Photosystem II Reaction Center Complex Research Articles

Rubredoxin 1 Is Required for Formation of the Functional Photosystem II Core Complex in Arabidopsis thaliana.

Chloroplast thylakoid protein rubredoxin 1 (RBD1) in Chlamydomonas and its cyanobacterial homolog RubA contain a rubredoxin domain. These proteins have been proposed to participate in the assembly of photosystem II (PSII) at early stages. However, the effects of inactivation of RBD1 on PSII assembly in higher plants are largely unclear. Here, we characterized an Arabidopsis rbd1 mutant in detail. A drastic reduction of intact PSII complex but relatively higher levels of assembly intermediates including PSII RC, pre-CP47, and pre-CP43 were found in rbd1. Polysome association and ribosome profiling revealed that ribosome recruitment of psbA mRNA is specifically reduced. Consistently, in vivo protein pulse-chase labeling showed that the rate of D1/pD1 synthesis is significantly reduced in rbd1 compared with WT. Moreover, newly synthesized mature D1 and pD1/D2 can assemble into the PSII reaction center (RC) complex but further formation of larger PSII complexes is nearly totally blocked in rbd1. Our data imply that RBD1 is not only required for the formation of a functional PSII core complex during the early stages of PSII assembly but may also be involved in the translation of D1 in higher plants.

Elevated NO2 damages the photosynthetic apparatus by inducing the accumulation of superoxide anions and peroxynitrite in tobacco seedling leaves.

This study aimed to further understand the toxicity of high concentrations of nitrogen dioxide (NO2) to plants, especially to plant photosynthesis. Tobacco plants in the six-leaf stage were exposed to 16.0μLL-1 NO2 to determine the activities of photosystem II (PSII) and photosystem I (PSI) reaction centers, the blocking site of PSII electron transport, the degree of membrane peroxidation and the relative expression of PsbA, PsbO and PsaA genes in the third fully expanded leaves by using gas exchange and chlorophyll fluorescence techniques, biochemical and RT-PCR analysis. The results showed that 16.0μLL-1 NO2 caused necrotic lesions to form on leaves and significantly increased the generation rate of superoxide anions (O2-) and the content of peroxynitrite (ONOO-) in leaves of tobacco seedling, leading to damage to cell membrane, chlorophyll content and net photosynthetic rate reduction, and photosynthetic apparatus destruction. Fumigation with 16.0μLL-1 NO2 decreased the activity of PSII reaction center and oxygen evolution complex, and the relative expression of PabA in leaves of tobacco seedlings to inhibit the electron transport from the donor side to the receptor side of PSII, especially blocking the electron transport from QA to QB on the receptor side. The activity of the PSI reaction center and the relative expression of PsaA decreased, weakening the ability to accept electrons and inhibiting the electron transfer from PSII to PSI, which further increased the damage of PSII of tobacco seedling leaves caused by 16.0μLL-1 NO2. Therefore, 16.0μLL-1 NO2 leaded to the accumulation of O2- and ONOO-, which damaged the cell membrane and thylakoid membrane, inhibit the electron transport, and destroyed the photosynthetic apparatus in leaves of tobacco seedlings. The results from this study emphasized the importance of reducing the NO2 concentration in the atmosphere.

LPA19, a Psb27 Homolog in Arabidopsis thaliana, Facilitates D1 Protein Precursor Processing during PSII Biogenesis

The biogenesis and assembly of photosystem II (PSII) are mainly regulated by the nuclear-encoded factors. To further identify the novel components involved in PSII biogenesis, we isolated and characterized a high chlorophyll fluorescence low psii accumulation19 (lpa19) mutant, which is defective in PSII biogenesis. LPA19 encodes a Psb27 homolog (At1g05385). Interestingly, another Psb27 homolog (At1g03600) in Arabidopsis was revealed to be required for the efficient repair of photodamaged PSII. These results suggest that the Psb27 homologs play distinct functions in PSII biogenesis and repair in Arabidopsis. Chloroplast protein labeling assays showed that the C-terminal processing of D1 in the lpa19 mutant was impaired. Protein overlay assays provided evidence that LPA19 interacts with D1, and coimmunoprecipitation analysis demonstrated that LPA19 interacts with mature D1 (mD1) and precursor D1 (pD1). Moreover, LPA19 protein was shown to specifically interact with the soluble C terminus present in the precursor and mature D1 through yeast two-hybrid analyses. Thus, these studies suggest that LPA19 is involved in facilitating the D1 precursor protein processing in Arabidopsis.

