*3.4.2.3. Sulfur donor ligands*

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 composed of 20 highly conserved cysteine residues based on mammalian MTs. Class II MTs have slightly flexible cysteine arrangements and are found in plants, fungi and invertebrates. A third class includes phytochelatins (Chaturvedi *et al.,* 2012). Based on the position and allocation of cysteine residues, class II plant MTs are additionally divided into four types (Cobbett and Goldsbrough 2002). Type 1 plant MT genes have been more highly expressed in roots compared to leaves while the reverse is observed for the expression of type 2 plant MT genes. Type 3 MT genes are highly expressed in ripening fruits or in leaves while the expression of type 4 plant MT gene is restricted to developing seeds (Sekhar *et al.,* 2011; Cobbett and Goldsbrough 2002).

The expression of MT genes in plants subjected to metal stress has been studied. *AtMT1* and *AtMT2* genes showed increased expression levels when *Arabidopsis* plants were exposed to high levels of Cu and Cd (Sekhar *et al.,* 2011). Van Hoof *et al.* (2001) reported that the copper tolerant *S. vulgaris* individuals showed higher *SvMT2b* expression in roots and shoots when exposed to high concentrations of copper compared to the copper sensitive plants. Huang and Wang (2009) reported an increase in BgMT2 mRNA expression in large-leafed mangrove plants (*Bruguiera gymnorrhiza*) when exposed to Zn, Cu or Pb. Similar results were described by Gonzalez-Mendoza *et al.* (2007) in black mangrove *(Avicennia germinans*) seedlings exposed to Cd or Cu, showing a significant increase in AvMT2. High levels of the *CcMT1* transcripts were also observed in pigeon pea (*Cajanus cajan* L.) exposed to Cd and Cu (Sekhar *et al.,* 2011).

In general, there are variations between species in the expression of MTs to various metals. The up and down regulation of MTs in response to metal stress is largely unknown in plants. The MT gene expression was shown to be strongly induced by Cu, Cd, Pb and Zn (Huang and Wang 2009; Gonzalez-Mendoza *et al.,* 2007; van Hoof *et al.,* 2001). MT gene expression is also influenced by other abiotic stressors including absciscic acid (ABA), drought, salinity, heat, cold light, wounding and senescence (Sekhar *et al.,* 2011).
