Dual-sensing genetically encoded fluorescent indicators, ABA and second messenger dynamics (Plant Cell)

Responses to a changing environment require an interplay between primary and secondary messengers. Hormones like abscisic acid (ABA), auxin etc. act as a primary messengers, whereas molecules like Ca²⁺, reactive oxygen species (ROS), other cations and anions act as a secondary messenger. In this paper, Waadt et al. generated a set of dual-reporting genetically encoded fluorescent indicators (2-in-1 GEFIs) that allow observation of the dynamics of two messengers at the same time in Arabidopsis roots. The indicators and ligands they recognize (in parenthesis) are: ABAleonSD1-3L21 (ABA), R-GECO1 (Ca²⁺), Arabidopsis codon optimized red-fluorescing (P)A-17 (H⁺), E²GFP (H⁺ and Cl^–), Grx1-roGFP2 (glutathione redox potential; E_GSH) and roGFP2-Orp1 (H₂O₂). These were linked by either a 14 amino acid peptide or a self-cleaving P2A linker that allows the two indicators to be expressed separately but in equal amounts. The sensors were imaged in different combinations, which showed the dynamics of ABA, Ca²⁺, H⁺ and H₂O₂ in response to various chemicals including ABA, IAA, glutamate, ATP, and H₂O₂. Thus, this paper reveals the interdependence of each signaling molecule and provides new tools for high resolution spatiotemporal information. (Summary by Vijaya Batthula @Vijaya_Batthula) Plant Cell 10.1105/tpc.19.00892