Hideg É Éva, Ogawa Ken'Ichi, Kálai Tamás, Hideg Kálmán
Institute of Plant Biology, Biological Research Center, H-6701 Szeged, PO Box 521, Hungary; Research Institute for Biological Sciences Okayama (RIBS), 7549-1 Yoshikawa, Kayou-cho, 716-1241 Okayama, Japan; Department of Organic and Medicinal Chemistry, University of Pécs, H-7643 Pécs, PO Box 99, Hungary.
Physiol Plant. 2001 May;112(1):10-14. doi: 10.1034/j.1399-3054.2001.1120102.x.
Arabidopsis thaliana leaves were infiltrated with DanePy (3-(N-diethylaminoethyl)-N-dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole), a double, fluorescent and spin sensor of singlet oxygen. DanePy fluorescence was imaged by laser scanning microscopy. We found that DanePy penetrated into chloroplasts but did not alter the functioning of the photosynthetic electron transport as assessed by chlorophyll fluorescence induction. In imaging, DanePy fluorescence was well distinct from chlorophyll fluorescence. 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 group in DanePy, there was no fluorescence quenching during photoinhibition. This shows that the fluorescence quenching of DanePy is caused by the conversion 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.
用丹吡（3-(N-二乙氨基乙基)-N-丹磺酰基）氨甲基-2,5-二氢-2,2,5,5-四甲基-1H-吡咯）对拟南芥叶片进行浸润处理，丹吡是一种单线态氧的双荧光和自旋传感器。通过激光扫描显微镜对丹吡荧光进行成像。我们发现丹吡能渗透到叶绿体中，但如通过叶绿素荧光诱导所评估的那样，它不会改变光合电子传递的功能。在成像过程中，丹吡荧光与叶绿素荧光明显不同。过量光合有效辐射引起的光抑制导致叶绿体中丹吡荧光猝灭，但在其他细胞区室中则不会。当用丹磺酰基（丹吡中的荧光基团）浸润叶片时，光抑制期间没有荧光猝灭现象。这表明丹吡的荧光猝灭是由其吡咯基团转化为氮氧化物引起的，即它是由单线态氧与双传感器的反应引起的，而非人为因素。这些数据为体内单线态氧产生定位于叶绿体提供了直接的实验证据。
