Background

Pollen germination and subsequent pollen tube growth is the critical step during sexual plant reproduction, as it provides passage for the delivery of two non-motitle sperm cells to finally effect double fertilization in flowering plants (McCormick, 2013). How pollen germination and pollen tube growth are regulated has been subject to intensive scrutiny in the past several decades. It was shown that actin is an essential regulator of pollen germination and pollen tube growth and morphogenesis (Gibbon et al., 1999; Vidali et al., 2001). As pollen germination and pollen tube growth are very sensitive to the perturbation on the actin cytoskeleton (Gibbon et al., 1999; Vidali et al., 2001), and considering along with the fact that Arabidopsis thaliana is amenable to genetic analysis, Arabidopsis pollen has increasingly becoming the great cellular system in documenting the organization and regulation of the actin cytoskeleton toward understanding how actin performs functions in pollen (Qu et al., 2015). Arabidopsis pollen cell is an excellent cellular system to study the organization and regulation of the actin cytoskeleton is also because the spatial organization of actin is very regular in pollen cells (Ren and Xiang, 2007; Cheung and Wu, 2008; Chen et al., 2009; Staiger et al., 2010; Fu, 2015; Qu et al., 2015), and it is therefore comparatively easy to quantify the organization of actin in pollen cells (Fu, 2015; Qu et al., 2015). The colleagues in this field have developed different methods in revealing the organization of actin in pollen cells, including staining with fluorescent-phalloidin or immune-staining probed with anti-actin antibody in fixed Arabidopsis pollen cells as well as decoration with different actin markers in living pollen cells. Compared to immuno-staining with anti-actin antibody in fixed pollen cells, actin staining with fluorescent phalloidin can yield more cleanly filamentous images as phalloidin specifically binds to actin filaments with high affinity that reduces the background noise, which will facilitate subsequent analysis and quantification of actin organization in pollen cells. In addition, the procedure of actin staining with fluorescent phalloidin is comparatively simple and straightforward. Furthermore, the whole procedure was completed on solid pollen germination medium, which minimizes the effect of various treatments on the morphology of pollen tubes. This method has been carried out routinely and successfully in our laboratory. The detailed method can be found in series of our previously published papers (Wu et al., 2010; Zhang et al., 2010; Qu et al., 2013; Zheng et al., 2013; Chang and Huang, 2015; Liu et al., 2015; Zhang et al., 2016; Jiang et al., 2017; Qu et al., 2017; Lan et al., 2018; Diao et al., 2019; Jiang et al., 2019) and the papers from our colleagues in this community (Gebert et al., 2008; Cao et al., 2013; Jia et al., 2013; Zhu et al., 2017).