draft - university of toronto t-space · like other organisms, ... (1980) classified metals into...

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Heavy metal detoxification and tolerance mechanisms in

plants: Its implications for Phytoremediation

Journal: Environmental Reviews

Manuscript ID er-2015-0010.R3

Manuscript Type: Review

Date Submitted by the Author: 28-Sep-2015

Complete List of Authors: Kushwaha, Anamika; Motilal Nehru National Institute of Technology, Biotechnology Rani, Radha; Motilal Nehru National Institute of Technology, Biotechnology Kumar, Sanjay; Motilal Nehru National Institute of Technology, Biotechnology Gautam, Aishvarya; Motilal Nehru National Institute of Technology,

Biotechnology

Keyword: Detoxification, Heavy metals, Phytoremediation, Phytochelatins, Metallothioneins

https://mc06.manuscriptcentral.com/er-pubs

Environmental Reviews

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Heavy metal detoxification and tolerance mechanisms in plants: Implications for 1

phytoremediation 2

3

Anamika Kushwahaa, Radha Rani

a*, Sanjay Kumar

a, Aishvarya Gautam

a 4

5

6

*Department of Biotechnology 7

Motilal Nehru National Institute of Technology, 8

Teliyar Ganj, Allahabad 9

Telephone: +91-532-2271240 10

Fax: +91-532-2271200 11

E-mail: [email protected]; 12

[email protected] 13

14 a Department of Biotechnology 15

Motilal Nehru National Institute of Technology, 16

Teliyar Ganj, Allahabad 17

18

*corresponding author 19

20

21

22

23

24

25

26

27

28

29

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Abstract 30

Heavy metals such as cobalt, copper, manganese, molybdenum, and zinc are essential in trace 31

amounts for growth by plants and other living organisms. However, in excessive amounts these 32

heavy metals have deleterious effects. Like other organisms, plants possess a variety of 33

detoxification mechanisms to counter the harmful effects of heavy metals. These include, the 34

restriction of heavy metals by mycorrhizal association, binding with plant cell wall and root 35

excretions, metal efflux from the plasma membrane, metal chelation by phytochelatins and 36

metallothioneins, and compartmentalization within the vacuole. Phytoremediation is an emerging 37

technology which uses plants and their associated rhizospheric microorganisms to remove 38

pollutants from contaminated sites. This technology is inexpensive, efficient and ecofriendly. 39

This review focuses on potential cellular and molecular adaptations by plants that are necessary 40

to tolerate heavy metal stress. 41

42

Keywords: Detoxification; Heavy metals; Phytoremediation; Phytochelatins; Metallothioneins; 43

Mechanism. 44

45

1. Introduction 46

A heavy metal is a member of ill-defined chemical elements that exhibit metallic properties 47

and occur naturally in the earth’s crust. Heavy metals cannot be degraded and are stable in the 48

environment, resulting in their accumulation over time causing soil pollution. Heavy metals such 49

as cobalt, copper, manganese, molybdenum, vanadium, strontium and zinc are required in trace 50

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amounts by plants and other living organisms, but in excessive amounts these heavy metals have 51

harmful effects. Heavy metals such as mercury, lead and cadmium have no known beneficial 52

effects to organisms. 53

Nieboer and Richardson (1980) classified metals into three groups based on ligand binding 54

preferences: 55

1. Class A metals prefer ligands with available oxygen (e.g. Li+, Na

+, K

+, Cs

+, Be

2+, Mg

2+, 56

Ca2+, Sr

2+, Ba

2+, Al

3+, Ga

3+, Sc

3+, and Y

3+). 57

2. Class B metals bind to ligands containing sulfur or nitrogen (e.g. Tl+, Tl

3+, Pb

4+, Bi

3+, 58

Pd2+, Pt

2+, Cu

+, Ag

+, Au

+, and Hg

2+). 59

3. Class C metals have binding properties that are immediate between those of classes A 60

and B (e.g. Ga3+, In

3+, Sn

4+, Pb

2+, As

3+, Sb

3+, Ti

2+, V

2+, Mn

2+, Fe

2+, Fe

3+, Co

2+, Ni

2+, Cu

2+, 61

Zn2+, Cd

2+). 62

Nieboer and Richardson (1980) hypothesized that, due to specific ligand binding 63

preferences, an identical effect was observed in different organisms. They further postulated that 64

metals of the highest toxicity were included in class B (e.g. Ag+, Tl

+, Hg

2+, Cd

2+). Class B 65

elements do not occur naturally and were found to bind strongly with cysteine (containing SH 66

group) and lysine (nitrogen containing groups) (Shaw et al. 2004). 67

The Industrial Revolution led to the increase in pollution by many folds and ultimately led 68

to the alteration of geochemical cycles as well as a shift in the balance of some heavy metals. 69

Heavy metal contamination of urban and agriculture soils is largely the result of anthropogenic 70

activities such as use of the pesticides, mining, nuclear wastes, disposal of munitions and agents 71

of war, combustion of fossil fuel, and industrial waste. However, natural processes such as 72

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volcanic eruptions, continental dusts, and other natural sources can add to the level of 73

contamination. 74

In the atmosphere, volatile heavy metals attach to particulates and can become widely 75

distributed throughout the atmosphere, traveling several miles from the site of release. Thus, the 76

lighter and smaller the particle, the greater its persistence in air (Department for Environment 77

Food and Rural Affairs, 2008). The accumulation of heavy metals in soil can be lethal for all 78

biota. Heavy metals are toxic as they can replace essential metals in pigments or enzymes, 79

thereby disrupting their natural function (Henry 2000). If a heavy metal is exposed for a long 80

period of time, its effect becomes chronic as it is transferred up the food chain. Long-term 81

exposure to humans of heavy metals like lead, cadmium and arsenic will lead to health issues 82

including mental lapse, skin poisoning, kidney and liver malfunction, gastro-intestinal tract and 83

central nervous system disorders (USDA NRCS, 2000). 84

Excessive accumulation of heavy metals in soil is toxic to most plants. Plant roots absorb 85

heavy metal ions from the environment in excessive concentration and translocate them to their 86

shoots, which affects metabolism and stunts growth (Bingham et al. 1986; Foy et al. 1978). 87

Elevated levels of metal concentrations in contaminated soil result in loss of soil fertility, 88

agricultural yield, and decrease in soil microbial activity (McGrath et al. 1995). Cadmium is 89

most commonly accumulated by agriculturally important crops and leads to a decrease in root 90

and shoot growth, and a decrease in nutrient uptake and homeostasis (di Toppi and Gabrielli 91

