**3.1 The effects of phytochelatin synthase overexpression on the accumulation of heavy metals in plants**

PC synthesis plays a critical role in heavy metal tolerance and accumulation. It is therefore no surprise that the breeding and engineering approaches for phytoremediation requiring heavy metal hyperaccumulators have focused on strategies to enhance PC biosynthetic capacity [48, 70–76]. Studies have shown that the transgenic plants expressing functional PCS usually have a higher tolerance to heavy metal stress. For example, the overexpression of AtPCS1 in Arabidopsis, tobacco or Indian mustard (*Brassica juncea*) enhanced Cd, Zn, and As tolerance and accumulation [71, 77–80]. Other PCS homologs e.g., CePCS, TaPCS1, NtPCS1, CdPCS from an aquatic As-accumulator plant (*Ceratophyllum demersum*), MaPCS1/MaPCS2 from mulberry (*Morus alba*), and VsPCS1 from legume *Vicia sativa* were also used to develop transgenic plants that accumulate higher concentrations of heavy metals than their natural variants [43, 46, 81–86]. These reports on improving heavy metal accumulation and tolerance of the plants indicate the potential applications of PCS on phytoremediation approaches.

Although PCS can be a molecular tool for phytoremediation of heavy metal-contaminated soils and waters, the overexpression of PCS promotes the catabolism of GSH, which also plays essential roles in redox reactions and heavy metal stress [87, 88]. If the metabolic pathways supplying GSH cannot maintain specific levels in the presence of highly expressed PCS, the consumption of GSH usually leads to changes in the GSH/GSSG ratio that exacerbate oxidative stress [62, 72]. The increased GSH demand driven by PC synthesis may also affect other metabolic pathways requiring GSH [89]. In these regards, the use of functional PCS with diminished catalytic activity could reduce the depletion of GSH, maintain redox homeostasis and supporting PC synthesis during exposure to heavy metals at the same time [62]. Indeed, the Arabidopsis and *Brassica juncea* transgenic lines expressing a partially deactivated AtPCS1 mutant, AtPCS1- Y186C, showed enhanced Cd2+ tolerance and higher GSH/GSSG ratio than the transgenic lines expressing wild-type AtPCS1 [62]. These results suggest that PC synthesis and redox homeostasis are both important for successful heavy metal resistance.

Besides the imbalance of cellular redox state, PCS overexpression could result in an unknown disruption in cellular metal homeostasis under heavy metal stress because PCS itself is a metalloenzyme and can bind a wide range of metal ions [90, 91]. Expressing synthetic genes encoding peptide analogs of PCs with a general structure of Met(Glu-Cys)nGly (n = 16–20) could be an alternative way to enhance the accumulation of heavy metals in the plants without the overexpression of PCS [92]. The Arabidopsis transgenic plants transformed with the artificial genes encoding these PC-like polypeptides resulted in hyperaccumulation of Cd2+ and As3+/5+ in the plants [92]. However, the impact of accumulating synthetic PC-like polypeptides on the overall metal homeostasis is yet to be determined.
