N-formylkynurenine as a marker of high light stress in photosynthesis

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganese-calcium active site. Light-dependent damage and repair occur in PSII under conditions of high light stress. The core reaction center complex is composed of the D1, D2, CP43, and CP47 intrinsic polypeptides. In this study, a new chromophore formed from the oxidative post-translational modification of tryptophan is identified in the CP43 subunit. Tandem mass spectrometry peptide sequencing is consistent with the oxidation of the CP43 tryptophan side chain, Trp-365, to produce N-formylkynurenine (NFK). Characterization with ultraviolet visible absorption and ultraviolet resonance Raman spectroscopy supports this assignment. An optical assay suggests that the yield of NFK increases 2-fold (2.2 ± 0.5) under high light illumination. A concomitant 2.4 ± 0.5-fold decrease is observed in the steady-state rate of oxygen evolution under the high light conditions. NFK is the product formed from reaction of tryptophan with singlet oxygen, which can be produced under high light stress in PSII. Reactive oxygen species reactions lead to oxidative damage of the reaction center, D1 protein turnover, and inhibition of electron transfer. Our results are consistent with a role for the CP43 NFK modification in photoinhibition.     

Light-induced oxidative stress, N-formylkynurenine, and oxygenic photosynthesis

Light stress in plants results in damage to the water oxidizing reaction center, photosystem II (PSII). Redox signaling, through oxidative modification of amino acid side chains, has been proposed to participate in this process, but the oxidative signals have not yet been identified. Previously, we described an oxidative modification, N-formylkynurenine (NFK), of W365 in the CP43 subunit. The yield of this modification increases under light stress conditions, in parallel with the decrease in oxygen evolving activity. In this work, we show that this modification, NFK365-CP43, is present in thylakoid membranes and may be formed by reactive oxygen species produced at the Mn(4)CaO(5) cluster in the oxygen-evolving complex. NFK accumulation correlates with the extent of photoinhibition in PSII and thylakoid membranes. A modest increase in ionic strength inhibits NFK365-CP43 formation, and leads to accumulation of a new, light-induced NFK modification (NFK317) in the D1 polypeptide. Western analysis shows that D1 degradation and oligomerization occur under both sets of conditions. The NFK modifications in CP43 and D1 are found 17 and 14 Angstrom from the Mn(4)CaO(5) cluster, respectively. Based on these results, we propose that NFK is an oxidative modification that signals for damage and repair in PSII. The data suggest a two pathway model for light stress responses. These pathways involve differential, specific, oxidative modification of the CP43 or D1 polypeptides.     

Revised scheme for the mechanism of photoinhibition and its application to enhance the abiotic stress tolerance of the photosynthetic machinery

When photosynthetic organisms are exposed to abiotic stress, their photosynthetic activity is significantly depressed. In particular, photosystem II (PSII) in the photosynthetic machinery is readily inactivated under strong light and this phenomenon is referred to as photoinhibition of PSII. Other types of abiotic stress act synergistically with light stress to accelerate photoinhibition. Recent studies of photoinhibition have revealed that light stress damages PSII directly, whereas other abiotic stresses act exclusively to inhibit the repair of PSII after light-induced damage (photodamage). Such inhibition of repair is associated with suppression, by reactive oxygen species (ROS), of the synthesis of proteins de novo and, in particular, of the D1 protein, and also with the reduced efficiency of repair under stress conditions. Gene-technological improvements in the tolerance of photosynthetic organisms to various abiotic stresses have been achieved via protection of the repair system from ROS and, also, by enhancing the efficiency of repair via facilitation of the turnover of the D1 protein in PSII. In this review, we summarize the current status of research on photoinhibition as it relates to the effects of abiotic stress and we discuss successful strategies that enhance the activity of the repair machinery. In addition, we propose several potential methods for activating the repair system by gene-technological methods.     

