Overexpression of Arabidopsis Phytochelatin Synthase Paradoxically Leads to Hypersensitivity to Cadmium Stress

Abstract

Phytochelatin (PC) plays an important role in heavy metal detoxification in plants and other living organisms. Therefore, we overexpressed an Arabidopsis PC synthase (AtPCS1) in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation, and metal tolerance in these plants. Transgenic Arabidopsis plants were selected, designated pcs lines, and analyzed for tolerance to cadmium (Cd). Transgenic pcs lines showed 12-to 25-fold higher accumulation of AtPCS1 mRNA, and production of PCs increased by 1.3-to 2.1-fold under 85 m CdCl 2 stress for 3 d when compared with wild-type plants. Cd tolerance was assessed by measuring root length of plants grown on agar medium containing 50 or 85 m CdCl 2 . Pcs lines paradoxically showed hypersensitivity to Cd stress. This hypersensitivity was also observed for zinc (Zn) but not for copper (Cu). The overexpressed AtPCS1 protein itself was not responsible for Cd hypersensitivity as transgenic cad1-3 mutants overexpressing AtPCS1 to similar levels as those of pcs lines were not hypersensitive to Cd. Pcs lines were more sensitive to Cd than a PC-deficient Arabidopsis mutant, cad1-3, grown under low glutathione (GSH) levels. Cd hypersensitivity of pcs lines disappeared under increased GSH levels supplemented in the medium. Therefore, Cd hypersensitivity in pcs lines seems due to the toxicity of PCs as they existed at supraoptimal levels when compared with GSH levels.Contamination of soils and waters with toxic heavy metals contributes to serious worldwide environmental and human health problems. These heavy metal pollutants have been discharged mainly by mining and combustion of fossil fuels during the period of global industrialization.A promising new technology, referred to as phytoremediation, offers promise for clean up of polluted areas in a cost-effective and environmentfriendly manner (Salt et al., 1995(Salt et al., , 1998Raskin et al., 1997). This technology involves removal of toxic heavy metals from contaminated soils and waters, or rendering them harmless by accumulating, chelating, or transforming these contaminants into biologically inactive forms through green plants. One possible approach for phytoremediation is to use "hyperaccumulators," plant species that have evolved to accumulate high concentrations of heavy metals in their biomass. However, most known hyperaccumulators tend to grow slowly, and produce relatively low biomass (Cunningham et al., 1995). Due to these limitations, genetic and molecular investigations of plant defense mechanisms involved in heavy metal stress have been under way to improve the efficiency of phytoremediation.Plants can produce Cys-rich peptides such as glutathione (GSH), phytochelatins (PCs), or metallothioneins (MTs) for detoxification or homeostasis of heavy metals (Rauser, 1999; Cobbett, 2000a Cobbett, , 2000b. PCs are a family of small enzymatically synthesized peptides having a general structure of (␥-Glu-Cys) nGly where n equals 2 to 11 (Rauser, 1990). These peptides are rapidly synthesized in response to ...

A critical review on the bio-removal of hazardous heavy metals from contaminated soils: Issues, progress, eco-environmental concerns and opportunities

“…Another new viewpoint towards phytoremediation is that adequate attention must be paid to the development of crop hyperaccumulators, termed by the author as "cropaccumulators", such as wheat, maize and rice [30][31][32][33]. In other words, investigations of other "noncropaccumulators" species would be of little significance in application.…”

“…Studies on this field are increasing and its mechanism is more and more clear [33][34][35][36][37][38][39][40][41]. Using transgenic technology is a tendency in the future to create an ideal species purposely.…”

Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis

“…In addition, a lot of key genes have been shown to be involved in Cd detoxification and tolerance in plants. These include ZNT1, OsHMA9, OsNRAMP5, ZntA, CAD2, AtPDR12, AtPDR8, AtATM3, ACBP1, Nramp5, EIN2, and MAN3 (Pence et al, 2000;Sharma et al, 2000;Shiraishi et al, 2000;Lee et al, 2003Lee et al, , 2007Kim et al, 2006Kim et al, , 2007Xiao et al, 2008;Cao et al, 2009;Lin and Aarts, 2012;Sasaki et al, 2012;Ishikawa et al, 2012;Chen et al, 2015). It was also found that Heat shock transcription factor A4a (HsfA4a) confers Cd tolerance by up-regulating MT gene expression in wheat (Triticum aestivum) and rice (Oryza sativa; Shim et al, 2009).…”

“…An important pathway of heavy metal detoxification in plants is the GSH-dependent PC synthesis pathway that confers plants to tolerable Cd accumulation (Grill et al, 1989;Li et al, 1997;Noctor et al, 2002;Lee et al, 2003;Kim et al, 2006;Verbruggen et al, 2009aVerbruggen et al, , 2009b. Therefore, we analyzed the growth of wild-type and ZAT6-OE plants in 1/2 MS media containing buthionine sulfoximine (BSO), an inhibitor of GSH synthesis (Kim et al, 2006).…”

“…It is well documented that glutathione (GSH) is essential for Cd detoxification (Zhu et al, 1999;Xiang et al, 2001;Cobbett and Goldsbrough, 2002;Jozefczak et al, 2012Jozefczak et al, , 2015Sobrino-Plata et al, 2014b;Hernández et al, 2015;Flores-Cáceres et al, 2015), and GSH-or phytochelatin (PC)-conjugated vacuolar sequestration is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants (Grill et al, 1989;Li et al, 1997;Cobbett and Goldsbrough, 2002;Lee et al, 2003;Lin and Aarts, 2012;Jozefczak et al, 2012). GSH is known to be synthesized from g-Glu, with Cys and Gly being successively catalyzed by g-glutamyl-Cys synthetase (encoded by GSH1) and glutathione synthetase (encoded by GSH2; May et al, 1998;Cobbett, 2000;Jozefczak et al, 2012), and PC is polymerized from GSH by phytochelatin synthase (PCS; encoded by PCS1 and PCS2 in Arabidopsis [Arabidopsis thaliana]), which is strongly induced by Cd stress Noctor et al, 2002;Semane et al, 2007).…”