Abstract
The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.
Highlights
Cadmium (Cd) is a heavy metal that exerts a detrimental to Cd exposure by producing phytochelatins (PCs), cysteineeffect on plants and on human health by interfering with bio- rich peptides with the general structure (Glu–Cys) n-Gly, chemical functions of essential metals
Seedlings from the wild type and these AtABCC3ox lines were grown in the presence of 60 μM CdSO4 with or without the inducer β-oestradiol, and AtABCC3 transcript levels were analysed after 9 d of growth
AtABCC3 expression is induced by Cd (Bovet et al, 2003), and the AtABCC3 protein is localized in the vacuolar membrane (Dunkley et al, 2006)
Summary
Cadmium (Cd) is a heavy metal that exerts a detrimental to Cd exposure by producing phytochelatins (PCs), cysteineeffect on plants and on human health by interfering with bio- rich peptides with the general structure (Glu–Cys) n-Gly, chemical functions of essential metals. PCs are able to bind cytoplasmic Cd, forming stable PC– Cd complexes, playing a major role in Cd detoxification: PC-deficient mutants of S. pombe and Arabidopsis—cad, mutated in AtPCS1—are hypersensitive to Cd (Ha et al, 1999); in most species, PCS overexpression leads to increased Cd tolerance (Vatamaniuk et al, 1999; Gisbert et al, 2003; Sauge-Merle et al, 2003; Martinez et al, 2006; Pomponi et al, 2006; Gasic and Korban, 2007; Guo et al, 2008; Wojas et al, 2010; Brunetti et al, 2011). Experiments on isolated vacuoles from Avena sativa roots suggested that transport of PC–Cd complexes is mediated by ATP-binding cassette (ABC)-type transporters (Salt and Rauser, 1995), ubiquitous transmembrane proteins that utilize ATP to translocate various substrates across membranes. It has been shown that in Saccharomyces cerevisiae, which lacks PCS and does not produce PCs, the ABCC-type transporter YCF1 is able to transport GSH–Cd complexes into the vacuole (Li et al, 1997), and overexpression of ScYCF1 increases Cd tolerance in Arabidopsis seedlings (Song et al, 2003)
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