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Knowledge of cellular metal homeostasis will provide a better understanding of the mechanisms involved in metal tolerance and hyperaccumulation in metal-hyperaccumulating plants. Energy dispersive X-ray spectrometry (EDS) was used to determine the localization of cadmium (Cd) in leaves of the Zn/Cd hyperaccumulator Picris divaricata which had a shoot Cd concentration of 565 mg kg−1 after 2 weeks of growth in solution culture supplying 10 mol L−1 CdCl2. The results indicated that Cd was distributed mainly in the trichomes, upper and lower epidermis and bundle sheath cells, with a relatively low level of Cd in mesophyll cells. Mesophyll protoplasts isolated from leaves remained viable after 24 h exposure to CdCl2 at a concentration up to 1 mmol L−1, indicating their high tolerance to Cd. The intracellular Cd was visualized by staining with Leadmium Green dye, a cellular permeable Cd fluorescence probe. The results showed that the majority of protoplasts (> 82%) did not accumulate Cd, with only a minority (< 18%) showing Cd accumulation. In the Cd-accumulating protoplasts, Cd accumulation was depressed by the addition of Fe2+, Mn2+ and the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), but not by Ca2+ or Zn2+. Furthermore, the entire process of Cd uptake from external solution into the cytoplasm and subsequent sequestration into vacuoles was successfully recorded by confocal images. These results suggested that reduced cellular Cd accumulation and efficient Cd vacuolar sequestration in mesophyll cells might be responsible for cellular Cd tolerance and distribution in the leaves of P. divaricata.

Using the differential centrifugation technique and sequential chemical extraction method, effects of Cd, Pb and different Zn salts on subcellular distribution and chemical form of Zn in Zn hyperaccumulator Potentilla griffithii var. velutina under nutrient solution culture were analyzed. Under all treatments except for the control, 46%-74% and 16%-33% of total Zn in the plants are distributed in cell wall and in soluble fraction, respectively. Further, 74%-95% of total Zn are localized in these two parts under all treatments, which suggest that cell wall and soluble fraction in the plant are major storage sites for Zn. Compared with the control, Zn percentage significantly increases by 9%-38% in the cell wall and decreases by 6%-40% in the soluble fraction with addition of Zn, Cd and Pb treatment (p0.05). Although the addition of Cd and Pb has no influence on the pattern of Zn subcellular distribution presenting cell wallsoluble fractionkaryon and chloroplastmitochondrion, it generally reduces Zn percentage in the chloroplast, karyon and mitochondrion and increases that in the cell wall or soluble fraction, suggesting that Cd and Pb promote the transferring processes of Zn from organelle to either cell wall or vacuole. As to the chemical forms, 61%-87% of total Zn exist as ethanol- and water-extractable forms in plants under control and only leaves under Zn addition treatment; while 62%-73% of total Zn exist as NaCl- and ethanol-extractable forms in leafstalks and roots under Zn addition treatment. NaCl-, ethanol- and water-extractable forms are also the main chemical forms in the plants, occupied almost 70%-89% of total Zn under Zn/Cd and Zn/Pb compound treatments. The addition of Zn, Cd and Pb generally increases the percentage of NaCl-extractable Zn forms, but decreases that of ethanol-extractable Zn, which facilitates Zn chemical form transferring from relatively higher active forms to less active ones. These results mentioned above indicate that cell wall binding, vacuolar compartmentalization and reduction of total percentage in higher active chemical forms are main tolerance mechanisms for Zn in Potentilla griffithii var. velutina in response to Zn, Zn/Cd and Zn/Pb treatments. Additionally, different Zinc salts have no obvious influence on Zn subcellular distribution in the plant, whereas the treatment of Zinc nitrate turns Zn ethanol-extraction to a dominant chemical form.

The kinetic characteristics of Zn uptake by Potentilla griffithii Hook. f. var. velutina Card. were studied with hydroponic culture under different Zn supply and its exposure time. When the Zn supply was 10 mg x L(-1), the Zn concentration in shoot and root reached the peak on the 8th day, being 2.49 x 10(3) mg x kg(-1) and 2.21 x 10(3) mg x kg(-1), respectively; while when the supply was 100 mg x L(-1), the Zn concentration in shoot reached the maximum (1.23 x 10(3) mg x kg(-1)) on the 16th day, but that in root did not exhibit saturation. The Zn concentration in leaf and leafstalk increased with increasing Zn supply from 0 to 160 mg x L(-1), but had no increase or even decreased when the Zn supply was higher than 160 mg x L(-1). The Zn concentration in root exhibited a positive correlation with Zn supply and its exposure time, and the kinetic curve of Zn uptake fitted linear-quadratic (LQ) model, i. e., fast linear uptake first and slow saturation uptake later, with the dividing point at about 1-2 hours' exposure, which could be related with the Zn adsorption on root cell wall and the Zn transportation across the cell membrane.

Hydroponic culture was conducted to study the effect of Cd on the growth, metal accumulation and nitrogen metabolism in Brassica chinensis. The enzymatic activities of nitrogen metabolism including nitrate reductase (NR), glutamine synthetase (GS) and GS-transferase as well as the concentrations of chlorophyll, free proline, soluble protein, NO3(-) -N, NH4+ -N and nutrients in Brassica chinensis were determined. Results indicated that the addition of Cd reduced the content of the soluble protein and the accumulation of Cu, Ca, Fe and Mg, but promoted the P uptake. Low level of Cd (1 mg x L(-1)) could significantly increase the biomass and the content of chlorophyll of Brassica chinensis and the activities of NR, GS and GS-transferase when compared to control plants. However, when the Cd levels were above 2.5 mg x L(-1) in the culture medium, the activities of these enzymes were inhibited. Accordingly, the contents of NO3(-) -N, NH4+ -N, free proline and the activity of protease in the leaf of Brassica chinensis increased significantly. These results suggested that Cd addition could interfere with the assimilation of N in Brassica chinensis. The increase of free proline might alleviate the toxicity of ammonium in Brassica chinensis.