*3.4.3.1. Metal uptake*

*3.4.2.2. Nitrogen donor ligands*

64 Environmental Change and Sustainability

metal (Wenzel *et al.,* 2003).

*3.4.2.3. Sulfur donor ligands*

This group consists of amino acids and their derivatives which have relatively high affinity for specific metals. Krämer *et al.,* (1996) revealed histidine to be involved in the Ni tolerance and translocation of the hyperaccumulator plant *Alyssum lesbiacum.* The majority of Zn in roots of the Zn hyperaccumulator, *T. caerulescens*, was complexed with histidine (Salt *et al.,* 1999). Studies have also shown histidine to be involved in the restriction of metal uptake. For example, plants chelate Ni with histidine in the rhizosphere which prevents the uptake of this

In plants, sulfur donor ligands are composed of two classes of metal chelating ligands which are phytochelatins (PCs) and metallothioneins (MTs). Phytochelatins are small metal binding peptides synthesized from the tripeptide glutathione (γ-Glu-Cys)2-11-Gly) (Solanki and Dhankhar 2011; Hall, 2002). Since there is a γ-carboxamide linkage between glutamate and cysteine, PCs are not synthesized by translation of mRNA, but rather it is a product of an enzymatic reaction involving the enzyme PC synthase (Yong and Ma 2002). The production of PCs is positively correlated with metal accumulation in plant tissues (Pal and Rai 2010). PCs are produced in cells immediately after heavy metal exposure, including Cd, Pb, Zn, Ag, Hg, As and Cu as seen in *Rubia tinctorum* (Maitani *et al.,* 1996). PC production can be induced in roots, shoots, and leaves as observed in *Sedum alfredii* when exposed to Cd (Pal and Rai 2010). Several research groups concurrently and independently cloned and characterized genes encoding PC synthase. These genes were isolated from *Arabidopsis thaliana*, *Schizosaccharomyces pombe*, and *T. aestivum*, and were designated *AtPSC1, SpPCS,* and *TaPCS1*, respectively. They encoded 50-55kDa sequences with 40-50% similarity. The polypeptides were found to be active in the synthesis of PCs from glutathione (GSH) (Yong and Ma 2002). In cultured *Silene cucubalis* cells, the presence of heavy metals, such as Cd, Cu, Zn, Ag, Hg and Pb, induce the synthesis of PCs by PC synthase from the GSH like substance (Pal and Rai 2010). Gaudet *et al.* (2011) did a comparative analysis of two *Populus nigra* genotypes from contrasting envi‐ ronments. They determined that both genotypes responded differently to Cd stress. The southern genotype (Poli) was more tolerant than the northern genotype (58-861). This variation was due to different adaptation strategies to Cd stress. The thiol and PC content, which was associated with the *glutathione S-transferase* gene, was higher in the southern genotype as compared to the northern genotype, which under Cd stress, revealed differences in the use of

phytochelatin pathway that might be related to the variation in their Cd tolerance.

The second class of sulfur donor ligands are metallotioneins (MTs). They are low molecular weight (4-14kDa), cysteine-rich, metal-binding proteins found in a wide range of organisms (animals, plants, eukaryotic microorganisms, and prokaryotes) (Huang and Wang 2010). Unlike PCs, they are encoded by structural genes (Yong and Ma 2002). They play essential roles in a variety of organisms including Cu, Cd and Hg detoxification by sequestration (Palmiter, 1998; Ecker *et al.,* 1989), Zn homeostasis (Coyle *et al.,* 2002) and also scavenging of reactive oxygen species (Wong *et al.,* 2004). MTs have been divided into two classes based on their cysteine residue arrangements. Class I MTs are widespread in vertebrates and are The uptake of metal from soil into roots is dependent on the bioavailabilty of the metal, as well as its mobility in the rhizosphere (Maestri *et al.,* 2010). The bioavailability of various metals greatly varies. No correlation exists between the metal content in soils and in plants (Clemens, 2006). The bioavailability of metals in the rhizosphere is affected by the chemical environment. For example, in *T. caerulescens,* the chemical form of nitrogen influences the plants ability to uptake Cd and Zn (Maestri *et al.,* 2010; Xie *et al.,* 2009). Metals present in the rhizosphere of hyperaccumulators are more bioavailable than for those of non hyperaccumulators. Plants can render metals mobile in their rhizosphere by excreting root exudates, such as organic acids and phytosiderophores and by acidification with protons (Maestri *et al.,* 2010; Marschner, 1995). Bacteria in the soil also affect metal mobility and availability by lowering the pH, producing hormones, organic acids, antifungals, antibiotics and metal chelators which all enhance the root growth (Maestri *et al.,* 2010; Wenzel *et al.,* 2003). Higher amounts of bacteria were found in the rhizosphere of hyperaccumulators. Microorganisms found in the rhizo‐ sphere were linked to an increased uptake of Cd, Z, and Pb in *Sedum alfredii* and an enhanced root growth (Maestri *et al.,* 2010; Xiong *et al.,* 2008).

