光合放氧复合物结构及其放氧机理的研究

光合水氧化是地球上最重要的生化过程之一.光合放氧生物包括光系统Ⅰ(PSⅠ)和光系统Ⅱ(PSⅡ)两种类型反应中心,光系统Ⅱ反应中心能以水作为电子给体,利用光能氧化水产生质子和氧气.对于水如何被氧化这个难题前人已做了大量的工作,但到目前为止放氧复合物(OEC)的结构及水氧化的机理仍不清楚.本文结合当前研究结果,就光合放氧复合物的结构及光合放氧机理进行了综述,希望能有助于推进这方面的工作.     

Synchronous cultures of the green alga Scenedesmus obliquus: Comparison of picosecond decay associated emission spectra and fluorescence induction kinetics

Decay-associated emission spectra of synchronized cultures of Scenedesmusobliquus have been studied at two stages of their life cycle corresponding to the maximum and minimum of photosynthetic capacity. These decay-associated spectra comprise three kinetic components. The two components which are assigned to photosystem II show variations in their relative amplitudes depending on the life cycle of the cells. From the correlations observed in the decay-associated fluorescence spectra on the one hand and the fluorescence induction parameters on the other hand we obtained further evidence that the two photosystem II fluorescence components are directly related to the two fluorescence induction phases. This correlation supports our previous assignment of the two photosystem II fluorescence decay components of about 0.3 ns and about 0.6 ns lifetimes at the F₀ level (open photosystem II reaction centres to photosystem II α units and photosystem II β units respectively. The most pronounced difference between cells at the 8th hour of the life cycle and those at the 16th hour consists in the size of the photosystem II β units which are about 30% larger for the latter. In agreement with previous studies it was found that at these two stages the photosystem I units do not differ in size.     

Chlamydomonas reinhardtii genetic variants as probes for fluorescence sensing system in detection of pollutants

The unicellular green alga Chlamydomonas reinhardtii is employed here for the setup of a biosensor demonstrator based on multibiomediators for the detection of herbicides. The detection is based on the activity of photosystem II, the multienzymatic chlorophyll–protein complex located in the thylakoid membrane that catalyzes the light-dependent photosynthetic primary charge separation and the electron transfer chain in cyanobacteria, algae, and higher plants. Several C. reinhardtii mutants modified on the D1 photosystem II protein are generated by site-directed mutagenesis and experimentally tested for the development of a biosensor revealing the modification of the fluorescence parameter (1 − V _J) in the presence of herbicides. The A250R, A250L, A251C, and I163N mutants are highly sensitive to the urea and triazine herbicide classes; the newly generated F255N mutant is shown to be especially resistant to the class of urea. It follows that the response of the multibiomediators is associated to a particular herbicide subclass and can be useful to monitor several species of pollutants.     

Function of the Reaction Center of Green Sulfur Bacteria

The reaction center (RC) of green sulfur bacteria belongs to the Fe-S type RC, as do the photosystem I of oxygenic photosynthetic organisms and the RC of heliobacteria. The core parts of the green sulfur bacterial and the heliobacterial RC are assumed to be homodimeric, in contrast to those of purple bacteria, photosystem I and photosystem II. This paper describes recent advances in the study of the function of the green sulfur bacterial RC.     

Auxiliary functions of the PsbO, PsbP and PsbQ proteins of higher plant Photosystem II: a critical analysis

Numerous studies over the last 25 years have established that the extrinsic PsbO, PsbP and PsbQ proteins of Photosystem II play critically important roles in maintaining optimal manganese, calcium and chloride concentrations at the active site of Photosystem II. Chemical or genetic removal of these components induces multiple and profound defects in Photosystem II function and oxygen-evolving complex stability. Recently, a number of studies have indicated possible additional roles for these proteins within the photosystem. These include putative enzymatic activities, regulation of reaction center protein turnover, modulation of thylakoid membrane architecture, the mediation of PS II assembly/stability, and effects on the reducing side of the photosystem. In this review we will critically examine the findings which support these auxiliary functions and suggest additional lines of investigations which could clarify the nature of the functional interactions of these proteins with the photosystem.     

