**2. Phytochelatins in metal/metalloid-exposed plants**

metals at toxic levels have the capability to interact with several vital cellular biomolecules such as nuclear proteins and DNA, leading to excessive augmentation of reactive oxygen species (ROS) [4–6]. In addition, these metals generate ROS which in turn can cause neuro‐ toxicity, hepatotoxicity and nephrotoxicity in humans and animals [7, 8]. Notably, higher plants, algae, certain yeasts and animals are equipped with a repertoire of mechanisms to counteract metal toxicity. The key elements of these are chelation of metals by forming phytochelatins (PCs) and related cysteine-rich polypeptides [9–11]. PCs are produced from glutamine, cysteine and glycine and the process is catalysed by PC synthases known as γglutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases [12, 11]. PCs have been identified in a wide variety of plant species, microorganisms and invertebrates. They are structurally related to glutathione (GSH) and were presumed to be the products of a biosynthetic pathway. Numerous physiological, biochemical and genetic studies have confirmed that GSH is the substrate for PC biosynthesis [13, 14]. The general structure of PCs is (γ-Glu-Cys)n-Gly, with increasing repetitions of the dipeptide Glu-Cys, where *n* can range from 2 to 11 but is typically no more than 5 [15]. Except glycine, other amino acid residues can be found on the C-terminal end of (γ-Glu-Cys)*n* peptides. In Figure 1, we show the general structure of PC and the major steps involved in its synthesis from GSH through PC synthase in response to high concentra‐ tions of toxic metals. Originally thought to be plant-specific, PC and PC synthases have now been reported in a few fungal taxa, such as the yeast *Schizosaccharomyces* sp. and the mycor‐ rhizal ascomycete *Tuber melanosporum* [16, 17] and invertebrates belonging to the nematodes,

In the light of recent literature, the PCs' role and modulation are overviewed separately in metal-exposed plants and animals/humans and major methods for the determination of PCs and the bioassays for enzymes involved in PC synthesis are discussed hereunder. Additionally, connection of PCs with bionanoparticles is evaluated, and finally, major aspects so far

annelids or plathyhelminths [18, 19, 4, 1, 20, 17, 21–24].

396 Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives

unexplored in the present context are briefly highlighted.

Step I

> **N H**

**Phytochelatins**

**NH O**

(GSH) through a PC synthase in response to high concentrations of toxic metals.

**2**

**O O**

**HO**

**SH**

γ-Glu-Cys-Gly γ-Glu-Cys+Gly γ-Glu-Cys+ (γ-Glu-Cys)<sup>n</sup>

**H N O**

**n**

**Figure 1.** General structure of phytochelatins (PCs) and the major steps involved in its synthesis from glutathione

**OH**


Step

(γ-Glu-Cys)n+1-Gly

Contamination by metals can be considered as one of the most critical threats to soil and water resources as well as to human health [25, 26]. In fact, the contamination of soils with toxic metals has often resulted from human activities, especially those related to accelerated rate of industrialization, intensive agriculture and extensive mining. Metal belongs to group of nonbiodegradable, persistent inorganic chemical having cytotoxic, genotoxic and mutagenic effects on humans or animals and plants through influencing and tainting food chains, soil, irrigation or potable water and aquifers [27, 28, 6]. Chelation and sequestration of metals by particular ligands are the major mechanisms employed by plants to deal with metal stress. The two best-characterized metal-binding ligands in plant cells are the PC and metallothioneins (MTs) [29–33, 6, 34].

Figure 2 shows the scheme of metal-detoxification by PCs in a plant cell. PC, which has a higher affinity for Cd, is formed by the polymerization of 2–11 γ-EC moieties via PC synthase. Several studies confirm that in plants, both GSH and PC synthesis are increased after exposure to Cd and other metals [12, 35–41]. In Figure 3, we show both general functions of the PC and a model of complex between Cd+2 ion and one molecule of PC.

