ABA enhances salinity tolerance in Chlamydomonas by reducing ROS formation in cells
We used the fluorescent probe H2DCFDA to estimate the cellular ROS-sensitive fluorescence in Chlamydomonas using confocal microscopy, where the green color indicates the presence of ROS and the red color indicates autofluorescence of chlorophyll (Fig. 4). ROS are natural by-products of cell metabolic processes (Mallick and Mohn, 2000), therefore non-stressed cells were also stained with H2DCFDA to determine background levels of ROS for quantifying salt-stressed ROS induction compared to the non-stressed control. A very weak ROS green fluorescent signal was observed under control growth conditions. After salt treatment, strong ROS fluorescent signals were detected in the chloroplast and the mock sample, whereas slight ROS signal was observed in the ABA-treated cells (Fig. 4A). These results indicate that ABA decreased the ROS level in cells.
Intracellular ROS accumulation was measured in control and ABA-treated cells following exposure to 125 mM NaCl by using flow cytometry. All treatments with 125 mM NaCl significantly increased intracellular ROS after 3 h in comparison to controls (Fig. 4B). In ABA-treated cells there was a comparative significant reduction in ROS accumulation. Cells treated with 50 μM ABA induced ROS to ~ 55% relative to stressed ABA-untreated cells.