Langmuir-Blodgett and X-ray Diffraction Studies of Isolated Photosystem II Reaction Centers in Monolayers and Multilayers: Physical Dimensions of the Complex*

Abstract— The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm²and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm³. This value is very close to the expected volume of 118 nm³, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. PL Physiol. 22,161–166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.     

Psb30 is a photosystem II reaction center subunit and is required for optimal growth in high light in Chlamydomonas reinhardtii

The psb30 (ycf12) gene is conserved in a wide variety of oxygenic-photosynthetic organisms except angiosperms and some marine cyanobacteria. Psb30 protein is found in cyanobacterial photosystem II (PSII) core complexes and is dispensable for PSII structure and function. The most recent three-dimensional structure of cyanobacterial PSII core complex has revealed that Psb30 is located in proximity of PsbJ, PsbK, and PsbZ. However Psb30 has not yet been detected in PSII complexes from eukaryotic photosynthetic organisms. Here we found the expression of the chloroplast psb30 gene in the green alga Chlamydomonas reinhardtii by immunoblotting and Psb30 is exclusively co-purifies with PSII core complex and is significantly reduced in PSII-deficient mutants. Partial disintegration of PSII core complex and subsequent fractionation of the resulting subcomplexes revealed that Psb30 is exclusively associated with PSII reaction center. We have generated chloroplast transformants in which the psb30 gene is disrupted and the resulting ΔPsb30 cells showed decreased oxygen evolution activity by 15%, grew photosynthetically under moderate light, and displayed increased sensitivity to high light relative to wild type. We conclude that Psb30 is a PSII reaction center subunit and is required for optimal PSII function under high light environments.     

PROTECTION OF THE D1 PHOTOSYSTEM II REACTION CENTER PROTEIN FROM DEGRADATION IN ULTRAVIOLET RADIATION FOLLOWING ADAPTATION OF Brassica napus L. TO GROWTH IN ULTRAVIOLET-B

As depletion of the stratospheric ozone layer continues, the biosphere will most likely be exposed to higher levels of ultraviolet-B (UV-B) irradiation (290–320nm). For plants, damage from UV-B can occur at several molecular targets with the photosynthetic apparatus being especially vulnerable. We are interested both in the mechanisms of UV-B-induced damage and identifying adaptation processes that can confer protection from UV-B. Toward this end, Brassica napus (oil seed rape) plants grown under visible light plus a low level of UV-B radiation (adapted plants) were compared to plants grown under visible light alone (control plants). Relative to the control plants, the adapted plants showed little evidence of damage at the levels of morphology or photosynthesis, indicating that B. napus has some tolerance of UV-B and that the plants may have protection mechanisms. Consistent with this, a strong UV-B adaptation process was observed in the plants-accumulation of flavonoids in the epidermis. These pigments seemed to screen a molecular target in the mesophyll. Namely, the D1 photosystem II reaction center protein, which is rapidly degraded in UV-B, was partially protected from degradation in UV-B in the adapted plants. Moreover, the extent that the half-life of the D1 protein increased in the adapted plants was on par with the elevation in total flavonoid concentrations. These experiments demonstrate that degradation of the D1 protein can be used as an in vivo assay of penetration of UV-B photons to the mesophyll.     

High Temperature Affects Photosynthetic and Molecular Processes in Field‐Cultivated Vitis vinifera L. × Vitis labrusca L.

