*3.4.4. Antioxidative defence involved in metal tolerance*

increase of Fe, Zn and Cu accumulation in *O. sativa* was associated with an overexpression of

Visioli *et al.* (2010) also showed that metallothioneins may be involved in the translocation of Ni in *T. caerulescens.* An increase in MT-1B in the individuals from the metal contaminated environment was observed when metallicolous *T. caerulescens* and non-metallicolous *T. caerulescens* individuals were grown in presence of high Ni concentrations, compared to non contaminated site. Additionally, Visioli *et al.* (2012) analyzed four *T. caerulescens* sub-popula‐ tion (MP1 to MP4) for their ability to accumulate and tolerate Ni. In four sub*-*populations analyzed*,* MP2p translocated the highest amount of Ni to the shoots. This sub-population also had the highest level of putative metallothionein protein (MT4C). Constitutively higher expressions of other MTs are also seen in the hyperaccumulators *A. halleri*, *S. paradoxa* and *S.*

Transporters are not only involved in the uptake of metals from the soil, but also in their transport out of the vacuole. These mobilized metals can then be translocated to aerial tissue. Visioli *et al*. (2012) subsequently found for sub-population MP2p, which exhibited the highest level of Ni translocation of the four sub-populations analyzed, significantly higher levels of the ABC27 transporter. This transporter is part of the ABC family of transporters which are involved in removing metals from the cytoplasm by pumping outside the cell wall, metals sequestered in vacuoles and other subcellular compartments (Visioli *et al.,* 2012; Martinoia *et al.,* 2002; Sanchez-Fernandez *et al.,* 2001). Hassinen *et al.* (2007) showed that the AtMRP10 homolog, also part of the ABC family of transporters, had different expression in roots of two *T. caerulescens* populations with contrasting Zn tolerance and accumulation. In addition, the AtNramp3 transporter was also involved in the mobilization of vacuolar Cd back into the cytosol. This was oberved when *AtNramp3* was overexpressed in *A*. *thaliana*. AtNramp3 was further hypothesized to play a role in the mobilization of Fe, Mn, and Zn in the vacuole

The passage of metal ions and/or metal ligand complexes from the cytosol of root cells into the vascular tissue requires their transport across the cell membrane. Transporters involved in this activity are the heavy metal transporting P-type ATPases (HMAs) (Clemens, 2006). The AtHMA2 and 4 are involved in translocation of Zn in *A. thaliana*. Stunted growth and chlorosis resulted in the *hma2hma4* double mutant from inadequate Zn supply to the leaves. The two genes were expressed in vascular tissue which indicates their hypothesized function in xylem loading (Hussain *et al.,* 2004). The AtHMA4 transporter was also involved in the transport of Cd2+ ions (Clemens, 2006). In *T. caerulescens*, the P-type ATPase, TcHMA4, was also involved in the translocation of Zn. When Zn and Cd levels were elevated or when Zn is deficient, the expression of *TcHMA4* was induced in the roots. This transporter was involved in the xylem loading of Zn in plant roots (Hassinen *et al.,* 2007; Papoyan and Kochian 2004). Milner *et al.* (2012) also determined that NcZNT1 in *T. caerulescens* was not only involved in Zn uptake from the soil but also could be involved in the long distance transport of Zn from root to shoot via

Metals have to undergo a xylem unloading process prior to their distribution and their detoxification in the shoot and their redistribution via the phloem (Schmidke and Stephan

the *NAS3* gene (Hassan and Aarts 2011; Kawachi *et al.,* 2009).

*vulgaris*.

70 Environmental Change and Sustainability

(Clemens, 2006).

the xylem.

*3.4.3.5. Metal storage*

In environments, where metals are present in toxic levels, the elevated activities of antioxidant enzymes and non-enzymatic constituents are important in the plant tolerance to stress. Metal tolerance may be enhanced by the plant's antioxidant resistant mechanisms. There is an indication that the alleviation of oxidative damage and increased resistance to stresses in the environment is often correlated with an effective antioxidative system. The minimization of damage due to oxidative stress is a universal feature of plants defense responses (Kachout *et al.,* 2009). The detrimental effect of heavy metals in plants is due to the production of ROS and induction of oxidative stress. Oxidative stress is expressed by the increase levels of reactive oxygen species such as singlet oxygen (1 O2), superoxide radical (O*<sup>−</sup>*<sup>2</sup> ), hydrogen peroxide (H2O2) and hydroxyl radical (OH*<sup>−</sup>* ) (Salin, 1988). ROS are strong oxidizing agents that lead to oxidative damage to biomolecules, for instance lipids and proteins and can eventually result in cell death (Gunes *et al.,* 2006). It is shown that plant tolerance to metals is correlated with a rise in antioxidants and activity of radical scavenging enzymes (Kachout *et al.,* 2009). Plants respond to oxidative stress by activating antioxidative defence mechanisms which involve enzymatic and non-enzymatic antioxidants. The enzymatic components include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and enzymes of ascorbate glutathione cycle whereas the non-enzymatic antioxidants include ascorbate and glutathione and atocoperol (Solanki and Dhankhar 2011; Kachout *et al.,* 2009). These antioxidants are responsible for elimination and destruction of the reactive oxygen species (Solanki and Dhankhar 2011).

