Production,Detection,and Signaling of Singlet Oxygen in Photosynthetic Organisms

Abstract

Significance: In photosynthetic organisms, excited chlorophylls (Chl) can stimulate the formation of singlet oxygen (¹O₂), a highly toxic molecule that acts in addition to its damaging nature as an important signaling molecule. Thus, due to this dual role of ¹O₂, its production and detoxification have to be strictly controlled. Recent Advances: Regulation of pigment synthesis is essential to control ¹O₂ production, and several components of the Chl synthesis and pigment insertion machineries to assemble and disassemble protein/pigment complexes have recently been identified. Once produced, ¹O₂ activates a signaling cascade from the chloroplast to the nucleus that can involve multiple mechanisms and stimulate a specific gene expression response. Further, ¹O₂ signaling was shown to interact with signal cascades of other reactive oxygen species, oxidized carotenoids, and lipid hydroperoxide-derived reactive electrophile species. Critical Issues: Despite recent progresses, hardly anything is known about how and where the ¹O₂ signal is sensed and transmitted to the cytoplasm. One reason for that is the limitation of available detection methods challenging the reliable quantification and localization of ¹O₂ in plant cells. In addition, the process of Chl insertion into the reaction centers and antenna complexes is still unclear. Future Directions: Unraveling the mechanisms controlling ¹O₂ production and signaling would help clarifying the specific role of ¹O₂ in cellular stress responses. It would further enable to investigate the interaction and sensitivity to other abiotic and biotic stress signals and thus allow to better understand why some stressors activate an acclimation, while others provoke a programmed cell death response. Antioxid. Redox Signal. 18, 2145–2162.

Singlet Oxygen in Photosynthetic Organisms

M olecular oxygen in its ground state is a triplet (³O₂) with two unpaired electrons, each located in a different π*-antibonding orbital (52). These two electrons have the same spin quantum number (parallel spins) that kinetically limits the reactivity of ³O₂. By contrast, the singlet form of oxygen (¹O₂), the superoxide anion radical (O₂ ^•−), hydrogen peroxide (H₂O₂), and hydroxyl radical (HO^•) are more reactive [for details on the chemistry of the three latter species, see for example (52)]. Singlet oxygen is the lowest electronic excited state of molecular oxygen. In ¹O₂, the spin restriction is removed, and since paired electrons are common in organic molecules, its oxidizing ability is greatly increased compared to ³O₂. In aqueous solutions, most ¹O₂ is quenched by H₂O resulting in a shorter lifetime (3.5 μs) compared to, for example, D₂O (39–42 μs). However, the lifetime of ¹O₂ is even shorter in H₂O-incubated cells (2 μs) (21) due to its high reactivity with many biomolecules such as proteins, nucleic acids, and lipids (especially polyunsaturated fatty acids [PUFA]). These reactions cause damage inside the cells and induce an oxidative stress.

All organisms living in an oxygen-rich atmosphere are frequently confronted with oxidative stress. Plants are especially exposed to oxidative stress evoked by ¹O₂ because of their pigments that act as photosensitizers. Some photosensitizers can transfer the absorbed light energy to ³O₂ and directly produce ¹O₂ (52). In photosynthetic organisms, ¹O₂ is most commonly produced by the photosensitizer chlorophyll (Chl), which acts as the main light-absorbing pigment in organisms able to perform oxygenic photosynthesis. Chl a has a very high extinction coefficient at about 670 nm in vivo, and its excited state is long-lived (up to a few nanoseconds). so that during photosynthesis, efficient conversion of the excitation energy into an electrochemical potential via charge separation in the photosystems (PS) is possible. If no charge separation takes place, the spins of the electrons in the excited state of the Chl a molecule can reorientate and give rise to the lowest energy-excited state, the Chl triplet state (³Chl) (123). This ³Chl can react with ³O₂ to produce ¹O₂ if no efficient quenchers are close by (68) (Fig. 1).

FIG. 1.

Generation of singlet oxygen (¹O₂) by excited chlorophyll (Chl), putative quenching sites and the induction of cellular responses in photosynthetic organisms.

Photosynthetic organisms have evolved a number of molecules that efficiently quench or scavenge ¹O₂ in the photosynthetic membranes and prevent damage under normal conditions. However, under some environmental conditions such as during cold or dry periods or exposure to high light intensities, these protection mechanisms can be overwhelmed, and ¹O₂ levels increase in the chloroplasts. Under these conditions, ¹O₂ was also shown to function as a signaling molecule that can trigger cellular responses such as acclimation, repair mechanisms, and activation of a programmed cell death (PCD) response.

Formation of Singlet Oxygen

Singlet-oxygen generation in the antenna

In plants and algae, Chl is located in the reaction centers of the PS, in the antenna systems, and in the cytochrome (Cyt) b₆f complex. The majority of Chl is found in the antenna, and ³Chl might be produced by an intersystem crossing from excited Chl (¹Chl). In isolated antenna complexes, the rate of intersystem crossing is significant (75), and the formation of ³Chl (131) and ¹O₂ (125) has been shown in vitro. However, in these studies, antenna complexes were isolated using detergents. In such preparations, a higher probability of ¹O₂ generation seems to be more likely than in the native complexes because of detergent-induced instabilities of the protein/pigment complexes. In the native antenna complexes, most of the formed ³Chl is efficiently quenched by nearby carotenoids such as xanthophylls (64). The nature and number of the carotenoids bound in the antenna complexes and in the reaction centers are well known from structural data (4, 26). The PSII complex contains 2 β-carotenes in each reaction center, 1 lutein and 1 violaxanthin in CP24 and 1 lutein, 1 neoxanthin, and 1 violaxanthin in CP29 and CP26, respectively. Further, depending on the light intensity and the length of the photoperiod, the variable number of light-harvesting complex (LHC)II trimers are associated with an individual PSII complex (87), each Lhcb polypeptide containing four carotenoids (1 neoxanthin, 2 luteins, and 1 violaxanthin). At PSI, about 20 carotenoids are located in the reaction center, and two xanthophylls are bound in each of the four Lhca polypeptides of the single LHCI per PSI. In addition, there is a pool of carotenoids that cannot be assigned to individual polypeptides on either the LHCI or PSI core. The physical quenching of ³Chl requires an edge-to-edge distance within the van der Waals distance (3.6 Å) between the Chl and carotenoid, so that the electron orbitals have some overlap. In this spin-exchange reaction, the triplet state of the carotenoid is formed, which can dissipate the excess energy as heat (30, 125).

The capacity of individual xanthophylls in the different locations of the PSII antenna complexes to quench ³Chl has been studied in detail by Mozzo et al. (100). These authors concluded that the xanthophylls bound in LHCII quench 95% of the ³Chl states. When measured, the carotenoid-to-Chl ratio of thylakoids from Arabidopsis thaliana grown in short-day photoperiods is 1.5-fold higher than expected from the above listing, even though this ratio can vary depending on the lipid composition of the membrane and between the A. thaliana ecotypes (163). Still, this indicates the presence of carotenoids also in the lipid matrix. Thus, the remaining 5% of ³Chl from LHCII may generate ¹O₂ that can either be quenched by carotenoids located at the interface between lipids and proteins or escape into the thylakoid membrane where it may be quenched by the free carotenoids in the lipid matrix (66).

Singlet-oxygen generation in the PS II

Most ¹O₂ is, however, produced in the reaction center of PS II (Fig. 2) [for extensive reviews, see (76, 77)]. Excitation of Chl leads to charge separations in the reaction center of PSII, and the electrons are transferred in the so-called linear electron flow through the whole electron transport chain to the final electron acceptors, mainly NADP⁺, at the acceptor side of PSI. The probability of ¹O₂ generation is very low when sufficient electron acceptors are available at a given light intensity and physiological state of a plant. However, when the light absorption exceeds the capacity of photosynthetic electron transport, the probability of ¹O₂ generation increases. Under these conditions, the plastoquinone pool is largely reduced, and charge recombination reactions within PSII take place, leading to the generation of ³P₆₈₀ (P₆₈₀, the Chl being the primary electron donor). The two β-carotene molecules present in the PSII reaction center are placed too far from P₆₈₀ to prevent this process [for location of the β-carotenes and P₆₈₀ in the reaction center, see Fig. 2 and (153)]. ³P₆₈₀ reacts subsequently with ³O₂ to form ¹O₂ and P₆₈₀ (Fig. 3).