#### *3.4.3.2. Metal uptake across the plasma membrane*

The uptake of heavy metals in plants is mediated by a group of metal transporter families which consists of iron-responsive transport proteins (ZIP-IRT), the heavy metal-transporting P1B-type subfamily of P-type ATPases, the natural resistance associated macrophage proteins (NRAMP) and the cation diffusion facilitators (CDF) (Baxter *et al.,* 2003). Transporters were originally identified for Fe2+ or Zn2+ homeostasis, but it was demonstrated that most trans‐ porters of essential metal ions can also carry non essential metals, such as Cd (Zhou *et al.,* 2012). The uptake of non essential metals may be the result of their close chemical character‐ istics or metal ion size to essential metals. Some metal transporters, present in the plasma membrane of root cells, exhibit low substrate specificity which can lead to the accumulation of other metals in plants (Schaaf *et al.,* 2006). For example, the non-functional metal Cd can be taken up via a Ca2+ transporter (Perfus-Barbeoch *et al.,* 2002) or also via the Fe2+ transporter IRT1 (Korshunova *et al.,* 1999). Plant tolerance to metal stress can be achieved with the modification of these transporter activities (Zhou *et al.,* 2012). Plants can prevent the uptake of certain metals by down-regulating the expression of such transporters, as observed in *S. vulgaris,* where the tolerant plants restrict the uptake of Cu by the down-regulation of Cutransporters (Assunçãno *et al.,* 2003; Harmens *et al.,* 1993). Since Fe and Ni belong to the group of transient metals and have similar chemical properties, Fe deficiency may be the result of Ni phytotoxicity. Ni competes with Fe in physiological and biochemical processes, and in turn roots, can uptake Ni by Fe transporters (Pandey and Sharma 2002).

Increased Zn uptake is driven by an overexpression of members of the ZIP family of trans‐ porters. Under Zn deficiency conditions, many members of the ZIP transporter family are overexpressed in non hyperaccumulator species, while in hyperaccumulators, they are independently expressed regardless of Zn supply (Verbruggen *et al.,* 2009). Nishida *et al.* (2011) and Schaaf *et al.* (2006) showed that *A. thaliana* can increase the uptake of Ni in roots when Fe levels are low by the Iron-Regulated Transporter 1 (AtIRT1; member of Zrt/IRT-like *ZIP* family of transporters). AtIRT1 has a wide specificity for divalent heavy metals including Ni, Zn, Mg, Co and Cd and mediates the accumulation of such metals under Fe-deficient conditions. Nakanishi *et al.* (2006) reported that Cd was uptaken in yeast by two *O. sativa* Fe2+ transporters, *OsIRT1* (Iron-Regulated Transporter 1) and *OsIRT2.*

The uptake of Ni of some Ni hyperaccumulator accessions of *Thlaspi goesingense*, *Thlaspi japonicum* and *T. caerulescens* has been reported to be inhibited in the presence of Zn. This demonstrated that Ni entered the cell via Zn uptake transporters, specifically the TcZNT1 transporter (Assunçãno *et al.,* 2008). In Zn deficiency conditions, the expression of AtZIP4, the orthologue of TcZNT1 in *A. thaliana,* can be induced but when additional Ni was added, the expression was repressed. This suggested that Zn and Ni competed for their uptake via AtZIP4/TcZNT1 transporters (Hassan and Aarts 2011). In addition, in presence of high Zn concentration, the expression of *ZNT1* was higher in Zn hyperaccumulator *T. caerulescens*roots than in the non hyperaccumulator *Thlaspi arvense* suggesting its involvement in the hyperac‐ cumulator phenotype (Hassinen *et al.,* 2007; Assunçãno *et al.,* 2001; Assunçãno *et al.,* 2001; Pence *et al.,* 2000). Milner *et al.* (2012) also determined that NcZNT1, isolated from *T. caerulescens*, played a role in Zn uptake from the soil which was based on its high expression in root.

