Stomatal Aperture Assay

The bioassay for stomatal aperture was performed as reported by Li et al. (2016) with a slight modification. In short, the leaves of 4-week-old plants were excised and incubated in closure buffer (20 mM KCl, 1 mM CaCl₂, 5 mM MES-KOH, pH 6.15) at 23°C for 2.5 h in light (100 μmol m⁻²s⁻¹ fluorescent lamp light), then added ABA (1, 10, 50 μM), 20 mM 3-amino-1,2,4-triazole (AT, an inhibitor of catalase) (Jannat et al., 2011), 100 μM H₂O₂ or absolute ethanol (solvent control) respectively for an additional 2.5 h in the same incubation place and condition. Subsequently, abaxial epidermal strips were peeled off and immediately photographed by a light inverted microscope. Stomatal aperture width and length were measured by the open access software ImageJ (v1.37, https://imagej.nih.gov/ij/). Each experiment included at least three biological replicates, with no fewer than 60 guard cells that were measured per each sample. The Student′s t-test was used to determine whether differences between mean values were statistically significant.