Studies on the mechanism of photosystem II photoinhibition II. The involvement of toxic oxygen species

In a previous paper it was shown that photoinhibition of reaction centre II of spinach thylakoids was predominantly caused by the degradation of D1-protein. An initial inactivation step at the Q_B-site was distinguished from its breakdown. The present paper deals with the question as to whether this loss of Q_B-function is caused by oxygen radical attack. For this purpose the photoinhibition of thylakoids was induced at 20°C in the presence of either superoxide dismutase and catalase or the antioxidants glutathione and ascorbic acid. This resulted in comparable though not total protection of D1-protein, photochemistry and fluorescence from photoinhibition. The combined action of both the enzymatic and the non-enzymatic radical scavenging systems brought about an even more pronounced protective effect against photoinhibition than did either of the two systems singularly at saturating concentrations. The results signify a major contribution of activated oxygen species to the degradation process of D1-protein and the related phenomena of photoinhibition. Thylakoids treated with hydroxyl radicals generated through a Fenton reaction showed a loss of atrazine binding sites, electron transport capacity and variable fluorescence in a similar manner, though not to the same extent, as usually observed following photoinhibitory treatment.Abbreviations Asc ascorbate - Fecy ferricyanide - GSH reduced glutathione - PQ plastoquinone - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SOD superoxide dismutase     

Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products

Illumination of a suspension of thylakoids with light at high intensity causes inhibition of the photosystem II electron transport activity and loss from the membrane of the D1 protein of the photosystem II reaction center. Impairment of the electron transport activity and depletion of D1 protein from the thylakoid membrane of pea were investigated with reference to the presence or absence of oxygen in the suspension. The breakdown products of the D1 protein were identified by immunoblotting with anti-D1 polyclonal antibodies which were proven to recognize mainly the C-terminal region of the protein. The results obtained show that (i) the light-induced inactivation of the photosystem II electron transport activity under anaerobic conditions is faster than in the presence of oxygen; (ii) depletion of D1 protein is observed on a longer time scale with respect to loss of electron transport activity and is faster when photoinhibition is performed in the presence of oxygen; (iii) C-terminal fragments of D1 are only observed when photoinhibition is carried out anaerobically and are mainly localized in the stroma-exposed regions; and (iv) the fragments observed after anaerobic photoinhibition are quickly degraded on further illumination of the thylakoid suspension in the presence of oxygen.     

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

Responses of photosystem I compared with photosystem II to high-light stress in tropical shade and sun leaves

Sun and shade leaves of several plant species from a neotropical forest were exposed to excessive light to evaluate the responses of photosystem I in comparison to those of photosystem II. Potential photosystem I activity was determined by means of the maximum P700 absorbance change around 810 nm (ΔA_810max) in saturating far-red light. Leaf absorbance changes in dependence of increasing far-red light fluence rates were used to calculate a ‘saturation constant’, K_s, representing the far-red irradiance at which half of the maximal absorbance change (ΔA_810max/2) was reached in the steady state. Photosystem II efficiency was assessed by measuring the ratio of variable to maximum chlorophyll fluorescence, F_v/F_m, in dark-adapted leaf samples. Strong illumination caused a high degree of photo-inhibition of photosystem II in all leaves, particularly in shade leaves. Exposure to 1800–2000 μ mol photons m⁻² s⁻¹ for 75 min did not substantially affect the potential activity of photosystem I in all species tested, but caused a more than 40-fold increase of K_s in shade leaves, and a three-fold increase of K_s in sun leaves. The increase in K_s was reversible during recovery under low light, and the recovery process was much faster in sun than in shade leaves. The novel effect of high-light stress on the light saturation of P700 oxidation described here may represent a complex reversible mechanism within photosystem I that regulates light-energy dissipation and thus protects photosystem I from photo-oxidative damage. Moreover, we show that under high-light stress a high proportion of P700 accumulates in the oxidized state, P700⁺. Presumably, conversion of excitation energy to heat by this cation radical may efficiently contribute to photoprotection.     

Scavenging of superoxide generated in photosystem I by plastoquinol and other prenyllipids in thylakoid membranes

We have examined scavenging of a superoxide by various prenyllipids occurring in thylakoid membranes, such as plastoquinone-9, alpha-tocopherolquinone, their reduced forms, and alpha-tocopherol, measuring oxygen uptake in hexane-extracted and untreated spinach thylakoids with a fast oxygen electrode under flash-light illumination. The obtained results demonstrated that all the investigated prenyllipids showed the superoxide scavenging properties, and plastoquinol-9 was the most active in this respect. Plastoquinol-9 formed in thylakoids as a result of enzymatic reduction of plastoquinone-9 by ferredoxin-plastoquinone reductase was even more active than the externally added plastoquinol-9 in the investigated reaction. Scavenging of superoxide by plastoquinol-9 and other prenyllipids could be important for protecting membrane components against the toxic action of superoxide. Moreover, our results indicate that vitamin K(1) is probably the most active redox component of photosystem I in the generation of superoxide within thylakoid membranes.     