sphere were linked to an increased uptake of Cd, Z, and Pb in *Sedum alfredii* and an enhanced

The uptake of heavy metals in plants is mediated by a group of metal transporter families which consists of iron-responsive transport proteins (ZIP-IRT), the heavy metal-transporting P1B-type subfamily of P-type ATPases, the natural resistance associated macrophage proteins (NRAMP) and the cation diffusion facilitators (CDF) (Baxter *et al.,* 2003). Transporters were originally identified for Fe2+ or Zn2+ homeostasis, but it was demonstrated that most trans‐ porters of essential metal ions can also carry non essential metals, such as Cd (Zhou *et al.,* 2012). The uptake of non essential metals may be the result of their close chemical character‐ istics or metal ion size to essential metals. Some metal transporters, present in the plasma membrane of root cells, exhibit low substrate specificity which can lead to the accumulation of other metals in plants (Schaaf *et al.,* 2006). For example, the non-functional metal Cd can be taken up via a Ca2+ transporter (Perfus-Barbeoch *et al.,* 2002) or also via the Fe2+ transporter IRT1 (Korshunova *et al.,* 1999). Plant tolerance to metal stress can be achieved with the modification of these transporter activities (Zhou *et al.,* 2012). Plants can prevent the uptake of certain metals by down-regulating the expression of such transporters, as observed in *S. vulgaris,* where the tolerant plants restrict the uptake of Cu by the down-regulation of Cutransporters (Assunçãno *et al.,* 2003; Harmens *et al.,* 1993). Since Fe and Ni belong to the group of transient metals and have similar chemical properties, Fe deficiency may be the result of Ni phytotoxicity. Ni competes with Fe in physiological and biochemical processes, and in turn

Increased Zn uptake is driven by an overexpression of members of the ZIP family of trans‐ porters. Under Zn deficiency conditions, many members of the ZIP transporter family are overexpressed in non hyperaccumulator species, while in hyperaccumulators, they are independently expressed regardless of Zn supply (Verbruggen *et al.,* 2009). Nishida *et al.* (2011) and Schaaf *et al.* (2006) showed that *A. thaliana* can increase the uptake of Ni in roots when Fe levels are low by the Iron-Regulated Transporter 1 (AtIRT1; member of Zrt/IRT-like *ZIP* family of transporters). AtIRT1 has a wide specificity for divalent heavy metals including Ni, Zn, Mg, Co and Cd and mediates the accumulation of such metals under Fe-deficient conditions. Nakanishi *et al.* (2006) reported that Cd was uptaken in yeast by two *O. sativa* Fe2+

The uptake of Ni of some Ni hyperaccumulator accessions of *Thlaspi goesingense*, *Thlaspi japonicum* and *T. caerulescens* has been reported to be inhibited in the presence of Zn. This demonstrated that Ni entered the cell via Zn uptake transporters, specifically the TcZNT1 transporter (Assunçãno *et al.,* 2008). In Zn deficiency conditions, the expression of AtZIP4, the orthologue of TcZNT1 in *A. thaliana,* can be induced but when additional Ni was added, the expression was repressed. This suggested that Zn and Ni competed for their uptake via AtZIP4/TcZNT1 transporters (Hassan and Aarts 2011). In addition, in presence of high Zn concentration, the expression of *ZNT1* was higher in Zn hyperaccumulator *T. caerulescens*roots than in the non hyperaccumulator *Thlaspi arvense* suggesting its involvement in the hyperac‐

root growth (Maestri *et al.,* 2010; Xiong *et al.,* 2008).

roots, can uptake Ni by Fe transporters (Pandey and Sharma 2002).

