Stomatal Aperture Regulation by Phytohormones and Elicitors

Leaf peels were collected from the abaxial side of fully expanded leaves and floated in stomatal buffer (10 mM MES-KOH, 30 mM KCl, pH 6.15) for 2.5 h under light (100 µmol m⁻² s⁻¹) to ensure that most of stomata were opened before treatments [4] (link). Purified chemical lipopolysaccharide (LPS from P. aeruginosa, Sigma), flg22 peptide (Biomer Technology, CA), elf26 (Biomer Technology, CA) or COR (Sigma) were used at indicated concentrations. flg22 and elf26 were diluted in 10 mM MgSO₄. COR and LPS were respectively diluted in milliQ water and in MES buffer containing 0.25 mM MgCl₂ and 0.1 mM CaCl₂[5] (link). ABA, methyl jasmonate (MeJA) and SA used at indicated concentrations were dissolved in 10% ethanol (Sigma). Diphenyleneiodonium chloride (DPI, Sigma) and K252a (Sigma) were dissolved in dimethylsulfoxide (DMSO). Ascorbic acid (ASC) and sodium butyrate (Butyrate, Sigma) were prepared in milliQ water. Mock controls were MES buffer containing 0.1% ethanol for MeJA, ABA and SA, 0.1% DMSO for DPI and K252a, and milliQ water for ASC and Butyrate. Bacterial concentration used was 1×10⁸ cfu.ml⁻¹ in 10 mM MgSO₄. Stomatal apertures were measured as described [43] (link).