When light energy absorbed by plants becomes excessive relative to the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly deepoxidized to zeaxanthin (violaxanthin cycle). The protective function of this phenomenon was investigated in a mutant of Arabidopsis thaliana, npq1, that has no functional violaxanthin deepoxidase. Two major consequences of the npq1 mutation are the absence of zeaxanthin formation in strong light and the partial inhibition of the quenching of singlet excited chlorophylls in the photosystem II light-harvesting complexes. Prolonged exposure of whole plants to bright light resulted in a limited photoinhibition of photosystem II in both npq1 and wild-type leaves, although CO 2 fixation and the linear electron transport in npq1 plants were reduced substantially. Lipid peroxidation was more pronounced in npq1 compared with the wild type, as measured by chlorophyll thermoluminescence, ethane production, and the total hydroperoxy fatty acids content. Lipid peroxidation was amplified markedly under chilling stress, and photooxidative damage ultimately resulted in leaf bleaching and tissue necrosis in npq1. The npq4 mutant, which possesses a normal violaxanthin cycle but has a limited capacity of quenching singlet excited chlorophylls, was rather tolerant to lipid peroxidation. The double mutant, npq4 npq1, which differs from npq4 only by the absence of the violaxanthin cycle, exhibited an increased susceptibility to photooxidative damage, similar to that of npq1. Our results demonstrate that the violaxanthin cycle specifically protects thylakoid membrane lipids against photooxidation. Part of this protection involves a mechanism other than quenching of singlet excited chlorophylls.

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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 ...

The ch1 mutant of Arabidopsis (Arabidopsis thaliana) lacks chlorophyll (Chl) b. Leaves of this mutant are devoid of photosystem II (PSII) Chl-protein antenna complexes and have a very low capacity of nonphotochemical quenching (NPQ) of Chl fluorescence. Lhcb5 was the only PSII antenna protein that accumulated to a significant level in ch1 mutant leaves, but the apoprotein did not assemble in vivo with Chls to form a functional antenna. The abundance of Lhca proteins was also reduced to approximately 20% of the wild-type level. ch1 was crossed with various xanthophyll mutants to analyze the antioxidant activity of carotenoids unbound to PSII antenna. Suppression of zeaxanthin by crossing ch1 with npq1 resulted in oxidative stress in high light, while removing other xanthophylls or the PSII protein PsbS had no such effect. The tocopherol-deficient ch1 vte1 double mutant was as sensitive to high light as ch1 npq1, and the triple mutant ch1 npq1 vte1 exhibited an extreme sensitivity to photooxidative stress, indicating that zeaxanthin and tocopherols have cumulative effects. Conversely, constitutive accumulation of zeaxanthin in the ch1 npq2 double mutant led to an increased phototolerance relative to ch1. Comparison of ch1 npq2 with another zeaxanthinaccumulating mutant (ch1 lut2) that lacks lutein suggests that protection of polyunsaturated lipids by zeaxanthin is enhanced when lutein is also present. During photooxidative stress, a-tocopherol noticeably decreased in ch1 npq1 and increased in ch1 npq2 relative to ch1, suggesting protection of vitamin E by high zeaxanthin levels. Our results indicate that the antioxidant activity of zeaxanthin, distinct from NPQ, can occur in the absence of PSII light-harvesting complexes. The capacity of zeaxanthin to protect thylakoid membrane lipids is comparable to that of vitamin E but noticeably higher than that of all other xanthophylls of Arabidopsis leaves.

Carotenoids are considered to be the first line of defense of plants against singlet oxygen ( 1 O 2 ) toxicity because of their capacity to quench 1 O 2 as well as triplet chlorophylls through a physical mechanism involving transfer of excitation energy followed by thermal deactivation. Here, we show that leaf carotenoids are also able to quench 1 O 2 by a chemical mechanism involving their oxidation. In vitro oxidation of b-carotene, lutein, and zeaxanthin by 1 O 2 generated various aldehydes and endoperoxides. A search for those molecules in Arabidopsis (Arabidopsis thaliana) leaves revealed the presence of 1 O 2 -specific endoperoxides in low-light-grown plants, indicating chronic oxidation of carotenoids by 1 O 2 . b-Carotene endoperoxide, but not xanthophyll endoperoxide, rapidly accumulated during high-light stress, and this accumulation was correlated with the extent of photosystem (PS) II photoinhibition and the expression of various 1 O 2 marker genes. The selective accumulation of b-carotene endoperoxide points at the PSII reaction centers, rather than the PSII chlorophyll antennae, as a major site of 1 O 2 accumulation in plants under high-light stress. b-Carotene endoperoxide was found to have a relatively fast turnover, decaying in the dark with a half time of about 6 h. This carotenoid metabolite provides an early index of 1 O 2 production in leaves, the occurrence of which precedes the accumulation of fatty acid oxidation products.