Quality control of photosystem II: reactive oxygen species are responsible for the damage to photosystem II under moderate heat stress

Moderate heat stress (40 degrees C for 30 min) on spinach thylakoid membranes induced cleavage of the reaction center-binding D1 protein of photosystem II, aggregation of the D1 protein with the neighboring polypeptides D2 and CP43, and release of three extrinsic proteins, PsbO, -P, and -Q. These heat-induced events were suppressed under anaerobic conditions or by the addition of sodium ascorbate, a general scavenger of reactive oxygen species. In accordance with this, singlet oxygen and hydroxyl radicals were detected in spinach photosystem II membranes incubated at 40 degrees C for 30 min with electron paramagnetic resonance spin-trapping spectroscopy. The moderate heat stress also induced significant lipid peroxidation under aerobic conditions. We suggest that the reactive oxygen species are generated by heat-induced inactivation of a water-oxidizing manganese complex and through lipid peroxidation. Although occurring in the dark, the damages caused by the moderate heat stress to photosystem II are quite similar to those induced by excessive illumination where reactive oxygen species are involved.     

5-Aminolevulinic Acid Induced Photodynamic Reactions in Thylakoid Membranes of Cucumber (Cucumis sativus L.) Cotyledons

The cucumber (Cucumis sativus L.) plants were sprayed with 20 mM 5-aminolevulinic acid or distilled water (control) and incubated in dark for 14 hr. The thylakoid membranes prepared from the intact chloroplasts, isolated from the above plants in dark, were illuminated with low light intensity (100 W/m2) for 30 min. Due 10 photodynamic reactions, the photochemical function of photosystem II was damaged by 50% in treated thylakoids whereas it was only slightly (8%) affected in control thylakoids. The photosystem I was, however, not affected. The exogenous electron donors, MnCl2, diphenyl carbazide and NH2OH failed to restore the photosystem II activity suggesting that the photodynamic damage had taken place very close to photosystem II reaction center. Singlet oxygen scavenger, histidine, could protect the photosystem II activity while superoxide radical scavengers, superoxide dismutase and 1, 2-dihydroxybenzene-3, 5-disulphonic acid disodium salt, and hydroxyl radical scavenger, formate, failed to protect the same.     

Inhibition of electron transport at the cytochrome b(6)f complex protects photosystem II from photoinhibition

Photoinhibition of photosystem II (PS II) activity was studied in thylakoid membranes illuminated in the presence of the inhibitor of the cytochrome b(6)f complex 2'iodo-6-isopropyl-3-methyl-2',4, 4'-trinitrodiphenylether (DNP-INT). DNP-INT was found to decrease photoinhibition. In the absence of DNP-INT, anaerobosis, superoxide dismutase and catalase protected against photoinhibition. No effect of these treatments was observed in the presence of DNP-INT. These data demonstrate that photoinhibition under these conditions is caused by reactive oxygen species which are formed most probably by the reduction of oxygen at photosystem I. The results are discussed in terms of the importance of photosynthetic control in protection against photoinhibition in vivo.     

Damage to photosystem II by lipid peroxidation products

BackgroundPhotosystem II proteins of higher plant chloroplasts are prone to oxidative stress, and most prominently the reaction center-binding D1 protein is damaged under abiotic stress. The reactive oxygen species produced under these stress conditions have been suggested to be responsible for the protein injury.Scope of reviewRecently, it has been shown that the primary and secondary products of non-enzymatic and enzymatic lipid peroxidation have a capability to modify photosystem II proteins. Here, we give an overview showing how lipid peroxidation products formed under light stress and heat stress in the thylakoid membranes cause oxidative modification of proteins in higher plant photosystem II.Major conclusionsDamage to photosystem II proteins by lipid peroxidation products represents a new mechanism underlying photoinhibition and heat inactivation.General significanceComplete characterization of photosystem II protein damage is of crucial importance because avoidance of the damage makes plants to survive under various abiotic stresses. Further physiological significance of photosystem II protein oxidation by lipid peroxidation product should have a potential relevance to plant acclimation because the oxidized proteins might serve as signaling molecules.     

The involvement of photosystem II-generated H2O2 in photoinhibition.

The involvement of H2O2 generated by photosystem II (PSII) in the process of photoinhibition of thylakoids with a functional oxygen-evolving complex (OEC) was investigated. The rate of photoinhibition was decreased to the rate of loss of activity in the dark when bovine Fe-catalase was present during the photoinhibitory illumination. Photoinhibition was accelerated for both Cl(-)-depleted and Cl(-)-sufficient thylakoids when KCN was present to inhibit the thylakoid-bound Fe-catalase. We propose that these preparations become photoinhibited by reactions with H2O2 produced via oxidation of water by the Cl(-)-depleted OEC and by reduction of O2 at the QB site when PSII is illuminated without an electron acceptor.     

