Fluo illuminator l4 23

Leaf epidermal peels from 4-week-old adult plants were incubated in stomatal opening solution for 2 h followed by an additional 2 h in 10 mM ABA.
The epidermis was incubated with 100 mM 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (H 2 DCF-DA) (Molecular Probes, Eugene, OR, USA) for 15 min. After washing dye off with distilled water to remove excess dye, ROS production in guard cells was visualized under a fluorescence microscope (Leica, DM2500 with a fluorescent module [Fluo Illuminator L4/23]) with the L5 filter system (excitation BP480/40, emission BP527/30). To prevent photo-oxidation of H 2 DCF-DA, all fluorescence images were collected with a single rapid capture (150.8 ms/frame) at 4003 magnification. To quantify ROS production, the brightness of fluorescence was measured using ImageJ software.

For interaction between BAK1 and OST1, and BAK1 and ABI1, full-length BAK1, OST1, and ABI1 cDNAs were amplified by PCR and cloned into p2YN (Yang et al., 2007) cut with PacI followed by calf intestinal alkaline phosphatase treatment or into p2YC cut with PacI/SpeI to make C-terminal fused YFP constructs of each protein, respectively (OST1-YFPn, OST1-YFPc, BAK1-YFPc, and ABI1-YFPn). For interaction between BAK1 and BIK1 or CERK1, full-length BAK1 cDNA was amplified by PCR and cloned into 326-YFP N cut with XbaI/BamHI C-terminal fused YFP construct (BAK1-YFPn), and the full-length BIK1 and CERK1 cDNAs were amplified by PCR and cloned into 326-YFP C cut with BamHI/SacI and XbaI/BamHI C-terminal fused YFP constructs, respectively (BIK1-YFPc, CERK1-YFPc). Transient expression of these proteins was performed with mesophyll cell protoplasts using the polyethylene glycol transfection method (Yoo et al., 2007) . After 16-28 h of transfection, fluorescent signals from the reconstituted YFP were taken for protoplast images with a fluorescence microscope (Leica, DM2500 with a fluorescent module [Fluo Illuminator L4/23]) using the YFP filter system (excitation BP500/20, emission BP535/30).