Cadmium uptake and interaction with phytochelatins in wheat protoplasts

In order to investigate the role of phytochelatins in short-time uptake of Cd2+ into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd2+ into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 μM CdCl2, than in the absence. Presence of CdCl2 in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd2+ and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd2+. Unidirectional flux analyses, using 109Cd2+, showed approximately the same uptake pattern of Cd2+ as did the fluorescence experiments showing the cytosolic uptake of Cd2+. Thus, the diminished uptake of Cd2+ into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd2+ causes a down-regulation of the short-term Cd2+ uptake, or an up-regulation of the Cd2+ extrusion. Moreover, since addition of Cd2+ to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd2+/H+-antiport mechanism is involved in the extrusion of Cd2+ from these protoplasts.