transporters, *OsIRT1* (Iron-Regulated Transporter 1) and *OsIRT2.*

*3.4.3.2. Metal uptake across the plasma membrane*

66 Environmental Change and Sustainability

Heavy metal-transporting P1B-type transporters are also involved in metal-ion homeostasis and tolerance in plants by transporting essential and non essential heavy metals such as Cu, Zn, Cd, Pb across cell membrane. Transporters located at the plasma membrane function as efflux pumps by removing toxic metals form cytoplasm. They have also been found in membranes of intracellular organelles for compartmentalization of metals for sequestration in vacuoles, golgi or endoplasmic reticulum (Yang *et al.,* 2005b). These ion pumps transport ions across a membrane by hydrolysing ATP (Benitez *et al.,* 2012). Eight P1B-ATPases, AtHMA1– AtHMA8, have been reported in *Arabidopsis* (Baxter *et al.,* 2003). AtHMA1, 2, 3, and 4 showed high similarity with Zn2+/Co2+/Cd2+/Pb2+ ATPases previously characterized in prokaryotes (Axelsen and Palmgren 2001). The AtHMA4 was located at the plasma membrane. The ectopic expression of AtHMA4 improved the growth of roots in the presence of toxic Zn, Cd and Co concentrations (Yang *et al.,* 2005b). The heterologous expression of AtHMA4 enhanced Cd tolerance in yeast (Mills *et al.,* 2003).

In addition, the gene *Nramp* encodes for another divalent metal transporter located at the plasma membrane. This transporter also removes toxic metals from the cytosol by efflux pumping. It has been reported to be expressed in roots of *Arabidopsis* and *O. sativa* (*OsN‐ ramp1*- expressed in rice roots where as *OsNramp2* is expressed in leaves and *OsNramp3* is expressed in both tissues). The *OsNramp1* gene was found to be involved in the uptake of Mn, while the *Nramp* genes in *Arabidopsis* and rice were involved in the uptake of Cd, and other divalent metals (Yang *et al.,* 2005b). The AtNRAMP1, 3, and 4 showed uptake of Cd2+ when they were expressed in the yeast *Saccharomyces cerevisiae*. In addition, Cd2+ hypersensitivity was observed in *A. thaliana* when AtNRAMP3 was overexpressed. This transporter was located in the vacuolar membrane where it is involved in the mobilization of metals from the vacuole (Clemens, 2006).

In bacteria and in some eukaryotes, Zn, Co and Cd are transported by the CDF transport proteins. Within the *Arabidopsis* genome, there are 12 nucleotide sequences that are predicted to encode members of CDF transporter family. However, these transporters might be involved in cation efflux out of the cytoplasm, by pumping ions out of the cytoplasm to the exterior of the cell or into intracellular compartments such as the vacuole (Yang *et al.,* 2005b).

Plants can make metal ions more available for uptake by acidifying the rhizosphere and pumping protons via plasma membrane-localized proton pumps; and also by exuding low molecular weight (LMW) compounds that act as metal chelators (Clemens, 2006). The secretion of organic acids can render heavy metals mobile and enhance their absorption by plant roots. Krishnamurti *et al.* (1997) reported that when Cd was complexed with organic acids, it was readily available for transport across the membrane, while free Cd ions were restricted for uptake. Cieśliński *et al.* (1998) revealed a higher acetic acid and succinate in the rhizosphere of the *T aestivum* (Kyle) Cd accumulating genotype compared to the non accumulating (Arcola) wheat genotype. The Zn/Cd hyperaccumulating *Sedum alfredii* was able to extract high levels of Zn and Pb from its contaminated environment because of the release of root exudates (Li *et al.,* 2005). In *Alyssum*, the Ni transport and accumulation was enhanced by secretion of histidine in the rhizosphere (Krämer *et al.,* 1996).

### *3.4.3.3. Sequestration/compartmentation*

Some metal tolerant plants can accumulate large amounts of metals within the cell without exhibiting toxicity symptoms (Entry *et al.,* 1999). These plants are able to store the surplus of accumulated metals where no sensitive metabolic activities occur such as organs or subcellular compartments (Ernst, 2006). This avoidance of metal poisoning involves the intracellular sequestration and apoplastic or vacuolar compartmentation of the toxic metal ions (Liu *et al.,* 2007). Compartmentation of metals can also be found in the cells central vacuole. This was observed in the Zn resistant *Deschampsia cespitosa* where the excess Zn ion was removed from the cytoplasm and actively pumped into the vacuoles of root cells where as Zn sensitive plants had a much lower capacity to do so (Brookes *et al.,* 1981).

