F-Actin Staining and Quantification

The actin cytoskeleton was visualized according to previously described methods with slight modifications (Zhang et al., 2011; Li et al., 2014). Briefly, pollen grains and pollen tubes were spread on the surface of pollen germination medium and fixed for 1 h in 300 μm 3-maleimidobenzoic acid N-hydroxysuccinimide ester in liquid pollen germination medium. The pollen grains were extracted subsequently with 0.05% Nonidet P-40 in liquid germination medium for 10 min. Fixed pollen grains and pollen tubes were incubated in PEM buffer (100 mm PIPES, 10 mm EGTA, 5 mm MgSO4, and 0.3 m mannitol, pH 6.9) that contained 2% (w/v) glycerol (Sigma-Aldrich) and 6.6 μm Alexa Fluor 488-phalloidin staining (Invitrogen). Images were collected with a Leica TCS SP5 confocal laser scanning microscope equipped with a ×63, 1.46 NA HC PLAN objective. The fluorescent phalloidin was excited using the 488-nm line of an argon laser, and optical sections were scanned and captured.

The angles formed between each actin cable and the growth axes of individual pollen tubes were analyzed using ImageJ. Curled pollen tubes were excluded from this analysis because their growth axes were difficult to define. As actin cables were nearly parallel to the growth axis in the shanks of wild-type pollen tubes, only this region was used for quantification. For each pollen tube, three to four optical sections were excluded between sections used for analysis to ensure that each actin cable was analyzed only once. For wave-like actin cables that formed more than one angle with the growth axis, only the largest angle was used for quantification. The average fluorescence intensity and actin bundles (skewness) were performed as described previously (Zhang et al., 2011; Li et al., 2014).