Summary
Autophagy is the major catabolic process in eukaryotes and a key regulator of plant fitness. It enables rapid response to stress stimuli, essential for plastic adaptation of plants to changes in the environment. Fluorescent reporters and confocal microscopy are among the most frequently used methods for assessing plant autophagic activity. However, detection of dynamic changes in the pathway activity has been hampered by stresses imposed on living plant tissues during sample mounting and imaging.
Here we implemented RoPod, a toolkit optimized for minimally-invasive time-lapse imaging of Arabidopsis roots, to reveal a time-resolved response of plant autophagy to drug treatments typically used for pathway modulation and discovered previously overlooked cell type-specific changes in the pathway response. These results not only give an insight into the complex dynamics of plant autophagy, but also provide necessary information for choosing sampling time for the end-point assays currently employed in plant autophagy research.
RoPods are inexpensive and easy-to-use devices that are based on commercial or custom designed chambers, compatible with inverted microscopes. We describe a detailed protocol for the fabrication and use of RoPods and provide a complete pipeline including semi-automated image analysis for root hair growth assays, demonstrating the broader applicability of the RoPod toolkit.
Competing Interest Statement
The authors have declared no competing interest.