Assessing the Applicability of Singlet Oxygen Photosensitizers in Leaf Studies

Singlet oxygen (1O2) is of special interest in plant stress physiology. Studies focused on internal, chlorophyll‐mediated production are often complemented with the use of artificial 1O2 photosensitizers. Here, we report a comparative study on the effects of Rose Bengal (RB), Methylene Violet (MVI), Neutral Red (NR) and Indigo Carmine (IC). These were infiltrated into tobacco leaves at concentrations generating the same fluxes of 1O2 in solution. Following green light‐induced 1O2 production from these dyes, leaf photosynthesis was characterized by Photosystem (PS) II and PSI electron transport and oxidative damage was monitored as degradation of D1, a PSII core protein. Cellular localizations were identified on the basis of the dyes’ fluorescence using confocal laser scanning microscopy. We found that RB and NR were both localized in chloroplasts but the latter had very little effect, probably due to its pH‐dependent photosensitizing. Both RB and intracellular, nonplastid MVI decreased PSII electron transport, but the effect of RB was stronger than that of MVI and only RB was capable of damaging the D1 protein. Intercellularly localized IC had no significant effect. Our results also suggest caution when using RB as photosensitizer because it affects PSII electron transport. Confocal microscopy imaging of tobacco leaf cells loaded with the photosensitizer Rose Bengal. Composite image shows Rose Bengal fluorescence as green and chlorophyll fluorescence as red.