Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves : implication of a transport function for cadmium-binding peptides

Abstract

The synthesis of Cd-binding peptides (CdBPs) was induced upon addition of 20 micromolar CdCl(2) (nonphytotoxic level) to the nutrient solution of hydroponically grown tobacco seedlings (Nicotiana rustica var Pavonii). Amino acid analysis showed that the main components were gamma-(Glu-Cys)(3)-Gly and gamma-(Glu-Cys)(4)-Gly. Seedlings exposed to the metal for 1 week contained similar glutathione levels as found in the controls (about 0.18 micromole per gram fresh weight). If, as has been proposed, CdBPs are involved in Cd-detoxification by chelation, both metal and ligand must be localized in the same cellular compartment. To directly determine the localization of Cd and CdBPs, protoplasts and vacuoles were isolated from leaves of Cd-exposed seedlings. Purified vacuoles contained virtually all of the CdBPs and Cd found in protoplasts (104% +/- 8 and 110% +/- 8, respectively). CdBPs were associated with the vacuolar sap and not with the tonoplast membrane. Glutathione was observed in leaves and protoplasts but not in vacuoles. The probability that CdBPs are synthesized extravacuolarly and our finding that they and Cd are predominantly located in the vacuole suggest that these molecules might be involved in transport of Cd to the vacuole. Our results also suggest that a simple cytoplasmic chelator role for CdBPs in Cd tolerance cannot be assumed.