Functional analysis of small Rab GTPases in cytokinesis in Arabidopsis thaliana

Rab proteins are key regulators of membrane transport in eukaryotes. Recent evidence from different species supports the notion that some Rab proteins are crucial for cytokinesis, a pivotal procedure for successful cell division. As a family of monomeric small GTPases of the Ras superfamily, the function of Rab proteins is modulated by guanine nucleotide binding and hydrolysis. To investigate the function of Rab proteins, creating dominant negative or constitutively active mutant forms of a Rab protein is a widely used approach. To study cytokinesis in plant cells, using fluorescent dye to highlight the cell shape and the nuclei, and to monitor the formation of the newly formed cell plate in mitotic cells, is easy and useful. In this chapter, we describe detailed methods for (1) generating transgenic plants expressing dominant negative or constitutively active form of RAB-A1c; (2) fluorescent staining of cell shape, cell wall, and nuclei of mitotic root tip cells; (3) fluorescent staining of newly formed cell plate; and (4) detecting fluorescent signals using Confocal Laser Scanning Microscopy in the genetic model plant species Arabidopsis thaliana.