Figure 1.

Subcellular localization and spectral behavior of E_GSH and H₂O₂ sensors in Arabidopsis. A–F (left panels), Confocal microscopy images of leaf epidermal cells from 7-d-old seedlings stably expressing Grx1-roGFP2, roGFP2-Grx1, or roGFP2-Orp1 targeted to plastids (A, B), cytosol (C, D), or mitochondria (E, F). All images show roGFP2 fluorescence recorded with λex = 488 nm and λem = 505–530 nm. Bars, 50 μm. A–F (right panels), Grx1-roGFP2, roGFP2-Grx1, or roGFP2-Orp1 fluorescence excitation spectra for nontreated seedlings (n.t., gray), and after reduction with 20 mM DTT (blue) or oxidation with 100 mM H₂O₂ (red). All spectra were recorded on a plate reader from 7-d-old seedlings with emission at 520 ± 5 nm and using the same gain for all lines. The curves show the mean of the fluorescence in AU + sd, with n ≥ 3 biological replicates, where each replicate is an independent pool of 4–5 seedlings. All spectra were corrected for the autofluorescence measured in nontransformed control seedlings (see Supplemental Figure S1). The dynamic range (δ) for the maximum change of the fluorescence ratio between the fully oxidized and fully reduced sensor was calculated from the fluorescence collected after sensor excitation at 410 and 480 nm. G, Schematic model of roGFP2 structure highlighting the disulfide bond formation upon reversible oxidation. H, Excitation spectrum of purified roGFP2 measured under similar conditions as the seedlings. To achieve full reduction and full oxidation, the purified protein was incubated in 10 mM DTT or 10 mM H₂O₂, respectively. Mean + sd, n = 6.