Effect of Reducing Nitric Oxide in <Emphasis Type="Italic">Rumex</Emphasis> K-1 Leaves on the Photoprotection of Photosystem II Under High Temperature with Strong Light

The purpose of this study was to explore the effect of reducing nitric oxide (NO) in Rumex K-1 leaves on the photoprotection of photosystem II (PSII) under high temperature with strong light. Reducing the content of NO in Rumex K-1 leaves significantly aggravated the PSII photoinhibition and net degradation of D1 protein under high temperature with strong light, but not under high temperature in the darkness. The reduction of NO remarkably inhibited the electron transport of PSII in the leaves under high temperature and strong light, which resulted in an increase in excitation pressure and an over-accumulation of reactive oxygen species (ROS). The over-accumulation of ROS further damaged PSII. However, when the synthesis of D1 protein was inhibited, the D1 protein content and PSII activity were no longer influenced by reducing NO content in the leaves. The reduction of NO in leaves decreased the activities of ROS scavenger enzymes after treatment with high temperature and strong light for 2 h, which enhanced the over-accumulation of ROS to damage photosynthetic apparatus severely. All of these results suggest that NO was involved in the synthesis of D1 protein. Maintaining physiologically appropriate NO content in leaves will alleviate net degradation of D1 protein under high temperature with strong light to keep photosynthetic electrons flowing smoothly, which mitigates the accumulation of ROS in photosystems to avoid damage to the photosynthetic apparatus. Therefore, NO plays an important role in maintaining higher PSII photosynthetic performance under high temperature with strong light.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the "classical" scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO(2) limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO(2) with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

Photoinhibition of photosystem II under environmental stress

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to an imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. In the “classical” scheme for the mechanism of photoinhibition, strong light induces the production of reactive oxygen species (ROS), which directly inactivate the photochemical reaction center of PSII. By contrast, in a new scheme, we propose that photodamage is initiated by the direct effect of light on the oxygen-evolving complex and that ROS inhibit the repair of photodamaged PSII by suppressing primarily the synthesis of proteins de novo. The activity of PSII is restricted by a variety of environmental stresses. The effects of environmental stress on damage to and repair of PSII can be examined separately and it appears that environmental stresses, with the exception of strong light, act primarily by inhibiting the repair of PSII. Studies have demonstrated that repair-inhibitory stresses include CO₂ limitation, moderate heat, high concentrations of NaCl, and low temperature, each of which suppresses the synthesis of proteins de novo, which is required for the repair of PSII. We postulate that most types of environmental stress inhibit the fixation of CO₂ with the resultant generation of ROS, which, in turn, inhibit protein synthesis.     

The desert green algae Chlorella ohadii thrives at excessively high light intensities by exceptionally enhancing the mechanisms that protect photosynthesis from photoinhibition

Although light is the driving force of photosynthesis, excessive light can be harmful. One of the main processes that limits photosynthesis is photoinhibition, the process of light-induced photodamage. When the absorbed light exceeds the amount that is dissipated by photosynthetic electron flow and other processes, damaging radicals are formed that mostly inactivate photosystem II (PSII). Damaged PSII must be replaced by a newly repaired complex in order to preserve full photosynthetic activity. Chlorella ohadii is a green microalga, isolated from biological desert soil crusts, that thrives under extreme high light and is highly resistant to photoinhibition. Therefore, C. ohadii is an ideal model for studying the molecular mechanisms underlying protection against photoinhibition. Comparison of the thylakoids of C. ohadii cells that were grown under low light versus extreme high light intensities found that the alga employs all three known photoinhibition protection mechanisms: (i) massive reduction of the PSII antenna size; (ii) accumulation of protective carotenoids; and (iii) very rapid repair of photodamaged reaction center proteins. This work elucidated the molecular mechanisms of photoinhibition resistance in one of the most light-tolerant photosynthetic organisms, and shows how photoinhibition protection mechanisms evolved to marginal conditions, enabling photosynthesis-dependent life in severe habitats.     