FIG. 2.

Structure of the reaction center of photosystem (PS) II showing only the main subunits D1 (dark gray), D2 (light gray), and cytochrome (Cyt) _b559, which carry the redox-active cofactors. The forward electron reactions are indicated by the orange arrows. Chl_Z, peripheral Chls; Car, carotenes; OEC, oxygen-evolving complex (Mn₄Ca complex); P, the Chls that become oxidized; Chl, accessory Chls, Ph, pheophytins; Q_A, primary quinone acceptor; Q_B, secondary quinone acceptor; Tyr_Z, redox-active tyrosine residue. The Chl molecules are colored differently to see more easily the position of the individual molecules. The sites of modification of the midpoint potential of the redox couple (Q_A/Q_A ⁻) and (Ph/Ph⁻) in vivo (different isoforms of D1 in cyanobacteria, absence of the Mn cluster in newly assembled PSII before photoactivation, and removal of the Ca²⁺ ion from the OEC in the presence of a large proton gradient) and in vitro (herbicides bound to the Q_B-binding pocket) and by mutagenesis of the Q_A-binding pocket are indicated. The model is based on the structure by Umena et al. (153).

FIG. 3.

Simplified scheme of the recombination pathway of the charge-separated state P⁺Q_A ⁻ (P stands for P₆₈₀). Dashed lines: different midpoint potentials of Ph/Ph⁻ and Q_A/Q_A ⁻ (Q_A, primary quinone acceptor). Gray pointed arrows: nonradiative charge recombination of P⁺Q_A ⁻ to the ground state or via indirect recombination involving the prepopulation of the primary charge pair P⁺Ph⁻. These pathways are safe and do not generate ³P and ¹O₂. Thick black arrows: charge recombination leading to the formation of ³P and ¹O₂. Thick gray dashed arrow indicates that the repopulation of P⁺Ph⁻ is unlikely when the redox potential of Q_A/Q_A ⁻ is shifted toward a more positive potential [for more details, see (77)].

The pathway of charge recombination depends on the energetics of the electron acceptors of PSII (for details, see Fig. 3). Charge recombination between the primary quinone acceptor, Q_A, and the positive charge at the donor side of PSII (P₆₈₀ ⁺) can proceed either via a repopulation of the primary radical pair (P₆₈₀ ⁺Ph⁻) (indirect pathway, radiative or nonradiative) or directly into the ground state of P₆₈₀ (nonradiative pathway). The indirect pathway can lead to the formation of the dangerous triplet states of the primary charge pair and finally to ³P₆₈₀, while the direct, nonradiative pathway is safe and does not lead to the generation of ¹O₂. It has been shown that the midpoint potential of the redox couple (Q_A/Q_A ⁻) controls the pathway of charge recombination and the yield of ¹O₂ formation (44, 45, 78). The modification of the midpoint potential of Q_A in PSII lacking or containing an inactive water-splitting complex without Ca²⁺ (79) or in the presence of a high proton gradient (81) is an important regulation mechanism to protect PSII from ¹O₂ generation and subsequent photo-oxidative damage. The influence of the midpoint potential of Q_A on the yield of ¹O₂ generation has also been demonstrated by the use of herbicides that bind to the Q_B-binding pocket (Fig. 2). 3-(3,4-Dichlorophenyl)-1,1-dimethylurea shifts the midpoint potential of Q_A to a more positive value and lowers thereby the yield of ¹O₂ generation. Phenolic herbicides lower the redox potential of Q_A and increase the yield of ¹O₂ formation (45, 78). In addition, the midpoint potential of Q_A was changed when the alanine at position 249 of the D2 protein was changed to a serine by site-directed mutagenesis, and this also affected ¹O₂ generation (44). The midpoint potential of the pheophytin-redox couple (Ph/Ph⁻) influences also the probability of the nonradiative pathway of charge recombination between the primary charge pair (126). Interestingly, cyanobacteria have two genes for distinct D1 proteins, a main subunit of the PSII reaction center, with different amino acids at positions D1–130. D1-E130 proteins are expressed only during high light conditions (73) and probably differently affect the redox potential of Ph to enhance the charge recombination via the nonradiative pathways and thereby lowering the yield of ¹O₂ generation (155). Similar to the high-light D1 protein in cyanobacteria, in all higher plants with known sequences, a glutamate occupies the position D1–130.

Photosynthetic organisms have evolved a number of protection mechanisms to prevent the formation of ¹O₂, including the dissipation of absorbed light energy as heat by the process of nonphotochemical quenching [for review, see (64, 102)]. Still, PSII is the main target of light-induced damage, the so-called photoinhibition, caused by unfavorable environmental conditions such as, drought, cold, and high light illumination. During this process the D1 protein is triggered for degradation, most likely by the action of ¹O₂ and/or other reactive oxygen species (ROS), even though direct experimental evidence for the oxidation of specific amino acid residues of D1 is still missing. During the turnover of the D1 protein, the pigments and other cofactors have to be stored in a safe way, so that no photosensitizing reactions take place by the free pigments. Free Chls may be bound by Early Light-Induced Proteins (ELIPs) and related proteins to prevent the formation of ¹O₂ (1). Heddad et al. showed that the transcripts of one isoform of ELIPs increased linearly with light intensity and the extent of PSII photodamage (54). In aging cauliflower leaves, a water-soluble Chl-binding protein (WSCP) is expressed. This protein is a tetramer, binds two Chl molecules, and does not contain any carotenoids. Upon excitation, ³Chl, but no ¹O₂, is produced; thanks to a diffusion barrier that prevents the interaction of ³Chl with ³O₂ (124, 132).

Singlet-oxygen generation by other components of the photosynthetic apparatus

It is generally accepted that PSI does not contribute to ¹O₂ generation in photosynthetic organisms. It has recently been proposed that back-reaction pathways in PSI leading to ³P₇₀₀ formation are specifically minimized by redox tuning to avoid ¹O₂ generation (126). Still, in PSI, illuminated under reducing conditions or when vitamin K1 is removed from the reaction center, charge recombination reactions do occur leading to the formation of ³P₇₀₀ [for a review, see (14)]. However, in PSI, the lifetime of the ³P₇₀₀ state is about 6 μs and is not shortened by the presence of ³O₂, indicating that P₇₀₀ is screened from O₂ (134). On the other hand, it has been shown recently in a mutant with low amounts of carotene that a higher amount of ¹O₂ is formed in PSI-LHCI supercomplexes compared to the wild-type, and that its PSI is more susceptible to photo-oxidative damage than PSII (17). In this mutant, enhanced photodamage of PSI may be either due to reduced quenching by β-carotene and higher ¹O₂ formation in the reaction center, less-stable PSI reaction centers lacking a structural component or due to a more vulnerable carotenoid-deficient LHCI. Further experimental evidence is needed to show whether any significant amount of ¹O₂ is generated via charge recombination reactions and ³P₇₀₀ formation in PSI.