Structural changes and lateral redistribution of photosystem II during donor side photoinhibition of thylakoids

The structural and topological stability of thylakoid components under photoinhibitory conditions (4,500 microE.m-2.s-1 white light) was studied on Mn depleted thylakoids isolated from spinach leaves. After various exposures to photoinhibitory light, the chlorophyll-protein complexes of both photosystems I and II were separated by sucrose gradient centrifugation and analysed by Western blotting, using a set of polyclonals raised against various apoproteins of the photosynthetic apparatus. A series of events occurring during donor side photoinhibition are described for photosystem II, including: (a) lowering of the oligomerization state of the photosystem II core; (b) cleavage of 32-kD protein D1 at specific sites; (c) dissociation of chlorophyll-protein CP43 from the photosystem II core; and (d) migration of damaged photosystem II components from the grana to the stroma lamellae. A tentative scheme for the succession of these events is illustrated. Some effects of photoinhibition on photosystem I are also reported involving dissociation of antenna chlorophyll-proteins LHCI from the photosystem I reaction center.     

Immunogold localization of light-harvesting and photosystem I complexes in the thylakoids ofFucus serratus (Phaeophyceae)

Summary The repartition of light-harvesting complex (LHC) and photosystem I (PS I) complex has been examined in isolated plastids ofFucus serratus by immunocytochemical labelling. LHC is distributed equally all along the length of thylakoid membranes, without any special repartition in the appressed membranes of the three associated thylakoids ofFucus. PS I is present on all the thylakoid membranes, but the external membranes of the three associated thylakoids are largely enriched relatively to the inner ones. This specific repartition of PSI on non-appressed membranes can be compared to the localization of PSI on stroma thylakoid membranes of higher plants and green algae. Consequently, although they share some common features with those of higher plants and green algae, the appressions of thylakoids in brown algae has neither the same structure nor the same functional role as typical grana stacked membranes in the repartition of the harvested energy.Abbreviations BSA bovine serum albumin - GAR goat anti-rabbit immunoglobulin G - LHC light-harvesting complex - PBS phosphatebuffered saline - PS I photosystem I - PS II photosystem II     

Light-induced D1-protein degradation in isolated photosystem II core complexes

Photoinhibitory illumination of isolated oxygen evolving photosystem II core complexes results in a substantial degradation of the D1-protein which is accompanied by the appearance of high amounts of at least 4 different degradation products. It is suggested that the degradation is due to a protease that is an integral part of the photosystem II complex.     

Quality control of photosystem II

Photosystem II is particularly vulnerable to excess light. When illuminated with strong visible light, the reaction center D1 protein is damaged by reactive oxygen molecules or by endogenous cationic radicals generated by photochemical reactions, which is followed by proteolytic degradation of the damaged D1 protein. Homologs of prokaryotic proteases, such as ClpP, FtsH and DegP, have been identified in chloroplasts, and participation of the thylakoid-bound FtsH in the secondary degradation steps of the photodamaged D1 protein has been suggested. We found that cross-linking of the D1 protein with the D2 protein, the alpha-subunit of cytochrome b(559), and the antenna chlorophyll-binding protein CP43, occurs in parallel with the degradation of the D1 protein during the illumination of intact chloroplasts, thylakoids and photosystem II-enriched membranes. The cross-linked products are then digested by a stromal protease(s). These results indicate that the degradation of the photodamaged D1 protein proceeds through membrane-bound proteases and stromal proteases. Moreover, a 33-kDa subunit of oxygen-evolving complex (OEC), bound to the lumen side of photosystem II, regulates the formation of the cross-linked products of the D1 protein in donor-side photoinhibition of photosystem II. Thus, various proteases and protein components in different compartments in chloroplasts are implicated in the efficient turnover of the D1 protein, thus contributing to the control of the quality of photosystem II under light stress conditions.     

Turnover of thylakoid photosystem II proteins during photoinhibition of Chlamydomonas reinhardtii

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.     

