Chemical Screening

Screening of chemicals was performed as described previously (Toh et al., 2018) with some modifications. Four-week-old C. benghalensis plants were transferred from a greenhouse to a dark room for incubation overnight to ensure complete stomatal closure on day 2. Leaf discs of 4 mm diameter were excised from dark-adapted C. benghalensis using a hole punch under dim light. The leaf discs were floated on basal reaction buffer [5 mM MES-BTP (Bis-trispropane), pH 6.5, 50 mM KCl and 0.1 mM CaCl₂] with chemical compounds and incubated in light (150 μmol m^–2 s^–1 red light and 50 μmol m^–2 s^–1 BL) or in the dark for 3 h. Leaf discs were incubated with compounds for 30 min before light exposure. Stomatal apertures of the abaxial epidermis were measured as described previously (Toda et al., 2018). Inhibition by PI1, PI2, and PI3 of stomatal opening was identified. To assay viability, abaxial epidermis was removed from leaf discs using forceps after chemical and light treatments and incubated in fresh basal reaction buffer containing 1 μg/mL fluorescein diacetate (FDA) for 30 min in the dark. Epidermis was washed three times with Milli-Q water to remove FDA. Fluorescence microscopy was used to detect fluorescence.