The Cyt b₆f complex is a dimer, and each monomer contains four major and four minor protein subunits (82, 143). Three of the major subunits bind the redox-active cofactors, the Cyt f with a c-type heme, the Cyt b₆ with two b-type hemes, and the so-called heme x, and the Rieske protein with a [2Fe-2S] cluster. In addition to these redox-active cofactors, the Cyt b₆f complex contains a carotene (β-carotene in most species) and a Chl a molecule that is potentially photoactive. However, structural analyses revealed that specific local protein structures involving the aromatic amino acid phenylalanine in subunit IV are responsible for shorting the excited ¹Chl lifetime and thus decreasing the probability of ¹O₂ formation (24, 161). Since this Chl a is not needed for the electron transfer reactions, the authors concluded that it seems to play rather a structural role in the Cyt b₆f complex (161). This is supported by the fact that Cyt b₆f complexes from etioplasts contain, instead of Chl a, a molecule of protochlorophyll a required for correct assembly of the complex (121). On the other hand, it has been demonstrated that the Chl a can generate ¹O₂ when the intact or the Rieske protein-depleted Cyt b₆f complex was exposed to high light (130). In this study, the neighboring carotene seemed unable to detoxify the ³Chl state by physical quenching; however, chemical quenching of ¹O₂ by β-carotene was demonstrated. The same authors showed that the level of ³Chl and ¹O₂ generation increased with an increased structural disturbance of the Cyt b₆f complex, indicating that in intact, enzymatically active complex ¹O₂ formation is rather low (92). Thus, ¹O₂ might not be produced in significant amounts by the Cyt b₆f complexes in vivo.

Singlet-oxygen generation by Chl precursors and catabolites

Assembly and disassembly of the protein/pigment complexes of the photosynthetic electron transport chain are critical processes that have to be strictly regulated to limit the danger of ¹O₂ formation by immature, not fully functional complexes and by free metabolites/catabolites that can act as photosensitizers (Fig. 4). Most of these metabolites are Chl precursors such as protoporphyrin IX and protochlorophyllide, even though it has been suggested recently that also carotenoid intermediates such as tetra-cis-lycopene might act as photosensitizers and generate ¹O₂ (53). In etiolated pea epicotyls, porphyrin-type Chl precursors were shown to be capable of photogenerating ¹O₂ (32). In a functional system, the accumulation of Chl precursors is prevented by metabolic control and by channeling the metabolites from one enzyme to the next (20, 147). Only the amount of pigments is synthesized, which is used in the specific pigment-binding sites of a given protein. In the A. thaliana fluorescent (flu) mutant a protein is mutated that plays a key role in the negative feedback regulation of Chl biosynthesis, and consequently, protochlorophyllide accumulates in the dark and leads to the generation of a high amount of ¹O₂ when the flu plants are transferred to the light (109). This mutant was extensively used to study the ¹O₂ response of A. thaliana (see Cellular Response to Singlet Oxygen). One has to keep in mind that in the flu mutant, protochlorophyllide accumulates in the chloroplast envelope and in the thylakoid membrane (8), and therefore ¹O₂ is partially produced at a site that is different from its production sites in a wild-type plant. Addition of herbicides that block the protoporphyrinogen oxidase can lead to the accumulation of protoporphyrin IX, a photodynamic pigment that also does produce high amounts of ¹O₂ in the light (11).

FIG. 4.

Schematic diagram showing possible pathways of ¹O₂ generation by Chl, its precursors, and catabolites. During light-induced damage of PSII (photoinhibition), nonfunctional PSII is formed. A high yield of ¹O₂ may be generated when the pigments are released during degradation of the D1 protein. Inactive PSII before protein degradation is protected by an upshift of the midpoint potential of the primary quinone acceptor Q_A and produces low amounts of ¹O₂ (see Figs. 2 and 3). Assembly and disassembly of the protein/pigment complexes of the photosynthetic electron transport chain are critical processes that have to be strictly controlled to limit the danger of ¹O₂ by immature complexes and by free metabolites that can act as photosensitizers.

To avoid ¹O₂ production and photo-oxidative damage during Chl synthesis, the precursors are not set free, but are bound to the enzymes involved in synthesis or to specialized protection proteins. Light-harvesting CP-like proteins (LIL) have been identified to participate in this process, and the isoform LIL3 is synthesized during de-etiolation (122). It carries the late metabolites of Chl biosynthesis (122) and interacts physically with geranylgeranyl reductase (CHLP) (146). It has been suggested that LIL3, CHLP, and other enzymes of late Chl biosynthesis form a protein complex close to the site of Chl insertion into its binding sites at the LHC proteins. The same may hold for the insertion of Chl into the reaction centers. Such a complex could allow the efficient channeling of Chl and its photo-reactive precursors into the PSs and thus prevent the generation of ¹O₂ during the greening and assembly process of the photosynthetic electron transport chain.

During leaf senescence, Chl is degraded in a controlled way to avoid the accumulation of phototoxic intermediates of Chl breakdown such as pheophorbide and the red Chl catabolite (RCC) [for a review on Chl degradation, see (63)]. It was recently shown that during senescence, the protein STAY-GREEN and five Chl catabolic enzymes, involved in the conversion of Chl a to a primary fluorescent Chl catabolite, localize to LHCII and form a complex (128). Metabolic control by channeling through this complex occurs in Chl breakdown at the LHCII (106), and it is likely that similar complexes exist at LHCI and other CP, so that the risk of accumulating Chl breakdown intermediates is minimized. Overexpression of STAY-GREEN proteins in A. thaliana and rice also stimulated ¹O₂ production (65, 104). Similarly, mutants deficient in the enzymes Pheide a oxygenase or RCC reductase accumulate phototoxic intermediates and promote light-dependent ¹O₂ production and cell death (114, 115). RCC reductase expression is induced during pathogen infection, and the protein might play a role during a pathogen-induced cell death response (162). Interestingly, the RCC reductase was recently found to be localized in both the chloroplasts and mitochondria, and increased ¹O₂ formation was detected in the mitochondria of RCC reductase-deficient mutants (110). The authors speculated that photoactive RCC might penetrate from the chloroplasts to the mitochondria, where a ¹O₂ burst stimulates pathogen-induced cell death response, and that RCC reductases might control this response. This implements an important signaling role of Chl catabolites and ¹O₂ in the cellular response to pathogen infection.

Detection of Singlet Oxygen

The importance of ¹O₂ in physiological stress response calls for a reliable means of detection. Direct detection and, what is more, quantification are hampered by the fact that ¹O₂ is reactive to a variety of biological molecules and is quenched by water. In this way, efficient ¹O₂ reporter molecules are to compete with target molecules of oxidative damage, ¹O₂-quenching antioxidants, as well as with solvent molecules. Upon oxidation by ¹O₂, chemical traps change their optical properties (absorption, fluorescence, or both) or their electron paramagnetic resonance (EPR) properties (Fig. 5).

FIG. 5.

A summary of ¹O₂-detecting methods reviewed in the present work. Reactions of pathway [1] are detailed in Figure 6. In addition to reactions with natural targets, externally added reporter molecules may also react with ¹O₂. These include non- (or low) fluorescent probes responding to oxidation by fluorescence increase [2]; probes with high initial fluorescence responding by fluorescence quenching [3]; or electron paramagnetic resonance (EPR)-inactive traps forming EPR-active ¹O₂ adducts [4]. An example of a triplet EPR absorption spectrum is shown in the lower left corner. Chemicals capable of acting as ¹O₂ probes are introduced in the text. Detecting ¹O₂ based on its infrared photoemission [5] does not require the addition of chemical probes.

EPR spectroscopy

Since its introduction as a ¹O₂ trap more than three decades ago, 2,2,6,6-tetramethylpiperidine (TEMP) and its derivatives have been used extensively. When reacting with ¹O₂, this sterically hindered amine is converted into a nitroxide radical that (unlike TEMP itself) is paramagnetic and thus detectable by EPR spectroscopy (90). A structurally similar, but more hydrophilic, ¹O₂ probe is TEMPD (2,2,6,6-tetramethyl-4-piperidone) (99). Because the product is more or less a stable free radical, the method is often referred to as ¹O₂ spin trapping, although ¹O₂ is not a radical itself. The first use in the isolated thylakoid membranes (60) showed that the resulting spin-adduct is prone to reduction into an EPR-silent hydroxylamine, and thus the method may underestimate the amount of ¹O₂ produced in the sample (62). It is also important to note that commercially available TEMP needs to be purified by vacuum distillation before use (45, 57), because even small amounts of contaminants inhibit the PSII electron transport (51, 57). TEMPD-HCl is preferred to TEMPD, because the hydrochloride form is more stable in storage and is only converted to the nonchlorinated TEMPD when dissolved in water-based measuring buffers (57). With care and adequate controls, ¹O₂ spin trapping is a sensitive tool that has been applied in a number of studies on isolated functional photosynthetic membranes (37, 44, 45, 60, 61, 80). However, the application of TEMP in vivo in leaves is hampered by the sensitivity of both trap and adduct to redox-active metabolites. Attempts to overcome this problem by replacing the piperidine ring with a theoretically less-sensitive pirrolydine were not successful (62), leaving ¹O₂ trapping by TEMP and its derivatives in the domain of in vitro experiments.

