Singlet oxygen is the major reactive oxygen species involved in photooxidative damage to plants

Reactive oxygen species act as signaling molecules but can also directly provoke cellular damage by rapidly oxidizing cellular components, including lipids. We developed a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-based quantitative method that allowed us to discriminate between free radical (type I)- and singlet oxygen (O-1(2); type II)- mediated lipid peroxidation (LPO) signatures by using hydroxy fatty acids as specific reporters. Using this method, we observed that in nonphotosynthesizing Arabidopsis (Arabidopsis thaliana) tissues, nonenzymatic LPO was almost exclusively catalyzed by free radicals both under normal and oxidative stress conditions. However, in leaf tissues under optimal growth conditions, O-1(2) was responsible for more than 80% of the nonenzymatic LPO. In Arabidopsis mutants favoring O-1(2) production, photooxidative stress led to a dramatic increase of O-1(2) (type II) LPO that preceded cell death. Furthermore, under all conditions and in mutants that favor the production of superoxide and hydrogen peroxide (two sources for type I LPO reactions), plant cell death was nevertheless always preceded by an increase in O-1(2)-dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent mutant, O-1(2) plays a major destructive role during the execution of reactive oxygen species-induced cell death in leaf tissues.