Evidence for the role of the oxygen-evolving manganese complex in photoinhibition of Photosystem II

Photoinhibition of PSII occurs at the same quantum efficiency from very low to very high light, which raises a question about how important is the rate of photosynthetic electron transfer in photoinhibition. We modulated electron transfer rate and light intensity independently of each other in lincomycin-treated pea leaves and in isolated thylakoids, in order to elucidate the specific effects of light and PSII electron transport on photoinhibition. Major changes in the rate of electron transport caused only small changes in the rate of photoinhibition, suggesting the existence of a significant photoinhibitory pathway that contains an electron-transfer-independent phase. We compared the action spectrum of photoinhibition with absorption spectra of PSII components that could function as photoreceptors of the electron-transfer-independent phase of photoinhibition and found that the absorption spectra of Mn(III) and Mn(IV) compounds resemble the action spectrum of photoinhibition, showing a steep decrease from UV-C to blue light and a low visible-light tail. Our results show that the release of a Mn ion to the thylakoid lumen is the earliest detectable step of both UV- and visible-light-induced photoinhibition. After Mn release from the oxygen-evolving complex, oxidative damage to the PSII reaction center occurs because the Mn-depleted oxygen-evolving complex cannot reduce P680+ normally.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO₂ is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO(2) is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.     

Mitochondrial alternative oxidase pathway protects plants against photoinhibition by alleviating inhibition of the repair of photodamaged PSII through preventing formation of reactive oxygen species in Rumex K-1 leaves

The purpose of this study was to explore how the mitochondrial AOX (alternative oxidase) pathway alleviates photoinhibition in Rumex K-1 leaves. Inhibition of the AOX pathway decreased the initial activity of NADP-malate dehydrogenase (EC 1.1.1.82, NADP-MDH) and the pool size of photosynthetic end electron acceptors, resulting in an over-reduction of the photosystem I (PSI) acceptor side. The over-reduction of the PSI acceptor side further inhibited electron transport from the photosystem II (PSII) reaction centers to the PSII acceptor side as indicated by an increase in V(J) (the relative variable fluorescence at J-step), causing an imbalance between photosynthetic light absorption and energy utilization per active reaction center (RC) under high light, which led to the over-excitation of the PSII reaction centers. The over-reduction of the PSI acceptor side and the over-excitation of the PSII reaction centers enhanced the accumulation of reactive oxygen species (ROS), which inhibited the repair of the photodamaged PSII. However, the inhibition of the AOX pathway did not change the level of photoinhibition under high light in the presence of the chloroplast D1 protein synthesis inhibitor chloramphenicol, indicating that the inhibition of the AOX pathway did not accelerate the photodamage to PSII directly. All these results suggest that the AOX pathway plays an important role in the protection of plants against photoinhibition by minimizing the inhibition of the repair of the photodamaged PSII through preventing the over-production of ROS.     

Heat stress: an overview of molecular responses in photosynthesis

The primary targets of thermal damage in plants are the oxygen evolving complex along with the associated cofactors in photosystem II (PSII), carbon fixation by Rubisco and the ATP generating system. Recent investigations on the combined action of moderate light intensity and heat stress suggest that moderately high temperatures do not cause serious PSII damage but inhibit the repair of PSII. The latter largely involves de novo synthesis of proteins, particularly the D1 protein of the photosynthetic machinery that is damaged due to generation of reactive oxygen species (ROS), resulting in the reduction of carbon fixation and oxygen evolution, as well as disruption of the linear electron flow. The attack of ROS during moderate heat stress principally affects the repair system of PSII, but not directly the PSII reaction center (RC). Heat stress additionally induces cleavage and aggregation of RC proteins; the mechanisms of such processes are as yet unclear. On the other hand, membrane linked sensors seem to trigger the accumulation of compatible solutes like glycinebetaine in the neighborhood of PSII membranes. They also induce the expression of stress proteins that alleviate the ROS-mediated inhibition of repair of the stress damaged photosynthetic machinery and are required for the acclimation process. In this review we summarize the recent progress in the studies of molecular mechanisms involved during moderate heat stress on the photosynthetic machinery, especially in PSII.     