1999). Thus, when these crops are consumed by organisms, it may cause severe health effects. 92

With continued increases in cadmium levels, agricultural soil will become unusable for crop 93

production. Similarly, soil contamination with cadmium leads to loss of biodiversity and activity 94

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of soil microbial communities (McGrath 1994). Some of the toxic effects of heavy metals on 95

plants are described in Table 1. 96

Many regulatory steps have been implemented and practiced to restrict the release of metal 97

pollutants into soils but they are typically not sufficient for fully evaluating contamination levels 98

(Ghosh and Singh 2005). Techniques employed for remediation of metal contaminated soil 99

include chemical, physical and biological methods (Baker and Walker 1990). Due to the high 100

cost and low efficiency of many methods, such as excavation and landfill, thermal treatment, 101

acid leaching and electroreclamation, most are not suitable for practical and fully effective 102

applications. Moreover, these methods may cause a pronounced destruction of soil structure, 103

fertility, and other properties. 104

Recently, work has been ongoing to develop cost-effective and high-efficiency 105

technologies for the remediation of heavy metal contaminated sites (Chatterjee et al. 2011). To 106

this end, plants can be used as a possible means for the remediation of heavy metals from 107

contaminated soil (termed as phytoremediation) and is considered to be a green solution. 108

109

2. PHYTOREMEDIATION: AN OVERVIEW 110

Phytoremediation has recently become the subject of more intense public and scientific 111

interest. For chemically polluted lands, vegetation plays an increasingly important ecological and 112

sanitary role. Proper management of plants in such areas may significantly contribute to restoring 113

the natural environment. The term phytoremediation comes from the Ancient Greek word phyto 114

meaning “plant” and the Latin word remedium meaning “restoring balance.” It is a technology 115

that uses plants to treat environmental pollution problems. Plants are used either to remove or to 116

stabilize (hold in place) pollution in the soil. 117

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Microbial populations are large in rhizospheric soils with high metabolic activity compared 118

to bulk soil (Anderson et al. 1993). Microbial populations are known to affect heavy metals 119

mobility and availability to the plant through the release of chelating agents, acidification, 120

phosphate solubilization, and redox changes (Abou-Shanab et al. 2003a; Smith and Read 1997). 121

When applied to seeds or incorporated into soils some plant growth-promoting bacteria that are 122

associated with plant roots may also exert beneficial effects on plant growth and nutrition 123

through a number of mechanisms such as N2 fixation, production of phytohormones and 124

siderophores, and the transformation of nutrient elements (Kloepper et al. 1989; Glick 1995; 125

Glick et al. 1999). The use of rhizobacteria in combination with plants increases 126

phytoremediation efficiency (Abou-Shanab et al. 2003a; Whiting et al. 2001). 127

Phytoremediation is often divided into four subsets as described in figure 1. Four subsets of 128

this technology, as applicable to toxic metal remediation from soil and water are; (i) 129

Phytoextraction – the use of metal–accumulating plants to remove toxic metals from soil; (ii) 130

Phytovolatilization – evaporation of certain metals from the aerial parts of the plant; (iii) 131

Phytostabilitzation - the use of plants to eliminate the bioavailability of toxic metals in soils; and 132

(iv) Rhizofiltration – the use of plant roots to remove toxic metals from polluted waters (Hooda 133

2007). Some plants capable of phytoremediation of heavy metals are described in Table 2. 134

135

2.1 Phytoextraction: 136

Phytoextraction, also termed phytoaccumulation, is the process of growing plants in metal-137

contaminated soil. Plant roots translocate the metals into the aboveground portions of the plant. 138

After plants have grown, they are harvested to recycle the metals or they are incinerated. If the 139

plants are incinerated, the ash can be deposited in a hazardous waste landfill (United States 140

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Department of Agriculture NRCS 2000). Some plants are referred to as hyperaccumulators as 141

they have the ability to accumulate heavy metals. For example,- some hyperaccumulators are 142

able to accumulate Zn in concentration higher than 1%, and Cu, Pb and Ni in concentration 143

higher than 0.1% of the tissue dry weight (Baker et al. 1994). The five most common 144

hyperaccumulator species, found associated with metal mining include arsenic 145

hyperaccumulators, Pteris vittata and Pteris cretica (Chen et al. 2002; Wei et al. 2002); Zn 146

hyperaccumulator, Sedum alfredii H (Yang et al. 2002); Cd hyperaccumulator, Viola 147

baoshanensis (Liu 2004); and Mn hyperaccumulator, Phytolacca acinosa (Xue et al. 2004). 148

Plants suitable for phytoextraction should ideally have the following characteristics 149

(Mejare and Bulow 2001; Tong et al. 2004; Adesodun et al. 2010; Sakakibara et al. 2011; 150

Shabani and Sayadi 2012): 151

152

i. High growth rate. 153

ii. Production of more above-ground biomass. 154

iii. Widely distributed and highly branched root system. 155

iv. More accumulation of the target heavy metals from soil. 156

v. Translocation of the accumulated heavy metals from roots to shoots. 157

vi. Tolerance to the toxic effects of the target heavy metals. 158

vii. Good adaptation to prevailing environmental and climatic conditions. 159

viii. Resistance to pathogens and pests. 160

ix. Easy cultivation and harvest. 161

x. Repulsion to herbivores to avoid food chain contamination. 162

163

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Hyperaccumulator plants have high metal accumulating capacity, but most of these plants 164

have a slow growth rate and often produce limited amounts of biomass when the concentration 165

of available metal in the contaminated soil is very high (Ali et al. 2012). 166

167

2.2 Phytovolatilization: 168

Phytovolatilization is the uptake of pollutants from soil by plants, transforming these 169

pollutants into a volatile form that is then released into the atmosphere. Phytovolatilization 170

occurs in growing trees and other plants when they take up water and the organic and inorganic 171

contaminants. These contaminants can pass through the plants to the leaves and volatilise into 172

the atmosphere at comparatively low concentrations (Mueller et al. 1999). For example P. 173

vittata, grown in a greenhouse was found to be effective at volatilizing As with the removal of 174

about 90% of the total uptake of As from contaminated soils (Sakakibara et al. 2010). 175