Schaaf *et al.* (2006) determined that the transporter AtIREG2, located at the tonoplast, was involved in Ni detoxification in roots. AtREG2, confined to roots, prevents heavy metal translocation to shoots restricting metals to roots. This transporter counterbalances the low substrate specificity of transporter AtIRT1 and other iron transporters in iron deficient root cells*.* The AtIREG2 transporter, found in *A. thaliana*, was involved in the detoxification of Ni in roots under Fe deficiency conditions at pH 5 (Schaaf *et al.,* 2006). The *T. caerulescens ZTP1* gene was involved in the intracellular sequestration of Zn. The expression of the *ZTP1* gene was higher in the roots and shoots of the Zn tolerant *T. caerulescens* compared to the non tolerant plant (Assunçãno *et al.,* 2001).

Members of the CDF protein play a role in tolerance to various metals including Cd, Co, Mn, Ni and Zn by their sequestration into vacuoles (Montanini *et al.,* 2007). Increased Zn tolerance and accumulation was reported in non accumulator *A. thaliana* when *AtMTP1*, *PtdMTP1*, *AtMTP3* and *TgMTP1* (members of the CDF family) were ectopically or heterologously expressed. This suggested that the function of these proteins was the creation of a sink of Zn in the vacuole of plant cells in instances of high intracellular Zn levels or as buffer in Zn deficiency situations (Hassan and Aarts 2011).

Phytochelatins are also thought to be involved in the restriction of metals to the roots (Zenk, 1996). When *Nicotiana tubacum* seedlings were exposed to excess Cd, the level of phytochelatin increased (Vogelilange and Wagner 1990). The metal-phytochelatin complexes are formed when plants are exposed to high heavy metal concentrations. They are then sequestered into vacuoles for detoxification. A group of organic solute transporters actively transport phyto‐ chelatin-metal complexes into the plant's vacuole (Solanki and Dhankhar 2011; Salt and Rauser 1995). In the presence of excess Cu and Cd, phytochelatins form complexes with these metals in *Zea mays* and in turn reduce the root to shoot translocation (Galli *et al.,* 1996). The synthesis of phytochelatins is catalyzed by the enzyme phytochelatin synthase (PCS), a constitutive enzyme which requires post-translational activation by heavy metals and/or metalloids that include Cd, Ag, Pb, Cu, Hg, Zn, Sn, As and Au (Solanki and Dhankhar 2011). Martínez *et al.* (2006) reported that the expression of a PCS gene isolated from *T. aestivum* improved the accumulation of Cd, Pb and Cu in *Nicotiana glauca.* The elevation of phytochelatin concentra‐ tion in roots might reduce the root to shoot transport required for accumulation in shoots.

### *3.4.3.4. Root to shoot translocation*

*et al.,* 2005). In *Alyssum*, the Ni transport and accumulation was enhanced by secretion of

Some metal tolerant plants can accumulate large amounts of metals within the cell without exhibiting toxicity symptoms (Entry *et al.,* 1999). These plants are able to store the surplus of accumulated metals where no sensitive metabolic activities occur such as organs or subcellular compartments (Ernst, 2006). This avoidance of metal poisoning involves the intracellular sequestration and apoplastic or vacuolar compartmentation of the toxic metal ions (Liu *et al.,* 2007). Compartmentation of metals can also be found in the cells central vacuole. This was observed in the Zn resistant *Deschampsia cespitosa* where the excess Zn ion was removed from the cytoplasm and actively pumped into the vacuoles of root cells where as Zn sensitive plants

