A new method to detect cadmium uptake in protoplasts
A new method to detect cadmium uptake in protoplasts
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DOI：
10.1007/s00425-004-1256-z
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发表时间：
2004-07-01
期刊：
影响因子：
4.3
通讯作者：
Greger, M
中科院分区：
文献类型：
--
作者：
Lindberg, S;Landberg, T;Greger, M
5 ~ 7日龄小麦（Triticum aestivum L. cv.）原生质体细胞质吸收镉（Cd2+）的机制。采用荧光显微镜和重金属特异性荧光染料5-硝基苯并噻唑香豆素BTC-5N对Kadett进行了研究。在外部培养基中反复添加CdCl2后，茎和根原生质体细胞质中镉荧光逐渐增强，反映了Cd2+的吸收。氯化钙和氯化钾以及钙和钾通道抑制剂维拉帕米和四乙基铵（TEA）分别抑制了摄取。钙竞争性地抑制镉的吸收。代谢抑制剂钒酸盐和二硝基苯酚部分抑制了摄取，表明它依赖于膜电位。结果表明，镉通过钙和钾均可渗透的通道被吸收。通过Cd-109（2+）单向通量分析，还检测了原生质体对镉的总吸收，并显示出与荧光测量值大致相同的最大Cd2+浓度。通过结合这两种方法，可以检测到细胞质和液泡的摄取情况。
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 Cd-109(2+), 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.