Oxidative damage could result when the balance between the detoxification of the ROS products and the antioxidative system is altered (Kachout *et al.,* 2009). The tolerance of deleterious environmental stresses, such as heavy metals, is correlated with the increased capacity to scavenge or detoxify activated oxygen species (Kachout *et al.,* 2009). Boominathan and Doran (2003a,b) determined the role of antioxidative metabolism of heavy metal tolerance in *T. caerulescens*. They determined that superior antioxidant defenses, mainly catalase activity, may have an important role in the hyperaccumulator phenotype of *T. caerulescens*. Kachout et al (2010) determined the effects of Cu, Ni, Pb and Zn on the antioxidative defense systems of *Atriplex* plants. They found that when the plants were exposed to different levels of metals, their dry matter production and shoot height decreased. Of the antioxidant enzymes, metal toxicity only diminished the levels of superoxide dismutase (SOD) and probably ascorbate peroxidase (APX) but increased the activity of catalase (CAT) and glutathione reductase (GR). The plants showed an intermediate level of tolerance to the metal stress conditions imposed. The antioxidative activity may be of fundamental significance for the *Atriplex* plants in their response against environmental stress.

#### **3.5. Problems associated with plant metal tolerance**

Soils enriched with metals are demanding on tolerant and accumulator plants. The costs associated with their adaptation to these sites are related to energy and resources alloca‐ tions. When a metal tolerant or accumulator plant is growing in a metal contaminated soil, there is an increase in cost because the organism has to spend energy to counter the effects of the metals (Maestri *et al.,* 2010). Slow growth and low reproduction are the main character‐ istics of plants growing on metal enriched soils (Ernst, 2006; Ernst *et al.,* 2000). Haldane (1954) stated that costs are associated with the natural selection of new alleles. More energy and resources are required for the maintenance of the tolerance mechanisms at the cellular level. It has been demonstrated that tolerant plants have increased synthesis of complexing molecules in the cytosol. For example, metallothioneins and phytochelatins for the detoxifica‐ tion of metals such as As, Cd, and Cu. ATP are also needed for the active transport of metals across the plasma membrane and tonoplast. The synthesis of these agents withdraws N, S and energy from the primary metabolism (Ernst, 2006; Verkleij *et al.,* 1998). Energy is also required for the translocation of metals from root to shoot as well as for their allocation to various tissues and cell types. The reduced biomass of metal tolerant plants compared to their non metal tolerant ancestors might also be the result of less than favourable environmental conditions such as low water and nutrient supply. The diminished biomass and seed production might be the result of all costs associated with their survival to these metal contaminated sites, such as adaptation and environmental constraints (Ernst, 2006). Plants have an advantages growing on metal contaminated soil. As previously mentioned, there is a lack of competitive species on these sites. With high metal accumulation of metals in their

aerial tissues, the "elemental hypothesis" speculates that hyperaccumulators can deter predators such as herbivores from feeding on them (Maestri *et al.,* 2010; Vesk and Reich‐ man 2009). However, some insects feed on hyperaccumulator plants and in turn accumu‐ late the metals in their tissue which then aid in their defence against predators (Maestri *et al.,* 2010). This contradiction may explain why there is a mix of excluders, accumulator and hyperaccumulators growing on metal contaminated sites. Another advantage of hyperaccu‐ mulation is the elimination of competitive plants by further contaminating the surrounding soil by shedding their metal contaminated leaves (Maestri *et al.,* 2010).

#### **3.6. Effects of metals on plant population diversity and structure**

dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and enzymes of ascorbate glutathione cycle whereas the non-enzymatic antioxidants include ascorbate and glutathione and atocoperol (Solanki and Dhankhar 2011; Kachout *et al.,* 2009). These antioxidants are responsible for elimination and destruction of the reactive oxygen species (Solanki and

Oxidative damage could result when the balance between the detoxification of the ROS products and the antioxidative system is altered (Kachout *et al.,* 2009). The tolerance of deleterious environmental stresses, such as heavy metals, is correlated with the increased capacity to scavenge or detoxify activated oxygen species (Kachout *et al.,* 2009). Boominathan and Doran (2003a,b) determined the role of antioxidative metabolism of heavy metal tolerance in *T. caerulescens*. They determined that superior antioxidant defenses, mainly catalase activity, may have an important role in the hyperaccumulator phenotype of *T. caerulescens*. Kachout et al (2010) determined the effects of Cu, Ni, Pb and Zn on the antioxidative defense systems of *Atriplex* plants. They found that when the plants were exposed to different levels of metals, their dry matter production and shoot height decreased. Of the antioxidant enzymes, metal toxicity only diminished the levels of superoxide dismutase (SOD) and probably ascorbate peroxidase (APX) but increased the activity of catalase (CAT) and glutathione reductase (GR). The plants showed an intermediate level of tolerance to the metal stress conditions imposed. The antioxidative activity may be of fundamental significance for the *Atriplex* plants in their