Schaaf *et al.* (2006) determined that the transporter AtIREG2, located at the tonoplast, was involved in Ni detoxification in roots. AtREG2, confined to roots, prevents heavy metal translocation to shoots restricting metals to roots. This transporter counterbalances the low substrate specificity of transporter AtIRT1 and other iron transporters in iron deficient root cells*.* The AtIREG2 transporter, found in *A. thaliana*, was involved in the detoxification of Ni in roots under Fe deficiency conditions at pH 5 (Schaaf *et al.,* 2006). The *T. caerulescens ZTP1* gene was involved in the intracellular sequestration of Zn. The expression of the *ZTP1* gene was higher in the roots and shoots of the Zn tolerant *T. caerulescens* compared to the non tolerant

Members of the CDF protein play a role in tolerance to various metals including Cd, Co, Mn, Ni and Zn by their sequestration into vacuoles (Montanini *et al.,* 2007). Increased Zn tolerance and accumulation was reported in non accumulator *A. thaliana* when *AtMTP1*, *PtdMTP1*, *AtMTP3* and *TgMTP1* (members of the CDF family) were ectopically or heterologously expressed. This suggested that the function of these proteins was the creation of a sink of Zn in the vacuole of plant cells in instances of high intracellular Zn levels or as buffer in Zn

Phytochelatins are also thought to be involved in the restriction of metals to the roots (Zenk, 1996). When *Nicotiana tubacum* seedlings were exposed to excess Cd, the level of phytochelatin increased (Vogelilange and Wagner 1990). The metal-phytochelatin complexes are formed when plants are exposed to high heavy metal concentrations. They are then sequestered into vacuoles for detoxification. A group of organic solute transporters actively transport phyto‐ chelatin-metal complexes into the plant's vacuole (Solanki and Dhankhar 2011; Salt and Rauser 1995). In the presence of excess Cu and Cd, phytochelatins form complexes with these metals in *Zea mays* and in turn reduce the root to shoot translocation (Galli *et al.,* 1996). The synthesis of phytochelatins is catalyzed by the enzyme phytochelatin synthase (PCS), a constitutive enzyme which requires post-translational activation by heavy metals and/or metalloids that include Cd, Ag, Pb, Cu, Hg, Zn, Sn, As and Au (Solanki and Dhankhar 2011). Martínez *et al.* (2006) reported that the expression of a PCS gene isolated from *T. aestivum* improved the

histidine in the rhizosphere (Krämer *et al.,* 1996).

had a much lower capacity to do so (Brookes *et al.,* 1981).

*3.4.3.3. Sequestration/compartmentation*

68 Environmental Change and Sustainability

plant (Assunçãno *et al.,* 2001).

deficiency situations (Hassan and Aarts 2011).

The translocation of metals to the aerial biomass can be an important biochemical process used by plants to remediate polluted areas. In some plants, the mobilization of metals from their roots to their above aerial organs can minimize the damage that could be exerted by these heavy metals on the root physiology and biochemistry (Zacchini *et al.,* 2009). Excluders prevent or limit the translocation of toxic metals or essential metals from roots to shoots. On the other hand, accumulators/hyperaccumulators translocate metals from roots to shoots via the xylem with the transpiration stream. This is accomplished by increasing the uptake of metals in roots, and by reducing the sequestration of metals in the root.

The chelation of metals with ligands, such as organic acids, amino acids and thiols facilitates the movements of heavy metals from roots to shoots (Zacchini *et al.,* 2009). The xylem cell wall has a high cation exchange capability, thus the movement of metal cations is severely retarded when the metals are not chelated by ligands. Organic acids are involved in the translocation of Cd in the species *Brassica juncea* (Salt *et al.,* 1995).

The chelation of Ni to histidine is involved in the long distance translocation of Ni in the hyperaccumulator *A. lesbiacum*, where a 36-fold increase was reported in the histidine content of the xylem sap upon exposure to nickel (Solanki and Dhankhar 2011; Krämer *et al.,* 1996). Richau *et al.* (2009) found that the Ni hyperaccumulator, *T. caerulescens*, had a higher free histidine concentration in roots compared to the non Ni hyperaccumulator *T. arvense.* Also, *T. caerulescens* had less Ni in root vacuoles than *T. arvense* because the histidine-Ni complexes were much less taken up by vacuoles than free Ni ions. Therefore, an increase in free histidine in roots inhibited the vacuolar sequestration of His-Ni in *T. caerulescens* compared to free Ni in *T. arvense* and also had enhanced histidine-mediated Ni xylem loading. The elevated free histidine in root cells appears to be involved in reduced vacuolar sequestration and enhanced xylem loading of Ni (Richau and Schat 2009). This was also the case for Zn and Cd for this hyperaccumulating species (Hassan and Aarts 2011). An increase in Ni accumulation was also observed in the Ni hyperaccumulator *Sebertia acuminata* where, when chelated to citrate, Ni was able to translocate to the shoot. In the absence of citrate, Ni was no longer accumulated in the aerial tissues (Lee *et al.,* 1977).