High‐temperature stress markedly influences grape growth and development. However, how high‐temperature stress response differs between controlled and field‐cultivated grape is poorly understood. In this study, the effects of heat treatment on grapevines were studied for changes in photosystem II (PSII) activity and expression levels of heat‐responsive genes and heat shock protein HSP21. July 31st, 2015 was considered as the post high‐temperature treatment (“42°C”; temperatures above 40°C for a period of time each day ranging from 1–7 h) under field cultivation in our experiment. The recovery of chlorophyll fluorescence indicators and the increasing expression of heat‐responsive genes and the heat shock protein HSP21 suggested the development of heat tolerance in the form of acclimation in grape. Changes in various parameters of photosynthetic pigment fluorescence and of the electron transport chain (Fv/Fm, PI_ABS, W_k, RC_QA, Φ_Po, and Φ_Eo) between “42°C” and the 45°C treatment demonstrated that the donor side, reaction center, and acceptor side of PSII were influenced by a critical high temperature. Furthermore, the difference between the two cultivation conditions studied was attributed to other environmental factors and inherent tree vigor.     

SINGLET OXYGEN IS NOT PRODUCED IN PHOTOSYSTEM I UNDER PHOTOINHIBITORY CONDITIONS

Abstract— Excess illumination of photosynthetic systems brings about the complex functional and structural damage known as photoinhibition. According to the generally accepted and experimentally confirmed model, photoinhibition involves singlet oxygen production and subsequent oxidative damage in the photosystem II reaction center. However, it was recently suggested that singlet oxygen is not necessarily produced in photosystem II itself but rather in the non-heme iron-containing Fe-S centers of photosystem I (Chung, S.K. & J. Jung, Photochem. Photobiol. 61, 383–389, 1995). Contrary to this suggestion, our electron paramagnetic resonance spectroscopy experiments with the singlet oxygen trap 2,2,6,6-tetramethylpiperidine demonstrate that under photoinhibitory conditions, singlet oxygen is present in thylakoids and photosystem II core complex preparations but is not produced in photosystem I particles.     

Bidirectional electron transfer in photosystem I: direct evidence from high-frequency time-resolved EPR spectroscopy

Efficient charge separation occurring within membrane-bound reaction center proteins is the most important step of photosynthetic solar energy conversion. All reaction centers are classified into two types, I and II. X-ray crystal structures reveal that both types bind two symmetric membrane-spanning branches of potential electron-transfer cofactors. Determination of the functional roles of these pairs of branches is of fundamental importance. While it is established that in type II reaction centers only one branch functions in electron transfer, we present the first direct spectroscopic evidence that both cofactor branches are active in the type I reaction center, photosystem I.     

Polymer-Modified Single-Walled Carbon Nanotubes Affect Photosystem II Photochemistry,Intersystem Electron Transport Carriers and Photosystem I End Acceptors in Pea Plants

Single-walled carbon nanotubes (SWCNT) have recently been attracting the attention of plant biologists as a prospective tool for modulation of photosynthesis in higher plants. However, the exact mode of action of SWCNT on the photosynthetic electron transport chain remains unknown. In this work, we examined the effect of foliar application of polymer-grafted SWCNT on the donor side of photosystem II, the intersystem electron transfer chain and the acceptor side of photosystem I. Analysis of the induction curves of chlorophyll fluorescence via JIP test and construction of differential curves revealed that SWCNT concentrations up to 100 mg/L did not affect the photosynthetic electron transport chain. SWCNT concentration of 300 mg/L had no effect on the photosystem II donor side but provoked inactivation of photosystem II reaction centres and slowed down the reduction of the plastoquinone pool and the photosystem I end acceptors. Changes in the modulated reflection at 820 nm, too, indicated slower re-reduction of photosystem I reaction centres in SWCNT-treated leaves. We conclude that SWCNT are likely to be able to divert electrons from the photosynthetic electron transport chain at the level of photosystem I end acceptors and plastoquinone pool in vivo. Further research is needed to unequivocally prove if the observed effects are due to specific interaction between SWCNT and the photosynthetic apparatus.     

Comparative Time-Resolved Photosystem II Chlorophyll a Fluorescence Analyses Reveal Distinctive Differences between Photoinhibitory Reaction Center Damage and Xanthophyll Cycle-Dependent Energy Dissipation*

Abstract— The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important pho-toprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (ΔpH) and the carotenoid pigment interconversions of the xanthophyll cycle. Co-incidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chi a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chi a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhi-bition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from ～2 ns to ～0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonra-diative decay path for the charge-separated state of the PSII reaction center. In contrast, the ΔpH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et ah, 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the ΔpH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.     

