H

300 vim

Fig. 2 Fluorescence probes that can be used to trace alteration in the pool of glutathione (monochlorobimane-MCB), oxidative stress (2',7'-dichlorofluorescin diacetate-H2DCFDA), and cell death (propidium iodide-PI). MCB (green) was visualized with epifluorescence microscopy (a-d), and H2DCFDA (green) and IP (red) with confocal microscopy (e-h). Alfalfa seedlings were treated with Cd, Hg and buthionine sulfoximine (BSO) for 24 h prior to staining with the different probes

Fig. 2 Fluorescence probes that can be used to trace alteration in the pool of glutathione (monochlorobimane-MCB), oxidative stress (2',7'-dichlorofluorescin diacetate-H2DCFDA), and cell death (propidium iodide-PI). MCB (green) was visualized with epifluorescence microscopy (a-d), and H2DCFDA (green) and IP (red) with confocal microscopy (e-h). Alfalfa seedlings were treated with Cd, Hg and buthionine sulfoximine (BSO) for 24 h prior to staining with the different probes

30 mM (observed as red fluorescence of PI in condensed nuclei; Fig. 2h), suggesting that oxidative stress only occurs in still viable cells with functional metabolism (Ortega-Villasante et al. 2005). A similar experiment with barley root tips showed that after 3-6 h exposure to 1 mM Cd or 0.5 mM Hg caused the overexpression of several genes encoding aquaporins and dehydrins, suggesting the onset of dehydration stress by heavy metal. These responses were accompanied by a significant inhibition of root growth and induction of oxidative stress (Tamas et al. 2010).

Another experimental alternative used frequently to study the direct effects of heavy metals on particular cellular components is the extraction of such materials from untreated plants, which are then tested with different treatments of metals in vitro. For example, it has been observed that direct exposure of tylakoid membranes caused the release of their components, especially proteins of the splitting water system and galactolipids probably connected with photosystem I (PSI) inhibition after heavy metals exposure (SkcSrzyiiska and Baszyiiski 1993; Nouari et al. 2006). These experiments showed that heavy metals bind to membranes through oxygen atoms or aminoacids such as histidine, tryptophan or tyrosine in proteins, leading to electron flow disturbance in photosystem II after illumination (Maksymiec 1997). Other studies indicated that high heavy metal concentration lead to substitution of the Mg in the chlorophyll molecules (Kowalewska et al. 1987). Similarly, Hg substitutes Cu in plastocyanin molecule, blocking electron passage to PSI (Radmer and Kok 1974), and Cd, Hg, and Pb may also bind to LHCII producing conformational changes. Inhibition of enzymes involved in chlorophyll production also produces a decrease in its synthesis (Boddi et al. 1995). In spite of these findings, it is not clear that alterations in the photosynthetic electron transport and dark phase reactions observed in vivo are the direct effect of metals, as these effects appear only after several days of exposure (Wang et al. 2009).

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