176

2.3 Phytostabilization: 177

Phytostabilization is the use of metal-tolerant plant species to immobilize heavy metals 178

through absorption and accumulation by roots, adsorption onto roots, or precipitation within the 179

rhizosphere. By this process, metal mobility and bioavailability of metals for its entry into the 180

food chain is greatly reduced (Wong 2003). Plants can immobilize heavy metals in soils through 181

sorption by roots, precipitation, complexation or metal valence reduction in the rhizosphere 182

(Barcelo and Poschenrieder 2003; Ghosh and Singh 2005; Yoon et al. 2006; Wuana and 183

Okieimen 2011). 184

Phytostabilization is a good option for large land areas where the removal of substantial 185

amounts of polluted soil is not a viable solution. In addition, wildlife can safely eat the plants, 186

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due to low accumulation of pollutants. Phytostabilization requires the growth of a healthy and 187

strong layer of plants before human activity can resume on the land being treated. 188

Yadav et al. (2009) reported that the use of organic amendments, such as dairy sludge 189

increases plant growth and reduces bioavailability of arsenic, chromium and zinc in soil, whereas 190

biofertilizers reduces the uptake of arsenic, chromium and zinc by plants. Jatropha curcas grows 191

in Cr-contaminated soil, and accumulates Cr in roots followed by shoots. Accumulation of Cr 192

induces oxidative stress in J. curcas and the plant is able to tolerate this stress through 193

hyperactivity of an antioxidant defense system (Yadav et al. 2010). Thus, non-edible and 194

economic plant species such as Jatropha curcas L. can be useful for the remediation of metalloid 195

and metal-contaminated soil. 196

197

2.4 Rhizofiltration: 198

Rhizofiltration is the removal of contaminants from flowing water; this can be achieved by 199

the plant itself or the microorganisms associated with the rhizosphere. Floating plants such as 200

water hyacinth and duckweed have been used in large-scale applications for the treatment of 201

municipal wastewater in Asia (Negri and Hinchman 1996). Roots of many hydroponically-grown 202

terrestrial plants, e.g., Indian mustard (Brassica juncea (L) Czern.), sunflower (Helianthus ennus 203

L), and various grasses, can effectively remove toxic metals such as Cu2+, Cd

2+, Cr

6+, Ni

2+, Pb

2+, 204

and Zn2+ from aqueous solution (Dushenkov et al. 1995). For example, Carex pendula 205

accumulates considerable amounts of lead, particularly in root biomass, and can be considered 206

for the cleanup of lead contaminated wastewaters in combination with proper biomass disposal 207

alternatives (Yadav et al. 2011). 208

209

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3. Factors influencing metals uptake by plants in soil 210

3.1 Soil Metal Concentration 211

Soils represent the major repository of trace elements over geologic time. Soils are formed 212

due to weathering of parent material and may contain different concentrations of heavy metals 213

near the surface of earth depending upon the climatic region (Krauskopf 1972). 214

The total metal concentration of soil includes both available and unavailable forms of 215

metals. Factors which influence the total metal concentration in soil include pH, organic matter, 216

clay, and redox conditions (as will be reviewed below). These factors along with available and 217

unavailable forms of metals determine how much of the soil pool will be available to plants 218

(Wolt 1994). Trace elements enter directly and pollute the soil and water by municipal wastes 219

and indirectly by industrial wastes (McCalla et al. 1977 and Thomas et al. 1977) and fertilizers, 220

or other soil additives (Allaway 1968) thereby increasing the concentration of metals in soil. 221

Lee et al. (2001) reported that there were particularly high levels of metals such Cd, Cu, Pb 222

and Zn in the tailings of Daduk Au–Ag–Pb–Zn mine in Korea. Elevated levels of Cd, Cu, Pb and 223

Zn were found in tailing with averages of 8.57, 481, 4,450 and 753 mg/kg, respectively. These 224

metals are continuously dispersed from the mine tailings and thus increase soil metal 225

concentration. The dispersion of metals may be due to clastic movement through wind and water, 226

especially during the wet season. According to the Korean Soil Environmental Conservation Act, 227

soils containing over 12 mg/kg of Cd, 200 mg/kg of Cu and 400 mg/kg of Pb extracted by 0.1N 228

HCl solution need to be continuously monitored and not used for agricultural purposes such as 229

crop planting (Lee et al 2001). Thus, these factors result in increasing metal concentrations in 230

soil and ultimately affect the phytoremediation process. 231

232

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3.2 Soil pH 233

The pH of the solution can greatly influence the equilibrium between speciation of metals, 234

solubility, adsorption and exchange on solid phase sites (Olomu et al. 1973; Kalbasi et al. 1978; 235

Cavallaro and McBride 1984; Sauve et al. 1997). Hence, various studies have found that metal 236

bioavailability is greatly affected by soil pH (Turner 1994; McBride et al. 1997). 237

The availability of uncomplexed ions for uptake by plants in soils mainly depends upon on 238

its solubility and thermodynamic activities (Jenne and Luoma 1977). Thus, for the successful 239

uptake by plant roots, the soluble species must occur near the vicinity of the root membrane. The 240

form of this soluble species present in soil will influence its persistence in soil solution, mobility, 241

and on the rate and extent of uptake, and most importantly its mobility and toxicity in plants 242

(Tiffin 1977). 243

EPA (1992) reported that pH directly or indirectly affects several mechanism of metal 244

retention and reported that adsorption of arsenic increased with pH. The adsorption of copper by 245

soils is also greatly dependent on pH (Cavallaro and McBride, 1980). McBride and Blasiak 246

(1979) reported that the concentration of Zn in solution increased with the increase in pH (i.e. 247

above pH 7.5). However, availability of zinc and magnesium is reported to increase with the 248

decrease in soil (Fergus 1954; McGrath et al. 1988; Turner 1994). Soil pH plays a crucial role in 249

directly or indirectly influencing in the sorption/desorption and complex formation. In general, 250

maximum retention of cationic metals occurs at pH >7 and for anion metals pH<7 (EPA 1992). 251

Thus, soil pH is known to affect plant uptake of most trace elements from soil. 252

253

3.3 Organic matter 254

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Ross (1994) reported that the organic matter in the solid phase, especially the humic 255

compounds of high molecular weight, strongly retain the metals in soils and reduces its 256

availability. Checkai et al. (1987) found that the increase in the formation of organo-metallic 257

compounds may increase the availability of trace metals to plants. Hence, bioavailability of 258

metals is inversely proportional to the organic matter in soils. For example, due to the formation 259

of strong complexes between soil organic matter and copper ions, the availability of copper 260

decreases with the increase in soil organic matter content (Stevenson 1976, 1991, del Castilho et 261

al. 1993) . In contrast, Mn2+ is less commonly associated with organic matter than is Cu and Zn 262

