Heavy Metal Detoxification and Tolerance Mechanisms in Plants: Implications For Phytoremediation

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

2 phytoremediation
4 Anamika Kushwahaa, Radha Rania*, Sanjay Kumara, Aishvarya Gautama
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Environ. Rev. Downloaded from www.nrcresearchpress.com by DALHOUSIE UNIVER on 10/11/15
7 *Department of Biotechnology
8 Motilal Nehru National Institute of Technology,
9 Teliyar Ganj, Allahabad
10 Telephone: +91-532-2271240
11 Fax: +91-532-2271200
12 E-mail: radharani@mnnit.ac.in;
13 raadharaani1982@gmail.com
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15 Department of Biotechnology
16 Motilal Nehru National Institute of Technology,
17 Teliyar Ganj, Allahabad
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19 *corresponding author
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For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 2 of 70
30 Abstract
31 Heavy metals such as cobalt, copper, manganese, molybdenum, and zinc are essential in trace
32 amounts for growth by plants and other living organisms. However, in excessive amounts these
33 heavy metals have deleterious effects. Like other organisms, plants possess a variety of
34 detoxification mechanisms to counter the harmful effects of heavy metals. These include, the
35 restriction of heavy metals by mycorrhizal association, binding with plant cell wall and root
36 excretions, metal efflux from the plasma membrane, metal chelation by phytochelatins and
37 metallothioneins, and compartmentalization within the vacuole. Phytoremediation is an emerging
38 technology which uses plants and their associated rhizospheric microorganisms to remove
39 pollutants from contaminated sites. This technology is inexpensive, efficient and ecofriendly.
40 This review focuses on potential cellular and molecular adaptations by plants that are necessary
41 to tolerate heavy metal stress.
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43 Keywords: Detoxification; Heavy metals; Phytoremediation; Phytochelatins; Metallothioneins;
44 Mechanism.
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46 1. Introduction
47 A heavy metal is a member of ill-defined chemical elements that exhibit metallic properties
48 and occur naturally in the earth’s crust. Heavy metals cannot be degraded and are stable in the
49 environment, resulting in their accumulation over time causing soil pollution. Heavy metals such
50 as cobalt, copper, manganese, molybdenum, vanadium, strontium and zinc are required in trace
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51 amounts by plants and other living organisms, but in excessive amounts these heavy metals have
52 harmful effects. Heavy metals such as mercury, lead and cadmium have no known beneficial
53 effects to organisms.
54 Nieboer and Richardson (1980) classified metals into three groups based on ligand binding
55 preferences:
56 1. Class A metals prefer ligands with available oxygen (e.g. Li+, Na+, K+, Cs+, Be2+, Mg2+,
57 Ca2+, Sr2+, Ba2+, Al3+, Ga3+, Sc3+, and Y3+).
58 2. Class B metals bind to ligands containing sulfur or nitrogen (e.g. Tl+, Tl3+, Pb4+, Bi3+,
59 Pd2+, Pt2+, Cu+, Ag+, Au+, and Hg2+).
60 3. Class C metals have binding properties that are immediate between those of classes A
61 and B (e.g. Ga3+, In3+, Sn4+, Pb2+, As3+, Sb3+, Ti2+, V2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+,
62 Zn2+, Cd2+).
63 Nieboer and Richardson (1980) hypothesized that, due to specific ligand binding
64 preferences, an identical effect was observed in different organisms. They further postulated that
65 metals of the highest toxicity were included in class B (e.g. Ag+, Tl+, Hg2+, Cd2+). Class B
66 elements do not occur naturally and were found to bind strongly with cysteine (containing SH
67 group) and lysine (nitrogen containing groups) (Shaw et al. 2004).
68 The Industrial Revolution led to the increase in pollution by many folds and ultimately led
69 to the alteration of geochemical cycles as well as a shift in the balance of some heavy metals.
70 Heavy metal contamination of urban and agriculture soils is largely the result of anthropogenic
71 activities such as use of the pesticides, mining, nuclear wastes, disposal of munitions and agents
72 of war, combustion of fossil fuel, and industrial waste. However, natural processes such as
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73 volcanic eruptions, continental dusts, and other natural sources can add to the level of
74 contamination.
75 In the atmosphere, volatile heavy metals attach to particulates and can become widely
76 distributed throughout the atmosphere, traveling several miles from the site of release. Thus, the
77 lighter and smaller the particle, the greater its persistence in air (Department for Environment
78 Food and Rural Affairs, 2008). The accumulation of heavy metals in soil can be lethal for all
79 biota. Heavy metals are toxic as they can replace essential metals in pigments or enzymes,
80 thereby disrupting their natural function (Henry 2000). If a heavy metal is exposed for a long
81 period of time, its effect becomes chronic as it is transferred up the food chain. Long-term
82 exposure to humans of heavy metals like lead, cadmium and arsenic will lead to health issues
83 including mental lapse, skin poisoning, kidney and liver malfunction, gastro-intestinal tract and
84 central nervous system disorders (USDA NRCS, 2000).
85 Excessive accumulation of heavy metals in soil is toxic to most plants. Plant roots absorb
86 heavy metal ions from the environment in excessive concentration and translocate them to their
87 shoots, which affects metabolism and stunts growth (Bingham et al. 1986; Foy et al. 1978).
88 Elevated levels of metal concentrations in contaminated soil result in loss of soil fertility,
89 agricultural yield, and decrease in soil microbial activity (McGrath et al. 1995). Cadmium is
90 most commonly accumulated by agriculturally important crops and leads to a decrease in root
91 and shoot growth, and a decrease in nutrient uptake and homeostasis (di Toppi and Gabrielli
92 1999). Thus, when these crops are consumed by organisms, it may cause severe health effects.
93 With continued increases in cadmium levels, agricultural soil will become unusable for crop
94 production. Similarly, soil contamination with cadmium leads to loss of biodiversity and activity
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95 of soil microbial communities (McGrath 1994). Some of the toxic effects of heavy metals on
96 plants are described in Table 1.
97 Many regulatory steps have been implemented and practiced to restrict the release of metal
98 pollutants into soils but they are typically not sufficient for fully evaluating contamination levels
99 (Ghosh and Singh 2005). Techniques employed for remediation of metal contaminated soil
100 include chemical, physical and biological methods (Baker and Walker 1990). Due to the high
101 cost and low efficiency of many methods, such as excavation and landfill, thermal treatment,
102 acid leaching and electroreclamation, most are not suitable for practical and fully effective
103 applications. Moreover, these methods may cause a pronounced destruction of soil structure,
104 fertility, and other properties.
105 Recently, work has been ongoing to develop cost-effective and high-efficiency
106 technologies for the remediation of heavy metal contaminated sites (Chatterjee et al. 2011). To
107 this end, plants can be used as a possible means for the remediation of heavy metals from
108 contaminated soil (termed as phytoremediation) and is considered to be a green solution.
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110 2. PHYTOREMEDIATION: AN OVERVIEW
111 Phytoremediation has recently become the subject of more intense public and scientific
112 interest. For chemically polluted lands, vegetation plays an increasingly important ecological and
113 sanitary role. Proper management of plants in such areas may significantly contribute to restoring
114 the natural environment. The term phytoremediation comes from the Ancient Greek word phyto
115 meaning “plant” and the Latin word remedium meaning “restoring balance.” It is a technology
116 that uses plants to treat environmental pollution problems. Plants are used either to remove or to
117 stabilize (hold in place) pollution in the soil.

