CHIMERIC FLUORESCENT FUSION PROTEINS TO MONITOR AUTOPHAGY IN PLANTS

Autophagy is induced under nutrient-deficient conditions in both growing tobacco BY-2 cultured cells as well as Arabidopsis and others intact plants. The fluorescent protein-tagged structural protein for autophagosomes, the Atg8 protein, allows nondestructive detection of autophagy induction in plant cells and tissues by fluorescence microscopy. Using this technique, the general operation of autophagy in growing root cells has been observed. A synthetic cargo protein for autophagy consisting of cytochrome b5 and the red fluorescence protein, DsRed, allows for the quantitative assay of autophagy in tobacco cells. This chapter describes methods for detecting autophagy in these plant cells using fluorescent protein fusions in situ with light microscopy, as well as quantification of autophagy.