al., 2015). Two important enzymes such GST and GPX involved in ROS and endogenous toxic compounds are dependent on endogenous GSH level (Foyer and Noctor, 2005; Anjum et al., 2014). GSH also an played important role in the detoxification of MG, a cytotoxic compound accumulated in large amounts under abiotic stress conditions, including HM stress (Hossain and Fujita, 2009; Hossain et al., 2010, 2012). Along with its oxidized form (GSSG), GSH acts as a redox couple vital for upholding cellular homeostasis and plays key roles in diverse signaling systems in plants. An increased level of GSH was found to detoxify ROS under Cd stress conditions in various crop plants (Qadir et al., 2004; Anjum et al., 2008; Metwally et al., 2003; Ahammed et al., 2013). Sharp increases in GSH level were found to increase in leaves, stems, and root tissues exposed to multiple HM (Cd2þ, Pb2þ, and Hg2þ) stress (Huang et al., 2010). Brassica juncea seedlings treated with Cd stress showed higher GSH and PC synthesis (Mohamed et al., 2012). The expression of genes associated with GSH metabolism responds to HM stress (Xiang and Oliver, 1998). However, depletion of the GSH pool has also been attributed to increased utilization of GSH (Pietrini et al., 2003). Decreased GSH levels ultimately resulted in deteriorated ROS metabolism; defenses against Cd have been found in sensitive genotypes of pea (Metwally et al., 2005). In-depth studies on Cd hyperaccumulating plants have validated the significance of GSH and its metabolizing enzymes for Cd tolerance (reviewed in Hossain et al., 2012). Carotenoids play a significant role as scavengers to protect the chloroplast against photo-oxidative damage. Carotenoids are simple unsaturated hydrocarbons based on lycopene and their oxygenated derivatives like xanthophylls. ROS detoxification is an essential mechanism of carotenoid that contributes toward the protection of enzymes under HM stress.

7. TRANSLOCATION OF HMS Micronutrients at optimum concentrations act as the sole energy regulators for the plant body. However, the processes associated with the movement of metals from root to shoot should be in a regulated manner. It involves the movement of toxic metals into the root epidermal cells from the soil solution where from ions move radially either through the root symplast and/or apoplast to the root stele where xylem loading occurs. These metal ions, including the solutes moving along with it, must cross the endodermis, whereby the Casparian strip effectively blocks the further radial apoplastic movement of ions and other solutes. At this point, the metal ions cross the plasma membrane and enter the root symplasm to reach the stele, allowing the plant to regulate the movement of ions into the xylem and restrict transport of ions to the rest of the plant. One of the important steps is loading of the ions into the xylem and subsequent translocation to the shoots via transpirational pull of water and

