Stomatal Opening Assay in Begonia

The day before the experiment, plants were transferred to constant darkness for at least 12 h to induce stomatal closure. Mature leaves from the second or third node position were collected, the major vein was removed, and the leaf blade was dissected into 1‐cm² squares and submerged into the opening buffer. To capture images from the same position of the leaf section, the microscope light source was used at a light intensity maintained to 100–200 μmol·m⁻²·s⁻¹ using a light meter (LI‐250A, LI‐COR) with a quantum sensor (LI‐190, LI‐COR). Leaf sections were observed under a Leica light microscope (DM 500). All images were recorded every 30 for 240 min (nine images taken per sample). The preparation of the opening buffer followed the method of Papanatsiou et al. (2017 (link)) with a few modifications, including 10 mM MES and 50 mM KCl. After testing for series of pH adjustments from 4.0 to 9.0, alkaline pH offered better responses for stomatal opening in the two Begonia spp. tested here. The opening buffer was thus adjusted to a pH of 8.86. All image recordings and quantification were performed as above for stomatal images for g_{s max}.