Changes in absorption or fluorescence of specific singlet oxygen probes

1,3-diphenylisobenzofuran (DPBF) reacts with ¹O₂ rapidly and irreversibly in a cycloaddition reaction, yielding an unstable endoperoxide that decomposes to give 1,2-dibenzoylbenzene. Products have lower blue fluorescence (excitation/emission 410/455 nm) than DPBF itself (159), and DPBF has been a popular ¹O₂ probe for decades [for examples, see (113, 164)]. DPBF can be useful in pure ¹O₂-yielding systems such as pure photosensitizers aimed at studying the properties of ¹O₂ (133), but it must be used with caution for the detection of ¹O₂ in complex biological samples, because it also reacts with different radical species, such as peroxyl or alkoxyl radicals (16, 158). The potential use of DPBF in plant ¹O₂ studies is limited by its low solubility in water, and also because its blue fluorescence may be absorbed by Chls.

The 440-nm absorption maximum of a water-soluble compound, p-nitroso-dimethylaniline (RNO), also appears disadvantageous from the perspective of Chl-rich samples. Nevertheless, a photometric technique based on observing RNO bleaching caused by a product of the reaction between ¹O₂ and histidine (74) has been successfully applied for detecting ¹O₂ production by PSII reaction centers isolated from Pisum sativum (148). Further, using an exogenous photosensitizer as an artificial ¹O₂ source, the RNO original method has also been developed into an assay measuring the ¹O₂-quenching capacity of a plant extract (94). The assessment of this parameter is not a direct indicator of actual ¹O₂ production rates, but a high quenching capacity certainly indicates a need for enhanced protection against putative high fluxes of ¹O₂ in the plant itself.

Other probes with more perspectives for in vivo studies include molecules comprised of a trapping moiety and a chromophore. Upon reaction with ¹O₂, the resulting oxygen adduct changes the intramolecular energetics and thus affects light emission of the chromophore. Such fluorescent or luminescent ROS probes have great perspectives in imaging techniques with potentials to identify ¹O₂ in situ. In one approach, developed by Kálai et al., ¹O₂ trapping converts the reporter molecule from a high to a low fluorescent form (67). Fluorescence quenching of 3-(N-diethylaminoethyl)-N-dansyl)aminomethyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole (DanePy) and its derivatives have been successfully applied in a variety of experiments to detect ¹O₂ in A. thaliana (59, 109), Vicia faba (58), spinach (7), or tobacco (55) leaves, and in Chlamydomonas reinhardtii cultures (39). Major disadvantages of this probe are that (i) fluorescence quenching by ¹O₂ is only partial and thus it is useful in detecting high fluxes of ROS only (58), and that (ii) it requires ultraviolet [345 nm, (67)] fluorescence excitation, which may interfere with plant metabolism. For successful delivery into photosynthetic tissues, DanePy needs to be infiltrated directly into the leaf (56); otherwise, it tends to concentrate in vascular tissues (43). Consequently, the use of this probe in hard leaf tissues such as of grapevine or Tilia was not found to be feasible (Hideg É, unpublished). Although europium (III) complexes have not been tested in plants so far, these probes that are converted into compounds emitting red light upon UV (294–335 nm) excitation by ¹O₂ (140) may also suffer from the problem of high-energy excitation.

A reverse chemistry governs the functioning of chemical ¹O₂ traps in which photoemission from the chromophore is quenched by electron transfer from the adjacent trapping moiety before a reaction with ¹O₂; therefore, these molecules have very low (if any) light emission in the absence of ROS. Upon reaction with ¹O₂, the resulting oxygen adduct is no longer an efficient intramolecular electron donor, and light emission occurs (140, 141, 145). The advantage of these chemical traps is a wide range of response, from virtually zero to intense light emission.

Nagano and coworkers developed a series of fluorescent probes in which fluorescein is fused with a 9,10-dimethylanthracene moiety as a chemical trap of ¹O₂. Endoperoxides formed from the probes upon reacting with ¹O₂ emit green fluorescence (with a maximum intensity at 516 nm) upon blue (492 nm) excitation (145, 154). While these probes have been successfully applied for detecting ¹O₂ in a variety of medicinal chemistry experiments (105), to our best knowledge, there is only one reported application in plants, in which ¹O₂ was found in isolated intact chloroplasts under photo-oxidative stress (160).

Singlet Oxygen Sensor Green^® [SOSG (141)] features fluorescence excitation and emission in the visible and has been applied to detect ¹O₂ in photoinhibited Arabidopsis leaves (42). However, the probe has shown sensitivity to both visible (55) and UV light (117), which is undesirable in most plant experiments. In addition, recent studies showed that both SOSG and the immediate product of the reaction between SOSG and ¹O₂ (the SOSG endoperoxide) are capable of sensitizing the production of ¹O₂ (47, 117). Although SOSG is marketed as a cell-impermeable compound (141), Gollmer et al. recently reported that it penetrated the living mammalian cells (47). In tobacco leaves infiltrated with SOSG, the probe was mainly found in the epidermal cells, but was also preferentially associated with the nucleus (55). Nevertheless, SOSG may be used for detection of ¹O₂ in in-vitro assays. SOSG has been applied successfully in isolated antenna (LHCII) and in PSII core complexes and PSII-LHCII, PSI-LHCI supercomplexes (17, 22). Differences in ¹O₂ generation depending on the pigment composition of these complexes were convincingly detected.

In addition to being prone to artifacts, another limitation of chemical probes is that they may also respond to ROS other than ¹O₂. Although excluding such cross-reactivity is a key aspect of all reports on new chemical ¹O₂ probes, testing for each and every reactive agent occurring in biological systems under oxidative stress (including radicals other than oxygen-centered ones) is certainly a formidable task. Another disadvantage of chemical ¹O₂ probes is that their versatility depends on the successful delivery into the very site of ¹O₂ production.

Near-infrared luminescence emission of singlet oxygen

The miniature fraction of ¹O₂ that undergoes radiative deactivation is a unique in situ signature of this molecule. In this way, this low-intensity near-infrared luminescence around 1270 nm provides the only undoubtedly direct method to detect ¹O₂. However, there are no gains without pains: detecting time-resolved ¹O₂ luminescence requires sensitive optical detectors with a low signal-to-noise ratio as well as careful signal analysis to separate the true ¹O₂ signal from scattered light and from phosphorescence and delayed fluorescence emissions from other molecules. In plant samples, where Chl is a major source of ¹O₂, the excited pigment may also relax through long-wavelength phosphorescence. Separation of infrared Chl phosphorescence (between 900 and 1000 nm) and ¹O₂ phosphorescence (at 1270 nm) is possible by measuring the emission spectra and also by fitting the time-resolved luminescence signal into components. In addition to its unambiguity, the advantage of detecting ¹O₂ using luminescence over chemical trapping is that with careful analysis, the luminescence method also reveals the lifetime of ¹O₂ in the studied system. This parameter is fundamental for studies on both the physiology of oxidative damage and on signaling events, since it has the information on putative distances to which ¹O₂ may reach from its production site. There are numerous studies on analyzing ¹O₂ luminescence decay in medical samples or model systems undisturbed by the presence of Chl, but these applications are not reviewed here. Interested readers are referred to a recent comprehensive study and references therein (21).