(McGrath et al. 1988) as Mn2+ forms weak coordination complexes with organic matter (Olomu 263

et al. 1973; McBride 1982). Thus, the complex of metal ions with organic matter greatly affects 264

its availability to plants. 265

266

3.4 Plant-bacteria interactions 267

For successful phytoremediation, the roots of plants are required to interact with a large 268

number of different microorganisms (Glick 1995). The functioning of associative plant-269

bacterial symbioses in heavy-metal-polluted soil can be affected by both micropartners (plant-270

associated bacteria) and the host plant. Soil microbes play crucial roles in the recycling of plant 271

nutrients, maintenance of soil structure, detoxification of noxious chemicals, and control of plant 272

pests and plant growth (Elsgaard et al. 2001; Filip 2002; Giller et al. 1998). Thus, the capacity of 273

plants used for remediation can be increased by the presence of bacteria in soil. For example, the 274

inoculation of Brassica napus seedlings with Pseudomonas chlororaphis SZY6, Azotobacter 275

vinelandii GZC24 and Microbacterium lactium YJ7 (EN) in the presence of copper, induced root 276

length promotion of both copper-treated and untreated seedlings (He et al. 2010). Ma et al. 277

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(2009) performed pot experiments by inoculating Brassica juncea with Achromobacter 278

xylosoxidans Ax10 (RS) in the presence of copper and found that this increased both Ni and Cr 279

uptake. Wu et al. (2006) reported the increase in plant growth and metal tolerance in Brassica 280

juncea when it was inoculated with Azotobacter chroococcum HKN-5, Bacillus megaterium 281

HKP-1 and Bacillus mucilaginosus HKK-1 in the presence of lead and zinc. Plants and bacteria 282

can form specific associations in which plants provide the bacteria with a carbon source that 283

helps bacteria to reduce the toxicity of the contaminated soil. Plants and bacteria can also form 284

nonspecific associations in which plant processes increases microbial community, which in the 285

course of normal metabolic activity, degrades contaminants in soil. Plant roots can provide root 286

exudate, as well as increase ion solubility. Thus, the remediation activity of plant roots 287

associated with bacteria is increased by this biochemical mechanism. 288

289

3.5 Heavy metal- bacteria interactions 290

The bioavailability of heavy metals can be altered by rhizobacteria (Lasat 2002; McGrath 291

et al. 2001; Whiting et al. 2001) through the release of chelating substances, acidification of the 292

microenvironment, and by changing the redox potential (Smith and Read 1997). For example, 293

the addition of Sphingomonas macrogoltabidus, Microbacterium liquefaciens, Microbacterium 294

arabinogalactanolyticum and Alyssum murale in serpentine soil was found to significantly 295

increase plant uptake of Ni as a result of soil pH reduction compared to control samples that 296

were not inoculated (Abou-Shanab et al. 2003a). Xian (1989) reported that the Eubacteria and the 297

Archaea are able to reduce Mn (IV), Fe (III), Co (II), AsO24- and SeO3

2 or oxidize Mn (II), Fe 298

(II), Co (III), AsO2-, Se

0 and conserve their energy in these reactions. Summers and Silver (1978) 299

found that prokaryotes were able to methylate metal and metalloid compounds, thereby 300

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producing volatile metal derivatives. The oxidation of AsO2- to AsO4

3- by Alcaligenes faeccalis 301

and reduction of CrO42- to Cr(OH)3 by Pseudomonas fluorescens LB 300 and Enterobacter 302

clocae are example of redox reactions (Wang and Shen 1995). However, excessive concentration 303

of heavy metals in soil are known to be toxic to both plants and most organisms. 304

305

4. Plants responses to heavy metal exposure 306

Plant tolerance to heavy metals is defined as the ability of plants to survive in soil that is 307

often toxic for other plants (Macnair et al. 2000). Plants have evolved different mechanisms to 308

tolerate excess concentrations of heavy metals with more than one mechanism often observed in 309

the same plant species (Hossain et al. 2011). The range of plant mechanisms to tolerate metal 310

stress is summarized in Figure 2 and can be divided into four stages (described in Table 3). 311

312

4.1 Immobilization by Mycorrhizal Associations: 313

The expectancy of mycorrhizal associations existing in heavy metal contaminated soils 314

has important implications for phytoremediation. Mycorrhizal associations increase the 315

absorptive surface area of the plant due to extra-matrical fungal hyphae exploring rhizospheres 316

beyond the root hair zone, which in turn enhance water and mineral uptake. Increase in water and 317

mineral uptake results in greater biomass production, an imperative for successful remediation. 318

The potential of phytoremediation can be enhanced by inoculating plants with mycorrhizal fungi. 319

Mycorrhizae have been found in plants growing on heavy metal contaminated sites and they play 320

a crucial role in phytoremediation as these fungi have evolved a heavy metal tolerance (Shetty et 321

al. 1995; Weissenhorn and Leyval 1995; Pawlowska et al. 1996; Chaudhry et al. 1998; Chaudhry 322

et al. 1999). Mycorrhizae provide an effective exclusion barrier to metal uptake by adopting 323

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absorption, adsorption or chelation mechanisms that restrict the entry of heavy metals into the 324

host plant (Hall 2002). In metal-contaminated soils, plant tolerance and accumulation depends 325

upon the mycorrhizal association between plant roots and fungi. According to Leyval et al. 326

(1997), the ectomycorrhizas (ECM) and arbuscular mycorrhizae (AM) are the most common 327

fungal association found in the plants growing in heavy-metal-contaminated soil. Mycorrhizae 328

particulary ECM that are characteristic of trees and shrubs, have been widely reported to be 329

effective in reducing the deleterious effects of heavy metals on the host plant (Marschner 1995; 330

Huttermann et al. 1999; Jentschke and Godbold 2000). 331

Establishment of native prairie grass community, consisting mostly of the seeded species 332

Elymus canadensis, was achieved on coarse taconite iron-ore tailing plots using amendments like 333

composted yard waste and arbuscular mycorrhizal fungi within one year, thus meeting-334

reclamation goals for the re-establishment of a sustainable native grass community (Noyd et al. 335

