Redox‐sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer

Abstract

SummaryThe cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H 2 O 2 or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mM cytosolic glutathione buffer would contain only 25 nM oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stressinduced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.

Glutathione – linking cell proliferation to oxidative stress

“…The distribution of GSH between different cellular compartments is important because it establishes and supports the redox environment in which metabolism and signalling events occur. The GSH redox potential of the cytosol of plant cells in vivo has been extensively monitored using redox-sensitive green fluorescent protein (roGFP) probes (92)(93)(94). Parallel analyses of the redox state of nucleus and cytosol using a yellow fluorescent protein-based redox sensor (rxYFP) in the yeast S. cerevisiae showed that nuclear GSH redox environment is highly reducing and similar to that of the cytosol (95), a situation that is also observed in plant cells (Figure 3; 44; 96).…”

Nuclear Accumulation of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Cadmium-Stressed Arabidopsis Roots

“…The roGFP2 is a redoxsensitive fluorescent probe that can exist in either reduced (dithiol, -SH HS-) or oxidized (disulfide, -SS-) forms, each with a different excitation spectrum. In vivo, the redox state of roGFP2 depends on the redox potential of glutathione (itself a function of the [GSH] 2 / [GSSG] ratio; Meyer et al, 2007), but possibly also by other molecules (e.g. ROS and RNS) that might react with roGFP2 redox-sensitive cysteines.…”

“…The pH of the media was adjusted to 6.0 6 0.1 with 0.5 N KOH before autoclaving at 121°C for 20 min. Seeds of the transgenic roGFP2 Arabidopsis line (Meyer et al, 2007) were provided by Markus Schwarzländer. Seeds of gapc1 and gapc2 Arabidopsis transfer DNA (T-DNA) insertional mutant lines were from the SALK Institute (SALK_010839 and SALK_016539, respectively).…”

“…1A). The effect of cadmium on the cellular redox state was assessed by using Arabidopsis transgenic seedlings expressing free redox-sensitive (ro)GFP2 (localized in the cytosol and nucleus [Meyer et al, 2007]). The roGFP2 is a redoxsensitive fluorescent probe that can exist in either reduced (dithiol, -SH HS-) or oxidized (disulfide, -SS-) forms, each with a different excitation spectrum.…”

Unraveling the Tapestry of Networks Involving Reactive Oxygen Species in Plants

“…Since then researchers have taken advantage of this tool to monitor the redox state involved in glutathione metabolism (Meyer et al, 2007) or the removal of ROS by manganese superoxide dismutase in mitochondria (Morgan et al, 2008), and additional papers are anticipated. Other valuable methods for in vivo monitoring of ROS were also introduced, including autoluminescence (Havaux et al, 2006) and biophotone imaging (Kobayashi et al, 2007) as well as luciferase imaging (Shao et al, 2007).…”