Structure–function relationships of the supramolecular assembly of the bacterial photosynthetic antenna complexes in lipid membranes

Appropriate experimental platforms are required to clarify the structure–function relationships of membrane protein assemblies. In photosynthetic bacteria, light-harvesting complex 2 and light-harvesting/reaction center core complex play key roles in capturing and transferring light energy and facilitating subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly in the photosynthetic membrane. However, the mechanism through which this assembly influences the efficiency of energy conversion remains to be clarified. We review our recent studies that were conducted to evaluate the structure–function relationship of the supramolecular assembly of photosynthetic antenna complexes in various lipid bilayer systems, as well as the construction of novel systems of planar lipid membranes for use as experimental platforms.     

Variants of photosystem II D1 protein in Thermosynechococcus elongatus

Cyanobacteria have several psbA genes encoding PsbA, the D1 reaction center protein of the photosystem II (PSII) complex which bears, with PsbD, the D2 protein, most of the cofactors involved in electron-transfer reactions. The thermophilic cyanobacterium Thermosynechococcus elongatus has three psbA genes differently expressed depending on the environmental conditions. Among the 344 residues constituting each of the three possible PsbA variants there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In this review, we briefly summarize the changes already identified in the properties of the redox cofactors depending on the D1 variant constituting PSII in T. elongatus.     

PICOSECOND FLUORESCENCE KINETICS and ENERGY TRANSFER IN THE ANTENNA CHLOROPHYLLS OF GREEN ALGAE*

Single-photon timing measurements on flowing samples of Chlorella vulgaris and Chlamydomonas reinhardtii at low excitation intensities at room temperature indicate two main kinetic components of the fluorescence at open reaction centers (F₀) of photosystem II with lifetimes of approx. 130 and 500 ps and relative yields of about 30 and 70%. Closing the reaction centers progressively by preincubation of the algae with increasing concentrations of 3-(3′,4′-dichlorophenyl)-l,l-dimethylurea (DCMU) and hydroxylamine gave rise to a slow component with a lifetime increasing from 1.4 to 2.2 ns (F_max) The yield of the slow component increased to 65-68% of the total fluorescence yield in parallel to a decrease in the yield of the fast component to a value close to zero at the f_max-level. The 130 ps lifetime of the fast component remained unchanged. The middle component showed an increase of its lifetime from 500 to 1100 ps and of its yield by a factor of 1.5. Spacing of the ps laser pulses by 12 μs allowed us to resolve a new long-lived fluorescence component of very small amplitude which is ascribed to a small amount of chlorophyll not connected to functional antennae. The opposite dependence of the yield of the fast and the slow component on the state of the reaction centers at almost constant lifetimes is consistent with a mechanism of energy conversion in largely separately functioning photosystem II units. Yields and lifetimes of these two components are in agreement with the high quantum yield of photosynthesis. The lower lifetime limit of 1.4 ns of the slow component is assigned to the average transfer time of an excited state from a closed to a neighboring open reaction center and the increase in the lifetime to 2.2 ns is evidence for a limited energy transfer between photosystems II. Relative effects of changing the excitation wavelength from 630 to 652 nm on the relative fluorescence yields of the kinetic components were studied at the fluorescence wavelengths 682, 703 and 730 nm. Our data indicate that (i) the middle component has its fluorescence maximum at shorter wavelength than the fast component and (ii) that the antennae chlorophylls giving rise to the middle component are preferentially excited by 652 nm light. It is concluded that the middle component originates from the light-harvesting chlorophyll alb protein complexes and the major portion of the fast component from the chlorophyll a antennae of open photosystem II reaction centers.     

