Transient expression and analysis of chloroplast proteins in Arabidopsis protoplasts

Although chloroplasts have their own genome, most chloroplast proteins are encoded in the nuclear genome and are targeted to chloroplasts posttranslationally. In vitro import studies with isolated chloroplasts have been widely used and have helped to elucidate the complex mechanisms involved in protein targeting to chloroplasts. Recently, an in vivo targeting method using protoplasts emerged as an alternative method to investigate protein targeting into chloroplasts. The present study describes a set of principles and methods, including polyethylene glycol-mediated reporter plasmid transformation, fluorescence microscopy, immunocytochemistry, and Western blotting, for studying chloroplast interior and envelope membrane protein targeting using protoplasts isolated from Arabidopsis thaliana leaf tissues.