Chloroplast movement provides photoprotection to plants by redistributing PSII damage within leaves

Plants use light to fix carbon through the process of photosynthesis but light also causes photoinhibition, by damaging photosystem II (PSII). Plants can usually adjust their rate of PSII repair to equal the rate of damage, but under stress conditions or supersaturating light-intensities damage may exceed the rate of repair. Light-induced chloroplast movements are one of the many mechanisms plants have evolved to minimize photoinhibition. We found that chloroplast movements achieve a measure of photoprotection to PSII by altering the distribution of photoinhibition through depth in leaves. When chloroplasts are in the low-light accumulation arrangement a greater proportion of PSII damage occurs near the illuminated surface than for leaves where the chloroplasts are in the high-light avoidance arrangement. According to our findings chloroplast movements can increase the overall efficiency of leaf photosynthesis in at least two ways. The movements alter light profiles within leaves to maximize photosynthetic output and at the same time redistribute PSII damage throughout the leaf to reduce the amount of inhibition received by individual chloroplasts and prevent a decrease in photosynthetic potential.     

Genomic investigation of the system for selenocysteine incorporation in the bacterial domain

The Photosystem II complex (PSII) is susceptible to inactivation by strong light, and the inactivation caused by strong light is referred to as photoinactivation or photoinhibition. In photosynthetic organisms, photoinactivated PSII is rapidly repaired and the extent of photoinactivation reflects the balance between the light-induced damage (photodamage) to PSII and the repair of PSII. In this study, we examined these two processes separately and quantitatively under stress conditions in the cyanobacterium Synechocystis sp. PCC 6803. The rate of photodamage was proportional to light intensity over a range of light intensities from 0 to 2000 microE m(-2) s(-1), and this relationship was not affected by environmental factors, such as salt stress, oxidative stress due to H2O2, and low temperature. The rate of repair also depended on light intensity. It was high under weak light and reached a maximum of 0.1 min(-1) at 300 microE m(-2) s(-1). By contrast to the rate of photodamage, the rate of repair was significantly reduced by the above-mentioned environmental factors. Pulse-labeling experiments with radiolabeled methionine revealed that these environmental factors inhibited the synthesis de novo of proteins. Such proteins included the D1 protein which plays an important role in the photodamage-repair cycle. These observations suggest that the repair of PSII under environmental stress might be the critical step that determines the outcome of the photodamage-repair cycle.     

Immunological detection of N-formylkynurenine in oxidized proteins

Reactions of tryptophan residues in proteins with radical and other oxidative species frequently lead to cleavage of the indole ring, modifying tryptophan residues into N-formylkynurenine (NFK) and kynurenine. Tryptophan modification has been detected in physiologically important proteins and has been associated with a number of human disease conditions. Modified residues have been identified through various combinations of proteomic analyses, tryptic digestion, HPLC, and mass spectrometry. Here we present a novel, immunological approach using polyclonal antiserum for detection of NFK. The specificity of our antiserum is confirmed using photooxidation and radical-mediated oxidation of proteins with and without tryptophan residues. The sensitivity of our antiserum is validated through detection of NFK in photooxidized myoglobin (two tryptophan residues) and in carbonate radical-oxidized human SOD1, which contains a single tryptophan residue. Analysis of photooxidized milk also shows that our antiserum can detect NFK residues in a mixture of proteins. Results from mass spectrometric analysis of photooxidized myoglobin samples corroborate the immunological data, detecting an increase in NFK content as the extent of photooxidation increases.     

Photoprotection in plants: a new light on photosystem II damage

Sunlight damages photosynthetic machinery, primarily photosystem II (PSII), and causes photoinhibition that can limit plant photosynthetic activity, growth and productivity. The extent of photoinhibition is associated with a balance between the rate of photodamage and its repair. Recent studies have shown that light absorption by the manganese cluster in the oxygen-evolving complex of PSII causes primary photodamage, whereas excess light absorbed by light-harvesting complexes acts to cause inhibition of the PSII repair process chiefly through the generation of reactive oxygen species. As we review here, PSII photodamage and the inhibition of repair are therefore alleviated by photoprotection mechanisms associated with avoiding light absorption by the manganese cluster and successfully consuming or dissipating the light energy absorbed by photosynthetic pigments, respectively.     

