Using Photoconvertible and Extractable Fluorescent Proteins to Study Autophagy in Plants

Several methodologies have been employed to understand the kinetics of induced autophagic degradation in plants, but most of them are not capable of distinguishing the autophagic cargo proteins before and after induction of autophagy in cells. Here, we designed a mass photoconverter that allowed us to simultaneously monitor protein synthesis and degradation in tobacco BY-2 cells using a photoconvertible fluorescence marker protein, Kikume Green Red (KikGR). An example of a new protocol for the analysis of autophagy progression using a fusion protein of cytochrome b5 and KikGR under phosphate starvation is described. The other example described is the analysis of the proliferation of Golgi apparatus in tobacco BY-2 cells using the fusion protein of a prolyl 4-hydroxylase NtP4H1.1 and monomeric KikGR. A detailed protocol on key analysis, as well as tips and notes for experiments using KikGR proteins, are described.