Fig. 2: Characterization of the effect of BITC on stomatal opening.

a Effect of BITC on PM H⁺-ATPase phosphorylation induced by blue light (left) or FC (right) in A. thaliana guard cells, as determined by immunohistochemistry. The leaf epidermis was treated for 30 min with DMSO (Ctrl) or 50 µM BITC under 50 m^–2 s^–1 red light. Red samples were taken immediately; +Blue, samples were taken after 2.5 min of superimposition of 10 mmol m^–2 s^–1 blue light; and +FC samples were taken after 5 min of treatment with 10 µM FC. The images show typical fluorescence of stomata using anti-phosphorylated PM H⁺-ATPase (pThr, upper) and quantification of fluorescence images of stomata (lower). Values are presented as mean ± SD (n = 3; 50 stomata in each experiment). Bar = 20 µm. b Immunoblotting analysis of the effect of BITC on PM H⁺-ATPase phosphorylation induced by FC in A. thaliana mesophyll cell protoplasts (MCPs). MCPs were pretreated with 50 µM BITC or the same volume of DMSO (−) for 30 min before being treated with 10 µM FC for 30 min. c Effect of BITC on the hydrolytic activity of PM H⁺-ATPase in isolated microsome from A. thaliana leaves. Values are presented as mean ± SD (n = 5; two or three technical replicates in each experiment). P values are indicated (unpaired, two-sided Student’s t-tests). d Effect of BITC on blue light-induced autophosphorylation of phot1 in A. thaliana MCP. MCP samples were incubated in the presence of chemicals in the dark for 20 min before being exposed to darkness (represented as D) or blue light (represented as B) (50 µmol m⁻² s⁻¹) for 5 min before sampling. ST, 50 µM staurosporine. Mobility shift of the bands to higher molecular weight (upper arrow) reflects autophosphorylation of phot1. e Effect of BITC on the phosphorylation status of AKS in a Vicia faba epidermis-rich fraction. As a loading control, 14-3-3 protein was used. For 20 min, the epidermis-rich fraction samples were treated with 20 µM ABA or 50 µM BITC. b, d, and e Representative data set from an experiment replicated three times with different biological samples are shown. f Signal components in the blue light-induced stomatal opening, as well as a working model for the mode of action of BITC. Arrows, positive regulation; dashed arrow, signaling cascade whose components have not been fully identified; T-bar, negative regulation; P, phosphorylation.