Chloroplast Cu/Zn-superoxide dismutase is a highly sensitive site in cucumber leaves chilled in the light

Light-chilling stress, the combination of low-light illumination and low temperature, preferentially inactivated photosystem I (PSI) of cucumber (Cucumis sativus L.) leaves, resulting in the photoinhibition of photosynthesis. The extent of PSI photoinhibition, determined in vivo by monitoring absorption changes around 810 nm (induced by far-red light), was closely correlated with the redox state of the PSII electron acceptor Q(A), measured as the chlorophyll fluorescence parameter, 1-qP, where qP is a photochemical quenching coefficient. In contrast, the decrease in the far-red-induced leaf absorptance signal was not well correlated with the limited fragmentation of the PsaA/B gene products in the PSI reaction center after the light-chilling stress. Amongst various enzymes involved in the photooxidative damage such as superoxide dismutase (SOD), ascorbate peroxidase, and NAD(P)H dehydrogenase, only SOD was inhibited by light-chilling treatment. Further, an approximately 3-fold increase in the leaf content of H(2)O(2), a potent inhibitor of Cu/Zn-SOD, was observed after light-chilling stress. From these results, we suggest that Cu/Zn-SOD is the primary target of the light-chilling stress, followed by subsequent inactivation of PSI by reactive oxygen species.     

Glu-69 of the D2 protein in photosystem II is a potential ligand to Mn involved in photosynthetic oxygen evolution

To probe the involvement of amino acid residues of the D2 protein in the water-splitting process in photosystem II, site-directed mutagenesis was applied to identify D2 residues that might contribute to binding the Mn cluster involved in oxygen evolution. Mutation of Glu-69 to Gln or Val in D2 of the cyanobacterium Synechocystis sp. PCC 6803 was found to lead to a loss of photoautotrophic growth. However, in cells of the Gln mutant (E69Q) a significant Hill reaction rate could be observed upon the start of illumination, but the oxygen evolution rate declined with a half-time of approximately 1 min. Addition of 1 mM Mn2+ stabilized oxygen evolution in E69Q thylakoids. Other divalent cations were ineffective in specific stabilization. When the water-splitting system was bypassed, the rate of electron transport remained stable during illumination, indicating that the inactivation of oxygen evolution is localized in the water-splitting complex. We interpret these observations to indicate that Glu-69 is a Mn ligand and that the loss of oxygen evolution in the E69Q mutant upon turnover of PS II is initiated by changes in the Mn cluster, possibly leading to Mn release from the water-splitting complex. The addition of exogenous Mn to E69Q thylakoids may help to keep the Mn cluster active for a longer time, perhaps by providing Mn to rebind in the cluster after release of one Mn and before the Mn cluster had disintegrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The PSI-K subunit of photosystem I is involved in the interaction between light-harvesting complex I and the photosystem I reaction center core

PSI-K is a subunit of photosystem I. The function of PSI-K was characterized in Arabidopsis plants transformed with a psaK cDNA in antisense orientation, and several lines without detectable PSI-K protein were identified. Plants without PSI-K have a 19% higher chlorophyll a/b ratio and 19% more P700 than wild-type plants. Thus, plants without PSI-K compensate by making more photosystem I. The photosystem I electron transport in vitro is unaffected in the absence of PSI-K. Light response curves for oxygen evolution indicated that the photosynthetic machinery of PSI-K-deficient plants have less capacity to utilize light energy. Plants without PSI-K have less state 1-state 2 transition. Thus, the redistribution of absorbed excitation energy between the two photosystems is reduced. Low temperature fluorescence emission spectra revealed a 2-nm blue shift in the long wavelength emission in plants lacking PSI-K. Furthermore, thylakoids and isolated PSI without PSI-K had 20-30% less Lhca2 and 30-40% less Lhca3, whereas Lhca1 and Lhca4 were unaffected. During electrophoresis under mildly denaturing conditions, all four Lhca subunits were partially dissociated from photosystem I lacking PSI-K. The observed effects demonstrate that PSI-K has a role in organizing the peripheral light-harvesting complexes on the core antenna of photosystem I.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

Formation of radicals from singlet oxygen produced during photoinhibition of isolated light-harvesting proteins of photosystem II

Electron spin resonance spectroscopy and liquid chromatography have been used to detect radical formation and fragmentation of polypeptides during photoinhibition of purified major antenna proteins, free of protease contaminants. In the absence of oxygen and light, no radicals were observed and there was no damage to the proteins. Similarly illumination of the apoproteins did not induce any polypeptide fragmentation, suggesting that chlorophyll, light and atmospheric oxygen are all participating in antenna degradation. The use of TEMP and DMPO as spin traps showed that protein damage initiates with generation of (1)O(2), presumably from a triplet chlorophyll, acting as a Type II photosensitizer which attacks directly the amino acids causing a complete degradation of protein into small fragments, without the contribution of proteases. Through the use of scavengers, it was shown that superoxide and H(2)O(2) were not involved initially in the reaction mechanism. A higher production of radicals was observed in trimers than in monomeric antenna, while radical production is strongly reduced when antennae were organized in the photosystem II (PSII) complex. Thus, monomerization of antennae as well as their incorporation into the PSII complex seem to represent physiologically protected forms. A comparison is made of the photoinhibition mechanisms of different photosynthetic systems.