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solutes in the xylem. The process is supposed to be mediated by the regulated efflux of metal ions from xylem parenchyma cells into the lumen of nonliving xylem vessels. The last step is movement of metal ions from the xylem sap into the leaf with subsequent transport across the plasma membrane of leaf cells. Plant transporters are classified into three main groupsdchannels, carriers, and pumpsdwith distinctive characteristics in terms of substrate specificity and energy source used to mediate the transport of different types of organic and inorganic solutes across biological membranes. Channels are described by their extremely high rate of transport across the membrane (Sanders and Bethke, 2000). Carriers, on the other hand, undergo conformational changes in protein structure that facilitate transport across the membrane. They bind to the substrate on one side of the membrane and after the conformational change; the substrate is released on the other side of the membrane. To protect the plants against HMs, roots secrete exudates into the soil matrix. The main role of root exudates is to chelate metals and thereby stops the uptake of HMs to prevent their uptake inside the cells (Marschner, 1995). For example, Ni-chelating histidine and citrate in root exudates reduce the uptake of Ni from soil. The binding of metal ions such as Cu and Zn in the apoplast also helps to control the metal content of root cells. Cation binding sites present on the root cell wall allows the exchange of metal thus influencing the availability of ions for uptake and diffusion into the apoplast. The cell wall acts as a central hub for immobilization of toxic HM ions by providing pectic sites and histidyl groups as well as extracellular carbohydrates such as callose and mucilage. Thus, different tobacco genotypes with chemically distinct root cell wall surfaces have different sensitivities to Mn toxicity. The data suggest that the chemical properties of the cell wall may alter and modify the plant metal uptake and hence metal tolerance. However, the role of the cell wall in metal tolerance is not completely understood. The cell wall is in direct contact with metal ions in the soil, but only a limited number of absorption sites are available, suggesting the cell wall has only a minor impact on metal tolerance. However, Silene vulgaris ssp. humilis is a HM–tolerant plant that accumulates different HMs by binding them to proteins or silicates in the epidermal cell walls (Bringezu et al., 1997). Metal ions absorbed by the roots are loaded into the xylem and translocated to the shoots as complexes with various chelators. Organic acids, especially citrate, are the major chelators for Fe and Ni in the xylem. Ni may also be chelated by histidine and translocated and the methionine derivative nicotianamine (NA) is involved in the transport of Cu. Several types of transporter proteins are involved in the root-to-shoot transport of metals. Whereas metal ions are translocated from source to sink tissue via phloem, NA is involved in the long-distance transport of metals inside the xylem and phloem, but other chelators are required for loading. High-molecular-weight compounds that chelate Ni, Co, and Fe are found in the phloem. Furthermore, transporters help to bind the complexes with the organic compounds and are transported as needed for a wide range of processes. Also, turgor pressure is accomplished by balancing the internal and external concentrations of inorganic and organic ions (Sanders and Bethke, 2000). HM transporter proteins are classified as zinc-regulated transporter, iron-regulated transporter–like protein ZIP family, ATP-binding cassette (ABC) transporters, the P-type metal ATPases, the natural resistance-associated macrophage protein family, ABC transporters of the mitochondria, cation diffusion facilitator family of proteins, copper transporter family proteins, and many others (Kramer et al., 2007). Plant cell walls are continuous and uninterrupted and act as a cation exchanger, holding large numbers of metals, and providing for some exclusion. Proper homeostasis in barley (Hordeum vulgare L.) leaves with the increase in cellular Zn with increasing exposure to external Zn was fully

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accounted by an increase in vacuolar Zn with the cytoplasm. To locate the position of Cd and the potential Cd-binding peptides, protoplasts and vacuoles were isolated from leaves of Cd-exposed tobacco (Nicotiana rustica var Pavonii) seedlings and purified vacuoles contained almost all of the Cd-binding peptides and Cd found in protoplasts. Computer simulation studies with Zn and Cd showed that the vacuole is the probable site of HM sequestration and detoxification in Nicotiana tabacum. However, many plant HM transporters remain to be identified at the molecular level; the transport function, specificity, and molecular location of most of these proteins in plants remain unknown. Mechanism of uptake and transportation helps to alleviate the HM toxicity in plants. Plants accumulate HM ions either by selective uptake of ions through roots or by diffusion of elements in the soil (Peralta-Videa et al., 2009). Concentration in the cortex is then moved apoplastically by concentration gradient and gets accumulated in the cell wall. Two types of uptake systems (i.e., active and passive transport) have been reported for Cd in roots of spruce and soybean. Transport process provides a central role for the mechanism of detoxification and tolerance. With the use of latest genetic and molecular techniques, a wide range of gene families have been found in transition metal transport. They are ATPase, natural resistance-associated macrophage protein family, cation diffusion facilitator family, ZIP family, and cation antiporters. These metal transporters maintain the internal metal concentration within the physiological limits. Resistance to Zn associated with ion transport was found to enhance expressions of the Zn transporter in Arabidopsis. ABC transporter AtPDR8 have shown to mediate the extrusion of Cd out of selective permeable membrane of root epidermal cells. Detoxification is also accomplished through members of the ZIP family. They are plasma proteins induced in roots and shoots of Arabidopsis under Zn-limiting uptake being potentially implicated in Cd root to shoot transport (Kramer et al., 2006).

8. SIGNALING IN PLANTS UNDER HM STRESS As with all other organisms, plants have also evolved different mechanisms to maintain physiological concentrations of essential metal ions and to reduce contact to nonessential HMs. Some mechanisms are required for homeostasis and are ubiquitous, whereas other mechanisms target individual metal ion for the exclusion of particular metals from the intracellular environment. When these two mechanisms are exhausted, then the plants activate oxidative stress defense mechanisms and synthesis of stressrelated proteins and signaling molecules such as heat shock proteins, hormones, and ROS and finally the transcriptional activation of specific metal-responsive genes to work against the stress (Maksymiec, 2007). However, different signaling pathways may be used to respond different HMs. The signal pathways comprise the calcium calmodulin system, hormones, ROS signaling, and the mitogen-activated protein kinase (MAPK) phosphorylation cascade which congregate by activating the previously mentioned stress-related genes. Shao et al. (2008, 2009) and Thapa et al. (2012) have reported that the HM stress causes calcium level changes, MAPK cascade, and transcriptional modulation of the stress-responsive genes.