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118 Microbial populations are large in rhizospheric soils with high metabolic activity compared
119 to bulk soil (Anderson et al. 1993). Microbial populations are known to affect heavy metals
120 mobility and availability to the plant through the release of chelating agents, acidification,
121 phosphate solubilization, and redox changes (Abou-Shanab et al. 2003a; Smith and Read 1997).
122 When applied to seeds or incorporated into soils some plant growth-promoting bacteria that are
123 associated with plant roots may also exert beneficial effects on plant growth and nutrition
124 through a number of mechanisms such as N2 fixation, production of phytohormones and
125 siderophores, and the transformation of nutrient elements (Kloepper et al. 1989; Glick 1995;
126 Glick et al. 1999). The use of rhizobacteria in combination with plants increases
127 phytoremediation efficiency (Abou-Shanab et al. 2003a; Whiting et al. 2001).
128 Phytoremediation is often divided into four subsets as described in figure 1. Four subsets of
129 this technology, as applicable to toxic metal remediation from soil and water are; (i)
130 Phytoextraction – the use of metal–accumulating plants to remove toxic metals from soil; (ii)
131 Phytovolatilization – evaporation of certain metals from the aerial parts of the plant; (iii)
132 Phytostabilitzation - the use of plants to eliminate the bioavailability of toxic metals in soils; and
133 (iv) Rhizofiltration – the use of plant roots to remove toxic metals from polluted waters (Hooda
134 2007). Some plants capable of phytoremediation of heavy metals are described in Table 2.
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136 2.1 Phytoextraction:
137 Phytoextraction, also termed phytoaccumulation, is the process of growing plants in metal-
138 contaminated soil. Plant roots translocate the metals into the aboveground portions of the plant.
139 After plants have grown, they are harvested to recycle the metals or they are incinerated. If the
140 plants are incinerated, the ash can be deposited in a hazardous waste landfill (United States
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141 Department of Agriculture NRCS 2000). Some plants are referred to as hyperaccumulators as
142 they have the ability to accumulate heavy metals. For example,- some hyperaccumulators are
143 able to accumulate Zn in concentration higher than 1%, and Cu, Pb and Ni in concentration
144 higher than 0.1% of the tissue dry weight (Baker et al. 1994). The five most common
145 hyperaccumulator species, found associated with metal mining include arsenic
146 hyperaccumulators, Pteris vittata and Pteris cretica (Chen et al. 2002; Wei et al. 2002); Zn
147 hyperaccumulator, Sedum alfredii H (Yang et al. 2002); Cd hyperaccumulator, Viola
148 baoshanensis (Liu 2004); and Mn hyperaccumulator, Phytolacca acinosa (Xue et al. 2004).
149 Plants suitable for phytoextraction should ideally have the following characteristics
150 (Mejare and Bulow 2001; Tong et al. 2004; Adesodun et al. 2010; Sakakibara et al. 2011;
151 Shabani and Sayadi 2012):
152
153 i. High growth rate.
154 ii. Production of more above-ground biomass.
155 iii. Widely distributed and highly branched root system.
156 iv. More accumulation of the target heavy metals from soil.
157 v. Translocation of the accumulated heavy metals from roots to shoots.
158 vi. Tolerance to the toxic effects of the target heavy metals.
159 vii. Good adaptation to prevailing environmental and climatic conditions.
160 viii. Resistance to pathogens and pests.
161 ix. Easy cultivation and harvest.
162 x. Repulsion to herbivores to avoid food chain contamination.
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164 Hyperaccumulator plants have high metal accumulating capacity, but most of these plants
165 have a slow growth rate and often produce limited amounts of biomass when the concentration
166 of available metal in the contaminated soil is very high (Ali et al. 2012).
167
168 2.2 Phytovolatilization:

169 Phytovolatilization is the uptake of pollutants from soil by plants, transforming these
170 pollutants into a volatile form that is then released into the atmosphere. Phytovolatilization
171 occurs in growing trees and other plants when they take up water and the organic and inorganic
172 contaminants. These contaminants can pass through the plants to the leaves and volatilise into
173 the atmosphere at comparatively low concentrations (Mueller et al. 1999). For example P.
174 vittata, grown in a greenhouse was found to be effective at volatilizing As with the removal of
175 about 90% of the total uptake of As from contaminated soils (Sakakibara et al. 2010).
176
177 2.3 Phytostabilization:
178 Phytostabilization is the use of metal-tolerant plant species to immobilize heavy metals
179 through absorption and accumulation by roots, adsorption onto roots, or precipitation within the
180 rhizosphere. By this process, metal mobility and bioavailability of metals for its entry into the
181 food chain is greatly reduced (Wong 2003). Plants can immobilize heavy metals in soils through
182 sorption by roots, precipitation, complexation or metal valence reduction in the rhizosphere
183 (Barcelo and Poschenrieder 2003; Ghosh and Singh 2005; Yoon et al. 2006; Wuana and
184 Okieimen 2011).
185 Phytostabilization is a good option for large land areas where the removal of substantial
186 amounts of polluted soil is not a viable solution. In addition, wildlife can safely eat the plants,
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187 due to low accumulation of pollutants. Phytostabilization requires the growth of a healthy and
188 strong layer of plants before human activity can resume on the land being treated.
189 Yadav et al. (2009) reported that the use of organic amendments, such as dairy sludge
190 increases plant growth and reduces bioavailability of arsenic, chromium and zinc in soil, whereas
191 biofertilizers reduces the uptake of arsenic, chromium and zinc by plants. Jatropha curcas grows
192 in Cr-contaminated soil, and accumulates Cr in roots followed by shoots. Accumulation of Cr
193 induces oxidative stress in J. curcas and the plant is able to tolerate this stress through
194 hyperactivity of an antioxidant defense system (Yadav et al. 2010). Thus, non-edible and
195 economic plant species such as Jatropha curcas L. can be useful for the remediation of metalloid
196 and metal-contaminated soil.
197
198 2.4 Rhizofiltration:
199 Rhizofiltration is the removal of contaminants from flowing water; this can be achieved by
200 the plant itself or the microorganisms associated with the rhizosphere. Floating plants such as
201 water hyacinth and duckweed have been used in large-scale applications for the treatment of
202 municipal wastewater in Asia (Negri and Hinchman 1996). Roots of many hydroponically-grown
203 terrestrial plants, e.g., Indian mustard (Brassica juncea (L) Czern.), sunflower (Helianthus ennus
204 L), and various grasses, can effectively remove toxic metals such as Cu2+, Cd2+, Cr6+, Ni2+, Pb2+,
205 and Zn2+ from aqueous solution (Dushenkov et al. 1995). For example, Carex pendula
206 accumulates considerable amounts of lead, particularly in root biomass, and can be considered
207 for the cleanup of lead contaminated wastewaters in combination with proper biomass disposal
208 alternatives (Yadav et al. 2011).
209
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210 3. Factors influencing metals uptake by plants in soil
211 3.1 Soil Metal Concentration
212 Soils represent the major repository of trace elements over geologic time. Soils are formed
213 due to weathering of parent material and may contain different concentrations of heavy metals
214 near the surface of earth depending upon the climatic region (Krauskopf 1972).
215 The total metal concentration of soil includes both available and unavailable forms of
216 metals. Factors which influence the total metal concentration in soil include pH, organic matter,
217 clay, and redox conditions (as will be reviewed below). These factors along with available and
218 unavailable forms of metals determine how much of the soil pool will be available to plants
219 (Wolt 1994). Trace elements enter directly and pollute the soil and water by municipal wastes
220 and indirectly by industrial wastes (McCalla et al. 1977 and Thomas et al. 1977) and fertilizers,
221 or other soil additives (Allaway 1968) thereby increasing the concentration of metals in soil.
222 Lee et al. (2001) reported that there were particularly high levels of metals such Cd, Cu, Pb
223 and Zn in the tailings of Daduk Au–Ag–Pb–Zn mine in Korea. Elevated levels of Cd, Cu, Pb and
224 Zn were found in tailing with averages of 8.57, 481, 4,450 and 753 mg/kg, respectively. These
225 metals are continuously dispersed from the mine tailings and thus increase soil metal
226 concentration. The dispersion of metals may be due to clastic movement through wind and water,
227 especially during the wet season. According to the Korean Soil Environmental Conservation Act,
228 soils containing over 12 mg/kg of Cd, 200 mg/kg of Cu and 400 mg/kg of Pb extracted by 0.1N
229 HCl solution need to be continuously monitored and not used for agricultural purposes such as
230 crop planting (Lee et al 2001). Thus, these factors result in increasing metal concentrations in
231 soil and ultimately affect the phytoremediation process.
232
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233 3.2 Soil pH
234 The pH of the solution can greatly influence the equilibrium between speciation of metals,
235 solubility, adsorption and exchange on solid phase sites (Olomu et al. 1973; Kalbasi et al. 1978;
236 Cavallaro and McBride 1984; Sauve et al. 1997). Hence, various studies have found that metal
237 bioavailability is greatly affected by soil pH (Turner 1994; McBride et al. 1997).
238 The availability of uncomplexed ions for uptake by plants in soils mainly depends upon on
239 its solubility and thermodynamic activities (Jenne and Luoma 1977). Thus, for the successful