Identification of tryptophan oxidation products in bovine alpha-crystallin.

Oxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal-catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of alpha-crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine alpha-crystallin sequence. After oxidative exposure, alpha-crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse-phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of alphaA- and tryptophan 60 of alphaB-crystallin were each converted into hydroxytryptophans (HTRP), N-formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of alphaB-crystallin. Oxidation of methionine 1 of alphaA- and methionine 1 and 68 of alphaB-crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact alpha-crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.     

Towards a critical understanding of the photosystem II repair mechanism and its regulation during stress conditions

Photosystem II (PSII) is vulnerable to high light (HL) illumination resulting in photoinhibition. In addition to photoprotection mechanisms, plants have developed an efficient PSII repair mechanism to save themselves from irreversible damage to PSII under abiotic stresses including HL illumination. The phosphorylation/dephosphorylation cycle along with subsequent degradation of photodamaged D1 protein to be replaced by the insertion of a newly synthesized copy of D1 into the PSII complex, is the core function of the PSII repair cycle. The exact mechanism of this process is still under discussion. We describe the recent progress in identifying the kinases, phosphatases and proteases, and in understanding their involvement in the maintenance of thylakoid structure and the quality control of proteins by PSII repair cycle during photoinhibition.     

Propagation of protein glycation damage involves modification of tryptophan residues via reactive oxygen species: inhibition by pyridoxamine

Nonenzymatic modification of proteins is one of the key pathogenic factors in diabetic complications. Uncovering the mechanisms of protein damage caused by glucose is fundamental to understanding this pathogenesis and in the development of new therapies. We investigated whether the mechanism involving reactive oxygen species can propagate protein damage in glycation reactions beyond the classical modifications of lysine and arginine residues. We have demonstrated that glucose can cause specific oxidative modification of tryptophan residues in lysozyme and inhibit lysozyme activity. Furthermore, modification of tryptophan residues was also induced by purified albumin-Amadori, a ribose-derived model glycation intermediate. The AGE inhibitor pyridoxamine (PM) prevented the tryptophan modification, whereas another AGE inhibitor and strong carbonyl scavenger, aminoguanidine, was ineffective. PM specifically inhibited generation of hydroxyl radical from albumin-Amadori and protected tryptophan from oxidation by hydroxyl radical species. We conclude that oxidative degradation of either glucose or the protein-Amadori intermediate causes oxidative modification of protein tryptophan residues via hydroxyl radical and can affect protein function under physiologically relevant conditions. This oxidative stress-induced structural and functional protein damage can be ameliorated by PM via sequestration of catalytic metal ions and scavenging of hydroxyl radical, a mechanism that may contribute to the reported therapeutic effects of PM in the complications of diabetes.     

UV-B resistance as a criterion for the selection of desert microalgae to be utilized for inoculating desert soils

The adaption capability of microalgae species to intense UV-B radiation is an important feature for their survival under the harsh growth conditions they have to face when used for inoculating unconsolidated sand soils in desert areas. In this study, the responses of photosynthetic activity, reactive oxygen species (ROS) generation, and DNA strand breaks to UV-B radiation in four microalgae isolated from artificially induced biological soil crusts were investigated. It was found that low UV-B doses easily inhibited the photosynthetic activity and induced serious DNA damage in Chlorella vulgaris. Microcoleus vaginatus showed the capability to withstand only moderate doses of UV-B, while Nostoc was capable of facing high doses of UV-B due to its lower generation of ROS and higher capability to repair photosystem II (PSII) and DNA damages. On the other hand, Scytonema javanicum showed additional strategies to survive UV-B irradiance, namely the closure of PSII when ROS generation increased rapidly, in addition to a high repair ability of PSII and DNA damage. The results obtained point out different resistance and defense mechanisms of the four microalgae in response to UV-B irradiance and suggest that the strain of Nostoc sp. tested is the most suitable for surviving under the high UV irradiation levels typical of desertified areas.