Live monitoring of ROS-induced cytosolic redox changes with roGFP2-based sensors in plants

Abstract

Plant cells produce reactive oxygen species (ROS) as by-products of oxygen metabolism and for signal transduction. Depending on their concentration and their site of production, ROS can cause oxidative damage within the cell, and must be effectively scavenged. Detoxification of the most stable ROS, hydrogen peroxide (H2O2), via the glutathione-ascorbate pathway may transiently alter the glutathione redox potential (EGSH). Changes in EGSH can thus be considered as a proxy of the oxidative load in the cell. Genetically encoded probes based on roGFP2 enable extended opportunities for in vivo monitoring of H2O2 and EGSH dynamics. Here, we report detailed protocols for live monitoring of both parameters in the cytosol with the probes Grx1-roGFP2 for EGSH and roGFP2-Orp1 for H2O2, respectively. The protocols have been adapted for live cell imaging with high lateral resolution on a confocal microscope and for multiparallel measurements in whole organs or intact seedlings in a fluorescence microplate reader. Elicitor-induced ROS generation is used as an example for illustration of the opportunities for dynamic ROS measurements that can easily be transferred to other scientific questions and model systems.

High robustness of cytosolic glutathione redox potential under combined salt and osmotic stress in barley as revealed by the biosensor Grx1-roGFP2

“…Using this approach, we determined that the in vivo dynamic range of cytosolic Grx1-roGFP is c. 1.3 -1.5 in leaves and c. 2.3 -2.7 in roots. The previously reported in vitro dynamic range of roGFP2 was c. 8.2 -9.2 (405/488 nm) (Schwarzländer et al 2008;Aller et al 2013) while in vivo samples often displayed decreased dynamic ranges of c. 5 in confocal microscopy or c. 3 in plate reader-based sensor read-out (Ugalde et al 2021(Ugalde et al , 2022. This difference in dynamic range between in vitro and in vivo sensor read-out can be explained by interference of plant compounds that overlay the changes in sensor absorption in the UV range (below the isosbestic point at c. 425 nm).…”

Endoplasmic reticulum oxidoreductin (ERO) provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

“…For sequential perfusion of different treatments, 4–5-day old seedlings were mounted in a RC-22 perfusion chamber mounted on a P1 platform using a steel anchor harp with a 1.5 mm grid mesh (Warner Instruments, Hamden CT). Imaging buffer, and the indicated solutions of DTT and H 2 O 2 , were loaded into 50 mL syringes connected to a VC-8M valve controller (Warner Instruments) with 1.5 mm polyethylene tubes (Ugalde et al, 2020).…”