240 uptake by plant roots, the soluble species must occur near the vicinity of the root membrane. The
241 form of this soluble species present in soil will influence its persistence in soil solution, mobility,
242 and on the rate and extent of uptake, and most importantly its mobility and toxicity in plants
243 (Tiffin 1977).
244 EPA (1992) reported that pH directly or indirectly affects several mechanism of metal
245 retention and reported that adsorption of arsenic increased with pH. The adsorption of copper by
246 soils is also greatly dependent on pH (Cavallaro and McBride, 1980). McBride and Blasiak
247 (1979) reported that the concentration of Zn in solution increased with the increase in pH (i.e.
248 above pH 7.5). However, availability of zinc and magnesium is reported to increase with the
249 decrease in soil (Fergus 1954; McGrath et al. 1988; Turner 1994). Soil pH plays a crucial role in
250 directly or indirectly influencing in the sorption/desorption and complex formation. In general,
251 maximum retention of cationic metals occurs at pH >7 and for anion metals pH<7 (EPA 1992).
252 Thus, soil pH is known to affect plant uptake of most trace elements from soil.
253
254 3.3 Organic matter
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255 Ross (1994) reported that the organic matter in the solid phase, especially the humic
256 compounds of high molecular weight, strongly retain the metals in soils and reduces its
257 availability. Checkai et al. (1987) found that the increase in the formation of organo-metallic
258 compounds may increase the availability of trace metals to plants. Hence, bioavailability of
259 metals is inversely proportional to the organic matter in soils. For example, due to the formation
260 of strong complexes between soil organic matter and copper ions, the availability of copper
261 decreases with the increase in soil organic matter content (Stevenson 1976, 1991, del Castilho et
262 al. 1993) . In contrast, Mn2+ is less commonly associated with organic matter than is Cu and Zn
263 (McGrath et al. 1988) as Mn2+ forms weak coordination complexes with organic matter (Olomu
264 et al. 1973; McBride 1982). Thus, the complex of metal ions with organic matter greatly affects
265 its availability to plants.
266
267 3.4 Plant-bacteria interactions
268 For successful phytoremediation, the roots of plants are required to interact with a large
269 number of different microorganisms (Glick 1995). The functioning of associative plant-
270 bacterial symbioses in heavy-metal-polluted soil can be affected by both micropartners (plant-
271 associated bacteria) and the host plant. Soil microbes play crucial roles in the recycling of plant
272 nutrients, maintenance of soil structure, detoxification of noxious chemicals, and control of plant
273 pests and plant growth (Elsgaard et al. 2001; Filip 2002; Giller et al. 1998). Thus, the capacity of
274 plants used for remediation can be increased by the presence of bacteria in soil. For example, the
275 inoculation of Brassica napus seedlings with Pseudomonas chlororaphis SZY6, Azotobacter
276 vinelandii GZC24 and Microbacterium lactium YJ7 (EN) in the presence of copper, induced root
277 length promotion of both copper-treated and untreated seedlings (He et al. 2010). Ma et al.
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278 (2009) performed pot experiments by inoculating Brassica juncea with Achromobacter
279 xylosoxidans Ax10 (RS) in the presence of copper and found that this increased both Ni and Cr
280 uptake. Wu et al. (2006) reported the increase in plant growth and metal tolerance in Brassica
281 juncea when it was inoculated with Azotobacter chroococcum HKN-5, Bacillus megaterium
282 HKP-1 and Bacillus mucilaginosus HKK-1 in the presence of lead and zinc. Plants and bacteria
283 can form specific associations in which plants provide the bacteria with a carbon source that
284 helps bacteria to reduce the toxicity of the contaminated soil. Plants and bacteria can also form
285 nonspecific associations in which plant processes increases microbial community, which in the
286 course of normal metabolic activity, degrades contaminants in soil. Plant roots can provide root
287 exudate, as well as increase ion solubility. Thus, the remediation activity of plant roots
288 associated with bacteria is increased by this biochemical mechanism.
289
290 3.5 Heavy metal- bacteria interactions
291 The bioavailability of heavy metals can be altered by rhizobacteria (Lasat 2002; McGrath
292 et al. 2001; Whiting et al. 2001) through the release of chelating substances, acidification of the
293 microenvironment, and by changing the redox potential (Smith and Read 1997). For example,
294 the addition of Sphingomonas macrogoltabidus, Microbacterium liquefaciens, Microbacterium
295 arabinogalactanolyticum and Alyssum murale in serpentine soil was found to significantly
296 increase plant uptake of Ni as a result of soil pH reduction compared to control samples that
297 were not inoculated (Abou-Shanab et al. 2003a). Xian (1989) reported that the Eubacteria and the
298 Archaea are able to reduce Mn (IV), Fe (III), Co (II), AsO24- and SeO32 or oxidize Mn (II), Fe
299 (II), Co (III), AsO2-, Se0 and conserve their energy in these reactions. Summers and Silver (1978)
300 found that prokaryotes were able to methylate metal and metalloid compounds, thereby
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301 producing volatile metal derivatives. The oxidation of AsO2- to AsO43- by Alcaligenes faeccalis
302 and reduction of CrO42- to Cr(OH)3 by Pseudomonas fluorescens LB 300 and Enterobacter
303 clocae are example of redox reactions (Wang and Shen 1995). However, excessive concentration
304 of heavy metals in soil are known to be toxic to both plants and most organisms.
305
306 4. Plants responses to heavy metal exposure
307 Plant tolerance to heavy metals is defined as the ability of plants to survive in soil that is
308 often toxic for other plants (Macnair et al. 2000). Plants have evolved different mechanisms to
