Detection of Singlet Oxygen and Superoxide with Fluorescent Sensors in Leaves Under Stress by Photoinhibition or UV Radiation

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Abstract
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 1O2 and O2–• by fluorescence quenching of two dansyl-based ROS sensors, the 1O2 specific DanePy and HO-1889NH, which was reactive with both 1O2 and O2–•. 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 1O2, but hardly any O2–•. On the other hand, the dominant ROS in UV-irradiated leaves was O2–•, while 1O2 was minor.