1996). Some researchers reported the presence of Glomus mosseae in heavy metal contaminated 336

sites (Debiane et al. 2008). It has been found that the external mycelium of certain AM fungi 337

produces a glycoprotein (Glomalin) which has heavy metal binding sites (Agely et al. 2005; 338

Citterio et al. 2005; Trotta et al. 2006; Vivas et al. 2003). Heavy metals present in soil 339

accumulate at these binding sites (Bano and Ashfaq 2013). Heavy metals are mostly accumulated 340

in fungal hyphae as well as in arbuscules. Joner et al. (2000) reported that the Glomus species 341

retained significant concentrations of Zn in mycelium when associated with clover or ryegrass. 342

Studies have indicated that the AM fungi release an extracellular, insoluble glycoprotein 343

(commonly known as glomalin) which can bind with Cu, Cd and Pb in polluted soils (Gonzalez-344

Chavez et al. 2004; Gohre and Paszkowski 2006). These authors reported that 1g of glomalin 345

was able to extract up to 4.3 mg copper, 0.08 mg cadmium and 1.12 mg lead from metal-346

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contaminated soils. Kaldorf et al. (1999) found that the Glomus species also immobilized metals 347

in maize. High concentration of metals is also stored in the mycorrhizal structures like arbuscules 348

and spores. For example, Chen et al. (2001) reported accumulation of Zn in fungal tissues of 349

Glomus mosseae up to 1200mgkg-1

and in G. versiforme up to 600 mgkg–1

. Colapaert and Van 350

Assche (1992) found that Zn content was reduced in Pinus sylestris as Zn is retained by the 351

ectomycorrhizal fungi Paxillus involutus. 352

Turnau et al. (2001) examined the AM fungi in roots of Fragaria vesca growing in Zn-353

contaminated soil and found that about 70% of the root sample was colonized with G. mosseae. 354

Griffioen et al. (1994) also reported the association of Scutellospora dipurpurascens with 355

Agrostis capillaries growing in the contaminated surroundings of a zinc refinery in the 356

Netherlands. Collectively, these examples provide strong evidence that fungi have evolved Zn 357

and Cd tolerance and play a crucial role in Zn and Cd tolerance in plants. 358

359

4.2 Role of plant cell wall in metal tolerance 360

Little is known about the role of the plant cell wall and its binding properties in relation to 361

metal tolerance. However, what is reported on has been somewhat controversial. In soil solution, 362

the root cell wall is in direct contact with the metals, so the adsorption of metals onto the cell 363

wall must be low. However, Mehes-Smith et al. (2013) reported that a significant amount of 364

metal accumulation occurred between the cell wall and the cell membrane. Divalent and trivalent 365

metal cations are able to bind to plants due to the presence of functional groups such as –COOH, 366

-OH and –SH in plant cell walls. The most important component of the plant cell wall is pectin 367

which consists of carboxyl groups. Under metal stress divalent and trivalent heavy metals ions 368

bind with the carboxyl group of pectin (Mehes-Smith et al. 2013). Other studies have also 369

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reported that the binding of the pectin carboxyl group with lead is the most important interaction 370

by which a plant can tolerate lead toxicity (Meyers et al. 2008; Jiang and Liu 2010). Bringezu et 371

al. (1999) reported that a range of metals accumulated in the epidermal cell walls of heavy-372

metal-tolerant Silene vulgaris ssp. humilis and these metals were found to be either bound to a 373

protein or to silicate. 374

The binding of lead to JIM5-P (within the cell wall) restrict the movement of metal to the 375

plasma membrane and act as a physical barrier in Funaria hygrometrica protonemata 376

(Krzesłowska et al. 2009). Krzesłowska et al. (2010) reported that the lead bound to JIM5-P 377

within the cell is either taken up or remobilized by endocytosis along with pectin epitope. 378

Elevated levels of Fe, Cu, Zn and Pb have been observed in cell walls of Minuartiaverna sp. 379

Hercynica growing on mine dumps (Solanki and Dhankhar 2011; Neumann et al. 1997). The 380

binding of copper can be prevented by decreasing pectin concentrations and increasing pectin 381

methylation in the cell walls of copper-tolerant Sileneparadoxa, thereby restricting copper 382

accumulation in roots (Colzi et al. 2012). 383

384

4.3 Role of root exudates 385

Roots exudates can be generally classified into two types, namely, high molecular weight 386

(HMW) (e.g. mucilage mainly polysaccharides and polyuronic acid and ectoenzymes) and low 387

molecular weight (LMW) (e.g. organic acids, sugars, phenols and various amino acids, including 388

non-protein amino acids such as phytosiderophores) materials. Root exudates play a significant 389

role in the processing of phytoremediation as an emerging in-situ, green remediation technology 390

using plants to absorb, accumulate, stabilize or volatilize contaminants from soil. 391

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Root exudates affect the metal solubility, mobility, and phytoavailability by reacting with 392

metal ions. The entry of metals in plants can be restricted by either immobilizing metals, thereby 393

limiting its entry to the plasma membrane (Colzi et al. 2011) or by mobilizing the metals from 394

root to shoot. Approximately half of the photosynthates produced in plants is retransported to 395

roots, whereas approximately 12-40% is released in the rhizophere during plant development as 396

exudates, including sugars and polysaccharides, organic and amino acids, peptides and proteins 397

(Lin et al. 2003; Hinsinger et al. 2006). The root secretions include carbohydrates, organic acids, 398

humic acids, polypeptides, proteins, amino acids, nucleic acids, etc. and the inorganic ligands 399

include Cl–, SO4

2–, NH4

+, CO3

2–, PO4

3– (Dong et al. 2007). These root secretions functions not 400

only as energy sources for microorganisms, but they also act as ligands which chelate heavy 401

metal ions and that ultimately influence the pH and Eh conditions in the rhizosphere. The change 402

in pH and Eh conditions is the main factor related to mobilization of metals in soils and their 403

accumulation in plants. Root exudates released from Echinochloa crusgalli releases citric acid 404

and oxalic acid, and increases the translocation of heavy metals such as Cd, Cu and Pb from 405

roots to shoots (Kim et al. 2010; Zhou et al. 2006). Fan et al. (2001) reported that in barley and 406

wheat there was reduced phytosiderophore production in the presence of Cd thereby enhancing 407

the transition metal. Root exudates could also increase solubility of metal ions in soils and 408

consequently increase their accumulation in plants. 409

Graminaceous plant species (e.g. paddy rice) secrete phytosiderophore (amino acids) that 410

form much more stable complexes than carboxylate with Fe, Cd, Zn and Cu (Römheld 1991; 411

