Confocal imaging of glutathione redox potential in living plant cells

Reduction–oxidation‐sensitive green fluorescent protein (roGFP1 and roGFP2) were expressed in different sub‐cellular compartments of Arabidopsis and tobacco leaves to empirically determine their performance as ratiometric redox sensors for confocal imaging in planta. A lower redox‐dependent change in fluorescence in combination with reduced excitation efficiency at 488 nm resulted in a significantly lower dynamic range of roGFP1 than for roGFP2. Nevertheless, when targeted to the cytosol and mitochondria of Arabidopsis leaves both roGFPs consistently indicated redox potentials of about –320 mV in the cytosol and –360 mV in the mitochondria after pH correction for the more alkaline matrix pH. Ratio measurements were consistent throughout the epidermal cell layer, but results might be attenuated deeper within the leaf tissue. Specific interaction of both roGFPs with glutaredoxin in vitro strongly suggests that in situ both variants preferentially act as sensors for the glutathione redox potential. roGFP2 targeted to plastids and peroxisomes in epidermal cells of tobacco leaves was slightly less reduced than in other plasmatic compartments, but still indicated a highly reduced glutathione pool. The only oxidizing compartment was the lumen of the endoplasmic reticulum, in which roGFP2 was almost completely oxidized. In all compartments tested, roGFP2 reversibly responded to perfusion with H2O2 and DTT, further emphasizing that roGFP2 is a reliable probe for dynamic redox imaging in planta. Reliability of roGFP1 measurements might be obscured though in extended time courses as it was observed that intense irradiation of roGFP1 at 405 nm can lead to progressive photoisomerization and thus a redox‐independent change of fluorescence excitation ratios.