The Cyanobacterial Homologue of HCF136/YCF48 Is a Component of an Early Photosystem II Assembly Complex and Is Important for Both the Efficient Assembly and Repair of Photosystem II in Synechocystis sp. PCC 6803

The role of the slr2034 (ycf48) gene product in the assembly and repair of photosystem II (PSII) has been studied in the cyanobacterium Synechocystis PCC 6803. YCF48 (HCF136) is involved in the assembly of Arabidopsis thaliana PSII reaction center (RC) complexes but its mode of action is unclear. We show here that YCF48 is a component of two cyanobacterial PSII RC-like complexes in vivo and is absent in larger PSII core complexes. Interruption of ycf48 slowed the formation of PSII complexes in wild type, as judged from pulse-labeling experiments, and caused a decrease in the final level of PSII core complexes in wild type and a marked reduction in the levels of PSII assembly complexes in strains lacking either CP43 or CP47. Absence of YCF48 also led to a dramatic decrease in the levels of the COOH-terminal precursor (pD1) and the partially processed form, iD1, in a variety of PSII mutants and only low levels of unassembled mature D1 were observed. Yeast two-hybrid analyses using the split ubiquitin system showed an interaction of YCF48 with unassembled pD1 and, to a lesser extent, unassembled iD1, but not with unassembled mature D1 or D2. Overall our results indicate a role for YCF48 in the stabilization of newly synthesized pD1 and in its subsequent binding to a D2-cytochrome b559 pre-complex, also identified in this study. Besides a role in assembly, we show for the first time that YCF48 also functions in the selective replacement of photodamaged D1 during PSII repair.

Role of the PsbI protein in photosystem II assembly and repair in the cyanobacterium Synechocystis sp. PCC 6803.

The involvement of the PsbI protein in the assembly and repair of the photosystem II (PSII) complex has been studied in the cyanobacterium Synechocystis sp. PCC 6803. Analysis of PSII complexes in the wild-type strain showed that the PsbI protein was present in dimeric and monomeric core complexes, core complexes lacking CP43, and in reaction center complexes containing D1, D2, and cytochrome b-559. In addition, immunoprecipitation experiments and the use of a histidine-tagged derivative of PsbI have revealed the presence in the thylakoid membrane of assembly complexes containing PsbI and either the precursor or mature forms of D1. Analysis of PSII assembly in the psbI deletion mutant and in strains lacking PsbI together with other PSII subunits showed that PsbI was not required for formation of PSII reaction center complexes or core complexes, although levels of unassembled D1 were reduced in its absence. However, loss of PsbI led to a dramatic destabilization of CP43 binding within monomeric and dimeric PSII core complexes. Despite the close structural relationship between D1 and PsbI in the PSII complex, PsbI turned over much slower than D1, whereas high light-induced turnover of D1 was accelerated in the absence of PsbI. Overall, our results suggest that PsbI is an early assembly partner for D1 and that it plays a functional role in stabilizing the binding of CP43 in the PSII holoenzyme.

Novel Evidence for a Reversible Dissociation of Light‐harvesting Complex II from Photosystem II Reaction Center Complex Induced by Saturating Light Illumination in Soybean Leaves

