title: A new method to detect cadmium uptake in protoplasts.
abstract: The mechanism for cadmium (Cd2+) uptake into the cytosol of protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) was investigated by a new method, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. Cadmium fluorescence gradually increased in the cytosol of shoot and root protoplasts upon repeated additions of CdCl2 to the external medium, reflecting an uptake of Cd2+. The uptake was inhibited by calcium and potassium chloride, as well as by Verapamil and tetraethylammonium (TEA), inhibitors of calcium and potassium channels, respectively. Calcium competitively inhibited the cadmium uptake. The metabolic inhibitors vanadate and dinitrophenol partly inhibited the uptake, suggesting it was dependent on membrane potential. The results indicate that cadmium is taken up by channels permeable to both calcium and potassium. The total uptake of cadmium into the protoplasts was also detected by unidirectional flux analyses using 109Cd2+, and showed approximately the same maximal concentration of Cd2+ as the fluorescence measurements. By combining the two methods it is possible to detect both uptake into the cytosol and into the vacuole.
authors.affiliation: Department of Plant Biology and Forest Genetics, SLU, Box 7080, 750 07, Uppsala, Sweden. sylvia.lindberg@vbsg.slu.se
authors.forename: Sylvia
authors.initials: S
authors.lastname: Lindberg
authors.forename: Tommy
authors.initials: T
authors.lastname: Landberg
authors.forename: Maria
authors.initials: M
authors.lastname: Greger
mesh_terms: D001693:Biological Transport, Active; D002104:Cadmium / Q000493:pharmacokinetics*; D003374:Coumarins; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D017136:Ion Transport; D008856:Microscopy, Fluorescence / Q000379:methods; D011523:Protoplasts / Q000378:metabolism*; D036226:Seedlings / Q000378:metabolism; D013844:Thiazoles; D014908:Triticum / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D013844:Thiazoles; D002104:Cadmium