FRAP of RLK-GFP-stained apical PM in tobacco pollen tubes. (A) PM localization of RLK-GFP in a tobacco pollen tube transiently expressing a LAT52∷RLK-GFP construct. GFP was fused to the C terminus of A. thaliana pollen-specific receptor kinase. Confocal images were taken from the midplane. (B) BFA effect on the localization of RLK-GFP. Transformed pollen tubes expressing RLK-GFP were treated with 1 μg/ml BFA for 1 h before imaging. BFA completely disrupted PM localization of RLK-GFP. The focus is on the midplane. Bars, 10 μm. (C and D) FRAP analysis of RLK-GFP in growing pollen tubes. Photobleaching was performed and recovery was analyzed in the apical area (C) and subapical area (D) of the pollen tube PM. A series of time-lapse images was recorded every 5 s for 3 min. The numbers in each image indicate elapsed time after photobleaching. The bleached area is marked by boxes. Note that photobleaching did not affect pollen tube elongation. The mean growth rate and oscillation period was 30.92 nm/s and 64 ± 6.5 s (n = 5), respectively, after photobleaching. These were similar to what was observed in the GFP-expressing pollen tube. These time series correspond to Videos 2 and 3 . Bars, 10 μm. (E) Quantitative analysis of FRAP for C and D. Fluorescence recovery was measured by calculating the mean GFP signal intensity on the PM of the region of interest. Fluorescence at the apical PM area recovered completely in 75 s, with a 35 ± 3-s half-time of recovery (▪), whereas little fluorescence recovery occurred in the subapical PM area (♦). Similar results were obtained from five individual experiments. (F) Quantitative FRAP analysis at four different regions of the membrane. The intensity of recovered GFP signal is plotted as a function of the time. Fluorescence recovery was fast at the apical PM. The rate of signal recovery gradually became slower away from the center of the apex. The recovered GFP signal was measured every 5 s for 80 s. The regions (1–4) where FRAP was measured are indicated in C.