Quantitatively Analyzing the Structural Organization at the Tip of Growing Tobacco Pollen Tubes.

Rapid, unidirectional pollen tube tip growth is a widely employed model for polar cell expansion, which is essential for plant morphogenesis. At the pollen tube tip, various proteins and lipids playing pivotal roles in regulating polar cell expansion are associated with distinct domains of the plasma membrane (PM) or accumulate within different cytoplasmic regions. These PM domains and cytoplasmic regions must be dynamically maintained during tip growth, which is contingent on massive secretory and endocytic membrane trafficking. The molecular and cellular mechanisms responsible for PM and cytoplasmic compartmentalization at the pollen tube tip are not well understood. To further characterize these mechanisms, standardized methods are required, which enable quantitative analysis of the structural organization of normally growing Nicotiana tabacum (tobacco) pollen tube tips, and enhance the comparability of data generated in independent studies. The application of stably transformed marker lines greatly facilitates quantitative structural analyses and enhances the reliability of results obtained. This chapter describes methods required for the generation of transgenic tobacco lines and for the expression level-independent quantitative characterization of PM domains and cytoplasmic regions at the pollen tube tip. Furthermore, a FM4-64 and brefeldin A-based method allowing quantitative analysis of endocytic lipid and protein recycling is discussed.