AbstractAfter saturating light illumination for 3 h the potential photochemical efficiency of photosystem II (PSII) (Fv/Fm, the ratio of variable to maximal fluorescence) decreased markedly and recovered basically to the level before saturating light illumination after dark recovery for 3 h in both soybean and wheat leaves, indicating that the decline in Fv/Fm is a reversible down‐regulation. Also, the saturating light illumination led to significant decreases in the low temperature (77 K) chlorophyll fluorescence parameters F685 (chlorophyll a fluorescence peaked at 685 nm) and F685/F735 (F735, chlorophyll a fluorescence peaked at 735 nm) in soybean leaves but not in wheat leaves. Moreover, trypsin (a protease) treatment resulted in a remarkable decrease in the amounts of PsbS protein (a nuclear gene psbS‐encoded 22 kDa protein) in the thylakoids from saturating light‐illuminated (SI), but not in those from dark‐adapted (DT) and dark‐recovered (DRT) soybean leaves. However, the treatment did not cause such a decrease in amounts of the PsbS protein in the thylakoids from saturating light‐illuminated wheat leaves. These results support the conclusion that saturating light illumination induces a reversible dissociation of some light‐harvesting complex II (LHCII) from PSII reaction center complex in soybean leaf but not in wheat leaf.

Effect of Eu(3+) on characterization of photosystem II particles from spinach by spectroscopy.

Photosystem II (PSII) particles were purified from Eu(3+)-treated spinach and studied by spectroscopy. The results showed that electron transport rate of PS II was accelerated by Eu3+ treating, that violet shift of the PSII Soret band or Q-band was 6 nm or 2 nm for the ultraviolet-visible (UV-Vis) spectrum, that the violet shift of the PSII fluorescence emission peak was 9 nm for fluorescence emission spectrum, that the PSII Signal II's of low-temperature electron paramagnetic resonance (EPR) spectrum was intensified under light, and that the PSII CD spectrum was similar to that of control. It is suggested that Eu(3+) might bind to the PSII reaction center complex and enhance the electron transport rate of PSII CD; however, Eu(3+) treatment does not change the configuration of the PSII reaction center complex.

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II.

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.

Assembly of the D1 Precursor in Monomeric Photosystem II Reaction Center Precomplexes Precedes Chlorophyll a–Triggered Accumulation of Reaction Center II in Barley Etioplasts

Assembly of plastid-encoded chlorophyll binding proteins of photosystem II (PSII) was studied in etiolated barley seedlings and isolated etioplasts and either the absence or presence of de novo chlorophyll synthesis. De novo assembly of reaction center complexes in etioplasts was characterized by immunological analysis of protein complexes solubilized from inner etioplast membranes and separated in sucrose density gradients. Previously characterized membrane protein complexes from chloroplasts were utilized as molecular mass standards for sucrose density gradient separation analysis. In etiolated seedlings, induction of chlorophyll a synthesis resulted in the accumulation of D1 in a dimeric PSII reaction center (RCII) complex. In isolated etioplasts, de novo chlorophyll a synthesis directed accumulation of D1 precursor in a monomeric RCII precomplex that also included D2 and cytochrome b(559). Chlorophyll a synthesis that was chemically prolonged in darkness neither increased the yield of RCII monomers nor directed assembly of RCII dimers in etioplasts. We therefore conclude that in etioplasts, assembly of the D1 precursor in monomeric RCII precomplexes precedes chlorophyll a-triggered accumulation of reaction center monomers.

Synthesis and assembly of the D1 protein into photosystem II: processing of the C-terminus and identification of the initial assembly partners and complexes during photosystem II repair.

In previous studies [van Wijk, K. J., Bingsmark, S., Aro, E.-M., & Andersson, B. (1995) J. Biol. Chem. 270, 25685-25695; van Wijk, K. J., Andersson, B., & Aro, E.-M. (1996) J. Biol. Chem 271, 9627-9636], we have demonstrated that D1 protein synthesized in isolated chloroplasts and thylakoids is incorporated into the photosystem II (PSII) core complex. By pulse-chase experiments in these in vitro systems, followed by sucrose gradient fractionation of solubilized thylakoid membranes, it was shown that this assembly proceeded stepwise; first the D1 protein was incorporated to form a PSII reaction center complex (PSII rc), and through additional assembly steps the PSII core complex was formed. In this study, we have analyzed this assembly process in more detail, with special emphasis on the initial events, through further purification and analysis of the assembly intermediates by nondenaturing Deriphat-PAGE and by flatbed isoelectric focusing. The D2 protein was found to be the dominant PSII reaction center protein initially associating with the new D1 protein. This strongly suggests that the D2 protein is the primary "receptor" or stabilizing component during or directly after synthesis of the D1 protein. After formation of the D1-D2 heterodimer, cyt b559 became attached, whereas the psbI gene product was assembled as a subsequent step, thereby forming a PSII reaction center complex. Subsequent formation of the PSII core occurred by binding of CP47 and then CP43 to the PSII rc. The rapid radiolabeling of a minor population of a PSII core subcomplex without CP43 indicated that an association of newly synthesized D1 protein with a preexisting complex consisting of D2/cyt b55q/psbI gene product/CP47 was possibly occurring, in parallel to the predominant sequential assembly pathway. The kinetics of synthesis and processing of the precursor D1 protein were followed in isolated chloroplasts and were compared with its incorporation into PSII assembly intermediates. No precursor D1 protein was found in PSII core complexes, indicating either that incorporation into the PSII core complex facilitates the cleavage of the C-terminus or, more likely, that processing is more rapid than the assembly into the PSII core.

