Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Application of low temperatures during photoinhibition allows characterization of individual steps in photodamage and the repair of photosystem II

Recent investigations of photoinhibition have revealed that photodamage to photosystem II (PSII) involves two temporally separated steps: the first is the inactivation of the oxygen-evolving complex by light that has been absorbed by the manganese cluster and the second is the impairment of the photochemical reaction center by light that has been absorbed by chlorophyll. Our studies of photoinhibition in Synechocystis sp. PCC 6803 at various temperatures demonstrated that the first step in photodamage is not completed at low temperatures, such as 10°C. Further investigations suggested that an intermediate state, which is stabilized at low temperatures, might exist at the first stage of photodamage. The repair of PSII involves many steps, including degradation and removal of the D1 protein, synthesis de novo of the precursor to the D1 protein, assembly of the PSII complex, and processing of the precursor to the D1 protein. Detailed analysis of photodamage and repair at various temperatures has demonstrated that, among these steps, only the synthesis of the precursor to D1 appears to proceed at low temperatures. Investigations of photoinhibition at low temperatures have also indicated that prolonged exposure of cyanobacterial cells or plant leaves to strong light diminishes their ability to repair PSII. Such non-repairable photoinhibition is caused by inhibition of the processing of the precursor to the D1 protein after prolonged illumination with strong light at low temperatures.     

PsbT polypeptide is required for efficient repair of photodamaged photosystem II reaction center.

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the photosystem II (PSII) core complex. A psbT-deficient mutant (Delta psbT) of the green alga Chlamydomonas reinhardtii grows photoautotrophically, whereas its growth is significantly impaired in strong light. To understand the photosensitivity of Delta psbT, we have studied the effect of strong illumination on PSII activity and proteins. It is shown that the level of PSII activity and proteins is reduced in the Delta psbT more significantly than in wild type under strong light. When recovery of the photodamaged PSII is inhibited by a chloroplast protein synthesis inhibitor, the light-induced inactivation and degradation of PSII occur similarly in wild-type and mutant cells. On the contrary, the recovery of PSII activity after partial photoinactivation is remarkably delayed in the Delta psbT cells, suggesting that PsbT is required for efficient recovery of the photodamaged PSII complex. These results therefore present the first evidence for involvement of this small PSII polypeptide in the recovery process. Partial disintegration of the purified PSII core complex and localization of PSII proteins in the resulting PSII subcore complexes have revealed that PsbT is associated with D1/D2 heterodimer. A possible role of PsbT in the recovery process is discussed.     

A Psb27 homologue in Arabidopsis thaliana is required for efficient repair of photodamaged photosystem II

Psb27 has been identified as a lumenal protein associated with photosystem II (PSII). To gain insight into the function of Psb27, we isolated a mutant Arabidopsis plant with a loss of psb27 function. The quantity of PSII complexes and electron transfer within PSII remained largely unaffected in the psb27 mutant. Our results also showed that under high-light-illumination, PSII activity and the content of the PSII reaction center protein D1 decreased more significantly in the psb27 mutant than in wild-type (WT) plant. Treatment of leaves with a chloroplast protein synthesis inhibitor resulted in similar light-induced PSII inactivation levels and D1 protein degradation rates in the WT and psb27 mutant plants. Recovery of PSII activity after photoinhibition was delayed in the psb27 mutant, suggesting that Psb27 is required for efficient recovery of the photodamaged PSII complex. Overall, these results demonstrated that Psb27 in Arabidopsis is not essential for oxygenic photosynthesis and PSII formation. Instead, our results provide evidence for the involvement of this lumenal protein in the recovery process of PSII. Hua Chen and Dongyuan Zhang contribute equally to this work.     

Cost and benefit of the repair of photodamaged photosystem II in spinach leaves: roles of acclimation to growth light

When visible light is excess, the photosynthetic machinery is photoinhibited. The extent of net photoinhibition of photosystem II (PSII) is determined by a balance between the rate of photodamage to D1 and some other PSII proteins and the rate of the turnover cycle of these proteins. It is widely believed that the protein turnover requires much energy cost. The aims of this study are to (1) evaluate the energy cost of PSII repair, (2) measure the benefit in terms of photosynthetic gain realized by the repairing of the photodamaged PSII, and (3) know whether acclimation of photosynthesis to growth light affects the rates of the photodamage and repair. We grew spinach in high-light (HL) and low-light (LL) and measured the rates of D1 photodamage and repair in these leaves. We determined the rate constants of photodamage (k (pi)) and repair (k (rec)) by the PAM fluorometry in the presence or in the absence of lincomycin, an inhibitor of 70S protein synthesis. HL leaves showed smaller k (pi) and greater k (rec) than LL leaves. The energy cost of the repairing of the photodamaged D1 protein was <0.5?% of ATP produced by photophosphorylation at PPFDs ranging from 400 to 1600?μmol?m(-2)?s(-1) and was greater in HL leaves than in LL leaves. The benefits brought about by the repair were more than from 35 to 270 times the cost at PPFDs ranging from 400 to 1600?μmol?m(-2)?s(-1). The benefits of HL leaves were greater than those of LL leaves because of the higher photosynthesis rates in HL leaves. Running a simple simulation of daily photosynthesis using the parameters obtained in this study, we discuss why the plants need to pay the cost of D1 protein turnover to repair the photodamaged PSII.     