309 tolerate excess concentrations of heavy metals with more than one mechanism often observed in
310 the same plant species (Hossain et al. 2011). The range of plant mechanisms to tolerate metal
311 stress is summarized in Figure 2 and can be divided into four stages (described in Table 3).
312
313 4.1 Immobilization by Mycorrhizal Associations:
314 The expectancy of mycorrhizal associations existing in heavy metal contaminated soils
315 has important implications for phytoremediation. Mycorrhizal associations increase the
316 absorptive surface area of the plant due to extra-matrical fungal hyphae exploring rhizospheres
317 beyond the root hair zone, which in turn enhance water and mineral uptake. Increase in water and
318 mineral uptake results in greater biomass production, an imperative for successful remediation.
319 The potential of phytoremediation can be enhanced by inoculating plants with mycorrhizal fungi.
320 Mycorrhizae have been found in plants growing on heavy metal contaminated sites and they play
321 a crucial role in phytoremediation as these fungi have evolved a heavy metal tolerance (Shetty et
322 al. 1995; Weissenhorn and Leyval 1995; Pawlowska et al. 1996; Chaudhry et al. 1998; Chaudhry
323 et al. 1999). Mycorrhizae provide an effective exclusion barrier to metal uptake by adopting
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324 absorption, adsorption or chelation mechanisms that restrict the entry of heavy metals into the
325 host plant (Hall 2002). In metal-contaminated soils, plant tolerance and accumulation depends
326 upon the mycorrhizal association between plant roots and fungi. According to Leyval et al.
327 (1997), the ectomycorrhizas (ECM) and arbuscular mycorrhizae (AM) are the most common
328 fungal association found in the plants growing in heavy-metal-contaminated soil. Mycorrhizae
329 particulary ECM that are characteristic of trees and shrubs, have been widely reported to be
330 effective in reducing the deleterious effects of heavy metals on the host plant (Marschner 1995;
331 Huttermann et al. 1999; Jentschke and Godbold 2000).
332 Establishment of native prairie grass community, consisting mostly of the seeded species
333 Elymus canadensis, was achieved on coarse taconite iron-ore tailing plots using amendments like
334 composted yard waste and arbuscular mycorrhizal fungi within one year, thus meeting-
335 reclamation goals for the re-establishment of a sustainable native grass community (Noyd et al.
336 1996). Some researchers reported the presence of Glomus mosseae in heavy metal contaminated
337 sites (Debiane et al. 2008). It has been found that the external mycelium of certain AM fungi
338 produces a glycoprotein (Glomalin) which has heavy metal binding sites (Agely et al. 2005;
339 Citterio et al. 2005; Trotta et al. 2006; Vivas et al. 2003). Heavy metals present in soil
340 accumulate at these binding sites (Bano and Ashfaq 2013). Heavy metals are mostly accumulated
341 in fungal hyphae as well as in arbuscules. Joner et al. (2000) reported that the Glomus species
342 retained significant concentrations of Zn in mycelium when associated with clover or ryegrass.
343 Studies have indicated that the AM fungi release an extracellular, insoluble glycoprotein
344 (commonly known as glomalin) which can bind with Cu, Cd and Pb in polluted soils (Gonzalez-
345 Chavez et al. 2004; Gohre and Paszkowski 2006). These authors reported that 1g of glomalin
346 was able to extract up to 4.3 mg copper, 0.08 mg cadmium and 1.12 mg lead from metal-
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347 contaminated soils. Kaldorf et al. (1999) found that the Glomus species also immobilized metals
348 in maize. High concentration of metals is also stored in the mycorrhizal structures like arbuscules
349 and spores. For example, Chen et al. (2001) reported accumulation of Zn in fungal tissues of
350 Glomus mosseae up to 1200mgkg-1 and in G. versiforme up to 600 mgkg–1. Colapaert and Van
351 Assche (1992) found that Zn content was reduced in Pinus sylestris as Zn is retained by the
352 ectomycorrhizal fungi Paxillus involutus.
353 Turnau et al. (2001) examined the AM fungi in roots of Fragaria vesca growing in Zn-
354 contaminated soil and found that about 70% of the root sample was colonized with G. mosseae.
355 Griffioen et al. (1994) also reported the association of Scutellospora dipurpurascens with
356 Agrostis capillaries growing in the contaminated surroundings of a zinc refinery in the
357 Netherlands. Collectively, these examples provide strong evidence that fungi have evolved Zn
358 and Cd tolerance and play a crucial role in Zn and Cd tolerance in plants.
359
360 4.2 Role of plant cell wall in metal tolerance
361 Little is known about the role of the plant cell wall and its binding properties in relation to
362 metal tolerance. However, what is reported on has been somewhat controversial. In soil solution,
363 the root cell wall is in direct contact with the metals, so the adsorption of metals onto the cell
364 wall must be low. However, Mehes-Smith et al. (2013) reported that a significant amount of
365 metal accumulation occurred between the cell wall and the cell membrane. Divalent and trivalent
366 metal cations are able to bind to plants due to the presence of functional groups such as –COOH,
367 -OH and –SH in plant cell walls. The most important component of the plant cell wall is pectin
368 which consists of carboxyl groups. Under metal stress divalent and trivalent heavy metals ions
369 bind with the carboxyl group of pectin (Mehes-Smith et al. 2013). Other studies have also
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370 reported that the binding of the pectin carboxyl group with lead is the most important interaction
371 by which a plant can tolerate lead toxicity (Meyers et al. 2008; Jiang and Liu 2010). Bringezu et
372 al. (1999) reported that a range of metals accumulated in the epidermal cell walls of heavy-
373 metal-tolerant Silene vulgaris ssp. humilis and these metals were found to be either bound to a
374 protein or to silicate.

