How to analyze autophagy in plant?

Autophagy is a process that cells use for degradation and intracellular recycling. In plant cell, autophagy occurs in vacuole, a large, membrane-bound organelle that contains hydrolytic enzymes. When the autophagy is activated, double membrane vesicles known as autophagosomes form in the cytoplasm to wrap the materials to be degraded. Then autophagosomes move to the vacuole.

To measure the level of autophagy in plant cells, several methods have been developed, which have mostly been optimized for use in the model plant Arabidopsis. These methods include non-invasive assays that can be used to monitor autophagy, mostly non-selective autophagy, in whole intact plants for live imaging.

For microscopic methods, ATG8, an autophagosome-localized protein and an ideal marker for tracking autophagy from early stage, is commonly used to detect autophagy in living cells. Fluorescence proteins such as green fluorescence protein or other color fluorescent protein variants can be fused to the ATG8 N-terminus and make this protein detectable under fluorescence microscope.

For fluorescent dyes methods, monodansylcadaverine (MDC) is a type of fluorescence dye used to stain autophagosomes in living plants. MDC accumulates and fluoresces inside autophagosomes, which include an acidic lumen and a lipid-rich membrane.

These methods allow us to do a quantification by counting the number of autophagosomes from live imaging of plant cells.


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