Arabidopsis guard cell vacuoles imaged after the induction of vacuole fusion

Wild type Arabidopsis thaliana plants from the Columbia-0 ecotype were grown in soil at 22C with a 16 h photoperiod.  Leaves from 4-week old plants were cut in the morning and immediately processed to generate epidermal peels. Briefly, a small leaf fragment was applied abaxially to a coverslip coated with medical adhesive and all but the bottommost layers of abaxial cells containing the stomata were scrapped away with a razor. 1 mm thick silicone isolators (GraceBio #664170) were used to create wells around the adhesive for incubations. Epidermal peels were immediately incubated in stomata buffer (MES pH 6.1). To induce stomatal closure, peels were incubated in closing buffer (10 mM MES pH 6.1, 40 mM malate, 5 mM CaCl2, 10 µM BCECF, 50 mM ABA (Sigma Aldrich A1049) at 22C in the dark for two hours. To induce stomatal opening or vacuole fusion, ABA-treated peels were incubated in opening buffer (10 mM MES pH 6.1, 50 mM KCl) supplemented with 10 µM BCECF (Fisher Scientific B1170) and either 3 µM fusicoccin (Sigma F0537) or 33 µM wortmannin (Sigma W3144). Wortmannin-treated peels were kept in the dark, while fusicoccin-treated peels were exposed to the light for up to 1 h. All concentrated stocks were first dissolved in DMSO. Images of leaf epidermal peels were acquired after ABA incubation and after 20, 40 and 60 min of fusicoccin or wortmannin treatments. Confocal laser scanning microscopy was carried out in a Zeiss LSM 980 confocal microscope. Images were taken with a 40× FCS water objective (1.1 N.A.). Acquisition of BCECF fluorescence was accomplished with 405 nm laser excitation and 495-550 nm emission filter set. Images were acquired with an Airyscan detector with a pinhole size of 2.5 airy units.