Systematic analysis of the relation of electron transport and ATP synthesis to the photodamage and repair of photosystem II in Synechocystis

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [(35)S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.     

A theoretical framework of the hybrid mechanism of photosystem II photodamage

Photosynthesis Research - Photodamage of photosystem II is a significant physiological process that is prevalent in the fields of photobiology, photosynthesis research and plant/algal stress. Since...     

Chlorophyll radical cation in photosystem II of chloroplasts. Millisecond decay at low temperature.

We compare the absorption changes, in the near infrared and in the green part of the spectrum, induced in spinach chloroplasts suspensions, at -- 170 degrees C, by continuous light and by flashes. (1) Following flash excitation, an absorption increase peaking at 825 nm which reverses rapidly (t 1/2 = 3.0 ms) is not affected by ferricyanide; it is suppressed when chloroplasts are preilluminated in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) and hydroxylamine. The reversion of that signal is simultaneous with a partial back reoxidation of C-550 (fully reduced by the flash) and with partial (about 25%) oxidation of cytochrome b559. The magnitude of the signal peaking at 825 nm (that we attribute to the radical cation of the trap chlorophyll of Photosystem II, acting as a primary electron donor) decreases progressively within a series of successive flashes. (2) An absorption increase (40% of which is slowly reversible) with a broad peak around 810 nm is induced by continuous light or by a flash. It is suppressed by pretreatment with ferricyanide, but it is little affected by the treatment with 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea and hydroxylamine. We attribute it to oxidized P700. (3) With chloroplasts pretreated with 10 mM ferricyanide, an absorption increase, whose magnitude is nearly independent of wavelength between 790 and 870 nm, can be induced by continuous light. One saturating flash produces only 20% of the signal. This absorption change (20% of which is reversible in 30 s) might be due to a secondary donor of Photosystem II.     

Role of charge recombination processes in photodamage and photoprotection of the photosystem II complex

Light-induced damage of the photosynthetic apparatus is an important and complex phenomenon, which affects primarily the photosystem II (PSII) complex. Here, the author summarizes the current state of understanding, which concerns the role of charge recombination reactions in photodamage and photoprotection. The main mechanism of photodamage induced by visible light appears to be mediated by acceptor side modifications, which develop under light intensity conditions when the capacity of light-independent photosynthetic processes limits the utilization of electrons produced in the initial photoreactions. This situation facilitates triplet chlorophyll formation and singlet oxygen production in the reaction center of PSII, which initiates the damage of electron transport components and protein structure. This mechanism is an important, but not exclusive, pathway of photodamage, and light-induced inactivation of the Mn cluster of water oxidation may occur in parallel with the singlet oxygen-dependent pathway.     

Tyrosine D oxidation at cryogenic temperature in photosystem II

Photosystem II (PSII) contains two redox-active tyrosines (TyrZ and TyrD) possessing very different properties. TyrD is stable in its oxidized form, while TyrZ is kinetically competent in the water oxidizing reaction. The temperature dependence of the formation of light-induced tyrosyl radical was studied as a function of pH in Mn-depleted PS II from cyanobacteria and plants. Tyrosyl radical formation was observed in the majority of the centers at pH 8.5 and 15 K. The use of two Synechocystis mutants where either TyrZ or TyrD was replaced by a phenylalanine residue allowed a clear demonstration that only TyrD and not TyrZ was oxidized under these conditions. By lowering the pH, the fraction of centers undergoing TyrD oxidation greatly diminished. This pH dependence could be fitted by assuming a single protonable group with a pK(a) of approximately 7.6. This pH dependence correlates with the unexpectedly rapid oxidation of TyrD at room temperature [Faller, P., Debus, R. J., Brettel, K., Sugiura, M., Rutherford, A. W., and Boussac, A. (2001) Proc. Nat. Acad. Sci. U.S.A. 98, 14368]. The equally unexpectedly ability to undergo oxidation at cryogenic temperatures reported here, puts limitations on the nature of the protonation reactions associated with electron transfer in this system, raising the possibility of proton tunneling along the hydrogen bond or alternatively the presence of deprotonated TyrD (tyrosinate) prior to oxidation.     