The first utilization of ¹O₂ infrared luminescence was the demonstration of ¹O₂ in isolated PSII reaction center under high-light stress (93). This was followed by a number of reports on ¹O₂ luminescence decay analyses in PSII reaction center preparations from leaves (88, 149), or PSII particles from Synechococcus (27) or Synechocystis (150), and one report identified the spectrum of ¹O₂ photoemission in the Cyt b₆f complex (92). Analyzing the time-resolved infrared emission spectra of PSII particles, Dědic et al. determined both the submicrosecond lifetime of Chl triplet states and the microsecond lifetime of ¹O₂ [18 μs and 3–4 μs in D₂O and in H₂O buffers, respectively (27)]. These authors also showed that the quantum efficiency of ¹O₂ generation in PSII particles substantially increased by addition of sodium dodecyl sulfate, which inactivated the complex and released Chl molecules from their specific binding sites on PSII proteins and thus abolished the effective interaction with quenchers. Although there are no experimental data available, the presence of a variety of additional quenchers in chloroplasts suggests a much shorter lifetime of ¹O₂ and thus a more difficult task of detection by infrared emission. Based simply on the concentration of ascorbate in the stroma of chloroplasts [20–300 mM, (139)] and pH-dependent rate constants for quenching of ¹O₂ by ascorbic acid/ascorbate (12), Li et al. estimate a short, ∼200-ns ¹O₂ lifetime and thus predict serious technical obstacles in detecting the luminescence of endogenously produced ¹O₂ in chloroplasts (88).

Reaction of Singlet Oxygen with Cellular Components

Main targets of singlet oxygen damage

The main targets of damage by ¹O₂ include lipids, proteins (25), and nucleic acids (137). Oxidized products are not only of impaired biological function, but may also initiate further biological damage, either as oxidizing (such as lipid peroxidation products or oxidized amino acid intermediates) or as genotoxic (oxidized DNA) agents. For example, ¹O₂ reacts with the guanine moiety in DNA to form 8-oxo-7-hydrodeoxyguanosine (8-oxodG; also called 8-hydroxydeoxyguanosine) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine, which stimulate the accumulation of point mutations (137). Further, ¹O₂ also causes alkali-labile sites and single-strand breaks in DNA (13).

Most reactions of ¹O₂ with amino acids occur via chemical quenching, although tryptophan has been reported to be capable of physical quenching too (95). During chemical quenching, primary oxidation mainly occurs on the side chains of tryptophan, tyrosine, histidine, methionine, and cysteine. The damage of a protein does depend not only on the number of sensitive side chains but also on the structure of the protein. A conformational change in a protein can either expose or hinder the access of ¹O₂ to specific amino acid residues. The major consequence of ¹O₂-mediated protein oxidation is cross-linking and aggregation (25). Oxidation of tryptophan, tyrosine, or histidine includes peroxide intermediates, and the relatively long lifetimes of these hydroperoxides may allow these species to diffuse considerable distances and thereby initiate damage at sites remote from their initial site of generation (49). In the photosynthetic apparatus, the main protein target of ¹O₂-mediated damage is the D1 protein. Attempts have been made to identify the amino acid residues that are oxidized by ¹O₂ (136). However, a study with the modern mass spectrometry technology is needed to identify the main targets.

Although oxidative damage of lipids is generally attributed to radical-mediated chain reactions initialized by hydroxyl radicals (type I reaction), studies with phospholipid model membranes showed that ¹O₂ is also capable of oxidizing PUFA (type II reaction) (Fig. 6). Linoleic acid is a major PUFA in photosynthetic membranes and also the main target of ¹O₂-induced lipid peroxidation, leading to the formation of different lipid hydroperoxides (LOOH). It was shown that 10′- and 12′-hydroperoxides of linoleic acids (hydroperoxy-octadecatrienoic acid [HPOTE]) are specifically generated by ¹O₂-stimulated peroxidation, whereas 9′- and 13′-hydroperoxides are formed by both type I and II reactions (142). On the other hand, the accumulation of LOOH formed by type II reactions was shown to stimulate radical-mediated lipid peroxidation in the presence of exited photosensitizers, leading to faster generation of type I LOOHs after a prolonged exposure to ¹O₂ (142). Moreover, LOOH produced during the oxidation by hydroxyl radicals can generate ¹O₂ by the Russell mechanism in vitro (97), and similar reactions have been suggested for DNA or protein hydroperoxides (98). Thus, it is important to efficiently detoxify LOOHs by either peroxidases or reductases, leading to the formation of hydroxy fatty acid isomers such as 13-hydroxy octadecatrienoic acid (13-HOTE) or 13-hydroxy octadecadienoic acid (13-HODE). Further, LOOHs can be converted to keto-forms such as 13-keto-octadecatrienoic acid (13-KOTE) and 13-keto-octadecadienoic acid or to other reactive electrophile species (RES), which were shown to play an important role in cellular signaling during ¹O₂ response (see “Reactive electrophiles as transmitters of singlet-oxygen signals”) (101).

FIG. 6.

Schematic diagram showing possible ¹O₂ reactions, including physical or chemical quenching and damage to biomolecules. 8oxodG, 8-oxo-7-hydro-deoxyguanosine; AA, amino acid; Asc, ascorbate; Car, carotenoids; DHAR, dehydroascorbate reductase; FapyG, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; GSH, glutathione; HODE, hydroxy octadecadienoic acid; HOTE, hydroxy octadecatrienoic acid; KODE, keto-octadecadienoic acid; KOTE, keto-octadecatrienoic acid; LOO•, lipid peroxyl radical; LOOH, lipid hydroperoxide (from type-I or type-II reactions, see text for details); •OH, hydroxyl radical; -OOH, hydroperoxide; -ox, oxidized; P, photosensitizer (*, excited); PUFA, polyunsaturated fatty acid; RES, reactive electrophile species. Letters in gray circles show antioxidants directly reacting with ¹O₂, and letters in squares indicate products considered as signaling molecules.

Singlet-oxygen quenchers

The high reactivity of ¹O₂ with essential biomolecules and the subsequent oxidative stress increase the need for efficient protection mechanisms. Protection of cells from oxidative stress generally includes some ROS-specific enzymes; however, there is no detoxification enzyme targeted to ¹O₂ only. Still, there are many antioxidants involved in ¹O₂ detoxification, and several excellent reviews summarize the ¹O₂-neutralizing pathways (5, 31, 72, 108, 151) of which a detail discussion would go beyond the scope of the present work. The aspect to be emphasized here is the variety of products yielded from reactions of ¹O₂ with target molecules, including antioxidants.

As illustrated in Figure 6, ¹O₂ that is produced by triplet-singlet energy-transfer reactions can undergo both physical and chemical quenching (scavenging) (30). The ability of carotenoids to quench ¹O₂ increases with the number of conjugated double bonds (27). In physical quenching, a singlet-triplet energy transfer dissipates the energy as heat and does not modify the reaction partner (e.g., β-carotene) chemically, while chemical quenching results in oxidized products. Depending on whether the molecule oxidized by ¹O₂ is regenerated (rereduced) or remains nonfunctional, chemical quenching can be beneficial or damaging. Although theoretically any reaction lessening the amount of ¹O₂ in the cell is antioxidative, in practice, only pathways including rapid replacement of the oxidized target molecules, are regarded as antioxidants. In plant cells, tocopherol (106), carotenoids (28), and ascorbate (12) are the most abundant ¹O₂ antioxidants. Tocopherol is oxidized to a free radical product and is regenerated by ascorbate, whereas oxidized ascorbate is reduced in the reactions involving specialized enzymes, glutathione, and NADPH. These regenerative pathways are also included in defense against ROS other than ¹O₂ (52).

