Figure 1: Selection of protein-kinase inhibitors that suppress blue light-dependent phosphorylation of guard cell H⁺-ATPase and stomatal opening.

(a,b) Effects of four inhibitors on blue light-dependent H⁺-ATPase phosphorylation in Arabidopsis guard cells. Immunohistochemical detection of the phosphorylated H⁺-ATPase was performed using anti-pThr antibody in the leaf epidermis²³. The fluorescent images (a) and relative intensities of the fluorescent signals (b) are shown. Epidermal fragments from dark-adapted plants were illuminated with red light (50 μmol m⁻² s⁻¹) for 20 min (Red), after which blue light was superimposed on the red right (10 μmol m⁻² s⁻¹) for 2.5 min (Blue). Each kinase inhibitor was administered at 50 μM to the epidermis under red light illumination. DMSO was used as a solvent control. The signal intensity was expressed as the ratio of the signal from the red light with DMSO or blue light with each inhibitor to that from blue light with DMSO. Scale bar represents 50 μm. Data indicate means ± SD (n = 3) with measurement of 30 stomata in each sample. *indicates values that statistically differ from blue light sample without inhibitor (Student’s t test; *p < 0.01). (c) Effects of the four inhibitors on blue light-dependent stomatal opening. The epidermal fragments were pre-treated with each inhibitor at 10 μM for 20 min in the dark, and then illuminated with or without mixed light (red light at 50 μmol m⁻² s⁻¹ and blue light at 10 μmol m⁻² s⁻¹) for 3 h. Values indicate means ± SD (n = 3); measurement of 30 stomata in each experiment. * indicates values that statistically differ from the light sample without inhibitor (Student’s t test; *p < 0.01). (d) Effects of the four inhibitors on phot1 activity. Blue light-dependent autophosphorylation of phot1 was determined by protein blotting using GST-14-3-3 protein (GF14ϕ) as a probe³². Etiolated seedlings were pre-treated with each of the inhibitors at 10 μM for 30 min and then illuminated with or without blue light at 100 μmol m⁻² s⁻¹ for 1 min.