375 The binding of lead to JIM5-P (within the cell wall) restrict the movement of metal to the
376 plasma membrane and act as a physical barrier in Funaria hygrometrica protonemata
377 (Krzesłowska et al. 2009). Krzesłowska et al. (2010) reported that the lead bound to JIM5-P
378 within the cell is either taken up or remobilized by endocytosis along with pectin epitope.
379 Elevated levels of Fe, Cu, Zn and Pb have been observed in cell walls of Minuartiaverna sp.
380 Hercynica growing on mine dumps (Solanki and Dhankhar 2011; Neumann et al. 1997). The
381 binding of copper can be prevented by decreasing pectin concentrations and increasing pectin
382 methylation in the cell walls of copper-tolerant Sileneparadoxa, thereby restricting copper
383 accumulation in roots (Colzi et al. 2012).
384
385 4.3 Role of root exudates
386 Roots exudates can be generally classified into two types, namely, high molecular weight
387 (HMW) (e.g. mucilage mainly polysaccharides and polyuronic acid and ectoenzymes) and low
388 molecular weight (LMW) (e.g. organic acids, sugars, phenols and various amino acids, including
389 non-protein amino acids such as phytosiderophores) materials. Root exudates play a significant
390 role in the processing of phytoremediation as an emerging in-situ, green remediation technology
391 using plants to absorb, accumulate, stabilize or volatilize contaminants from soil.
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392 Root exudates affect the metal solubility, mobility, and phytoavailability by reacting with
393 metal ions. The entry of metals in plants can be restricted by either immobilizing metals, thereby
394 limiting its entry to the plasma membrane (Colzi et al. 2011) or by mobilizing the metals from
395 root to shoot. Approximately half of the photosynthates produced in plants is retransported to
396 roots, whereas approximately 12-40% is released in the rhizophere during plant development as
397 exudates, including sugars and polysaccharides, organic and amino acids, peptides and proteins
398 (Lin et al. 2003; Hinsinger et al. 2006). The root secretions include carbohydrates, organic acids,
399 humic acids, polypeptides, proteins, amino acids, nucleic acids, etc. and the inorganic ligands
400 include Cl–, SO4 2–, NH4+, CO32–, PO43– (Dong et al. 2007). These root secretions functions not
401 only as energy sources for microorganisms, but they also act as ligands which chelate heavy
402 metal ions and that ultimately influence the pH and Eh conditions in the rhizosphere. The change
403 in pH and Eh conditions is the main factor related to mobilization of metals in soils and their
404 accumulation in plants. Root exudates released from Echinochloa crusgalli releases citric acid
405 and oxalic acid, and increases the translocation of heavy metals such as Cd, Cu and Pb from
406 roots to shoots (Kim et al. 2010; Zhou et al. 2006). Fan et al. (2001) reported that in barley and
407 wheat there was reduced phytosiderophore production in the presence of Cd thereby enhancing
408 the transition metal. Root exudates could also increase solubility of metal ions in soils and
409 consequently increase their accumulation in plants.
410 Graminaceous plant species (e.g. paddy rice) secrete phytosiderophore (amino acids) that
411 form much more stable complexes than carboxylate with Fe, Cd, Zn and Cu (Römheld 1991;
412 Hinsinger 1998; Chaignon et al. 2002; Xu et al. 2005). Plants secrete low molecular weight
413 organic acids that play a crucial role in solubility and availability of heavy metals. Organic acids
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414 (e.g. oxalic acid, malic acid and citric acid) secreted by plants, such as wheat and buckwheat
415 prevent the entrance of Cd2+ into roots (Meach and Martin 1991).
416 In a study by Nair et al. (2007), they found that P. azotoformans produced a mixed type of
417 siderophore including both catecholate and hydroxamate that was able to remove almost 92.8%
418 of accumulated arsenic as compared to control plants which only removed 33.8%. Nair et al.
419 (2008) studied the speciation of metals in an industrial sludge and highlighted the prospect of the
420 use of siderophores for bioremediation due to their biodegradable and ecofriendly characteristics.
421 Other studies have shown that the Ni-chelating histidine and citrate accumulate in root
422 exudates and help to reduce Ni uptake, thereby playing a crucial role in Ni-detoxification
423 strategies (Salt et al. 2000; Hall 2002). Persans et al. (1999) proposed that in Thlaspi
424 goesingense, Ni hyperaccumulation is not determined by the overproduction of histidine in
425 response to Ni. Krämer et al. (2000) found that in T. goesingense the uptake of Ni and its
426 accumulation across the cytoplasm into the vacuole is restricted by the free histidine, which
427 could be responsible for Ni tolerance and accumulation. Kerkeb and Krämer (2003) also showed
428 that in A. lesbiacum and B. juncea the release of Ni from roots to the xylem is enhanced by
429 histidine.
430 Salt et al. (2000) provided evidence for Zn-histidine complexes in the roots of Zn
431 hyperaccumulator Thlaspi caerulescens. However, this is in contrast to other studies that did not
432 find any such complex related to the accumulation of Zn by T. caerulescens (Knight et al. 1997;
433 McGrath et al. 1997; Zhao et al. 2001). Other studies proposed that the mechanism of Cu
434 exclusion in T. aestivum involve phytochelatins citrate and malate (Yang et al. 2005; Bálint et al.
435 2007).
436
18
437 4.5 Restriction of metal uptake through plasma membrane
438 The plant plasma membrane functions as the first line of defence for heavy metal
439 contamination as it is the first cell structure that is exposed to heavy metals. The plasma
440 membrane restricts the uptake and accumulation of metals by inhibiting its entry into the
441 cytoplasm (Mehes-Smith et al. 2013). The restriction of metals to the plasma membrane can be
442 achieved by changing the cell wall binding capacity to metal ions or by modifying the ion
443 channels present on the membrane or by modifying the efflux pumps or with the root exudates
444 (Tong et al. 2004). Wainwright and Woolhouse (1977) found that Cu increases the efflux of K+
445 from the excised roots of Agrostis capillaries but this is not the case with Zn. Iwasaki et al.
446 (1990) showed that 60% of Cu in Italian ryegrass was found to be bound by the cell wall and the
447 plasma membrane.
448 In Holcus lanatus the suppression of high affinity arsenate transport system absorbs less
449 arsenate (Meharg and Macnair 1992) along with the synthesis of phytochelatins (Hartley-
450 Whitaker et al. 2001). According to Manara (2012) a heavy metal efflux pump in plant is most
451 likely to be P1B-ATPases and the CDF families of transporters (based on the sequence similarity
452 to microbial and animal proteins). Further work has suggested that the P1B-type ATPases belong
453 to P-type ATPase superfamily and to translocate the metal ions across the membrane it uses ATP
454 as an energy source (Axelsen and Palmgren 2001).
455
456 4.6 Phytochelatins (PCs)
457 The toxic effect of metals ions in cytosol can be eliminated by specific high affinity ligands
458 such as phytochelatins (PCs). PCs were first discovered in fission yeast as cadmium-binding
459 “cadystins A and B”. Various studies have confirmed that PCs are synthesized from GSH by the
19
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460 help of enzyme γ-glutamyl cysteinyl dipeptidyl transpeptidase (PC synthase) (Zenk 1996).
461 Phytochelatins are rich in cysteine non-protein metal binding peptides produced by plants (Schat
462 and Kalif 1992; Zenk 1996). PCs belong to the family of metal-complexing peptides having a
463 general structure of (c-Glu-Cys)nGly (n = 2–11) (Cobbett and Goldsbrough 2002). In the
464 cytosol, PCs are synthesized and then transported to the vacuole as a complex. In the presence of
465 heavy metals such as Cd, Cu, Zn, Ag, Au, Hg, and Pb PCs are rapidly induced (Rauser 1995;
466 Cobbett 2000), with Cd being the strongest inducer (Grill et al. 1987, 1989). PCs complex with
467 Cd ions with the aid of the thiolic group (-SH) of cys and this complex is accumulated in the
468 vacuole by the activity of ABC transporters (Di Toppi and Gabbrielli 1999).The relationship
469 between PC synthesis and Cd accumulation in Sedum alfredii was studied by Zhang et al. (2010)
470 and their results suggested that PCs act as an intercellular Cd detoxification mechanism in shoots
471 rather than roots.
472 Galli et al. (1996) reported that Zea mays synthesizes PCs in the presence of excess Cu and
473 Cd. However, this was not tested against a control or sensitive cultivar. Maitani et al. (1996)
474 showed that Rubia tinctorum root cultures initiated the synthesis of PCs when exposed to a
