**4.2 Phytochelatin synthase may participate in initiating the first step of glutathione-***S***-conjugates degradation in the cytosol**

Monochlorobimane (MCB) is widely used as a model xenobiotic for Arabidopsis to study the catabolism of GS conjugates [14, 15, 17–20, 98]. The bimane-labeled thiols can be analyzed by high performance liquid chromatography [99]. In addition, the fluorescent GS-bimane can be directly monitored *in situ*, which indicates the compartmentation and the turnover of GS-conjugates [15, 18, 20]. Data have shown that AtPCS1 initiates the first step of GS-bimane degradation in cytosol by removing the Gly residue and providing substrates for the vacuolar GGT [17, 19–21] (**Figure 1**). This detour could be a functionally alternative route to detoxify xenobiotics when the major pathway is blocked [17–20].

The direct evidence showing the involvement of PCS in GS-conjugate metabolism is the defects in the turnover of GS-bimane shown in the Arabidopsis AtPCS1-deficient mutants [21, 47]. The AtPCS1-deficient mutant, *ΔPCS1*, and the AtPCS1/AtPCS2 double-deficient mutant, *ΔPCS*, are impaired in the degradation of GS-bimane to γ-Glu-Cys-bimane [19, 20]. Blum et al. (2007) report that in the absence of Cd2+, the abundance of the γ-Glu-Cys-bimane in both *ΔPCS1* and *ΔPCS* mutants was significantly reduced compared to the wild type after the plants were challenged by the xenobiotic bimane [19]. Moreover, the induction of γ-Glu-Cysbimane was not observed in AtPCS1-deficient lines in the plants treated with Cd2+, which resulted in a > 10-fold lower γ-Glu-Cys-bimane accumulation compared with the wild type grown in the same conditions [19]. The GS-baimane concentration could be rescued by transfecting AtPCS1 cDNA into PCS-deficient protoplasts, suggesting that this process is indeed PCS-dependent [19]. The inhibited γ-Glu-Cysbimane accumulation in the mutant lines indicates that AtPCS1 efficiently catalyzes GS-conjugates in the presence of Cd2+ [19, 20].

Although the GS-bimane conversion is altered in the AtPCS1-deficient mutants, the GS-bimane in these mutants still can be degraded through the major detoxification pathway in the vacuoles [18–20]. Besides, the overall turnover of GS-bimane in the mutants is only slightly affected without blocking the vacuolar transport pathway [18–20]. These findings underline that the vacuolar GGT-initiated GS-conjugates degradation is the major pathway among two compensatory routes responsible for the turnover of the xenobiotics [18].

In plant cells, both the cytosolic PCS and the vacuolar carboxypeptidase can catalyze the formation of γ-Glu-Cys-bimane [16–19]. However, the vacuolar carboxypeptidase tends to catalyze the Cys-Gly-conjugates following the cleavage of γ-Glu-residue initiated by GGT [15]. In this regard, PCS is supposed to be the primary component responsible for the γ-Glu-Cys-bimane formation observed in the process of GS-conjugate conversion. Another example showing the importance of PCS in the initiation of the cytosolic xenobiotic compound is the metabolism of the herbicide safener fenclorim [100]. Fenclorim enhances GST activity in Arabidopsis

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

and is subsequently degraded via the GS-conjugation pathway [97, 100]. In the Arabidopsis suspension cells, GS-fenclorim was sequentially processed to γ-Glu-Cys-fenclorim and Cys-fenclorim, suggesting that deglycination is the initial step to the catabolism of fenclorim [21, 100]. However, more evidence is needed to confirm the direct involvement of PCS in this process.

#### **4.3 The glutathione-***S***-conjugate conversion via phytochelatin synthase is metal-dependent**

The presence of metal ions is a critical requirement for PCS-dependent catalysis of GS-bimane [17–20]. Intriguingly, the efficiency of GS-bimane hydrolysis activated by different metal ions is separate from that of metal-stimulated PC synthesis [17]. For example, the PC formation of AtPCS1 activated by Cd2+ is usually 2–5 times more efficient than the PC synthesis rate measured in the presence of Cu+/2+ [4, 6, 53]. On the other hand, AtPCS1 could catalyze the deglycination of GS-bimane 60% more efficiently in Cu+/2+ solutions than in the presence of Cd2+ [17]. It was suggested that *in vivo* AtPCS1 is a Cu-containing metalloenzyme in unstressed conditions, and consequently, the Cu-bound PCS favors the catalysis of GS-conjugate over PC synthesis in the normal growth conditions [17]. Evidence supporting this hypothesis is that in Arabidopsis, the deglycination of GS-bimane was PCS-dependent in the absence of heavy metals [19]. However, considering AtPCS1 binds Cu2+ only at a low affinity [6, 21], and the majority of cytosolic Cu is usually associated with Cu chaperons [101], it is possible that the concentration of free cytosolic Cu ions is not sufficient to fully activate PCS for the catalysis of GS-conjugates.
