Photoinhibition of photosynthesis in vivo results in singlet oxygen production detection via nitroxide-induced fluorescence quenching in broad bean leaves

In plants experiencing environmental stress, the formation of reactive oxygen is often presumed. In this study, singlet oxygen was detected in broad bean (Vicia faba) leaves that were photoinhibited in vivo. Detection was based on the reaction of singlet oxygen with DanePy (dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole) yielding a nitroxide radical (DanePyO) which is EPR active and also features lower fluorescence compared to DanePy. The two (fluorescent and spin) sensor fuctions of DanePy are commensurate, which makes detecting singlet oxygen possible with a spectrofluorimeter in samples hard to measure with EPR spectroscopy [Kalai, T., Hideg, E., Vass, I., and Hideg, K, (1998) Free Radical Biol, Med. 24, 649-652]. We found that in leaves saturated with DanePy, the fluorescence of this double sensor was decreased when the leaves were photoinhibited by 1500 mu mol m(-2) s(-1) photosynthetically active radiation. This fluorescence quenching is the first direct experimental evidence that photoinhibition of photosynthesis in vive is accompanied by O-1(2) production and is, at least partly, governed by the process characterized as acceptor side-induced photoinhibition in vitro.