Stomatal aperture measurement

Fully expanded young leaves from 4- to 5-week-old plants were excised for stomatal aperture measurements as described previously (Ye et al., 2013). For assays of light-induced stomatal opening, leaves were floated on assay solution containing 5 mM KCl, 50 μM CaCl₂, and 10 mM MES–Tris (pH 6.15) with their adaxial surface upward in the dark for 2 h to close the stomata. After adding AITC, the leaves were kept in the light (80 μmol m⁻² s⁻¹) for 2 h before measurement. For assays of stomatal closure, leaves were floated on the assay solution in the light for 2 h to open the stomata. Then AITC was added, and the leaves were kept in the light for 2 h before measurement. Inhibitors were added 30 min before AITC treatment. For measurement of stomatal apertures, the leaves were shredded for 30 s, and epidermal tissues were collected using nylon mesh. Thirty stomatal apertures were measured for each sample.