Hinsinger 1998; Chaignon et al. 2002; Xu et al. 2005). Plants secrete low molecular weight 412

organic acids that play a crucial role in solubility and availability of heavy metals. Organic acids 413

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(e.g. oxalic acid, malic acid and citric acid) secreted by plants, such as wheat and buckwheat 414

prevent the entrance of Cd2+ into roots (Meach and Martin 1991). 415

In a study by Nair et al. (2007), they found that P. azotoformans produced a mixed type of 416

siderophore including both catecholate and hydroxamate that was able to remove almost 92.8% 417

of accumulated arsenic as compared to control plants which only removed 33.8%. Nair et al. 418

(2008) studied the speciation of metals in an industrial sludge and highlighted the prospect of the 419

use of siderophores for bioremediation due to their biodegradable and ecofriendly characteristics. 420

Other studies have shown that the Ni-chelating histidine and citrate accumulate in root 421

exudates and help to reduce Ni uptake, thereby playing a crucial role in Ni-detoxification 422

strategies (Salt et al. 2000; Hall 2002). Persans et al. (1999) proposed that in Thlaspi 423

goesingense, Ni hyperaccumulation is not determined by the overproduction of histidine in 424

response to Ni. Krämer et al. (2000) found that in T. goesingense the uptake of Ni and its 425

accumulation across the cytoplasm into the vacuole is restricted by the free histidine, which 426

could be responsible for Ni tolerance and accumulation. Kerkeb and Krämer (2003) also showed 427

that in A. lesbiacum and B. juncea the release of Ni from roots to the xylem is enhanced by 428

histidine. 429

Salt et al. (2000) provided evidence for Zn-histidine complexes in the roots of Zn 430

hyperaccumulator Thlaspi caerulescens. However, this is in contrast to other studies that did not 431

find any such complex related to the accumulation of Zn by T. caerulescens (Knight et al. 1997; 432

McGrath et al. 1997; Zhao et al. 2001). Other studies proposed that the mechanism of Cu 433

exclusion in T. aestivum involve phytochelatins citrate and malate (Yang et al. 2005; Bálint et al. 434

2007). 435

436

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4.5 Restriction of metal uptake through plasma membrane 437

The plant plasma membrane functions as the first line of defence for heavy metal 438

contamination as it is the first cell structure that is exposed to heavy metals. The plasma 439

membrane restricts the uptake and accumulation of metals by inhibiting its entry into the 440

cytoplasm (Mehes-Smith et al. 2013). The restriction of metals to the plasma membrane can be 441

achieved by changing the cell wall binding capacity to metal ions or by modifying the ion 442

channels present on the membrane or by modifying the efflux pumps or with the root exudates 443

(Tong et al. 2004). Wainwright and Woolhouse (1977) found that Cu increases the efflux of K+ 444

from the excised roots of Agrostis capillaries but this is not the case with Zn. Iwasaki et al. 445

(1990) showed that 60% of Cu in Italian ryegrass was found to be bound by the cell wall and the 446

plasma membrane. 447

In Holcus lanatus the suppression of high affinity arsenate transport system absorbs less 448

arsenate (Meharg and Macnair 1992) along with the synthesis of phytochelatins (Hartley-449

Whitaker et al. 2001). According to Manara (2012) a heavy metal efflux pump in plant is most 450

likely to be P1B-ATPases and the CDF families of transporters (based on the sequence similarity 451

to microbial and animal proteins). Further work has suggested that the P1B-type ATPases belong 452

to P-type ATPase superfamily and to translocate the metal ions across the membrane it uses ATP 453

as an energy source (Axelsen and Palmgren 2001). 454

455

4.6 Phytochelatins (PCs) 456

The toxic effect of metals ions in cytosol can be eliminated by specific high affinity ligands 457

such as phytochelatins (PCs). PCs were first discovered in fission yeast as cadmium-binding 458

“cadystins A and B”. Various studies have confirmed that PCs are synthesized from GSH by the 459

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help of enzyme γ-glutamyl cysteinyl dipeptidyl transpeptidase (PC synthase) (Zenk 1996). 460

Phytochelatins are rich in cysteine non-protein metal binding peptides produced by plants (Schat 461

and Kalif 1992; Zenk 1996). PCs belong to the family of metal-complexing peptides having a 462

general structure of (c-Glu-Cys)nGly (n = 2–11) (Cobbett and Goldsbrough 2002). In the 463

cytosol, PCs are synthesized and then transported to the vacuole as a complex. In the presence of 464

heavy metals such as Cd, Cu, Zn, Ag, Au, Hg, and Pb PCs are rapidly induced (Rauser 1995; 465

Cobbett 2000), with Cd being the strongest inducer (Grill et al. 1987, 1989). PCs complex with 466

Cd ions with the aid of the thiolic group (-SH) of cys and this complex is accumulated in the 467

vacuole by the activity of ABC transporters (Di Toppi and Gabbrielli 1999).The relationship 468

between PC synthesis and Cd accumulation in Sedum alfredii was studied by Zhang et al. (2010) 469

and their results suggested that PCs act as an intercellular Cd detoxification mechanism in shoots 470

rather than roots. 471

Galli et al. (1996) reported that Zea mays synthesizes PCs in the presence of excess Cu and 472

Cd. However, this was not tested against a control or sensitive cultivar. Maitani et al. (1996) 473

showed that Rubia tinctorum root cultures initiated the synthesis of PCs when exposed to a 474

number of metals such as Zn, Cu and Cd, and also tested it against controls plants in which no 475

phytochelatin production was observed. 476

In a study by Schat and Kalif (1992) tolerant and non-tolerant strains of Silene vulgaris 477

were exposed to different concentrations of Cu did not show any PCs synthesis and thus they 478

concluded that PCs are not involved in the tolerance mechanism in S. vulgaris. This could be due 479

to the lower ratio of PCs to Cu. de Knecht et al. (1994) also found that PCs do not play any role 480

in Cd tolerance in S. vulgaris. 481

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Techniques such as X-ray absorption spectroscopy (XAS), high performance liquid 482

chromatography-mass spectrometry (HPLC-MS), and inductively coupled plasma optical 483

emission spectrometer (ICP- OES) have been used to understand the role of phytochelatins in 484

cadmium tolerance in plants. Salt et al. (1997) showed that in Indian mustard seedlings the 485

percentage of Cd bound to PCs increased by 34% after 6 hours of Cd exposure and by 60% after 486