SURFACE-ENHANCED RESONANCE RAMAN SCATTERING SPECTROSCOPY AS A SURFACE TOPOGRAPHY PROBE IN PLANT PHOTOSYNTHETIC MEMBRANES

Strong resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) signals from carotenoids were detected from thylakoid (stromal-side out) vesicles and inside-out (lumenal-side out) vesicles isolated from spinach chloroplasts. The intensity of the signals from both types of membranes was comparable, indicating that plant carotenoids are exposed on or close to both surfaces or sides of the thylakoid membrane. This is in contrast to previous studies with bacterial photosynthetic membranes (Picorel et al., 1988, J. Biol. Chem. 263 , 4374–4380; and 1990, Biochemistry 29 , 707–712) that show carotenoids selectively located on the cytoplasmic side. In addition_; strong RR and SERRS signals were detected from stacked and unstacked photosystem-II-enriched membrane fragments, demonstrating that carotenoids are also exposed on both surfaces of the appressed region of the thylakoid membrane. Antibodies against the photosystem (PS) II extrinsic proteins blocked SERRS signals from stacked PS II membrane fragments, but only partially affected the SERRS signals from unstacked membranes. The results indicate that these antibodies, which preferentially cover the surface of the original lumenalside of the appressed region, act as spacers between the membrane and SERRS electrode surfaces. The original stromal-side of the appressed region is unaffected. These findings verify the distance sensitivity of the SERRS technique and underscore the above conclusion about the location of carotenoids in the appressed regions. Finally, SERRS signals are sensitive to membrane aging and storage temperature; caution is suggested to those applying SERRS spectroscopy to intact membrane systems.     

Mass spectrometric characterization of photooxidative protein modifications in Arabidopsis thaliana thylakoid membranes

Oxidative and nitrosative stress leaves footprints in the plant chloroplast in the form of oxidatively modified proteins. Using a mass spectrometric approach, we identified 126 tyrosine and 12 tryptophan nitration sites in 164 nitrated proteolytic peptides, mainly from photosystem I (PSI), photosystem II (PSII), cytochrome b(6) /f and ATP-synthase complexes and 140 oxidation products of tyrosine, tryptophan, proline, phenylalanine and histidine residues. While a high number of nitration sites were found in proteins from four photosynthetic complexes indicating that the nitration belongs to one of the prominent posttranslational protein modifications in photosynthetic apparatus, amino acid oxidation products were determined mostly in PSII and to a lower extent in PSI. Exposure of plants to light stress resulted in an increased level of tyrosine and tryptophan nitration and tryptophan oxidation in proteins of PSII reaction center and the oxygen-evolving complex, as compared to low light conditions. In contrast, the level of nitration and oxidation of these amino acid residues strongly decreased for all light-harvesting proteins of PSII under the same conditions. Based on these data, we propose that oxidative modifications of proteins by reactive oxygen and nitrogen species might represent an important regulatory mechanism of protein turnover under light stress conditions, especially for PSII and its antenna proteins.     

UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities

The reaction center of photosystem II is susceptible to photodamage. In particular the D1 protein located in the photosystem II core has a rapid, light-dependent turnover termed the photosystem II repair cycle that, under illumination, degrades and resynthesizes D1 protein to limit accumulation of photodamaged photosystem II. Most studies concerning the effects of UVB (280-320 nm) on this cycle have been on cyanobacteria or specific phytoplankton species rather than on natural communities of phytoplankton. During a 5-year multidisciplinary project on the effects of UV radiation (200-400 nm) on natural systems, the effects of UVB on the D1 protein of natural phytoplankton communities were assessed. This review provides an overview of photoinhibitory effects of light on cultured and natural phytoplankton, with an emphasis on the interrelation of UVB exposure, D1 protein degradation and the repair of photosystem II through D1 resynthesis. Although the UVB component of the solar spectrum contributes to the primary photoinactivation of photosystem II, we conclude that, in natural communities, inhibition of the rate of the photosystem II repair cycle is a more important influence of UVB on primary productivity. Indeed, exposing tropical and temperate phytoplankton communities to supplemented UVB had more inhibitory effect on D1 synthesis than on the D1 degradation process itself. However, the rate of net D1 damage was faster for the tropical communities, likely because of the effects of high ambient light and water temperature on mechanisms of protein degradation and synthesis.