Singlet Oxygen Is the Major Reactive Oxygen Species Involved in Photooxidative Damage to Plants

Abstract

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 ( 1 O 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, 1 O 2 was responsible for more than 80% of the nonenzymatic LPO. In Arabidopsis mutants favoring 1 O 2 production, photooxidative stress led to a dramatic increase of 1 O 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 1 O 2 -dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent mutant, 1 O 2 plays a major destructive role during the execution of reactive oxygen species-induced cell death in leaf tissues.Plant leaves capture sun-derived light energy to drive CO 2 fixation during photosynthesis. During this process, leaves need to cope with photooxidative stress when the balance between energy absorption and consumption is disturbed. Excess excitation energy in the photosystems (PSI and PSII) leads to the inhibition of photosynthesis via the production of various reactive oxygen species (ROS) at different spatial levels of the cell (Apel and Hirt, 2004;Asada, 2006;Van Breusegem and Dat, 2006). Both exposure to high light intensities and decreased CO 2 availability direct linear electron transfer toward the reduction of molecular oxygen, generating superoxide radicals (O 2 2. ) at PSI (the Mehler reaction). Superoxide dismutation generates hydrogen peroxide (H 2 O 2 ), which is detoxified in the chloroplast by ascorbate peroxidases. As such, this so-called water-water cycle participates in the dissipation of excess energy (Asada, 2006). Decreased CO 2 availability affects the first step in CO 2 fixation by shifting the carboxylation of Rubisco by the Rubisco carboxylase-oxygenase enzyme toward oxygenation, a process called photorespiration. This leads, through the action of glycolate oxidase, to peroxisomal H 2 O 2 production that is counteracted by catalases. Finally, when the intersystem electron carriers are overreduced, triplet excited P680 in the PSII reaction center as well as triplet chlorophylls in the light-harvesting antennae are produced, with the production of singlet oxygen ( 1 O 2 ) as a consequence (Krieger-Liszkay, 2005). In photosynthetic membranes, 1 O 2 ...

Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses

“…Singlet oxygen has previously been implicated as the major toxicity factor in chloroplast-driven photooxidative cell death. It is also a causal agent in hypersensitive immune responses that occur in the light (Triantaphylidès et al, 2008;Triantaphylidès and Havaux, 2009;Kim et al, 2012;Zoeller et al, 2012). In addition, singlet oxygen is the major ROS agent produced in the flu mutant during dark-to-light transition (op den Camp et al, 2003).…”

“…The chlorophyll triplet state can react with ground state oxygen to make the highly chemically reactive singlet state ( 3 Chl* + 3 O 2 → Chl + 1 O 2 ). Activated chlorophyll can give rise to the triplet state when light energy is inefficiently used, although even normal light conditions may result in detectable formation of singlet oxygen (Triantaphylidès et al, 2008;Ramel et al, 2012a). Singlet oxygen can participate in lipid peroxidation resulting in diagnostic chemical signatures.…”

“…Peroxidation might result from direct assault of membrane lipids by ROS or it can be the outcome of enzymatic activity. Type I lipid peroxidation consists of free radical peroxidation, whereas type II is the outcome of peroxidation by singlet oxygen (Triantaphylidès et al, 2008). Type II reactions are considered to have a major role in the execution of ROS-induced photooxidative cell death of photosynthetic tissue (Triantaphylidès et al, 2008).…”

“…Type I lipid peroxidation consists of free radical peroxidation, whereas type II is the outcome of peroxidation by singlet oxygen (Triantaphylidès et al, 2008). Type II reactions are considered to have a major role in the execution of ROS-induced photooxidative cell death of photosynthetic tissue (Triantaphylidès et al, 2008). In addition to photoactive stress, the Arabidopsis (Arabidopsis thaliana)-Pseudomonas syringae hypersensitive response results in lipid peroxidation signatures corresponding to singlet oxygen and the enzymatic activity of lipoxygenase2.…”

Reactive oxygen species and plant resistance to fungal pathogens

“…In plants, PCD takes place during leaf senescence, photosynthesis and as part of the innate immune response triggered by plant-pathogen interactions, called hypersensitive response (HR). ROS-induced PCD has been described as a mechanism of photooxidative damage during photosynthesis, since plants under severe light stress show PCD induced by an increase in the level and toxicity of singlet oxygen 1 O 2 (Triantaphylides et al, 2008). Interestingly, a recent analysis indicates that under non-stress light conditions 1 O 2 can also play a role as signalling molecule, regulating the PCD pathway and generating microscopic lesions in the leaf (Kim et al, 2012).…”