475 number of metals such as Zn, Cu and Cd, and also tested it against controls plants in which no
476 phytochelatin production was observed.
477 In a study by Schat and Kalif (1992) tolerant and non-tolerant strains of Silene vulgaris
478 were exposed to different concentrations of Cu did not show any PCs synthesis and thus they
479 concluded that PCs are not involved in the tolerance mechanism in S. vulgaris. This could be due
480 to the lower ratio of PCs to Cu. de Knecht et al. (1994) also found that PCs do not play any role
481 in Cd tolerance in S. vulgaris.
20
482 Techniques such as X-ray absorption spectroscopy (XAS), high performance liquid
483 chromatography-mass spectrometry (HPLC-MS), and inductively coupled plasma optical
484 emission spectrometer (ICP- OES) have been used to understand the role of phytochelatins in
485 cadmium tolerance in plants. Salt et al. (1997) showed that in Indian mustard seedlings the
486 percentage of Cd bound to PCs increased by 34% after 6 hours of Cd exposure and by 60% after
487 70 hours.
488 In Brassica juncea it has been shown that PC synthesis increases as Cd accumulation is
489 increased intracellularly, thereby protecting the photosynthetic unit without decreasing the
490 transpiration rate (Haag-Kerwer et al. 1999). Xiang and Oliver (1998) showed that treatment of
491 A. thaliana with Cd and Cu results in an increase of transcriptional genes that are involved in the
492 synthesis of PC precursor, glutathione.
493 Jambhulkar and Juwarkar (2009) found that Cassia siamea accumulate Ni, Cr and Pb at
494 higher concentrations compared to other species. Higher accumulation of all the metals was
495 observed in C. siamea because this plant that was grown on a fly ash dump contains non-protein
496 thiols, which is a marker of phytochelatin synthesis and is responsible for metal accumulation.
497 Thus it could be grown easily on fly ash dumps as it acts as a hyperaccumulator plant.
498 Jatropha curcus accumulates high concentrations of Cr in roots. Despite this high
499 accumulation in roots, the level of free Cr ions in roots may remain low since most of the Cr ions
500 are either immobilized or compartmentalized in vacuoles or form Cr–phytochelatin complexes
501 (Yadav et al. 2010).
502
503 4.7 Metallothioneins (MTs)
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504 Evidence for the role of metallothioneins (MTs) in plant metal tolerance was given by
505 Rauser (1984), who found Cu-binding low molecular weight protein in Agrostis gigantea. MTs
506 are low molecular weight cysteine-rich metal-binding peptides and can be classified into two
507 classes: Class 1: Consist of 61 amino acids and lack aromatic amino acid or histidines that are
508 related to mammals; Class 2: These MTs are related to Candida albicansa yeast or cyanobacteria
509 (Winge et al. 1985).
510 MTs are not only expressed under various abiotic stresses in plants but also expressed
511 during plant development (Rauser 1999). Plant MTs sequester metal ions by complexing metals
512 ions with multiple cysteine thiol groups (Robinson et al. 1993). Various stimuli have been found
513 which can upregulate the expression of MT genes in plants. Natural senescence (Bhalerao et al.
514 2003), hormones such as ABA (Reynolds and Crawford 1996), ethylene (Coupe et al. 1995),
515 wound and infection by virus (Choi et al. 1996), heat shock proteins (Hsieh et al. 1995), nutrient
516 starvation such as sucrose (Hsieh et al. 1996), UV-light (Foley and Singh 1994), cold and salt
517 stress (Reid and Ross 1997) were found to increase the expression of MT genes.
518 In Brassica rapa three different MT genes are regulated differently under various heavy
519 metal stress. When Brassica rapa seedlings were treated with Fe, there was an increased
520 expression of BrMTat after 6 and 24h. When Brassica rapa seedlings were treated with Zn the
521 expression of BrMT2 was downregulated and BrMT1 expression was increased whereas BrMT3
522 remained unchanged. In Mn-treated seedlings, BrMT1 and BrMT3 genes were upregulated until
523 12h and then downregulated. The expression of BrMT2 was affected for the first 12h, after it was
524 downregulated (Ahn et al. 2012).
525
526 4.8 Compartmentalization of heavy metal in vacuoles
22
527 Once a heavy metal has entered the cell, the plant copes with the toxicity of heavy metal
528 either by transporting the heavy metal out of the cell or by sequestrating it into the vacuole,
529 thereby limiting the contact of heavy metals with the sensitive metabolic activities taking place
530 in the cytosol or other cellular compartments (DalCorso et al. 2008; Dalcorso et al. 2010;
531 Clemens 2001). Heavy metal uptake has been driven by either channels or transporters. Some
532 examples of heavy metal transporters include protein ZIP family, ABC transporters (ATP-
533 binding cassette), P-type metal ATPases, members of the CDF transporter family (also called
534 MTPs in plants), zinc regulated transporter AtMTP1 (Kramer et al. 2007), the natural resistance-
535 associated macrophage protein (NRAMP) family, CAX family involved in the vacuolar
536 accumulation of Cd, copper transporter (COPT) family proteins, pleiotropic drug resistance
537 (PDR) transporters, and yellow-stripe-like (YSL) transporter (Lee et al. 2005, Chiang et al. 2006;
538 Kramer et al. 2007).
539 Earlier reports showed that a number of heavy metals including Zn and Cd have been
540 accumulated in the vacuole (Ernst et al. 1992; De 2000). For example Alyssum serpytllifolium, is
541 a nickel hyperaccumulator that accumulated 72% of cellular Ni in the vacuole (Brooks et al.
542 1980). Davies et al. (1991b) reported the increased vacuolation of Zn in Festuca rubra roots.
543 Phragmites australis showed increased Zn sequestration in the vacuole or that it was
544 immobilized in the apoplast (Jiang and Wang 2008). CDF transporter family (also called MTPs
545 in plants) are reported to be involved in efflux of transition metals such as Zn2+, Cd2+, Co2+, Ni2+
546 or Mn2+ from cytoplasm. Van der Zaal et al. (1999) reported ZAT1 transporter, a member of
547 CDF family, found in A. thaliana to sequester zinc in the vacuole. The transporter AtHMA3, a
548 member of P1B-ATPases, may play a role in the detoxification Cd, Pb, Co and Zn through
549 storage in the vacuoles (Morel et al. 2009).
23
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550 5. Conclusions
551 The problem of heavy metal pollution is continuously increasing due to the acceleration of
552 many human activities, leading to an intensification of research dealing with the phytotoxicity of
553 these contaminants as well as the mechanisms used by plants to counter their harmful effects. It
554 is clear that plants play a crucial role in the remediation of metal-enriched soils. This review
555 focused on the potential cellular and molecular adaptations by plants that are necessary to
556 tolerate heavy metal stress. Significant progress has been made in the identification and
557 understanding the role of key components like metal transporters, hyper accumulation,
558 phytochelatins and metallothionein proteins that ensure heavy metal tolerance to plants.
559 Phytoremediation is a fast developing field, and over the past ten years field applications
560 have been initiated world-wide, including phytoremediation of organic, inorganic and
561 radionuclides. This sustainable and inexpensive process is fast emerging as a viable alternative to
562 more conventional remediation methods, and will be most suitable for developing countries such
563 as India. Although phytoremediation offers some advantages over more commonly used
564 conventional technologies such as being cost-effective and eco-friendly, this technique requires
565 careful consideration of several factors in order to accomplish effective, high performance
566 results. The most important factor is the use of a suitable plant species which can be used to
567 uptake the particular contaminant. It is important to understand that although the
568 phytoremediation technique is often the best alternative, like all technologies it does have
569 limitations. These include a restricted surface area and, depth occupied by the roots, and the
570 inherent slow growth and low biomass requires a long-term commitment to the remediation
571 project. Continued research is required to minimize these limitations so that this technique can be
572 applied most effectively.
24
573 Acknowledgement
574 The authors are grateful to TEQIP-II and MNNIT, Allahabad for financial support for this
575 research. Ms. Anamika Kushwaha and Ms. Aishvarya Gautam, acknowledge TEQIP-II for their
576 financial support in the form of fellowship. The authors are also thankful to editors and reviewers
577 for improving the manuscript.