Gonzalez-Mendoza et al. showed that PC synthase gene (in coordination with the expression of metallothionein gene) is present in *Avicennia germinans* leaves, and that their expression increases in response to metal exposure, which supports the hypothesis that PC synthase and metallothionein are part of the metal-tolerance mechanisms in this species. In addition, these authors found that *A. germinans* has the ability to express both genes (*AvMT2* and *AvPCS*) as a coordinated response mechanism to avoid the toxic effects caused by non-essential metals. However, for essential metals such as Cu2+, the results showed that *AvPCS* was the most active gene involved in the regulation of this metal in the leaves [42]. Recent study showed that *Lunularia cruciata* compartmentalizes Cd+2 in the vacuoles of the photosynthetic parenchyma by means of a PC-mediated detoxification strategy, and possesses a PC synthase that is activated by Cd and homeostatic concentrations of Fe(II) and Zn. *Arabidopsis thaliana* PC synthase displays a higher and broader response to several metals (such as Cd, Fe(II), Zn, Cu, Hg, Pb, As(III)) than *L. cruciata* PC synthase [35].

Naturally hyperaccumulating plants do not overproduce PCs as a part of their mechanism against toxic metals. This appears to be an inducible rather than a constitutive mechanism, observed especially in metal non-tolerant plants [43]. Some reports have argued against the roles of PC in some metal-tolerant plants based on the effects of buthionine-S-sulphoximine and PCs/metal concentrations [44]. Several studies on plants overexpressing γ-glutamylcysteine synthetase or transgenic plants expressing bacterial γ-glutamyl-cysteine synthetase evaluated its effect on metal tolerance based on the assumption that higher levels of GSH and PCs will lead to more efficient metal sequestration [45]. *Bacopa monnieri*, a wetland macrophyte, is well known for its accumulation potential of metals and metal tolerance and thus is suitable for phytoremediation. Aquatic plants respond to metal stress by increasing the production of PC as well as other antioxidants. The accumulation potential of *B. monnieri* for various metals warrants its evaluation for metal tolerance and detoxification mechanism and for its suitability

**Figure 2.** The scheme of heavy metal (HM) detoxification by phytochelatins (PC) in a plant cell. HM activates phyto‐ chelatin synthase (PCS) and the HM–PC complexes are established. These complexes are consequently transported through tonoplast to vacuole by ATP-binding-cassette and P1B-ATPase transporter (ABC-P1B). HM is chelated in the cytosol by ligands such as PC. Induction of PC synthesis by HM and a large flux of GSH is further achieved by in‐ creased activity of the GSH metabolic enzymes, γ-ECS and GS. It is possible that the enzyme activation is not directed through effects of HM but due to H2O2 produced as a result of HM-presence. Transport of HM through the plasma membrane (ZIP). Vacuolar transport of HM (NRAMP: natural resistance associated macrophage protein). Heavy met‐ als are shown as black dots. Figure adapted and modified from [26].

**Figure 3.** General functions of phytochelatins (PCs) and the model of complex between cadmium (Cd+2) ion and one molecule of PC2. Cys, cysteine; Glu, glutamic acid; Gly, glycine; S, sulphur.

in phytoremediation [38]. In a study on *Arabidopsis thaliana* showed that Cd is immediately scavenged by thiols in root cells, in particular PC, at the expense of GSH. At the same time, a redox signal is suggested to be generated by a decreased GSH pool in combination with an altered GSH:GSSG ratio in order to increase the antioxidant capacity [46]. Overexpression of PCs synthetase in Arabidopsis led to 20–100 times more biomass on 250 and 300 μM arsenate than in the wild type. Also, the accumulation of thiol-peptides was 10 times higher after the exposure to Cd and arsenic, compared to the wild type. Gamma-glutamyl cysteine, which is a substrate for PC synthesis, increased rapidly after arsenate or Cd-exposure. Overexpression of PC synthase gene can be useful for phytoremediation [47]. Additionally, legumes are also capable of synthesizing homo-PCs in response to metal stress [45]. Citrus plants were also reported to synthesize PC in response to metal intoxication [48]. In wheat (*Triticum aestivum*), PC–metal complexes have been reported to accumulate in the vacuole. Retention of Cd in the root cell vacuoles might influence the symplastic radial Cd transport to the xylem and further transport to the shoot, resulting in genotypic differences in grain Cd accumulation [49].
