Effects related to the adaptation of wheat to Mn excess were determined by structural and biochemical characterization of chloroplasts obtained from three-leaf seedlings of Mn-treated wheat. Chloroplasts were isolated from two wheat genotypes: sensitive (Raweta) and tolerant (Parabola) cultivated in hydroponic conditions in Hoagland nutrient supplemented with 0 (control), 5, 10, and 20 mmol dm -3 MnSO 4 . Microscopic observations of the chloroplast structure revealed differences in the size and starch presence between both objects. Changes indicating the stresogenic influence of Mn on Raweta seedlings appeared already at the Mn dose of 10 mmol dm -3 , whereas on Parabola, only at 20 mmol dm -3 . Biochemical analysis indicated the differences in starch content. Results of measurements of lipid peroxidation, polarity, and electrokinetic potential of chloroplasts point to Mn-stimulated modifications of chloroplast membranes which occurred to be larger for Raweta. The activation of antioxidative enzymes (SOD and POX) shows that ROS are generated under Mn-excess conditions. The content of Mn and Cu, Fe, Mo, and Zn (microelements) as well as Ca, Mg, K, P, and S (macroelements) in chloroplasts was determined by mass (ICP MS) and plasma optic emission (ICP OES) spectrometry. Raweta accumulated greater amount of Mn in comparison with Parabola at all Mn doses in media. Increased concentration of Mn was accompanied with a decrease of uptake other investigated elements (except for K).
Manganese, a microelement important for plant metabolism, when accumulated at higher doses, may act as a stress factor. Such action of this element is not fully recognized and is currently being intensively studied. The influence of manganese, at high (1, 2 and 3 mM) concentrations, on the induction of oxidative stress in wheat cells was studied under in vitro conditions. Calli of two wheat cultivars, different in terms of stress tolerance, were cultured for 7 days on Murashige-Skoog media with or without auxin (2,4-D) and with additional Mn supplementation. Changes of malondialdehyde (MDA) concentration and the activity of enzymes (SOD, CAT and POX) involved in oxidative response as well as the accumulation of Mn and elements essential for plant development (Mn, Mg, S, Ca, K, P, Na, Zn, Mo, Cu, Fe) were detected. Moreover, proline and carbohydrate contents were determined to check the induction of osmotic stress. An increase of lipid peroxidation (expressed by MDA content), induced by ROS generation, was smaller in the tolerant ('Parabola') cultivar than in the sensitive one ('Raweta'). The activation of antioxidative enzymes was more effective in the cells of tolerant wheat, where a lower quantity of Mn was accumulated. Mn uptake was correlated with a decrease of an amount of almost all the investigated elements. Auxin presence in the culture media accelerated the stressogenic effect of Mn. Keywords Manganese Á Oxidative stress Á In vitro cells Á Wheat Á Micro-and macroelements Communicated by R. Baczek-Kwinta.
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