Live-Cell Monitoring of Piecemeal Chloroplast Autophagy

When plants undergo senescence or experience carbon starvation, leaf cells degrade proteins in the chloroplasts on a massive scale via autophagy, an evolutionarily conserved process in which intracellular components are transported to the vacuole for degradation to facilitate nutrient recycling. Nonetheless, how portions of chloroplasts are released from the main chloroplast body and mobilized to the vacuole remains unclear. Here, we developed a method to observe the autophagic transport of chloroplast proteins in real time using confocal laser-scanning microscopy on transgenic plants expressing fluorescently labeled chloroplast components and autophagy-associated membranes. This protocol enabled us to track changes in chloroplast morphology during chloroplast-targeted autophagy on a timescale of seconds, and it could be adapted to monitor the dynamics of other intracellular processes in plant leaves. Key features • This protocol enables real-time monitoring of chloroplast morphology in living Arabidopsis leaves. • The method is based on confocal microscopy of transgenic plants that express fluorescent protein markers for specific organelles or suborganellar compartments. • We used this protocol to monitor the piecemeal autophagic degradation of chloroplasts, but it could also be extended to other intracellular phenomena.