Relative Membrane Potential Measurements Using DISBAC 2 (3) Fluorescence in Arabidopsis thaliana Primary Roots.

Abstract

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC 2 (3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC 2 (3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.

Plant phenotyping relevance

植物根の膜電位を蛍光画像から定量する非侵襲的手法の開発と画像解析プロトコルが中心であり、植物表現型計測法に該当する。

Code and data availability

The protocol explicitly states that the raw imaging data re-analyzed in the paper are deposited on Zenodo and that all analysis scripts (R and Python) are available in a public SourceForge repository. Both are paper-specific, public, and actionable.