In the reaction center of PSII, β-carotene does not quench the P₆₈₀ triplet physically (29), but it was shown to be a chemical quencher (148). Recently, specific endoperoxide molecules that resulted from ¹O₂ oxidation of β-carotene, lutein, and zeaxanthin in Arabidopsis leaves were characterized (118). This carotenoid oxidation results in products such as β-cyclocitral and β-ionone (118, 119), and oxidized carotenoids have to be replaced by de novo synthesis. When plants are exposed to high-light stress, the level of β-carotene endoperoxides increases rapidly and much faster than the level of lipid peroxidation products, indicating that these carotenoid oxidation products are an early index for ¹O₂ production (152). Plastoquinol has been shown recently to be a more-efficient ¹O₂ quencher than tocopherol during exposure of plants to high light. Plastoquinone-9 reacts with ¹O₂ to form plastoquinone-C (107). However, the concentration of plastoquinol is much lower than that of tocopherol, indicating that overall tocopherol may be the more important quencher.

Cellular Response to Singlet Oxygen

The high reactivity of ¹O₂ with biomolecules such as proteins and lipids is one reason for its cytotoxicity during harsh environmental conditions. Further, analyses of the specific ¹O₂ reaction products 10-HOTE and 15-HOTE revealed that it is also the major ROS responsible for lipid peroxidation in plants exposed to simultaneous high-light and cold conditions (152). Thus, large amounts of ¹O₂ produced during strong photo-oxidative stress conditions can cause an irreversible damage to cellular components and cause cytotoxic effects (116).

Induction of PCD responses

Singlet oxygen can also induce cell death in plants at lower, noncytotoxic levels by inducing a genetically PCD response. This cellular response has been extensively studied by Apel and coworkers taking advantage of the Arabidopsis flu mutant [for recent reviews see (70, 103, 151)]. Strong generation of ¹O₂ in flu after a dark-to-light shift causes growth arrest of mature plants, bleaching and cell death in seedlings accompanied by the accumulation of enzymatically formed lipid peroxidation products, and the induction of ¹O₂-specific changes in gene expression (109). A similar cell death phenotype for seedlings has also been observed for a mutant in the barley flu-orthologous called tigrina-d.12 and for another Arabidopsis mutant called oep16. This oep16 mutant is deficient in the import of the NADPH:protochlorophyllide oxidoreductase A involved in the protochlorophyllide conversion during Chl synthesis what leads to the accumulation of this photoactive Chl precursor (112, 156). In the flu system, the isolation of two suppressor mutants of the cell death and growth arrest phenotype, executer 1 (ex1) and executer 2 (ex2), confirmed a signaling role of ¹O₂ in this genetically controlled cellular response (85, 116, 157). Detailed analyses revealed that the transcriptional response of A. thaliana to increased ¹O₂ formation is distinct from the response to other ROS such as O₂ ^•− and H₂O₂, and that H₂O₂ even antagonizes the ¹O₂ response and induction of PCD (83, 109). On the other hand, several genes associated with the biosynthesis of or signal transduction through the phytohormones ethylene (ET), salicylic acid (SA), 12-oxo phytodienoic acid (OPDA), and jasmonic acid (JA) were upregulated by ¹O₂ produced in the flu system or by exogenous photosensitizers such as rose bengal (Fig. 7) (23, 91). This includes the genes of the 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and of several ET-responsive element-binding factors (ERFs) involved in ET signaling, the enhanced disease susceptibility gene EDS1 required for the accumulation of SA, and the genes for lipoxigenase (LOX3), allene oxide synthase (AOS) or cyclase (AOC3), and OPDA reductase (OPR3) involved in OPDA and/or JA synthesis, respectively. Thus, increased synthesis of ET, SA, and JA might be involved in the PCD response after increased ¹O₂ formation.

FIG. 7.

Model for the role of various phytohormones in the different cellular responses of plants to increased ¹O₂ formation. (A) The level of ¹O₂ production might determine whether plants induce an acclimation, programmed cell death (PCD), or cytotoxic response by causing a direct damage or controlling the production of the phytohormones ethylene (ET), 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), or salicylic acid (SA). Direct measurements or indirect lines of evidence by gene expression profiles (see B) indicated that the levels of these hormones either do (arrows) or do not increase (block arrows) by specific ¹O₂ levels. Individual hormone might then either stimulate (arrow) or even inhibit (block arrows) a cellular response. (B) Induction of genes involved in ET, OPDA, JA, or SA synthesis under different ¹O₂-generating conditions is presented in a gray scale-based induction profile. Low ¹O₂ levels are considered to be produced in wild-type plants (71), and Arabidopsis cell suspension cultures (ACSC) (48) exposed to high light conditions (HL) at moderate temperature, low-to-medium levels are produced in wild-type plants, and the npq1lut2 mutants exposed to HL under cold conditions (2) and medium levels are produced in wild-type plants exposed to rose bengal (91) and the flu mutant after a dark-to-light shift (85).

Further analyses confirmed a link between these phytohormones and ¹O₂-induced PCD response in A. thaliana (Fig. 7). Cell death in the flu mutant after the dark-to-light shift depended on a functional synthesis of ET and SA, both shown to be involved in various physiological responses, including pathogen response (23). SA plays a role in defense against biotrophic pathogens. ET is, together with JA, usually associated with defense against necrotrophic pathogens and herbivorous insects and is mutually antagonistic with the SA response (6). Interestingly, JA was also found to promote ¹O₂-mediated cell death in flu. Further, the levels of JA as well as of its precursor OPDA and related oxylipins such as 13-HOTE and 13-HODE strongly increase after increased ¹O₂ formation (23, 116). On the other hand, mutants deficient in JA and OPDA synthesis revealed that these hormones are not essential for the PCD response, showing that they do not function directly as second messengers for ¹O₂ signaling. An opr3 mutant, disable to convert OPDA to JA, showed a much lower ¹O₂ response in the flu background compared to the wild-type allele, which could be partially complemented by the addition of exogenous JA (23). This indicates that not JA itself but rather the ratio of JA and OPDA influences the PCD response. Thus, increased OPDA levels might exhibit an antagonistic effect on the ¹O₂-induced PCD response, which seems to be controlled by the level of JA (Fig. 7). This idea is supported by the abolished induction of OPR3 under conditions producing lower levels of ¹O₂ compared to flu and rose bengal such as the exposure of wild-type plants or cell cultures to high-light conditions, whereas the genes for OPDA synthesis (LOX3 and AOC3) are still upregulated.

Beside the induction of a PCD response, ¹O₂ was also shown to function as a signal for other cellular responses. An ¹O₂-induced transcriptional response in immature Arabidopsis seeds before the onset of dormancy was shown to affect plastid differentiation later during seed germination (69). This long-term effect of ¹O₂ signaling depended on the recruitment of another plant hormone, abscisic acid. The role of this pregermination ¹O₂ response in plastid development is unknown. However, mutants disturbing plastid homeostasis were found to have constitutive high expression of ¹O₂-specific stress-response genes, suggesting a linkage between the chloroplast function and ¹O₂ response (138). It was argued that the disturbance of the chloroplast homeostasis induces a molecular stress response that stimulates acclimation to the environmental stressors. This acclimation response suppressed the normal ¹O₂-induced PCD response in the flu background and enhanced the pathogen resistance (138). Similar phenotypes were found for a second class of suppressor mutants of the ¹O₂-induced bleaching in flu seedlings, called singlet-oxygen-linked death activator (soldat), and for happy-on-norflurazon (hon) mutants. The hon mutants are resistant to the bleaching herbicide norflurazon, which blocks carotenoid synthesis. In hon, norflurazon treatment still increased ¹O₂ accumulation in the light, but the expression of the nuclear Lhcb gene, normally downregulated by the herbicide, was derepressed (127). It was shown that resistance to norflurazon was due to the induction of an acclimation response caused by a disturbed plastid protein homeostasis in the hon mutants. Similarly, deletion of the SIGMA6 factor of plastid RNA polymerase in the soldat8 mutant and of a gene coding for a putative transcription termination factor in the soldat10 mutant disturbed plastid protein synthesis and stimulated acclimation at the beginning of the seedling development. This acclimation process suppressed the ¹O₂-induced PCD response in the flu mutant (19, 96). All these examples show that acclimation to a disturbed plastid homeostasis can suppress the normal cellular response to increased ¹O₂ levels.

