One of the promising approaches to enhance the ability of metal ions to enter plant cells is the identification of the metal transporter proteins and introducing genes encoding transporter molecules. These are generally proteins found in the cell membrane, that either have an affinity for metal ions, or that create favorable energetic conditions to allow metals to enter the cell. Till date, several plant metal transporters have been reported and more remain to be recognized. Some of the transporters identified so far include the Arabidopsis IRT1 gene that encodes a protein that regulates the uptake of iron and other metals (Eide et al. 1996) and the MRP1 gene encoding, an Mg-ATPase transporter, also from Arabidopsis (Lu et al. 1997).

Further, success in this approach is achieved by identifying proteins such as ZNT1, ZIP1-4, IRT1, COPT1, LCT1, and tVramp-1/3/4 on the plasma membrane-cytosol interface; ZAT, ABC type, HMT1, AtMRP, CAX2 seen in vacuoles; and RAN1 in Golgi bodies. Manipulations of these transporters to achieve the removal of metal ions from the cell hold great potential (Tong et al. 2004). The natural resistance-associated macrophage proteins (Nramp) family of transporters has been recently characterized from rice and Arabidopsis.

The principal classes of metal chelators include phytochelatins, metallothioneins, organic acids, and amino acids. Phytochelatins (PCs) are small metal-binding peptides found in plants. Iso-PCs, a series of PC-like homologous chelating peptides, are reported with varying terminal amino acids and have a C-terminal modified residue other than glycine. The PC and iso-PC molecules form complexes with heavy metals like Cd. In addition to PC-Cd complex other PC-metal complexes include Ag, Cu, and As (Shah and Nongkynrih 2007).

In vitro experiments have shown that a series of metal-sensitive plant enzymes can tolerate a 10- to 1,000-fold concentration of Cd in the form of a PC complex than as free radical ion. PC reactivate metal poisoned plant enzymes such as nitrate reductase up to 1,000-fold better than chelators such as glutathione (GSH) or citrate, signifying the extraordinary sequestering potential of these peptides.

Metallothioneins (MTs) are metal-binding proteins that confer heavy metal tolerance and accumulation (Hamer 1986; Malin and Bülow 2001). These are low molecular mass cysteine rich proteins that were originally isolated as Cu, Cd, and Zn binding proteins in mammals. MTs are able to bind a variety of metals by the formation of mercaptide bonds between the numerous cysteine (Cys) residues present in the proteins and the metal, and it is the arrangement of these residues that in part determines the metal-binding properties of the MT proteins. Researchers successfully reported more than 50 MTs in different plants categorized into four types, types 1–4. These are based on the Cys arrangement. Although MTs are expressed throughout the plant, some have been found to be expressed in a tissue-specific manner. Transgenic plants expressing MTs have been created, and although these plants exhibited enhanced tolerance to high metal concentrations, the uptake of metals was not enhanced. To enhance higher plant metal sequestration, the yeast MT CUP1 was introduced into tobacco plants, and the cup 1 gene expression and Cu and Cd phytoextraction were determined. Overexpression of Cu inducible MT cup 1 also enhanced Cu tolerance in plants. In plants, a wide range of MT genes from various sources have been overexpressed including those from human, mouse, Chinese hamster, and yeast.

The vacuole is generally considered to be the main storage site for metals in yeast and plant cells and there is evidence that phytochelatin-metal complexes are pumped into the vacuole. The best characterized of the known vacuolar transporters and channels involved in metal tolerance are YCF1 from yeast, Saccharomyces cerevisiae. YCF1 is an MgATP-energized glutathione S-conjugate transporter responsible for vacuolar sequestration of compounds after their S-conjugation with glutathione. Overexpression of the YCF1 gene in Arbidobsis thaliana and the YCF1 proteins were found to be associated with the tonoplast and the plasma membrane. The vacuoles of the YCF1-transgenic plants exhibited a 4-fold higher rate of glutathione-Cd uptake than those of wild-type plants, indicating that expression of YCF1 strongly increases Cd transport activity. The transgenic plants showed improved resistance to both Cd and Pb and elevated metal content, characteristics desirable for phytoremediation.

Genes may be introduced that code for enzymes to change the oxidation state of heavy metals like Hg and selenium (Se). For instance, introduction of the bacterial merA gene encoding mercuric oxide reductase (Rugh et al. 1996), or that which converts metals into less toxic forms, such as enzymes that can methylate Se into dimethylselenate (Hansen et al. 1998). In both these cases, the resulting form of the metal is volatile, so that one can create a plant capable of metal remediation by phytovolatilization, another type of phytoremediation.

Several researchers have already reported encouraging results using plants bioengineered with increased heavy metal tolerance and uptake of heavy metals for the purpose of phytoremediation. The majority of these novel plants have only been tested under limited laboratory conditions and very few have been grown in the field (Table 13.1).