Parameterization of photosystem II photoinactivation and repair

The photoinactivation (also termed photoinhibition or photodamage) of Photosystem II (PSII) and the counteracting repair reactions are fundamental elements of the metabolism and ecophysiology of oxygenic photoautotrophs. Differences in the quantification, parameterization and terminology of Photosystem II photoinactivation and repair can erect barriers to understanding, and particular parameterizations are sometimes incorrectly associated with particular mechanistic models. These issues lead to problems for ecophysiologists seeking robust methods to include photoinhibition in ecological models. We present a comparative analysis of terms and parameterizations applied to photoinactivation and repair of Photosystem II. In particular, we show that the target size and quantum yield approaches are interconvertible generalizations of the rate constant of photoinactivation across a range of incident light levels. Our particular emphasis is on phytoplankton, although we draw upon the literature from vascular plants. This article is part of a Special Issue entitled: Photosystem II.     

Photoinhibition of hydroxylamine-extracted photosystem II membranes: identification of the sites of photodamage.

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P680+ were made of NH2OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D.J., & Cheniae, G.M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K3Fe(CN)6, gave evidence for rapid (t1/2 = 2-3 min) and slow (t1/2 = 3-4) losses of formation of the tyrosyl radicals YZ+ and YD+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 degrees C in subsequent darkness in rather constant abundance (approximately 1/2 spin per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH2OH greater than or equal to NH2NH2 greater than DPC much greater than Mn2+) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P680+, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P680, via pheophytin (Pheo), to the first stable electron acceptor, QA. However, a 4-microsecond decay of P680+, reflecting YZ----P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140 microsecond decay, presumably reflecting QA-/P680+ back-reaction. Photoinhibition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O2 evolution is impaired is Chl/Car greater than YZ greater than YD much greater than P680, Pheo, QA.     

Ammonia triggers photodamage of photosystem II in the cyanobacterium Synechocystis sp. strain PCC 6803

Ammonia has long been known to be toxic for many photosynthetic organisms; however, the target for its toxicity remains elusive. Here, we show that in the cyanobacterium Synechocystis sp. strain PCC 6803, ammonia triggers a rapid photodamage of photosystem II (PSII). Whereas wild-type cells can cope with this damage by turning on the FtsH2-dependent PSII repair cycle, the FtsH2-deficient mutant is highly sensitive and loses PSII activity at millimolar concentration of ammonia. Ammonia-triggered PSII destruction is light dependent and occurs already at low photon fluence rates. Experiments with monochromatic light showed that ammonia-promoted PSII photoinhibition is executed by wavebands known to directly destroy the manganese cluster in the PSII oxygen-evolving complex, suggesting that the oxygen-evolving complex may be a direct target for ammonia toxicity.     

Herbicide effect on the photodamage process of photosystem II: fourier transform infrared study

The photodamage process of photosystem II by strong illumination was investigated by examining the herbicide effects on the photoinactivation of redox cofactors. O(2)-evolving photosystem II membranes from spinach in the absence of herbicide and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and bromoxynil were subjected to strong white-light illumination at 298K, and the illumination-time dependence of the activities of Q(A), the Mn cluster, and P680 were monitored using light-induced Fourier transform infrared (FTIR) difference spectroscopy. The decrease in the Q(A) activity was suppressed and accelerated by DCMU and bromoxynil, respectively, in comparison with the sample without herbicide. The intensity change in the S(2)/S(1) FTIR signal of the Mn cluster exhibited a time course virtually identical to that in the Q(A) signal in all the three samples, suggesting that the loss of the S(1)→S(2) transition was ascribed to the Q(A) inactivation and hence the Mn cluster was inactivated not faster than Q(A). The decrease in the P680 signal was always slower than that of Q(A) keeping the tendency of the herbicide effect. Degradation of the D1 protein occurred after the P680 inactivation. These observations are consistent with the acceptor-side mechanism, in which double reduction of Q(A) triggers the formation of (1)O(2)* to promote further damage to other cofactors and the D1 protein, rather than the recently proposed mechanism that inactivation of the Mn cluster initiates the photodamage. Thus, the results of the present study support the view that the acceptor-side mechanism dominantly occurs in the photodamage to PSII by strong white-light illumination.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

A relationship between the midpoint potential of the primary acceptor and low temperature photochemistry in photosystem II

Redox titrations of the photo-induced pheophytin EPR signal in Photosystem II show two transitions which reflect the redox state of Q. The high potential wave (E_m ? ?50 mV) can be photo-induced at 5 K and 77 K. The low potential wave (E_m ? ?275 mV) required illumination at 200 K. This indicates the presence of two kinds of PS-II reaction centres differing in terms of the competence of their donors at low temperature and the E_m-values of their acceptors. Measurements of the semiquinone-iron acceptor also demonstrate functional heterogeneity at low temperature. This is the first observation of the semiquinone-iron acceptor in a non-mutant species.