Induction of acclimation responses

Acclimation to stressful environmental conditions usually is stimulated by pre-exposure to low levels of the stressor that changes the expression levels of responsive genes helping to protect the organism from the harmful effects of high stress levels. Such an acclimation response to ¹O₂ has been found in the green alga C. reinhardtii. In this alga pre-exposure to low concentrations of exogenous photosensitizers such as rose bengal or neutral red or to high-light illumination can increase the tolerance to normally toxic levels of the ¹O₂-producing chemicals (35, 84). This increased tolerance could be linked to the stimulated expression of nuclear defense genes, including the glutathione peroxidase homologous gene GPXH/GPX5 and the glutathione S-transferase gene GSTS1. Overexpression of either of these genes was sufficient to increase the tolerance to ¹O₂-producing chemicals (84). Both genes were also induced in C. reinhardtii exposed to high-light illumination (41). This indicates that the same response as induced by the exogenous photosensitizers might stimulate acclimation to ¹O₂ under natural photo-oxidative stress conditions.

Even though no increased tolerance to photo-oxidative stress was shown yet for plants after acclimation to low ¹O₂ levels, changes in the gene expression profiles associated with an acclimation process have also been detected in A. thaliana. The double-mutant npq1lut2 is deficient in the two ¹O₂-scavenging xanthophylls lutein and zeaxanthin. This enables the plant to induce an ¹O₂-specific signal upon a high light shift in the cold, which is weaker than in the flu system (2). As a result, many genes encoding for chloroplast proteins were differently expressed in the mutant compared to wild-type plants. It also increased the expression and synthesis of carotenoids, tocopherol, and other antioxidants and caused a rearrangement of the photosynthetic apparatus (2). Thus, it was claimed that in this mutant ¹O₂ induced an acclimation response. However, in another study, similar stress treatments of wild-type A. thaliana were able to induce a PCD response after prolonged exposure (96). Thus, in agreement with a rather strong ¹O₂ response, a high induction of EDS1, AOC3, and OPR3 was detected in npq1lut2 exposed to photo-oxidative stress conditions (Fig. 7B). On the other hand, genes involved in ET response (ERFs and ACS) were not upregulated under these conditions what might explain the missing PCD response. Alternatively, ET response and stimulation of PCD might be delayed under the slightly weaker ¹O₂ stress conditions than in the flu system.

In Arabidopsis cell suspension cultures (ACSC) exposed to high-light conditions, the ¹O₂-specific transcriptional response was also similar to the one in the flu system, but no PCD response was detected (48). Thus, under these conditions, lower ¹O₂ levels might similar to npq1lut2 induce an acclimation response. However, different to npq1lut2, in ACSC exposed to high-light conditions, genes involved in ET response were strongly induced, as were genes of OPDA and/or JA synthesis (LOX3 and AOC3) (Fig. 7B). On the other hand, no evidence for increased SA synthesis such as the induction of EDS1 could be detected in ACSC, supporting an essential role of SA synthesis in PCD response (48). Further, transcripts encoding peroxisome-located transporters for OPDA or enzymes for the synthesis of JA from OPDA such as OPR3 were also not upregulated. This suggests that OPDA, but not JA, synthesis was induced. In agreement with that, a much higher correlation of the high-light response in ACSC with the response of A. thaliana to OPDA than to JA was found (50). OPDA was shown to trigger a transcriptional response different from JA, but more similar to wounding, including the specific induction of many stress response and detoxification genes (144). This indicates that OPDA rather than JA might trigger the acclimation response to low ¹O₂ levels. Together with the fact that the ratio of JA and OPDA affects the induction of PCD, these findings suggest that the rates of ¹O₂ production determine the relative levels of OPDA and JA, which might play a key role in the decision between acclimation and PCD responses (50).

Activation of Signal Cascades by Singlet Oxygen

In photosynthetic organisms, ¹O₂ is produced in the plastid as described above, but it activates the expression of many genes in the nucleus. The induction of nuclear gene expression by plastid signals is called retrograde signaling, and several components of the chloroplast-to-nucleus signal transduction pathways have been identified [for reviews, see (18, 33, 46, 89, 111)]. Singlet oxygen is one of the plastid signals shown to activate nuclear gene expression; however, due to its high reactivity, ¹O₂ is unlikely to be the signaling molecule that transmits the signal from the chloroplast to the cytoplasm. Still, it has been shown in C. reinhardtii that ¹O₂ generated in PSII can be detected in the cytoplasm (39). In that study, it was not possible to distinguish whether ¹O₂ diffuses from its site of production to the cytoplasm or is produced by secondary reactions of LOOH at the outer membrane of the chloroplast envelope (Russell mechanism). However, it is most likely that the ¹O₂ signal has to be detected by a specific sensor in the thylakoids or the stroma of the chloroplast, and that this sensor than activates a signal transduction pathway directed to the nucleus (39). Several studies were conducted attempting to identify the components of the ¹O₂ signaling pathways, most of them applying screens to identify mutants affected in the physiological or molecular response to the signal.

Components involved in singlet-oxygen signaling

In A. thaliana, the two plastid-localized proteins EX1 and EX2 have been identified in a mutant screen for plant deficient in the ¹O₂-induced PCD response (157). The exact function of these two homologous proteins is not known, but they play a crucial role in the ¹O₂-mediated changes in gene expression and stimulation of phytohormone synthesis in the flu mutant as shown by the loss of ¹O₂ response in the ex1 single or ex1/ex2 double mutant (Fig. 8) (85, 116). Another approach was used by screening for mutants deficient in the ¹O₂-specific transcriptional response. The promoter of an AAA-ATPase gene was used for reporter gene construction and screening for constitutive activator of AAA-ATPase (caa) or non-activator of AAA-ATPase (naa) mutants (9). Characterization of these mutants revealed that there is probably not a single linear signaling pathway for the response of this gene that could be easily mutated. In agreement with that, identification of the mutant caa13, containing a mutation in the pleiotropic response locus 1 (PRL1) gene, suggested that ¹O₂ signaling is strongly interconnected with other signaling pathways by the function of the PRL1 protein (10). Similarly, other ¹O₂-response mutants (caa33, soldat8, and soldat10) are depleted in processes generally affecting plastid homeostasis and consequently stimulating ¹O₂ response and acclimation (19, 96, 138). All these examples indicate that the ¹O₂ signal transduction in plants forms an integral part of a complex signaling network that is modified by other signaling routes (9).

FIG. 8.

Schematic diagram of a plant/algal cell showing the locations and interactions of some components considered to be involved in ¹O₂-mediated induction of either Arabidopsis thaliana (At, black) or Chlamydomonas reinhardtii (Cr, gray) genes. Full arrows indicate activation or conversion, and dashed arrows indicate diffusion or transport of molecules. Red upward arrows show the components whose levels were shown to increase upon formation, and pink letters indicated reactive electrophile species (RES). 13-HPOTE, 13-hydroperoxy octadecatrienoic acid; 13-KODE, 13-keto-octadecadienoic acid.

¹O₂ signaling does not only change gene expression on the transcriptional level, but was also shown to affect translation (120). A reduced expression of many chloroplast proteins as a response to ¹O₂-producing conditions was detected in the barley flu-orthologous mutant tigrina-d.12 (68). This suppression of protein synthesis was caused by an arrest in translation initiation at the 80S cytoplasmic ribosomes what correlated with a declined phosphorylation level of the ribosomal protein S6. A strong effect of ¹O₂ production on translation was also observed in the A. thaliana oep16 mutant deficient in the protochlorophyllide conversion. Even though upon the dark-to-light shift oep16 exhibits a similar PCD phenotype to flu, translation was differently affected in the two mutants. This shows that again multiple signaling mechanisms might control translational by ¹O₂ production (129).

