A comparative study of fluorescent singlet oxygen probes in plant leaves

Abstract

Four fluorescent singlet oxygen sensors: DanePy, its oxalate salt, Singlet Oxygen Sensor Green and MVP, were infiltrated into tobacco leaves and tested for toxicity, subcellular localization, light sensitivity and capacity to trap the singlet oxygen produced in photoinhibition. For reference, a broad sensitivity free radical probe, TEMPO-9-AC, was also included. Photochemical yield was approximately 15% and 10% inhibited by Singlet Oxygen Sensor Green and MVP, respectively, but was not significantly affected by the other probes. Under photoinhibitory conditions, brought about by irradiating lincomycin-treated leaves with strong photosynthetically active radiation, DanePy and Singlet Oxygen Sensor Green were responsive. Singlet Oxygen Sensor Green was also reactive to low, non-photoinhibitory light exposure of the leaf, which was not characteristic to the other probes. MVP did not respond to singlet oxygen which can partly be explained by a possible attenuation of its blue emission in the leaf, as shown by the example TEMPO-9-AC. DanePy-oxalate did not respond to photosynthetic singlet oxygen due to lack of its penetration into photosynthetic tissue and hence could be useful in detecting any singlet oxygen which escapes from a chloroplast initiation site. DanePy was localized in the chloroplasts, while Singlet Oxygen Sensor Green was mainly found in the epidermal cells preferentially associated with the nucleus.

Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses

“…4C, top row). Nuclear localization of SOSG was previously reported for mesophyll cells (Hideg, 2008). Although the signal in the nucleus was variable in some cases, it could clearly be detected in Figure 4.…”

Reactive oxygen species: Reactions and detection from photosynthetic tissues

“…A disadvantage of SOSG is that when illuminated with wavelengths below 600 nm, the fluorescence yield of SOSG increases similarly as during exposure to 1 O 2 ( Fig. 3; [92,109]). Furthermore, the reaction product of SOSG and 1 O 2 sensitizes 1 O 2 production [112].…”

“…Both imidazole and RNO are commercially available. According to the manufacturer's data, SOSG is cell-impermeable, but a study with tobacco leaves showed that a pinhole administration of SOSG to tobacco leaves leads to preferential association of the dye with the nuclei of epidermal cells [109]. Gollmer et al [110] showed that SOSG is also able to penetrate into human cells; therefore SOSG can be used in confocal microscopy [111].…”

“…Furthermore, the reaction product of SOSG and 1 O 2 sensitizes 1 O 2 production [112]. Surprisingly few researchers have taken the photosensitivity of SOSG into account (for correct use of SOSG, see [92,109,113]). SOSG caused 15 % inhibition of the photochemical yield of PSII in tobacco leaves [109].…”

“…Surprisingly few researchers have taken the photosensitivity of SOSG into account (for correct use of SOSG, see [92,109,113]). SOSG caused 15 % inhibition of the photochemical yield of PSII in tobacco leaves [109]. It is unclear if SOSG can be used with live cyanobacteria, as SOSG seems to penetrate only to a small fraction of the incubated cells and fluorescence from both SOSG and Chl a is only rarely detected from the same cell [114].…”