Design and Application of a Rotatory Device for Detecting Transient Ca²⁺ Signals in Response to Mechanical Stimulation Using an Aequorin-Based Ca²⁺ Imaging System

Elevation of the cytosolic free calcium ion (Ca²⁺ ) concentration ([Ca²⁺ ]_cyt ) is one of the earliest responses to biotic and abiotic stress in plant cells. Among the various Ca²⁺ detection systems available, aequorin-based luminescence Ca²⁺ imaging systems provide a relatively amenable and robust method that facilitates large-scale genetic-mutant screening based on [Ca²⁺ ]_cyt responses. Compared to that mediated by chemical elicitors, mechanical stimulation-induced elevation of [Ca²⁺ ]_cyt is considerably more rapid, occurring within 10 s following stimulation. Therefore, its assessment using aequorin-based Ca²⁺ imaging systems represents a notable challenge, given that a time interval of ≥1 min is required to reduce the background light before operating the photon imaging detector. In this context, we designed a device that can rotate automatically within the confines of an enclosed dark box, and using this, we can record [Ca²⁺ ]_cyt dynamics immediately after plants had been rotated to induce mechanical stimulation. This tool can facilitate the study of perception and early signal transduction in response to mechanical stimulation on a large scale based on [Ca²⁺ ]_cyt responses. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Detection of background luminance signals in aequorin-transformed Arabidopsis seedlings using a photon imaging detector Basic Protocol 2: Construction of the rotatory device Basic Protocol 3: Calcium measurement in Arabidopsis seedlings after rotatory stimulation Basic Protocol 4: Data analysis and processing.