SUMMARYCarotenoids are known to play important roles in plants as antioxidants, accessory light-harvesting pigments, and attractants for pollinators and seed dispersers. A new function for carotenoids has recently emerged, which relates to the response of plants to environmental stresses. Reactive oxygen species, especially singlet oxygen, produced in the chloroplasts under stress conditions, can oxidize carotenoids leading to a variety of oxidized products, including aldehydes, ketones, endoperoxides and lactones. Some of those carotenoid derivatives, such as volatile b-cyclocitral, derived from the oxidation of b-carotene, are reactive electrophile species that are bioactive and can induce changes in gene expression leading to acclimation to stress conditions. This review summarizes the current knowledge on the non-enzymatic oxidation of carotenoids, the bioactivity of the resulting cleavage compounds and their functions as stress signals in plants.

Singlet oxygen ( 1 O 2 ) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, 1 O 2 -induced transcriptomic changes result in acclimation to 1 O 2 . Here, using a chlorophyll b-less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of 1 O 2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to 1 O 2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing 1 O 2 formation, indicating acclimation to 1 O 2 . Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in 1 O 2 -induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonatetreated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2.

The early light-induced proteins (ELIPs) belong to the multigenic family of light-harvesting complexes, which bind chlorophyll and absorb solar energy in green plants. ELIPs accumulate transiently in plants exposed to high light intensities. By using an Arabidopsis thaliana mutant (chaos) affected in the posttranslational targeting of light-harvesting complex-type proteins to the thylakoids, we succeeded in suppressing the rapid accumulation of ELIPs during high-light stress, resulting in leaf bleaching and extensive photooxidative damage. Constitutive expression of ELIP genes in chaos before light stress resulted in ELIP accumulation and restored the phototolerance of the plants to the wild-type level. Free chlorophyll, a generator of singlet oxygen in the light, was detected by chlorophyll fluorescence lifetime measurements in chaos leaves before the symptoms of oxidative stress appeared. Our findings indicate that ELIPs fulfill a photoprotective function that could involve either the binding of chlorophylls released during turnover of pigment-binding proteins or the stabilization of the proper assembly of those proteins during high-light stress.L ight is essential for plants through photosynthetic carbon assimilation. However, when absorbed light exceeds the photosynthetic capacities, reactive O 2 species are generated in the chloroplasts, causing oxidative damage to proteins, lipids, and photosynthetic pigments (1, 2). This effect is amplified by environmental stresses such as low temperature or drought, for example, that inhibit the photosynthetic activity, leading to strong yield reduction in crops. In green plants, solar energy is collected by chlorophyll-and carotenoid-binding lightharvesting complexes (LHCs), which are encoded by a multigene family of LHC genes. The expression of these genes is tightly regulated by light (2-4). High light intensities inhibit transcription of LHC genes and activate synthesis of the early lightinduced proteins (ELIPs), a class of proteins structurally related to the LHCs (5). The ELIPs are predicted to have three transmembrane helices, and they have sequence similarity to the LHCs in the central pair of helices (6, 7). The similarity is not only at the sequence level, however, because both LHCs and ELIPs bind chlorophyll and carotenoids (8). The ELIPs differ from the LHCs by their transient expression under high-light stress (5). Recently, a number of ELIP-type polypeptides, containing LHC motifs and inducible by high light, have been discovered in vascular plants: the one-helix high-light-induced proteins (9) and the two-helix stress-enhanced proteins (10).The physiological role of the ELIPs in vascular plants has not yet been elucidated, although there have been several suggestions (11)(12)(13)(14). The induction of ELIPs by high light intensities suggests a role in the acclimation to light stress rather than a light-harvesting function, but this has not yet been demonstrated. ELIP antisense transgenic tobacco plants did not exhibit any phenotype of sensitivity to high ...