Soils enriched with metals are demanding on tolerant and accumulator plants. The costs associated with their adaptation to these sites are related to energy and resources alloca‐ tions. When a metal tolerant or accumulator plant is growing in a metal contaminated soil, there is an increase in cost because the organism has to spend energy to counter the effects of the metals (Maestri *et al.,* 2010). Slow growth and low reproduction are the main character‐ istics of plants growing on metal enriched soils (Ernst, 2006; Ernst *et al.,* 2000). Haldane (1954) stated that costs are associated with the natural selection of new alleles. More energy and resources are required for the maintenance of the tolerance mechanisms at the cellular level. It has been demonstrated that tolerant plants have increased synthesis of complexing molecules in the cytosol. For example, metallothioneins and phytochelatins for the detoxifica‐ tion of metals such as As, Cd, and Cu. ATP are also needed for the active transport of metals across the plasma membrane and tonoplast. The synthesis of these agents withdraws N, S and energy from the primary metabolism (Ernst, 2006; Verkleij *et al.,* 1998). Energy is also required for the translocation of metals from root to shoot as well as for their allocation to various tissues and cell types. The reduced biomass of metal tolerant plants compared to their non metal tolerant ancestors might also be the result of less than favourable environmental conditions such as low water and nutrient supply. The diminished biomass and seed production might be the result of all costs associated with their survival to these metal contaminated sites, such as adaptation and environmental constraints (Ernst, 2006). Plants have an advantages growing on metal contaminated soil. As previously mentioned, there is a lack of competitive species on these sites. With high metal accumulation of metals in their

Dhankhar 2011).

72 Environmental Change and Sustainability

response against environmental stress.

**3.5. Problems associated with plant metal tolerance**

Elevated accumulations of metals in soil and vegetation have been documented within short distances of the smelters compared to control sites (Nkongolo *et al.,* 2008; Gratton *et al.,* 2000). Several authors have reported differences in genetic structure of plants growing in contami‐ nated areas (Vandeligt *et al.,* 2011; Nkongolo *et al.,* 2008; Scholz and Bergmann 1984). Enzymatic studies of Norway spruce (*Picea abies*) revealed genetic differences between groups of sensitive trees in polluted areas (Scholz and Bergmann 1984). It has been demonstrated that the evolution of heavy metal tolerant ecotypes occurs at an unexpectedly rapid rate (Wu *et al.,* 1975) and that despite founder effect and selection, in several cases, the recently established tolerant populations maintain a high level of variation and appear to be at least as variable as non tolerant populations. Observations of higher heterozygosity in tolerant plants of European beech (*Fagus sylvatica*) in Germany (Muller and Starck 1985), scots pine (*Pinus* sylvestris) in Germany and Great Britain (Geburek *et al.,* 1987), trembling aspen (*Populus tremuloides*) and red maple (*Acer rubrum*) in the United States (Berrang *et al.,* 1986) have been reported. Several studies, however, have reported the detection of bottleneck effects (Nordal *et al.,* 1999; Vekemans and Lefebre 1997; Mejnartowicz, 1983). Mejnatowicz (1983) presented evidence of loss of genes and heterozygosity in tolerant Scots pines. The frequent lack of a bottleneck effect has been explained by different hypotheses: successive colonization events, a high number of tolerant plants in the primary populations, pollen flow from the neighboring populations, environmental heterogeneity and human disturbance (Nkongolo *et al.,* 2007).

Molecular analyses of several conifer and hardwood species clearly indicated that the exposure to metals for more than 30 years has no effect on genetic structure and diversity of early generations of *Picea mariana, P. glauca, Pinus banksiana, P. rubens, P. strobus,* and several hardwood populations in Northern Ontario (Narendrula *et al.,* 2012; Nkongolo *et al.,* 2012; Dobrzeniecka *et al.,* 2011; Vandeligt *et al.,* 2011). This lack of association between the level of genetic variation and metal content can be attributed to the long life span of these tree species. Table 1 shows similar level of genetic variabilities in pine populations growing in metal contaminated sites for more than 30 years compared to control in Northern Ontario, Canada. This is in contrast to data observed in herbaceous species such as *D. cespitosa* where a high level of metal accumulation reduced significantly the level of genetic variation (Table 2) (Nkongolo *et al.,* 2007). Metals impose severe stress on plants, especially in the rooting zone, which has led to the evolution of metal resistant ecotypes in several herbaceous species like *D. cespitosa* (Cox and Hutchinson 1980).


**Table 1.** Genetic variability parameters of *Pinus banksiana* populations growing in the Sudbury, Ontario (Canada) area based on ISSR data.

P represents percentage of polymorphic loci; h, Nei's gene diversity; I, Shannon's information index; Ne, effective number of alleles; Na, observed number of alleles.


**Table 2.** Genetic variability within *Deschampsia cespitosa* populations from Northern Ontario generated with ISSR primers.

P represents percentage of polymorphic loci; Sudbury and Cobalt regions were moderately and highly contaminated with metals, respectively. Manitoulin Island region was not conta‐ minated with metals and was used as a control region.
