Tyrphostin 23

For inhibitor treatment, leaves were infiltrated on the abaxial side using a needleless syringe and then incubated in an inhibitor solution [10 μM brefeldin A (BFA; Sigma), 50–200 μM tyrphostin23 (Sigma), 10 μM wortmannin (Sigma)] for 30/60min, respectively, before microscopic examination. As a control, water with 0.5% methanol/dimethylsulphoxide (DMSO) was infiltrated into leaves.

Cells were perfused with bath solution containing the following (mM): NaCl 145.0, MgCl₂ 1.2, CaCl₂ 1.0, KCl 5.6, glucose 10.0, and HEPES 10.0. The pH of the bath solution was adjusted to 7.4. For recording the BKCa currents, 3 mM 4-aminopyridine (Sigma-Aldrich, USA) was used to block the other outward K⁺ currents. The pipette solution consisted of (mM) KCl 140.0, CaCl₂ 0.686, MgCl₂ 1.651, EGTA 1.0, K₂ATP 2.0, and HEPES 1.0 (pH 7.3). The patch electrode resistance was 4–7 MΩ after filling with pipette solution. Whole-cell BKCa currents were activated with 500 ms voltage steps from -40 to +80 mV in 10 mV increments. The currents were recorded at a holding potential of -70 mV. After the currents were stabilized, 50 μmol/L genistein, tyrphostin 23, or daidzein (Sigma-Aldrich, USA) was added into the bath solution, and the change of the currents was recorded accordingly. The current density was presented as current amplitude/cell capacitance (pA/pF). Axopatch 700B Amplifier (Axon Instrument, USA), Signal 3.06 software, and CED Power 1401 A/D interface (Cambridge Electronic Design Limited, Cambridge, UK) were used in this study to record whole-cell BKCa currents.