Functional Characterization of an Unusual Phytochelatin Synthase, LjPCS3, of Lotus japonicus

Abstract

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins from glutathione in the presence of certain metals and metalloids. We have used budding yeast (Saccharomyces cerevisiae) as a heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of phytochelatin production for each metal tested were normalized with respect to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.

Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

“…This work shows evidence of a connection of PC biosynthesis induction in connection with iron ions. Similar results were obtained in Lotus japonicus ( Fabaceae ) , where the ability of iron to induce phytochelatin synthase 3 (LjPCS3) was detected [92]. Induction of phytochelatin synthase (SmPCS) by iron ions was detected in in vitro culture of Schisostoma mansonii , however, this species belongs to the kingdom Animalia [93].…”

Arsenite Elicits Anomalous Sulfur Starvation Responses in Barley

“…Many plants respond to high concentrations of AsIII by increasing the synthesis of a range of thiol-containing compounds (Raab et al, 2005;Schulz et al, 2008), including phytochelatins (PCs; Schmöger et al, 2000). PC synthase genes have now been identified in a number of species, including Arabidopsis (Arabidopsis thaliana; Vatamaniuk et al, 1999), wheat (Triticum aestivum; Clemens et al, 1999), Brassica juncea (Heiss et al, 2003), Pteris vittata (Dong et al, 2005), and Lotus japonicus (Ramos et al, 2008). The importance of these genes in arsenic tolerance in Arabidopsis is underlined by the sensitivity of mutants deficient in PC synthase (Ha et al, 1999) and the increased tolerance in plants overexpressing PC synthase (Gasic and Korban 2007).…”

Homologous cloning, characterization and expression of a new halophyte phytochelatin synthase gene in Suaeda salsa

“…In L . japonicus , Pb and Hg signifi cantly but diff erentially induced the PCS1 and PCS3 genes, and such a variance was ascribed to the diff erences in the C-terminal domains of the two PCS proteins (Ramos et al, 2008). In a similar manner, the diff erent responses of SsPCS to Pb, Zn and Hg might mirror the metal activation patterns of the SsPCS C-terminal domain and metal accumulation in plants.…”

“…PCs are a series of peptides containing a landmark structure (γ-Glu-Cys) n -Gly ( n =2-11) that play principal roles in the detoxifi cation of heavy metals, such as lead (Pb), mercury (Hg), copper (Cu), zinc (Zn) and cadmium (Cd) (Tsuji et al, 2002;Ramos et al, 2008). However, PCs are not synthesized directly but by transferring the glutamylcysteine dipeptide moiety of glutathione to another glutathione until an oligomer chain is formed, which contains 2 to 11 γ-Glu-Cys monomers (Grill et al, 1989).…”