engleski

Physiological and biochemical aspects of cadmium interaction with copper and zinc in Lemna minor L.

Background and aims: Heavy metals are natural elements present in soil and water due to natural processes as well as human activities. At low concentrations some heavy metals (e.g. Cu, Mo, Ni, Zn, Mn) are essential for plant metabolism, but at higher concentrations they can be toxic. Other heavy metals are nonessential (e.g. Cd, Pb, As, and Hg) and toxic even in low concentrations. In the majority of studies only single additions of heavy metals have been investigated and there is no much information about the mechanisms induced when plants are exposed to more heavy metals simultaneously. In the present study the specific effect of Cd, Zn and Cu was compared to effects of Cd in combination with Cu or Zn. For the assessment of plant growth, metal uptake, oxidative stress, antioxidative enzymes and photosynthetic performance the aquatic free-floating plant Lemna minor L. was exposed to tested metals. Methods: Duckweed (Lemna minor L.) was exposed for 7 days to different concentrations of tested metals added into modified Steinberg medium (ISO 20079:2001). The effect of series of Zn and Cu concentrations in combination with Cd on growth and morphological parameters was evaluated in preliminary experiments and for further investigations the following concentrations were selected: 5 µM CdCl2 along with ZnCl2 (25 µM or 50 µM) as well as 5 µM CdCl2 along with CuCl2 (2.5 µM or 5.0 µM CuCl2). Growth rate was estimated according to ISO 20079:2001. Cd, Zn and Cu content in plant tissue were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (ISO 11885:1998). Photosynthetic pigments were quantified spectrophotometrically (Lichtenthaler 1987). Chlorophyll fluorescence analysis was performed in vivo with pulse-modulated chlorophyll fluorometer (Maxwell and Johnson, 2000). Parameters of protein oxidation (carbonyl content) and lipid peroxidation (malondialdehyde content) were measured according to Levine et al. (1990) and Heath and Packer (1968), respectively. Activity of antioxidative enzymes catalase and peroxidase was investigated by methods of Aebi (1984) and Chance and Maehly (1955), respectively. Isoenzyme analysis was done by polyacrilamide gel electrophoresis (PAGE) in native conditions and bands visualised according to Woodbury et al. (1971) and Chance and Maehly (1955). Key results: In comparison to control plants, all treatments caused significant reduction of duckweed growth rate. The most pronounced reduction (~ 67% inhibition) was observed after treatments with following combinations of metals: 50 μM Zn + 5 μM Cd, 25 μM Zn + 5 μM Cd and 5.0 μM Cu + 5.0 μM Cd. Plant growth expressed as doubling time of duckweed showed 3.3 days for control plants, 5.4 – 7.0 days for Cd (5.0 μM), Zn (25 μM and 50 μM), and Cu (2.5 μM) while Cu (5.0 μM) and all combinations tested showed 8 -10.4 days. Duckweed accumulated all three tested metals. In plants cultivated on media containing Cd + Zn or Cd + Cu combination, Cd uptake was suppressed by Zn or Cu, respectively. Furthermore, in the presence of Cd the amount of accumulated Zn was lower than its content in plants treated with Zn only. Considering accumulation of Cu in plants that was simultaneously exposed to Cd and Cu, such effect was not so pronounced, but the reason could be lower concentration of Cu in comparison to Zn. After 7 days of experiment chlorophyll content was reduced, especially in plants cultivated on media containing 5 μM Cd, 25 μM Zn and 50 μM Zn, as well as both combinations of Cd and Cu (5 μM Cd + 2.5 μM Cu and 5 μM Cd + 5.0 μM Zn). The same treatments caused significant reduction of carotenoids concentration on the 7th day of treatment. The maximum quantum yield of PSII (Fv/Fm value) was lower in all treated plants on the 4th day while after prolonged 7-day exposure this parameter was affected only by single Cd and Cu treatments as well as by their combinations. Cd treated plants and those exposed to combination of Cd and Cu showed oxidative stress which was indicated by high levels of lipid peroxidation, while Zn as well as its combination with Cd had milder effect, significantly higher only at day 7. Carbonyl content, a consequence of protein oxidation, was significantly increased on day 7 by treatments of Zn, Cu and combination Cd + 5 μM Cu. Antioxidant enzyme catalase showed increased activity in Cd and Cd + Cu treated plants. Peroxidase was decreased by all treatments on day 4, but on the 7th day its activity in plants treated with Cd, Cu and their combinations showed values similar to those of control. Conclusions: Excess amount of zinc or copper in nutrient medium reduced cadmium accumulation in duckweed tissue but it did not alleviate inhibitory effect of cadmium on plant growth. When applied separately, all three tested metals significantly diminished photosynthetic performance. Considering results obtained by their combinations, zinc lowered adverse effect of cadmium, while copper in applied concentrations increased it. Cadmium-induced oxidative stress was inhibited by zinc supplementation, while copper did not showed such effect. References: Aebi H. (1984). Methods in Enzymology 105, 121–126. Chance, B., Maehly, A. C., 1955. Methods in Enzymology 2, 764–775. ISO 11885:1998 Water quality – Determination of selected elements by inductively coupled plasma optical emission spectrometry (ICP-OES) Heath R. L., Packer L. (1968). Archives of Biochemistry and Biophysics 125, 189–198. ISO 20079:2001 Water quality - Determination of the toxic effect of water constituents and waste water to duckweed (Lemna minor) - Duckweed growth inhibition test. Levine R. L., Garland D., Oliver C. N., Amici A., Climent I., Lenz A. G., Ahn B. W., Shaltiel S., Stadtman E. R. (1990). Methods in Enzymology 186, 464–478. Lichtenthaler H. K. (1987). Methods in Enzymology 148, 350–382. Maxwell K., Johnson G. N. (2000). Journal of Experimental Botany 41, 659-668. Woodbury W, Spencer A. K., Stahmann M. A. (1971). Analytical Biochemistry 44, 301–305.

duckweed; Lemna minor; cadmium; zinc; copper; oxidative stress

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