Detection of singlet oxygen and superoxide with fluorescent sensors in leaves under stress by photoinhibition or UV radiation

In order to understand the physiological functions of reactive oxygen species (ROS) generated in leaves, their direct measurement in vivo is of special importance. Here we report experiments with two dansyl-based ROS sensors, the singlet oxygen specific DanePy and HO-1889NH, which is reactive to both singlet oxygen and superoxide radicals. Here we report in vivo detection of O-1(2) and O2-. by fluorescence quenching of two dansyl-based ROS sensors, the O-1(2) specific DanePy and HO-1889NH, which was reactive with both O-1(2) and O-2(-.). The ROS sensors were administered to spinach leaves through a pinhole, and then the leaves were exposed to either excess photosynthetically active radiation or UV (280-360 nm) radiation. Microlocalization of the sensors' fluorescence and its ROS-induced quenching was followed with confocal laser scanning microscopy and with fluorescence imaging. These sensors were specifically localized in chloroplasts. Quenching analysis indicated that the leaves exposed to strong light produced O-1(2), but hardly any O-2(-.). On the other hand, the dominant ROS in UV-irradiated leaves was O-2(-.), while O-1(2) was minor.