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sunflower plants. Plant and Soil. 208: 221–226.

Zornoza, P., Robles, S., Martin, N. 1999. Alleviation of nickel toxicity by ammonium supply to
60
al use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version
Figure 1: Types of Phytoremediation

Page 62 of 70
Page 63 of 70
HEAVY METAL
1.Immobilization by Mycorrhizal 2. Binding to plant cell wall 3. Chelation by plant root exudates
Associations
6. Chelation by various ligands: PCs, MTs, acids
4. Reduced influx by
Ligand-metal complex
plasma membrane
CYTOSOL 5. Active efflux of

VACUOLE HM metal
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).
al use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version Page 64 of 70
Table 1: Toxic effects of heavy metals on plants

Zinc Limit the growth of both root and shoot; Chlorosis; Choi et al. 1996; Ebbs and Kochian 1997; Fontes and
Senescence; Inhibit metabolic functions Cox 1998.
Copper Growth retardation; Leaf chlorosis; oxidative Lewis et al. 2001; Stadtman and Oliver 1991.
stress and ROS formation
Nickel Chlorosis; Necrosis; Nutrient imbalance; Disorder of cell Zornoza et al. 1999; Pandey and Sharma 2002;
membrane functions Rahman et al. 2005.
Mercury Obstruction of water flow in plants; interfere the mitochondrial Zhou et al. 2007; Zhang and Tyerman 1999.
activity and induces oxidative stress; disruption of
biomembrane lipids and cellular metabolism
Lead Effect morphology, growth and photosynthetic processes; Sharma and Dubey 2005; Reddy et al. 2005.
enzyme inhibition; water imbalance; Alteration in membrane
permeability, Oxidative stress
1
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Chromium Chlorosis; Nutrient and water imbalance; Inhibition of Chatterjee and Chatterjee 2000; Dixit et al. 2002;
chlorophyll biosynthesis; wilting of tops; root injury; enzyme Sharma et al. 2003; Scoccianti et al. 2006; Vajpayee et
inhibition; oxidative stress; inhibition of plant growth al. 2000; Shanker et al. 2005.
Arsenic Inhibits photosynthesis, Inhibits growth, Biomass and yield, Marques and Anderson 1986; Carbonell et al. 1998.
Death
Cobalt Affect shoot growth and biomass; Decrease chlorophyll, Li et al. 2009; Chatterjee and Chatterjee 2000.
protein and catalase activity; decrease water potential and
transpiration rate
Cadmium Reduction in photosynthesis, water uptake, and nutrient Wójcik and Tukiendorf 2004;
Uptake, chlorosis, growth inhibition, browning of root tips, and Mohanpuria et al. 2007.
finally death
Copper Growth retardation, leaf chlorosis, Oxidative stress, damage to Lewis et al. 2001; Stadtman and Oliver 1991; Hegedus
macromolecules et al. 2001.
2
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 Pb and Zn Yanqun et al. 2005.
Corydalis pterygopetata
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 Zn
tatrense, and Thlaspi caerulescens
3
Page 67 of 70
Psycotria vanhermanni and Streptanthus Ni

polygaloides
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.
4
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.
5
Page 69 of 70
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
Occur in Symplast
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

Occur at whole plant level
Repair mechanism
Plant level detoxification
6
7
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Heavy Metal Detoxification and Tolerance Mechanisms in Plants: Implications For Phytoremediation

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

4 Anamika Kushwahaa, Radha Rania*, Sanjay Kumara, Aishvarya Gautama

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

41 to tolerate heavy metal stress.

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

57 Ca2+, Sr2+, Ba2+, Al3+, Ga3+, Sc3+, and Y3+).

59 Pd2+, Pt2+, Cu+, Ag+, Au+, and Hg2+).

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

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

96 plants are described in Table 1.

104 fertility, and other properties.

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

110 2. PHYTOREMEDIATION: AN OVERVIEW

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

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

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

136 2.1 Phytoextraction:

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

151 Shabani and Sayadi 2012):

153 i. High growth rate.

154 ii. Production of more above-ground biomass.

155 iii. Widely distributed and highly branched root system.

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

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

160 viii. Resistance to pathogens and pests.

161 ix. Easy cultivation and harvest.

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

168 2.2 Phytovolatilization:

177 2.3 Phytostabilization:

184 Okieimen 2011).

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

196 and metal-contaminated soil.

198 2.4 Rhizofiltration:

208 alternatives (Yadav et al. 2011).

210 3. Factors influencing metals uptake by plants in soil

211 3.1 Soil Metal Concentration

231 soil and ultimately affect the phytoremediation process.

233 3.2 Soil pH

243 (Tiffin 1977).

254 3.3 Organic matter

265 its availability to plants.

267 3.4 Plant-bacteria interactions

288 associated with bacteria is increased by this biochemical mechanism.

290 3.5 Heavy metal- bacteria interactions

294 the addition of Sphingomonas macrogoltabidus, Microbacterium liquefaciens, Microbacterium

306 4. Plants responses to heavy metal exposure

313 4.1 Immobilization by Mycorrhizal Associations:

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

352 ectomycorrhizal fungi Paxillus involutus.

360 4.2 Role of plant cell wall in metal tolerance

374 protein or to silicate.

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

385 4.3 Role of root exudates

409 consequently increase their accumulation in plants.

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

437 4.5 Restriction of metal uptake through plasma membrane

447 plasma membrane.

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

456 4.6 Phytochelatins (PCs)

471 rather than roots.

476 phytochelatin production was observed.

481 in Cd tolerance in S. vulgaris.

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

492 synthesis of PC precursor, glutathione.

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