8.1 THE CALCIUM-CALMODULIN SYSTEM Calcium is an essential macronutrient taken by the plants through roots and is delivered to the shoots via xylem (Tuteja et al., 2009; Ahmad et al., 2012). Calcium ions regulate a range of activities within the cell such as cell division and elongation, cytoplasmic streaming, photomorphogenesis, and plant

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defense against environmental stresses (Tuteja et al., 2009; Kader and Lindberg, 2010). They function as the central node in overall signaling web and have a promising role in stress tolerance (Tuteja and Sopory, 2008). Surplus HM alters the stability of Ca channels by increasing the calcium flux into the cell. Intracellular Ca is a secondary messenger that interacts with calmodulin to transmit the signal and regulate the downstream flux genes involved in HM transport, metabolism, and tolerance (Yang and Poovaiah, 2003; Choong et al., 2014). In plants exposed to Cd, higher intracellular Ca levels have been observed, inducing adaptive mechanisms that alleviate the toxic effects of the HM (Yang and Poovaiah, 2003). Calmodulin, an important Ca2þ-binding protein, is a small acidic protein and is responsible for the regulation of intracellular Ca2þ levels. Increased Ca2þ concentration activates calmodulin that then induces specific kinases. Calmodulin is a very important calcium-binding protein in Ca2þ signaling and has been found to be involved in biotic and abiotic stresses (Tuteja and Sopory, 2008). In the response of other HM toxicity such as Ni and Pb, the Ca-calmodulin system is also involved. Transgenic tobacco plant expressing NtCBP4 (Nicotiana tobacum calmodulin-binding protein) can tolerate higher levels of Ni2þ but are sensitive to Pb2þ, showing the omission of Ni2þ and the accumulation of more Pb2þ than wild-type plants.

8.2 CONNECTION WITH HORMONE SIGNAL PATHWAYS In many physiological and developmental processes, plant hormones play a vital role in the adaptation to abiotic stress, as shown by the regulation of hormone synthesis in the presence of HMs (Ahmad et al., 2011). For instance, an increase in ethylene biosynthesis in response to Cd stress has been observed in mustard (Masood et al., 2012), pea (Rodriguez-Serrano et al., 2009), and in soybean (Chmielowska-Bak et al., 2013). Arteca and Arteca (2007) also reported that, except for Zn, both Cu and Cd were found to elicit the greatest amount of ethylene produced by Arabidopsis plants. Maksymiec and Krupa (2006) have reported that ethylene may be involved in the inhibitory action of copper on roots and leaves of dicotyledonous plants. Some researchers have reported that the plants exposed to Cd, Cu, Fe, and Zn stresses produce the higher levels of ethylene, but the copper may not have the same effect. In Phaseolus coccineus, copper and cadmium induce the accumulation of jasmonic acid (Maksymiec et al., 2005; Maksymiec, 2011), and the copper has shown this effect on rice (Rakwal et al., 1996) and Arabidopsis thaliana (Maksymiec et al., 2005) as well. However, decrease in jasmonic acid content in Cd-stressed roots of soybean leads to an increase in abscisic acid (ABA) and metabolites at different times of Cd stress, suggesting their involvement in Cd response (Perez Chaca et al., 2014) and showing that in some experimental conditions jasmonate accumulation after HM stress may not occur. Under Cd stress, pea seedlings showed increase in salicylic acid (SA) and its exogenous ability helps to protect leaves from lipid peroxidation caused by Cd toxicity (Popova et al., 2009). They suggested that high endogenous levels of SA after treatment with Cd may act directly as an antioxidant to scavenge the ROS and/or indirectly modulate redox balance through activation of antioxidant responses. Zhang and Chen (2011) also showed that exogenously applied SA prevented the Cd-induced photochemical efficiency decrease and mitigated Cd toxicity in Arabidopsis. Additionally, SA pretreatment could alleviate Cd-induced ROS overproduction. Very recently, Mostofa and Fujita (2013) showed Cu stress leads to severe oxidative stress in rice seedlings, as indicated by chlorosis, necrosis, and rolling of leaves and higher levels of oxidative parameters such as level of hydrogen peroxide, lipid peroxidation, and lipoxygenase activity. Importantly, SA pretreatment restrained the ROS and MG detoxification systems and rendered the plants more tolerant to Cd-induced oxidative

