**Abstract**

Phytochelatin synthase (PCS) is well-known for its role in heavy metal detoxification in plants, yeasts and non-vertebrate animals. It is a protease-like enzyme that catalyzes glutathione (GSH) to form phytochelatins (PCs), a group of Cys-rich and non-translational polypeptides with a high affinity to heavy metals. In addition, PCS also functions in xenobiotic metabolism by processing GS-conjugates in the cytosol. Because PCS is involved in GSH metabolism and the degradation of GS-conjugates, it is one of the important components in GSH homeostasis and GSH-mediated biodegradation. This chapter reviews the biochemical mechanism of PCS, how the enzyme activity is regulated, and its roles in heavy metal detoxification as well as GS-*S*-conjugate metabolism. This chapter also highlights the potential applications of PCS in the improvement of plant performance under combined stresses.

**Keywords:** Phytochelatin synthase, heavy metal stress, GS-conjugate metabolism, glutathione, combined pollution

### **1. Introduction**

Phytochelatins (PCs, (γGlu-Cys)n-Gly, n = 2–11) are cysteine-rich polypeptides that are synthesized non-translationally from the tripeptide glutathione (GSH, γGlu-Cys-Gly); this process is catalyzed by phytochelatin synthase (PCS, EC 2.3.2.15) [1–4]. PCs play essential roles in heavy metal detoxification because of their high affinities to a broad range of metal ions, e.g. cadmium (Cd), mercury (Hg), arsenic (As), zinc (Zn), lead (Pb), silver (Ag), nickel (Ni) and copper (Cu) [1–3]. Upon exposure to heavy metals, PCs are synthesized in the cytosol to chelate free metal ion and to prevent the generation of hydroxyl radicals [4–6] (**Figure 1**). These PC-metal complexes eventually are transferred into the vacuole through specific tonoplast ABCC-type transporters for sequestration [22–26] (**Figure 1**). In plants, PCS is constitutively expressed in the cytosol and can be activated by multiple types of metal ions [1, 3, 6]. For example, AtPCS1 isolated from *Arabidopsis thaliana* can be activated by the metal ions mentioned above [6]. In addition, some PCS homologs, such as the model legume *Lotus japonicus* LjPCS1 and LjPCS3, can be activated by iron (Fe) and aluminum (Al) [27].

PCS can be found in plants, yeasts and non-vertebrate animals and plays a critical role in responding to heavy metal stress in these organisms [28–32]. It was first partially purified from the suspension cells of bladder campion (*Silene cucubalus*) for its ability to synthesize PCs from GSH in the presence of Cd2+ [4]. Soon after the isolation of the enzyme, the genes coding PCS were cloned from plant and yeast

#### **Figure 1.**

*The involvement of phytochelatin synthase in glutathione metabolism, heavy metal detoxification, and glutathione-S-conjugate degradation.*

*An overview of the roles of phytochelatin synthase (PCS) in the metabolic pathways of glutathione (GSH, γ-Glu-Cys-Gly). The brief pathway of GSH biosynthesis and the major route of glutathione-S-conjugate (GS-conjugate) degradation are also shown in the figure [7–9]. The presence of xenobiotic compounds (X, marked as green circles) and free heavy metal ions (red circles) induces ROS generation and causes oxidative stress. The cytosolic xenobiotic compound is transferred to GSH by glutathione S-transferase (GST) to initiate the detoxification, and then the GS-conjugates enter vacuoles for further degradation [10–13]. In the vacuoles, GS-conjugates are first catalyzed to Cys-Gly-conjugates by γ-glutamyl transpeptidase (GGT) before the final deglycination catalyzed by carboxypeptidase (CP)[14–16]. In the presence of heavy metals, PCS uses GSH as substrates to synthesize phytochelatins (PCs), which chelate free metal ions in the cytosol [4]. Heavy metals also activate PCS to initiate the cleavage of GS-conjugates [17–20]. The cytosolic γ-Glu-Cys-conjugates then enter vacuoles and serve as the substrates of GGT for the second step of degradation [21]. The blue cylinders represent tonoplast ABCC transporters that facilitate the import of GS-metabolites and PC-metal complexes [12, 13, 22–24]. In the brief biosynthetic pathway of GSH, the rate-limiting enzymes are indicated in bold. Note that the cellular compartmentation of GSH synthesis or redox reactions is not included in this figure. SAT, serine acetyltransferase; γ-ECS, γ-glutamylcysteine synthetase; GSH-S, GSH synthetase. GP, GSH peroxidase; GR, GSH reductase. The figure was created with BioRender.com.*

sources, including AtPCS1, TaPCS1 from wheat (*Triticum aestivum*), and SpPCS from *Schizosaccharomyces pombe* by three independent research groups [5, 33, 34]. Since then, PCS sequences from various model organisms have been largely characterized, such as *Caenorhabditis elegans* (CePCS1) [31], the Cd hyperaccumulator *Thlaspi caerulescens* (TcPCS1) [35], and *Oryza sativa* (OsPCS1, OsPCS2, OsPCS5, OsPCS15) [36–38]. Besides eukaryote PCS sequences, a gene encoding a PCS-like protein, NsPCS, was identified from the genome of cyanobacterium *Nostoc* sp. [39–42].

PCS is a key component for the heavy metal tolerance in plants. Its importance was first confirmed in the Arabidopsis mutants locking AtPCS1 activity, as these mutants show severe growth defects when challenged by heavy metals such as Cd2+, Hg2+, Zn2+, Pb2+, and As3+ [28, 43–45]. The synthesis of PCs is crucial to the local response to heavy metal stress and is also involved in the roots-to-shoots translocation of heavy metals [26, 37, 43, 46]. The first evidence of the long-distance transfer of PC-metal complexes is that the PCs synthesized in the roots can be translocated to the shoots via phloem loading and *vice versa* [26, 43, 46]. Additionally, plants defective in PC

*Phytochelatin Synthase in Heavy Metal Detoxification and Xenobiotic Metabolism DOI: http://dx.doi.org/10.5772/intechopen.99077*

synthesis show altered patterns of heavy metal accumulation at the whole-plant level while being sensitive to heavy metal stress. For example, the Arabidopsis AtPCS1 deficient mutant, *cad1–3*, accumulated significantly less Cd in the shoots than the wild type or the transgenic line heterologously overexpressing TaPCS1 [43], and the rice *OsPCS2* RNAi plants failed to transfer As3+ from the roots to the shoots [37]. Overall, the phenotypes of these PCS-deficient mutants suggest heavy metal ions absorbed through the roots can be loaded into the shoots in the form of PC-chelates.

PCS is a well-known multitasker involved in different biological processes [21, 47]. Besides its significant role in synthesizing PCs from GSH, PCS can catalyze the deglycination of GSH-*S*-conjugates (GS-conjugates), and thus, it is involved in the GS-conjugate catabolism [17–21, 48]. In addition, PCS is also associated with indole glucosinolates metabolism and immune responses [49–51]. Intriguingly, the catalytic-site mutants of PCS are still functional in this pathway, which suggests that the role of PCS in the indole glucosinolate metabolism is independent of PC synthesis and GS-metabolism [51]. Among these PCSinvolving biological processes, this chapter focuses on the catalytic mechanism of PCS and its functions in both heavy metal stress and GSH metabolism. The potential applications of PCS in combating multiple stresses are also discussed.
