These seedlings had a smaller size, leaves with chlorotic symptoms, and a root length reduced by 24% (Figure 1(b)). To determine whether NaCl selleck chem inhibitor could affect the cell-membrane integrity of the root cells, a histochemical method based on the Evans Blue staining was used. Thus, an intense blue color appeared in roots of seedlings exposed to 100mM NaCl, indicating the loss of cell-membrane integrity (Figure 1(c)).Figure 1Effect of salinity in Arabidopsis seedlings growth. (a) Appearance of 14-day-old Arabidopsis seedling growth in MS medium supplemented and nonsupplemented with 100mM NaCl. (b) Primary root length. Results are the mean of three different experiments …

To establish whether our experimental salinity conditions affect ROS metabolism, the activity of the first line of antioxidant enzymes was analyzed, including superoxide dismutase (SOD) and catalase, and some key enzymes of the photorespiratory pathway (NADH-hydroxypyruvate reductase and glycolate oxidase). The analysis of SOD activity in native gel showed the presence of four isozymes which differed according to their susceptibility to the inhibitor, whether cyanide or hydrogen peroxide: one Mn-SOD, two Fe-SODs, and one CuZn-SOD, which displayed increasing electrophoretic mobility (Figure 2). As can also be seen, only the CuZn-SOD isozyme was strongly induced by salinity without significantly affecting the other SOD isozymes (Figure 2(a)). On the other hand, the catalase activity increased 2.9-fold under salinity conditions (Figure 2(b)).

However, the hydroxypyruvate reductase (HPR) and the glycolate oxidase activities were not affected after 100mM NaCl treatment (Figures 2(c) and 2(d), resp.).Figure 2Enzyme activities in Arabidopsis seedlings exposed to 100mM NaCl. (a) Superoxide dismutase (SOD) isoenzymes activities in native gels incubated in the absence and the presence of specific inhibitors, either cyanide or hydrogen peroxide. (b) Catalase …3.2. Cellular Analysis of Superoxide Radical (O2??), NO, and ONOO? Production Induced by SalinityFigure 3(a)to 3(f) shows the analysis by confocal laser scanning microscope (CLSM) of the content of O2??, NO, and ONOO? in the root tips of Arabidopsis seedlings exposed to 100mM NaCl. The cellular production of O2?? was analyzed using the fluorescent probe DHE, which is specific for this radical. In control seedlings, the green fluorescence corresponding to O2?? was slightly detected in the root tips (Figure 3(a)). However, in roots from NaCl-treated seedlings, the green fluorescence was intensified in the root Drug_discovery tips (Figure 3(b)).