Abstract
Autophagy is a major catabolic process in eukaryotes with a key role in homeostasis, programmed cell death, and aging. In plants, autophagy is also known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Despite its significance, there is still a shortage of reliable tools modulating plant autophagy. Here, we describe the first robust pipeline for identification of specific plant autophagy-modulating compounds. Our screening protocol comprises four phases: (1) high-throughput screening of chemical compounds in cell cultures of tobacco (Nicotiana tabacum); (2) confirmation of the identified hits in planta using Arabidopsis (Arabidopsis thaliana); (3) further characterization of the effect using conventional molecular biology methods; and (4) verification of chemical specificity on autophagy in planta. The methods detailed here streamline the identification of specific plant autophagy modulators and aid in unraveling the molecular mechanisms of plant autophagy.
Highlights
In plants, autophagy is known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield
firefly luciferase (Fluc)-ATG8 was delivered to the lytic vacuole, leading to the loss of its activity and resulting in an increase of the ratio of Renilla luciferase (Rluc) to Fluc
The dual-luciferase assay permits measurement of both response (Fluc-ATG8 for the reporter line and Fluc-nuclear localization signal (NLS) for the control line) and reference (Rluc-NLS) luminescence in the same cells, removing possible bias originating from cell-to-cell fluctuations of expression or differences in cell number per sample
Summary
Autophagy is known to regulate agronomically important traits such as stress resistance, longevity, vegetative biomass, and seed yield. Rapamycin was originally thought to be ineffective in Arabidopsis (Arabidopsis thaliana; Sormani et al, 2007), potentially due to the low capacity of plant FKBP12 proteins to form the complex, a more recent report suggests that this is not the case (Xiong and Sheen, 2012) These studies illustrate the importance and need for precise and robust screening strategies to identify autophagy modulators. The assays utilize a combination of reporter and control transgenic lines to optimize the treatment conditions for cell cultures and in planta to ensure that the compounds influence autophagic flux without causing considerable stress to other endomembrane compartments. Using this new method, we have identified several promising autophagy-modulating compounds
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
