New sensors for monitoring reactive oxygen in plants under light stress

Abstract

Reactive oxygen species (ROS) play a key role in a variety of biotic and abiotic stress conditions. Double (spin and fluorescent) ROS sensors contain a fluorophore and a spin trap: conversion of the latter to an EPR active nitroxide also causes partial fluorescence quenching. In this way, double sensors can be applied for monitoring ROS in experiments when EPR measurements are hard to carry out [1]. Using a dansyl-based double sensor, DanePy, we detected singlet oxygen in vivo, in leaves exposed to photoinhibition by excess photosynthetically active radiation [2]. Because the stress-inducible blue-green fluorescence may obstruct the detection of DanePy fluorescence quenching at 535-545 nm in leaves, we also investigated the application of phtalimide and rhodamine fluorophores in double sensors. Besides these molecules, we also studied the versatility (specificity, penetration, stability) of double sensors in which the fluorophore was combined with a spin trap instead of the singlet oxygen reactive moiety. These latter enabled us to compare oxygen (superoxide, hydroxyl) radical and singlet oxygen production in leaves exposed a variety of biotic stress conditions, such as light- (UV-B, photoinhibition), water- or temperature-stress. This work was supported by the Hungarian National Research Foundation (OTKA T030362) and the Hungarian Ministry of Education (FKFP 0252/1999). [1] Hideg É, Vass I, Kálai T and Hideg K (2000) Meth. Ezymol., 319, 77-85. [2] Hideg É, Kálai T, Hideg K and Vass I (1998) Biochemistry 37, 11405-11411.