Abstract

Autophagy is the main catabolic process in eukaryotes and plays a key role in cell homeostasis. In vivo measurement of autophagic activity (flux) is a powerful tool for investigating the role of the pathway in organism development and stress responses. Here we describe a significant optimization of the tandem tag assay for detection of autophagic flux in planta in epidermal root cells of Arabidopsis thaliana seedlings. The tandem tag consists of TagRFP and mWasabi fluorescent proteins fused to ATG8a, and is expressed in wildtype or autophagy-deficient backgrounds to obtain reporter and control lines, respectively. Upon autophagy activation, the TagRFP-mWasabi-ATG8a fusion protein is incorporated into autophagosomes and delivered to the lytic vacuole. Ratiometric quantification of the low pH-tolerant TagRFP and low pH-sensitive mWasabi fluorescence in the vacuoles of control and reporter lines allows for a reliable estimation of autophagic activity. We provide a step by step protocol for plant growth, imaging and semi-automated data analysis. The protocol presents a rapid and robust method that can be applied for any studies requiring in planta quantification of autophagic flux.

Keywords

Plant autophagy; Autophagic activity; Arabidopsis; Tandem tag; Autophagic activity in vivo; In planta autophagic activity assay; High-throughput autophagic activity measurement; Autophagic flux

Published in

Bio-protocol
2020, Volume: 10, number: 5, article number: e3535

SLU Authors

• Dauphinee, Adrian

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

• Ohlsson, Jonas

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    

• Minina, Alyona

  • Department of Molecular Sciences, Swedish University of Agricultural Sciences
    
  • University of Heidelberg

UKÄ Subject classification

Agricultural Science


Publication identifier

DOI: https://doi.org/10.21769/BioProtoc.3535

Permanent link to this page (URI)

https://res.slu.se/id/publ/105810