Measuring ROS in Guard Cells

ROS production in guard cells was analysed using a chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes, Invitrogen) as previously described [4 (link)] with slight modifications. Seven-day-old WT and Atcuaoβ mutant seedlings were incubated for 3 h in the assay solution containing 5 mM KCl, 50 μM CaCl₂ and 10 mM MES-Tris (pH 6.15), and then 10 μM CM-H₂DCFDA was added to the sample. Seedlings were incubated for 30 min at room temperature and then the excess dye was washed out twice with the assay solution. After this period, the assay solution was replaced by liquid medium in the absence or presence of treatment performed as follows: 50 μM MeJA, 50 μM MeJA + 100 μM DMTU, 50 μM MeJA + 5 mM 2-BrEtA. Seedlings were incubated for 1 h. Images were acquired by Laser Scanning Confocal Microscopy (LSCM), using a Leica TCS-SP5 equipped with an Argon laser (Excitation/Emission: ~492–495/517–527 nm) and the Leica Application Suite Advanced Fluorescence (LAS-AF; Leica Microsystems, Wetzlar, Germany).