Roles of calcium and cadmium on Cd-containing intra- and extracellular formation of Ca crystals in tobacco

Growth was severely inhibited when tobacco plants were exposed to toxic levels of cadmium (0.2 mM). However, when this treatment was combined with a high concentration of calcium (30 mM), the Cd-induced damage was strongly alleviated. Under these enhanced conditions, i.e., 30 mM Ca and 0.2 mM Cd, Ca crystals not only were heavily deposited in the leaves but were also actively excreted from the trichomes. The X-ray spectrum from our Energy Dispersive analysis revealed that both intra- and extracellullar Ca crystals contained detectable amounts of Cd. Moreover, intracellular Ca deposition in the leaves was stimulated only by a high Ca concentration (30 mM); moderate levels of Ca (3 mM) or a toxic amount of Cd (0.2 mM) alone resulted in crystal deposition that was undetectable under a light microscope. In contrast, extracellular crystal formation on the trichomes was stimulated by toxic Cd treatment but not by high Ca concentrations alone. Finally, Inductively Coupled Plasma Spectroscopy revealed that a high level of Ca (30 mM) suppressed Cd accumulation while also increasing the endogeneous Ca concentration in the leaves. These observations imply that the amelioration of Ca against toxic Cd in tobacco plants is a result of not only the inhibition of Cd uptake, but also the extra- and intracellular sequestration of cadmium via Ca crystallization.