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stress tolerance. Yan et al. (2013) studied the effects of methyl jasmonate (MeJA) on Capsicum frutescens seedlings exposed to Cd stress. Cd stress caused an impaired growth and increased lipid peroxidation and decreased chlorophyll b, whereas the negative effects of Cd stress were significantly mitigated by MeJA. Exogenous application of MeJA increases the level of endogenous MeJA and the activities of APX and CAT at low MeJA concentrations. These results suggested that low exogenous MeJA concentrations exhibited protective effects on the growth and physiology of Capsicum frutescens seedlings under Cd stress. Taken together, all this evidence could explain to some extent the protective role of SA on photochemical activity of chloroplast membranes and photosynthetic carboxylation reactions in Cd-stressed pea plants. Recently, Chen et al. (2014) studied the roles of exogenous MeJA in Cd stress tolerance in the leave of Kandelia obovata seedlings. Cd stress lead to a decrease in chlorophyll content and increase in lipid peroxidation. Exogenous MeJA alleviates oxidative stress by increasing the activities of CAT and APX and also modulated the expression of type-2 metallothionein gene (KoMT2) expression in the leaves of K. obovata seedlings exposed to Cd stress. Methyl jasmonate also inhibited the uptake of Cd to the aboveground part (leaves) of the seedlings, which helped to reduce the direct damages caused by Cd to the photosynthetic organ of the plant. The reduced uptake of Cd might be a result of stomatal closure and decreased transpiration by exogenous MeJA.

8.3 ROLE OF ROS One of the major consequences of the HM accumulation is the production of ROS as stated previously, leading to oxidative stress and the effect of HMs on oxidative processes is the base for other connections with signaling response (Tuteja and Sopory, 2008). HMs such as Cd can produce ROS via Fenton and Haber–Weiss reactions and indirectly inhibit the antioxidant enzymes (Romero-Puertas et al., 2007). Hydrogen peroxide (H2O2) acts as a signaling molecule in response to HMs and other stresses (Dat et al., 2000). In A. thaliana, Cu and Cd treatment increases the level of H2O2 (Maksymiec and Krupa, 2006). Lin et al. (2005) have reported that Cu can act through changes in H2O2-dependent peroxidase activity by stiffening the cell wall from the formation of cross-linking among the cell wall polymers. As per reports, Cd also enhances H2O2 accumulation (Maksymiec and Krupa, 2006). Thus, H2O2 accumulation changes the redox status of the cell (Foyer and Noctor, 2005) and induces the production of antioxidants and the activation of antioxidant mechanism. H2O2 priming was also found to increase the plant tolerance to HM stress. Hu et al. (2009) showed H2O2 pretreatment induced Cd tolerance in rice seedlings. Plant growth (length and biomass of shoot and root) was significantly repressed by Cd exposure. However, pretreatment with 100 mM H2O2 for 1 day mitigated Cd stress by inducing the antioxidant enzymes (SOD, CAT, GPX, APX, and GST) and elevated the contents of GSH and AsA. As a result, the H2O2 and MDA contents decreased in plants and seedling growth was less inhibited. In contrast, H2O2 pretreatment decreased Cd concentration in shoots, thus lowering the ratio of Cd concentration in shoots and roots, indicating that H2O2 may affect Cd distribution in rice seedlings. The improved Cd tolerance is partly from an enhanced antioxidant system that efficiently prevents the accumulation of H2O2 during Cd stress. Increased Cd sequestration in rice roots may contribute to the decline of Cd translocation. Xu et al. (2010) showed H2O2-induced AsA and GSH metabolism in inducing Al-induced oxidative stress tolerance in wheat seedlings. Al stress increased the O2  and H2O2 levels leading to more predominant lipid peroxidation, programmed cell death, and inhibited root elongation in both Al-tolerant and sensitive genotypes. Al-stress increased the activities