Langmuir-Blodgett and X-ray diffraction studies of isolated photosystem II reaction centers in monolayers and multilayers: physical dimensions of the complex.

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 nm2 and 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.

Role of PSII-L protein (psbL gene product) on the electron transfer in photosystem II complex. 1. Over-production of wild-type and mutant versions of PSII-L protein and reconstitution into the PSII core complex.

To establish a system for over-production of PSII-L protein which is a component of photosystem II (PSII) complex, a plasmid designated as pMAL-psbL was constructed and expressed in Escherichia coli JM109. A fusion protein of PSII-L and maltose-binding proteins (53 kDa on SDS-PAGE) was accumulated in E. coli cells to a level of 10% of the total protein upon isopropyl-beta-D-thiogalactopyranoside (IPTG) induction. The carboxyl-terminal part of 5.0 kDa was cleaved from the fusion protein and purified by an anion exchange column chromatography in the presence of detergents. This 5.0 kDa protein was identified as PSII-L by amino-terminal amino acid sequence analysis and the chromatographic behavior on an anion exchange gel. A few types of mutant PSII-L were also prepared by the essentially same procedure except for using plasmids which contain given mutations in psbL gene. Plastoquinone-9 (PQ-9) depleted PSII reaction center core complex consisting of D1, D2, CP47, cytochrome b-559 (cyt b-559), PSII-I and PSII-W was reconstituted with PQ-9 and digalactosyldiglyceride (DGDG) together with the wild-type or mutant PSII-L produced in E. coli or isolated PSII-L from spinach. Significant difference between the wild-type PSII-L proteins from E. coli and spinach was not recognized in the effectiveness to recover the photo-induced electron transfer activity in the resulting complexes. The analysis of stoichiometry of PQ-9 per reaction center in the PQ-9 reconstituted PS II revealed that two molecules of PQ-9 were reinserted into a reaction center independent of the presence or absence of PSII-L. These results suggest that PSII-L recovers the electron transfer activity in the reconstituted RC by a mechanism different from the stabilization of PQ-9 in the Q(A) site of PSII. Ubiquinone-10 (UQ-10), but not plastoquinone-2 (PQ-2), substituted PQ-9 for recovering the PSII-L supported electron transfer activity in the reconstituted PSII reaction center complexes. The results obtained with the mutant PSII-L proteins revealed that the carboxyl terminal part rather than amino terminal part of PSII-L is crucial for recovering the electron transfer activity in the reconstituted complexes.

Kinetic Resolution of the Incorporation of the D1 Protein into Photosystem II and Localization of Assembly Intermediates in Thylakoid Membranes of Spinach Chloroplasts