The chelation of metals with nicotianamine (NA) also contributes to improved tolerance. Nicotianamine can chelate and transport divalent Ni, Cu and Zn (Takahashi *et al.,* 2003; Pich *et al.,* 2001; Ling *et al.,* 1999). The nicotianamine synthase (NAS) enzyme is responsible for the synthesis of NA by trimerization of S-adenosylmethionine (Shojima *et al.,* 1990). When exposed to high levels of Zn, Cd, and/or Ni, all four *NAS* genes were highly expressed in *T. caerules‐ cens* compared to non hyperaccumulator *A. thaliana* (van de Mortel *et al.,* 2006). In the presence of elevated Mn, Zn, Fe and Cu concentrations, Kim *et al.* (2005) reported an increased expres‐ sion of the *NAS* gene, as well as NA levels for *A. thaliana* and *N. tubacum*. In addition, Pianelli *et al.* (2005) showed that the over-expression of the *T. caerulescens NAS3* gene in the Ni excluder *A. thaliana* resulted in improved Ni tolerance and Ni accumulation in their aerial organs. An increase of Fe, Zn and Cu accumulation in *O. sativa* was associated with an overexpression of the *NAS3* gene (Hassan and Aarts 2011; Kawachi *et al.,* 2009).

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. vulgaris*.

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 (Clemens, 2006).

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 the xylem.

#### *3.4.3.5. Metal storage*

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 1995). Once unloaded, the metals are either taken up into surrounding cells and are symplas‐ tically transported through the leaf tissues or they are apoplastically distributed over the leaf (Hassan and Aarts 2011; Marschner, 1995). NA is important in the chelation of metals for their symplastic transport through the leaf. This occurs through the Yellow Stripe Like proteins (YLS) (Hassan and Aarts 2011; DiDonato *et al.,* 2004). In the hyperaccumulator *T. caerulescens*, three *YSL* genes (*TcYLS3*, *TcYSL5* and *TcYSL7*) were highly expressed in shoots around vascular tissues. This high level of expression was not observed in the excluder plant *A. thaliana* orthologues (Hassan and Aarts 2011; Gendre *et al.,* 2007). For the *TcYSL3*, it was suggested that its function was to unload Ni-NA complexes from the xylem into leaf cells and to distribute it to storage cells. Using yeast complementation and uptake measurement studies, it was determined that TcYSL3 was also a Fe/Ni-NA influx transporter. Considering that YSL proteins have a role in the transport of Fe-NA complexes, it was proposed that they might also be involved in the hyperaccumulation of Fe-NA in some plants (Hassan and Aarts 2011; Curie *et al.,* 2009).

The sequestration of excess essential and non essential metals is localized in various parts of the aerial tissue, such as trichomes, leaf epidermal cell vacuole and mesophyll vacuole. Broadhurst *et al.* (2004) grew five *Alyssum* hyperaccumulator species/ecotypes on Ni-enriched soil and determined that the majority of hyperaccumulated Ni was stored in either leaf epidermal cell vacuoles or in the basal section of stellate trichomes. They also found that the metal concentration in the basal part of the trichome was 15% to 20% of dry weight. This was among the highest metal concentrations reported in healthy vascular plant tissues. In *A. halleri*, the majority of Zn ions were stored in the vacuoles of mesophyll cells, while for *T. caerulescens*, most Zn ions were located in the vacuoles of epidermal cells (Verbruggen *et al.,* 2009). The transport of metals through the phloem sap is less documented. The sole molecule identified as a phloem metal transporter is nicotianamine which is involved in the transport of Fe, Cu, Zn and Mn (Stephan *et al.,* 1994).
