RISAP Accumulates Subapically Directly Behind the CZ Filled with Apical Vesicles in Normally Elongating Tobacco Pollen Tubes.(A) Single confocal optical sections through pollen tubes transiently expressing YFP:RISAP at different levels 6 h after gene transf... RISAP Accumulates Subapically Directly Behind the CZ Filled with Apical Vesicles in Normally Elongating Tobacco Pollen Tubes.(A) Single confocal optical sections through pollen tubes transiently expressing YFP:RISAP at different levels 6 h after gene transfer revealed three clearly discernable distribution patterns. Subapical YFP:RISAP accumulation was seen in pollen tubes elongating at normal growth rates ([A], top image; [B]). Apical YFP:RISAP accumulation was seen in pollen tubes, which were elongating at reduced rates ([A], central image; [B]) or which had stopped growing ([A], bottom image). Pollen tubes that had stopped growing in addition showed tip swelling (cf. Figure 7A, right column) and often contained YFP:RISAP-associated large organelles in the granular cytoplasm ([A], bottom image; arrowheads). Bars = 20 μm.(B) Statistical analysis of the growth rates of pollen tubes expressing YFP or YFP:RISAP at low (distribution pattern as shown in [A], top image) or intermediate levels (distribution pattern as shown in [A], central image]. Error bars: 95% confidence interval (n > 10).(C) FM4-64 labeling of apical membrane traffic in pollen tubes transiently expressing YFP:RISAP at different levels 6 h after gene transfer. Simultaneously acquired single confocal optical sections displaying YFP:RISAP (green channel) and FM4-64 (red channel) fluorescence are shown. In normally elongating pollen tubes (left column), YFP:RISAP accumulated subapically directly behind the CZ, which was labeled by FM4-64 as reported in the literature (Parton et al., 2001). In pollen tubes displaying apical YFP:RISAP fluorescence and reduced growth rates, FM4-64-labeled cytoplasmic vesicles accumulated subapically (central column, arrowhead). High-level YFP:RISAP expression, which stopped pollen tube growth and induced tip swelling, also inhibited endocytic FM4-64 internalization (right column). Bars = 10 μm. Octavian Stephan et al. Plant Cell 2014;26:4426-4447 ©2014 by American Society of Plant Biologists