Singlet oxygen imaging in Arabidopsis thaliana leaves under photoinhibition by excess photosynthetically active radiation

Arabidopsis thaliana leaves were infiltrated with DanePy (3-(N -diethylamin oethyl) -N- dansyl)aminomethyl - 2,5-dihydro - 2, 2,5,5-tetramethyl-1H-pyrr ole), a double, fluorescent and spin sensor of singlet oxygen, DanePy fluor escence was imaged by laser scanning microscopy. We found that DanePy penet rated into chloroplasts but did not alter the functioning of the photosynth etic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluoresc ence. Photoinhibition by excess photosynthetically active radiation caused quenching of DanePy fluorescence in the chloroplasts but not in other cell compartments. When leaves were infiltrated with dansyl, the fluorescent gro up in DanePy, there was no fluorescence quenching during photoinhibition, T his shows that the fluorescence quenching of DanePy is caused by the conver sion of its pyrrol group into nitroxide, i.e. it was caused by the reaction of singlet oxygen with the double sensor and not by artifacts, These data provide direct experimental evidence for the localization of singlet oxygen production to chloroplasts in vivo.