Identification of the form of Cd in the leaves of a superior Cd-accumulating ecotype of Thlaspi caerulescens using ¹¹³Cd-NMR

Thlaspi caerulescens (Ganges ecotype) is a known Cd hyperaccumulator, however, the ligands which coordinate to Cd ions in the leaves have not been identified. In the present study, the chemical form of Cd was investigated by using ¹¹³Cd-nuclear magnetic resonance (NMR) spectroscopy. Plants were grown hydroponically with a highly enriched ¹¹³Cd stable isotope. Measurements of ¹¹³Cd-NMR with intact leaves showed a signal at the chemical shift of around -16 ppm. Crude leaf sap also gave a similar chemical shift. Purification by gel filtration (Sephadex G-10), followed by cationic and anionic exchange chromatography, showed that Cd occurred only in the anionic fraction, which gave the same chemical shift as intact leaves. Further purification of the anionic fraction, combined with ¹¹³Cd- and ¹H-NMR studies, revealed that only the fraction containing malate showed a chemical shift similar to the intact leaves. These results indicate that Cd was coordinated mainly with malate in the leaves of T. caerulescens. The malate concentration in the leaves was not affected by increasing Cd concentration in the solution, suggesting that malate synthesis is not induced by Cd. Because the Cd-malate complex is relatively weak, we suggest that the complex forms inside the vacuoles as a result of an efficient tonoplast transport of Cd and a constitutively high concentration of malate in the vacuoles, and that the formation of the Cd-malate complex may lead to a decrease of subsequent Cd efflux to the cytoplasm.