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of SOD, POD, CAT, MDHAR, DHAR, GR, GPX, AsA, and GSH content and their redox state. However, Al-stressed seedlings pretreated with H2O2 showed higher SOD, POD, CAT, MDHAR, DHAR, GR, and GPX activities and AsA and GSH content and their redox state than nontreated Alstressed seedlings. Importantly, antioxidant capacity was more enhanced in the Al-sensitive genotype than in the tolerant one. Therefore, H2O2 pretreatment makes the plant more tolerant to Al-induced oxidative stress by inducing AsA and GSH levels and their metabolizing enzymes. To support these findings, Bai et al. (2011) studied the effect of H2O2 pretreatment on Cd tolerance and translocation by using two rice cultivars (N07-6 and N07-63) differing in Cd tolerance. Imposition of Cd stress significantly inhibited rice growth, enhanced the production of GSH, non-protein thiols (NPT), PCs, and MDA, and increased the activity of GST. The H2O2 pretreatment alleviated Cd toxicity by further increasing the GSH, NPT, and PCs contents as well as the GST activity in root tissues. These findings indicated that endogenous H2O2 act as a stress signal and regulated the HM stress tolerance by modulating multiple stress signaling pathways and genes.

8.4 THE MAPK CASCADE When plants come across with excessive HMs, the gene expression pattern changes. HM responsive changes to Cd and Cu have shown to code for signal transduction components in rice, maize, and Arabidopsis mitogen-activated protein kinase kinase kinase (MAPKKK), transcription factors, stressinduced proteins, proteins participating in protein folding, and sulfur and GSH metabolism (Yeh et al., 2007; Liu et al., 2010; Wang et al., 2010). The MAPK class plays a remarkable role in plant signaling of a variety of abiotic and biotic stresses and is an essential step in the establishment of resistance to pathogens (Pitzschke et al., 2009). This pathway is also used in hormone signal transduction and in response to developmental stimuli (Jonak et al., 2002). The MAPK cascade consists of three kinases consecutively activated by phosphorylation: MAPKKK, MAPKK, and MAPK. Last, in this cascade of phosphorylation, the MAPKs phosphorylate different substrates in different cellular compartments including transcription factors in the nucleus. The MAPKKK is a Ser/Thr protein kinase that phosphorylates and thereby activates the MAPKKs. These MAPKKs are dual-specific kinases that phosphorylate MAPKs on a Thr and Tyr residue. And the dual phosphorylation of MAPKs renders the enzyme active and MAPKs are predirected Ser/Thr kinases phosphorylating numerous substrates in various cellular compartments. In this way, this pathway allows the transduction of the information to downstream targets. In alfalfa (Medicago sativa) seedlings, four isoforms of MAPK were activated when exposed to Cu or Cd stress (Jonak et al., 2004) and the MAPK gene is also activated by Cd treatments in rice (Yeh et al., 2007). Hence, recognition of these MAPKs and their expression kinetics led researchers to propose the distinct metal-specific signaling pathways exist in plants (Jonak et al., 2004). Finally, all of these pathways congregate in the regulation of transcription factors that activate genes required for stress adaptation, mainly in the perspective of HMs meaning genes for the activation of metal transporters and the biosynthesis of chelating compounds.

9. CONCLUSIONS AND FUTURE PERSPECTIVES Global detrimental effect on health because of the HM exposure is a matter of serious concern. Nowadays, researchers are much more concerned in developing new technologies at low-cost and ecofriendly land retrieval techniques. Understanding the mechanisms that help plants to cope with

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HM stress would help in making new tools that are used in phytoremediation. Hence, in response to HM stress, molecular and cellular adaptation of plant cells seems obligatory to improve HM plant tolerance, which will reduce the chances of HM entrance into the food chain. Being a multigenic trait, recent findings have revealed besides other mechanisms plants protect themselves from HM toxicity through an elevated level of enzymatic and nonenzymatic components of antioxidant system. In our chapter, it is clear that the HMs irrespective of their redox-related mode of action are also efficient in disturbing the equilibrium of antioxidants in cells and slanting the balance that induces the ROS reacting with the functioning of cellular macromolecules and organelles. Consequently, functional diversity and molecular adaptability of PCs and MTs are becoming fascinating regarding the HM detoxification and sustaining cellular ion balance. They act as cellular homeostatic agents and interact directly or indirectly with antioxidant defense system and get tangled in translocating and distributing excessive metal ion between root and shoot in a time- or tissue-specific manner. GSH also plays a vital role in HM uptake, transport, sequestration, and formation of specific metal-binding ligands such as PCs. MAPK cascades transduce environmental and developmental signals into adaptive and programmed responses. Physiological and developmental processes including stress and hormonal responses and innate immunity are regulated by MAPK cascades. More research is needed to identify substrates of MAPKs and cross-talk with other signaling molecules.

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