70 hours. 487

In Brassica juncea it has been shown that PC synthesis increases as Cd accumulation is 488

increased intracellularly, thereby protecting the photosynthetic unit without decreasing the 489

transpiration rate (Haag-Kerwer et al. 1999). Xiang and Oliver (1998) showed that treatment of 490

A. thaliana with Cd and Cu results in an increase of transcriptional genes that are involved in the 491

synthesis of PC precursor, glutathione. 492

Jambhulkar and Juwarkar (2009) found that Cassia siamea accumulate Ni, Cr and Pb at 493

higher concentrations compared to other species. Higher accumulation of all the metals was 494

observed in C. siamea because this plant that was grown on a fly ash dump contains non-protein 495

thiols, which is a marker of phytochelatin synthesis and is responsible for metal accumulation. 496

Thus it could be grown easily on fly ash dumps as it acts as a hyperaccumulator plant. 497

Jatropha curcus accumulates high concentrations of Cr in roots. Despite this high 498

accumulation in roots, the level of free Cr ions in roots may remain low since most of the Cr ions 499

are either immobilized or compartmentalized in vacuoles or form Cr–phytochelatin complexes 500

(Yadav et al. 2010). 501

502

4.7 Metallothioneins (MTs) 503

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Evidence for the role of metallothioneins (MTs) in plant metal tolerance was given by 504

Rauser (1984), who found Cu-binding low molecular weight protein in Agrostis gigantea. MTs 505

are low molecular weight cysteine-rich metal-binding peptides and can be classified into two 506

classes: Class 1: Consist of 61 amino acids and lack aromatic amino acid or histidines that are 507

related to mammals; Class 2: These MTs are related to Candida albicansa yeast or cyanobacteria 508

(Winge et al. 1985). 509

MTs are not only expressed under various abiotic stresses in plants but also expressed 510

during plant development (Rauser 1999). Plant MTs sequester metal ions by complexing metals 511

ions with multiple cysteine thiol groups (Robinson et al. 1993). Various stimuli have been found 512

which can upregulate the expression of MT genes in plants. Natural senescence (Bhalerao et al. 513

2003), hormones such as ABA (Reynolds and Crawford 1996), ethylene (Coupe et al. 1995), 514

wound and infection by virus (Choi et al. 1996), heat shock proteins (Hsieh et al. 1995), nutrient 515

starvation such as sucrose (Hsieh et al. 1996), UV-light (Foley and Singh 1994), cold and salt 516

stress (Reid and Ross 1997) were found to increase the expression of MT genes. 517

In Brassica rapa three different MT genes are regulated differently under various heavy 518

metal stress. When Brassica rapa seedlings were treated with Fe, there was an increased 519

expression of BrMTat after 6 and 24h. When Brassica rapa seedlings were treated with Zn the 520

expression of BrMT2 was downregulated and BrMT1 expression was increased whereas BrMT3 521

remained unchanged. In Mn-treated seedlings, BrMT1 and BrMT3 genes were upregulated until 522

12h and then downregulated. The expression of BrMT2 was affected for the first 12h, after it was 523

downregulated (Ahn et al. 2012). 524

525

4.8 Compartmentalization of heavy metal in vacuoles 526

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Once a heavy metal has entered the cell, the plant copes with the toxicity of heavy metal 527

either by transporting the heavy metal out of the cell or by sequestrating it into the vacuole, 528

thereby limiting the contact of heavy metals with the sensitive metabolic activities taking place 529

in the cytosol or other cellular compartments (DalCorso et al. 2008; Dalcorso et al. 2010; 530

Clemens 2001). Heavy metal uptake has been driven by either channels or transporters. Some 531

examples of heavy metal transporters include protein ZIP family, ABC transporters (ATP- 532

binding cassette), P-type metal ATPases, members of the CDF transporter family (also called 533

MTPs in plants), zinc regulated transporter AtMTP1 (Kramer et al. 2007), the natural resistance-534

associated macrophage protein (NRAMP) family, CAX family involved in the vacuolar 535

accumulation of Cd, copper transporter (COPT) family proteins, pleiotropic drug resistance 536

(PDR) transporters, and yellow-stripe-like (YSL) transporter (Lee et al. 2005, Chiang et al. 2006; 537

Kramer et al. 2007). 538

Earlier reports showed that a number of heavy metals including Zn and Cd have been 539

accumulated in the vacuole (Ernst et al. 1992; De 2000). For example Alyssum serpytllifolium, is 540

a nickel hyperaccumulator that accumulated 72% of cellular Ni in the vacuole (Brooks et al. 541

1980). Davies et al. (1991b) reported the increased vacuolation of Zn in Festuca rubra roots. 542

Phragmites australis showed increased Zn sequestration in the vacuole or that it was 543

immobilized in the apoplast (Jiang and Wang 2008). CDF transporter family (also called MTPs 544

in plants) are reported to be involved in efflux of transition metals such as Zn2+, Cd

2+, Co

2+, Ni

2+ 545

or Mn2+ from cytoplasm. Van der Zaal et al. (1999) reported ZAT1 transporter, a member of 546

CDF family, found in A. thaliana to sequester zinc in the vacuole. The transporter AtHMA3, a 547

member of P1B-ATPases, may play a role in the detoxification Cd, Pb, Co and Zn through 548

storage in the vacuoles (Morel et al. 2009). 549

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5. Conclusions 550

The problem of heavy metal pollution is continuously increasing due to the acceleration of 551

many human activities, leading to an intensification of research dealing with the phytotoxicity of 552

these contaminants as well as the mechanisms used by plants to counter their harmful effects. It 553

is clear that plants play a crucial role in the remediation of metal-enriched soils. This review 554

focused on the potential cellular and molecular adaptations by plants that are necessary to 555

tolerate heavy metal stress. Significant progress has been made in the identification and 556

understanding the role of key components like metal transporters, hyper accumulation, 557

phytochelatins and metallothionein proteins that ensure heavy metal tolerance to plants. 558

