Measuring Cytosolic pH in Arabidopsis Flowers

The 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence analysis was carried out to measure cytosolic pH. According to the previously described studies (Sundaresan et al., 2014 (link); Ying et al., 2016 (link)), Arabidopsis flowers were removed from the plant body and soaked into 10 μM BCECF-AM (B1150, Thermo Scientific) solution for 20 min under darkness. Then, phosphate-buffered saline (PBS, pH 7.4) was used to remove the excess BCECF-AM. Images were taken using the confocal laser scanning microscope (LSM 7 DUO, ZEISS, Germany). BCECF fluorescence and chlorophyll autofluorescence were detected under 488 nm and 633 nm light, respectively.