The green alga C. reinhardtii provides another powerful tool to screen for ¹O₂-response mutants, because its haploid genome and asexual reproduction allow the fast identification of loss-of-function mutations. Further, several ¹O₂-specific responses have been identified in C. reinhardtii, including acclimation and induction of the defense genes (84, 86). The HSP70A gene was shown to be induced by ¹O₂ and different HSP70A-based reporter constructs could be created which specifically responded to either ¹O₂ or H₂O₂ (135). The GPXH/GPX5 gene is strongly induced by ¹O₂, but only moderately by other ROS, and it was shown to be specifically induced by ¹O₂ during exposure to high-light conditions (37, 38). Further, the transcriptional activation of GPXH is dependent on multiple regulatory elements that are distinct from an RES-responsive element (34, 40). A reporter construct containing an ¹O₂-specific GPXH promoter fragment was used to screen more than 5,000 UV-mutagenized clones for their response to ¹O₂. The fact that no mutants deficient in signal transduction could be isolated indicates that in C. reinhardtii, similar to plants, the ¹O₂ signal is not transmitted through a single linear pathway (36). On the other hand, an independent screen for mutants deficient in the response of a GPXH-dependent reporter construct resulted in the isolation of a psbP2 mutant (15). The PSBP2 gene is paralogous to the oxygen-evolving enhancer gene PSBP1, and it was hypothesized that it might function as a sensor for ¹O₂ to activate GPXH expression. However, induction of the wild-type GPXH gene and acclimation to ¹O₂ are not depleted completely in psbP2, supporting the fact that multiple signaling pathways might be involved in a specific ¹O₂ response in C. reinhardtii (Fig. 8).

Reactive electrophiles as putative transmitters of singlet-oxygen signals

An alternative signaling pathway in the response of C. reinhardtii to ¹O₂ was recently identified in a screen for mutants that are constitutively acclimated to ¹O₂ and thus more tolerant to high concentrations of ¹O₂-generating photosensitizers (40). One of these singlet oxygen-resistant (sor) mutants was affected by a transcription factor called SOR1. SOR1 seems to regulate the expression of a number oxidative stress response and detoxification genes, including GPXH and GSTS1. Most of the SOR1-regulated genes are also strongly induced by lipophilic RES and contain a RES-response element (Fig. 8). This indicates a link between the response to RES and the acclimation to ¹O₂. Such a link was confirmed by the increased tolerance of RES-acclimated C. reinhardtii cells to subsequent exposure to toxic ¹O₂ levels (40).

RES can be formed after increased ¹O₂ formation by direct oxidation of PUFA (see “Main targets of singlet oxygen damage”) or enzymatically by the activation of lipid-oxidizing enzymes called LOXs. In A. thaliana, ¹O₂ was shown to activate enzymatic lipid oxidation by inducing the expression of LOX2 and LOX3 coding for two 13-LOXs catalyzing the synthesis of 13-HPOTE (Fig. 8). This hydroperoxide is a precursor for OPDA synthesis but can also be reduced to 13-HOTE or converted to 13-KOTE. Due to their α/β-unsaturated keto group, 13-KOTEs participate in nucleophilic Michael additions and therefore are RES (101). Similarly, OPDA also belongs to the RES, whereas JA is a saturated ketone and not as electrophilic. Different RES have been shown to activate similar response mechanisms causing comparable gene expression profiles that were also related to the response to other stressors such as pathogens and wounding (3, 101, 144). Exogenous RES can even stimulate the accumulation of OPDA without affecting the JA levels (3). Thus, the much higher overlap of low ¹O₂-induced genes with the OPDA- than with JA-responsive genes indicates that many genes induced during ¹O₂ acclimation are responding to reactive electrophilic oxylipins such as 13-KOTE and OPDA (50). Direct requirement of OPDA accumulation was shown for the ¹O₂ response of the CYP94C and VSP1 genes in the OPDA synthesis mutant aos (116). On the other hand, other OPDA-responsive genes such as OPR1 and HSP17.6 were derepressed in aos, what might be due to released repression by JA, which is also depleted in aos mutants. Finally, a lox1lox5 mutant deficient in 9-HPOTE synthesis was more sensitive to the ¹O₂ photosensitizer rose bengal. This supports an important role of reactive electrophilic oxylipins such as OPDA, HPOTE, and KOTE in ¹O₂ signaling and acclimation in photosynthetic organisms (91).

Another group of RES that can be formed by ¹O₂ are carotenoid oxidation products (118). Carotenoids are the main ¹O₂ quenchers in the photosynthetic apparatus, and therefore increased levels of the carotenoid oxidation products β-cyclocitral and β-ionone could be measured in A. thaliana exposed to a high light intensity in the cold (119). Exposure to β-cyclocitral induced a very similar response pattern to the flu mutant. It stimulated the expression of many genes specifically induced by ¹O₂ or ¹O₂ and RES such as HSP17.6, GST1, and GST6 (Fig. 8). Further, β-cyclocitral also induced the expression of genes for OPDA synthesis (LOX2, AOC1 and AOS), indicating an interaction with oxylipin signaling and the induction of RES responses. The induction of genes by β-cyclocitral increased the tolerance to high light and cold treatment (119). Because the responses to β-cyclocitral were EX1 independent, it was suggested that this carotenoid oxidation product might function as second messenger to stimulate the ¹O₂-induced acclimation response in A. thaliana. Induction experiments with β-cyclocitral in C. reinhardtii revealed no transcriptional response similar to the specific ¹O₂ induction, but rather a stimulated expression of detoxification mechanisms similar to other RES treatments (Fischer BB, unpublished). Thus, β-cyclocitral might not be an universal signaling molecule transmitting the ¹O₂ signal to the nucleus in photosynthetic organisms. However, it might be a part of a larger group of RES molecules that individually or in combination stimulate part of the ¹O₂-specific response and activate acclimation to photo-oxidative stress conditions (Fig. 8).

Conclusions and Outlook

Despite the increasing body of knowledge in the literature on ¹O₂ generation and signaling, many important questions remain open for future research. The mechanism of safe transport and storage of Chl during degradation of CP to prevent ¹O₂ formation is still unidentified. Even though first lines of evidence are provided on how Chl is inserted into the LHC by LIL proteins, it is still unclear how Chl is inserted into the reaction centers of the PSs. Further, the fate of Chl during the turnover of the D1 protein is not known. Proteins of the ELIP family may play a role in this process, and the water-soluble CP may be involved in the protection of old leaves against destruction through ¹O₂ produced by free Chl; however, the exact protection mechanisms still need to be solved.

Beside its deleterious effects, ¹O₂ plays an important function in cell signaling by activating a cascade to transmit a chloroplast signal to the nucleus and stimulate a specific gene expression response. However, hardly anything is known about how and where the ¹O₂ signal is sensed and transmitted to the cytoplasm. Such specific questions also increase the need to establish reliable ¹O₂ detection methods that allow to detect ¹O₂ quantitatively and selectively, to localize it inside the cell compartments, and to determine its lifetime in Chl-containing organisms. Studies on ¹O₂ signaling also revealed that different and multiple mechanisms might be involved in ¹O₂ response in different photosynthetic organisms, and that strong interactions with other ROS and RES-signaling cascades occur. Unraveling these different mechanisms will help to clarify the specific role of ¹O₂ in the response to photo-oxidative stress and in the response to other stressors such as pathogen infection. By knowing all the factors, their interaction and sensitivity to abiotic and biotic stressors, we might start to understand under which conditions plants respond to stressors by activating an acclimation or a PCD response.

Footnotes

Acknowledgments

We would like to thank Rik Eggen (Eawag) for critical reading of the manuscript and Roberto Bassi (University Verona) for providing detailed information on the carotenoid composition of the different protein subunits. A. K.L. was supported by the Agence Nationale de Recherche reference ANR-09-BLAN-0005-01. É. H. acknowledges support from the Hungarian Scientific Research Fund (OTKA K 101430).

Author Disclosure Statement

No competing financial interests exist.

Abbreviations Used

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