The chloroplast-encoded D1 protein of photosystem II (PSII) has a much higher turnover rate than the other subunits of the PSII complex as a consequence of photodamage and subsequent repair of its reaction center. The replacement of the D1 protein in existing PSII complexes was followed in two in vitro translation systems consisting of isolated chloroplasts or isolated thylakoid membranes with attached ribosomes. By application of pulse-chase translation experiments, we followed translation elongation, release of proteins from the ribosomes, and subsequent incorporation of newly synthesized products into PSII (sub)complexes. The time course of incorporation of newly synthesized proteins into the different PSII (sub)complexes was analyzed by sucrose density gradient centrifugation. Immediately after termination of translation, the D1 protein was found both unassembled in the membrane as well as already incorporated into PSII reaction center complexes, possibly due to a cotranslational association of the D1 protein with other PSII reaction center components. Later steps in the reassembly of PSII were clearly post-translational and sequential. Different rate-limiting steps in the assembly process were found to be related to the depletion of nuclear encoded and stromal components as well as the lateral migration of subcomplexes within the heterogeneous thylakoid membrane. The slow processing of precursor D1 in the thylakoid translation system revealed that processing was not required for the assembly of the D1 protein into a PSII (sub)complex and that processing of the unassembled precursor could take place. The limited incorporation into PSII subcomplexes of three other PSII core proteins (D2 protein, CP43, and CP47) was clearly post-translational in both translation systems. Radiolabeled assembly intermediates smaller than the PSII core complex were found to be located in the stroma-exposed thylakoid membranes, the site of protein synthesis. Larger PSII assembly intermediates were almost exclusively located in the appressed regions of the membranes.

Oligonucleotide-directed mutagenesis of psbB, the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803

A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl alpha-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II.

Isolation of the photosystem II reaction center complex from barley. Characterization by circular dichroism spectroscopy and amino acid sequencing

A photosystem II (PSII) reaction center complex containing Chl a -protein 2, Chl a -protein 3, cytochrome b559, the herbicide-binding polypeptide and the 33 kD polypeptide from the oxygen-evolving site of PSII was isolated by sucrose gradient centrifugation of the dodecyl-β,d-maltoside extract of grana membranes highly enriched in PSII. The reaction center complex contained ca 19 lipid molecules per reaction center and sedimented slightly faster than catalase. It mediated light-induced, DCMU-sensitive electron transport from 1,5-diphenylcarbazide to dichlorophenolindophenol. The N-terminal amino acid sequence of a proteolytic fragment of Chl a -protein 2 was determined. Eighteen out of 20 amino acid residues were identical with the deduced amino acid sequence of the 51 kD Chl a -apoprotein from spinach. In contrast to grana membranes, which had a 77 K fluorescence emission maximum at 685 nm with a pronounced shoulder at 695 nm, the PSII reaction center complex had a single peak at 685 nm. Circular dichroism measurements in the visible range showed that the pigments associated with the isolated PSII reaction center complex and the light-harvesting complex (LHC) were highly oriented. The secondary structure of the PSII reaction center complex and LHC was investigated by circular dichroism measurements in the ultraviolet range. Both had a high α-helix content, suggesting that the chlorophyll molecules may be oriented between transmembrane helices in a similar way as in bacterial reaction centers and light harvesting systems.

Fluorescence Properties Indicate that Photosystem II Reaction Centers and Light-Harvesting Complex Are Modified by Low Temperature Growth in Winter Rye

Thylakoids isolated from winter rye (Secale cereale L. cv Puma) grown at 20 degrees C (nonhardened rye, RNH) or 5 degrees C (cold-hardened rye, RH) were characterized using chlorophyll (Chl) fluorescence. Low temperature fluorescence emission spectra of RH thylakoids contained emission bands at 680 and 695 nanometers not present in RNH thylakoids which were interpreted as changes in the association of light-harvesting Chl a/b proteins and photosystem II (PSII) reaction centers. RH thylakoids also exhibited a decrease in the emission ratio of 742/685 nanometers relative to RNH thylakoids.Room temperature fluorescence induction revealed that a larger proportion of Chl in RH thylakoids was inactive in transferring energy to PSII reaction centers when compared with RNH thylakoids. Fluorescence induction kinetics at 20 degrees C indicated that RNH and RH thylakoids contained the same proportions of fast (alpha) and slow (beta) components of the biphasic induction curve. In RH thylakoids, however, the rate constant for alpha components increased and the rate constant for beta components decreased relative to RNH thylakoids. Thus, energy was transferred more quickly within a PSII reaction center complex in RH thylakoids. In addition, PSII reaction centers in RH thylakoids were less connected, thus reducing energy transfers between reaction center complexes. We concluded that both PSII reaction centers and light-harvesting Chl a/b proteins had been modified during development of rye chloroplasts at 5 degrees C.