Review Article

Techniques to Study Autophagy in Plants

Table 2

Advantages and disadvantages of techniques used to study autophagy.

TechniqueAdvantagesDisadvantages

Electron microscopyGolden standard.Morphological characterization of autophagosomes, autolysosomes and their cargo.Equipment and expertise required.Difficult to make quantitative analyses.
Atg8/LC3 conjugation to lipidRapid detection and quantification of autophagy.Amenable to high throughput techniques.Used to create transgenic organisms for in vivo study of autophagy.Dots do not always reflect autophagic activity.Molecular weight shift tests need careful interpretation.
Other molecular markers (Atg5, Atg6, Atg16 and Atg18 detection)Detection of various stages of autophagic vesicle formation.Most of them need further evaluation.
PI3P detectionReflects the activity of Vps34 kinase.Quantitative analysis possible.PI3P accumulation in phenomena not directly related to autophagy (vesicular transport).
Atg1 and Atg4 activityDetermination of enzymatic activity.So far no clear kinetic studies were published.
P62/SQSTM1 degradationActivated especially by protein aggregates.Not all stimuli activate its degradation.Orthologue in plants?
Lysotracker and acridine orange stainingDetermination and quantification of autophagy-related lytic activity (lysosomal/vacuolar).FACscan analysis possible.Autophagosomes are not detected.Lytic activity induced by other conditions as well.
MDC stainingDetermination and quantification of autophagy-related lytic activity (lysosomal/vacuolar).Not all autophagosomes are detected.Lytic activity induced by other conditions as well.
Long-lived protein degradationMeasures autophagic degradation of proteins.Kinetic measurements possible.Nonspecific degradation of proteins by mechanisms other than autophagy. Radioactive technique.
Sequestration of sugarsMeasures autophagic sequestration phase.Quantification may be possible.Sugars may be metabolized.
Phosphorylcholine accumulationPromising plant autophagy technique.Quantification may be possible.Quantification requires special equipment (NMR spectroscopy).
Nonselective and selective degradation of proteinsPromising techniques for plant autophagy.Detection of both sequestration and degradation phases.Quantification may be possible.Autophagy target proteins need further characterization.
Test of mitophagy or chloroplast autophagyDetection of autophagy target organelle degradation.Various organelle-specific proteins or organelle-tagged may be used.Quantification not always possible.