Phytoremediation is a fast developing field, and over the past ten years field applications 559

have been initiated world-wide, including phytoremediation of organic, inorganic and 560

radionuclides. This sustainable and inexpensive process is fast emerging as a viable alternative to 561

more conventional remediation methods, and will be most suitable for developing countries such 562

as India. Although phytoremediation offers some advantages over more commonly used 563

conventional technologies such as being cost-effective and eco-friendly, this technique requires 564

careful consideration of several factors in order to accomplish effective, high performance 565

results. The most important factor is the use of a suitable plant species which can be used to 566

uptake the particular contaminant. It is important to understand that although the 567

phytoremediation technique is often the best alternative, like all technologies it does have 568

limitations. These include a restricted surface area and, depth occupied by the roots, and the 569

inherent slow growth and low biomass requires a long-term commitment to the remediation 570

project. Continued research is required to minimize these limitations so that this technique can be 571

applied most effectively.572

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Acknowledgement 573

The authors are grateful to TEQIP-II and MNNIT, Allahabad for financial support for this 574

research. Ms. Anamika Kushwaha and Ms. Aishvarya Gautam, acknowledge TEQIP-II for their 575

financial support in the form of fellowship. The authors are also thankful to editors and reviewers 576

for improving the manuscript. 577

578

579

580

581

582

583

584

585

586

587

588

589

590

591

592

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596

597

598

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Figure 1: Types of Phytoremediation

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Figure 2: Summary of cellular mechanism for detoxification and tolerance of metals by plants. 1. Immobilization by mycorrhizal

associations. 2. Restriction of heavy metal by binding to plant cell wall. 3. Chelation of heavy metals by root exudates such as sugars

and polysaccharides, organic and amino acids, peptides and proteins. 4. Reduced influx of heavy metals by plasma membrane. 5.

Active efflux of HM. 6. Chelation of HM by PCs, MTs, acids. (PCs= Phytochelatins; MTs= Metallothionein; HM= Heavy metals).

HEAVY METAL

1.Immobilization by Mycorrhizal

Associations

2. Binding to plant cell wall 3. Chelation by plant root exudates

6. Chelation by various ligands: PCs, MTs, acids

Ligand-metal complex

VACUOLE

4. Reduced influx by

plasma membrane

5. Active efflux of

metal HM

CYTOSOL

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Table 1: Toxic effects of heavy metals on plants

Zinc Limit the growth of both root and shoot; Chlorosis;

Senescence; Inhibit metabolic functions

Choi et al. 1996; Ebbs and Kochian 1997; Fontes and

Cox 1998.

Copper Growth retardation; Leaf chlorosis; oxidative

stress and ROS formation

Lewis et al. 2001; Stadtman and Oliver 1991.

Nickel Chlorosis; Necrosis; Nutrient imbalance; Disorder of cell

membrane functions

Zornoza et al. 1999; Pandey and Sharma 2002;

Rahman et al. 2005.

Mercury Obstruction of water flow in plants; interfere the mitochondrial

activity and induces oxidative stress; disruption of

biomembrane lipids and cellular metabolism

Zhou et al. 2007; Zhang and Tyerman 1999.

Lead Effect morphology, growth and photosynthetic processes;

enzyme inhibition; water imbalance; Alteration in membrane

permeability, Oxidative stress

Sharma and Dubey 2005; Reddy et al. 2005.

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Chromium Chlorosis; Nutrient and water imbalance; Inhibition of

chlorophyll biosynthesis; wilting of tops; root injury; enzyme

inhibition; oxidative stress; inhibition of plant growth

Chatterjee and Chatterjee 2000; Dixit et al. 2002;

Sharma et al. 2003; Scoccianti et al. 2006; Vajpayee et

al. 2000; Shanker et al. 2005.

Arsenic

Inhibits photosynthesis, Inhibits growth, Biomass and yield,

Death

Marques and Anderson 1986; Carbonell et al. 1998.

Cobalt Affect shoot growth and biomass; Decrease chlorophyll,

protein and catalase activity; decrease water potential and

transpiration rate

Li et al. 2009; Chatterjee and Chatterjee 2000.

Cadmium Reduction in photosynthesis, water uptake, and nutrient

Uptake, chlorosis, growth inhibition, browning of root tips, and

finally death

Wójcik and Tukiendorf 2004;

Mohanpuria et al. 2007.

Copper Growth retardation, leaf chlorosis, Oxidative stress, damage to

macromolecules

Lewis et al. 2001; Stadtman and Oliver 1991; Hegedus

et al. 2001.

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Table 2: Plants capable for phytoremediation of heavy metals

S.No. Techniques used Plant Species Metals References

1 Phytoextraction

Arabidopsis halleri Zn Reeves and Backer 2000.

Sonchus asper and

Corydalis pterygopetata

Pb and Zn

Yanqun et al. 2005.

Alyssum bertolonii Ni Chaney et al. 2000; Li et al. 2003b.

Pteris vittata

As Ma et al. 2001.

Haumaniastrum robertii Co Reeves and Baker 2000.

Aeollanthus subacaulis Cu

Maytenus bureaviana Mn

Minuartia verna and Agrostis tenuis Pb

Dichapetalum gelonioides, Thlaspi

tatrense, and Thlaspi caerulescens

Zn

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Psycotria vanhermanni and Streptanthus

polygaloides

Ni

Lecythisollaria

Se

Sedum alfredii Cd Yang et al. 2004.

Pteris vittata As Ma et al. 2001.

2 Phytovolatilization Brassica juncea

Se

Masayuki Sakakibara et al. 2007.

Brassica napus Se Banuelos and Meek 1990; Banuelos et

al. 1993.

Chenopodium album L.; Zn Banuelos et al. 1997.

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Solanum nigrum

3 Phytostabilization Carex pendula Pb Walker et al. 2004; Marques et al.

2007a.

4 Rhizofilteration Helianthus annuus L. Cd and Pb Niu et al 2007.

Sedum alfredii Zn, Cd, Pb Yang et al. 2004; He et al. 2002.

Sesbania drummondii Pb Sahi et al. 2002.

Fagopyrum esculentum Cr Kleiman and Cogliatti 1998.

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Table 3: Cellular mechanism for detoxification of metals in plants

Stages Mechanism

1 Exclusion

AOS Scavenging Occurs in plant Apoplast

Signal perception

2 Phytochelatins- compartmentalization

AOS Scavenging

3 Metallothioniene synthesis

Stress protein synthesis

Cell wall lignification Occur at whole plant level

Organic acid synthesis

Sulphur metabolism

4 Mycorrhizal protection

Development of new root

Repair mechanism

Plant level detoxification

Occur in Symplast

Occur at whole plant level

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draft - university of toronto t-space · like other organisms, ... (1980) classified metals into...

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