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The availability of neither D2 nor CP43 limits the biogenesis of photosystem II in tobacco.

ABSTRACT: The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5'-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.

SUBMITTER: Fu HY

PROVIDER: S-EPMC8133689 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Posttranslational modifications in the CP43 subunit of photosystem II.

Project description:Photosystem II (PSII) catalyzes the light-driven oxidation of water and the reduction of plastoquinone; the oxidation of water occurs at a cluster of four manganese. The PSII CP43 subunit functions in light harvesting, and mutations in the fifth luminal loop (E) of CP43 have established its importance in PSII structure and/or assembly [Kuhn, M. G. & Vermaas, V. F. J. (1993) Plant Mol. Biol. 23, 123-133]. The sequence A(350)PWLEPLR(357) in luminal loop E is conserved in CP43 genes from 50 organisms. To map important posttranslational modifications in this sequence, tandem mass spectrometry (MS/MS) was used. These data show that the indole side chain of Trp-352 is posttranslationally modified to give mass shifts of +4, +16, and +18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (+4), a keto-/amino-/hydroxy- (+16) derivative, and a dihydro-hydroxy- (+18) derivative of the indole side chain. Peptide synthesis and MS/MS confirmed the kynurenine assignment. The +16 and +18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The site-directed mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.

| S-EPMC137478 | biostudies-literature

Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II.

Project description:Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680(+), was similar to that of PSII complexes lacking the Mn(4)CaO(5) cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

| S-EPMC3497071 | biostudies-literature

Psb27, a transiently associated protein, binds to the chlorophyll binding protein CP43 in photosystem II assembly intermediates.

Project description:Photosystem II (PSII), a large multisubunit pigment-protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, mediates light-driven evolution of oxygen from water. Recently, a high-resolution X-ray structure of the mature PSII complex has become available. Two PSII polypeptides, D1 and CP43, provide many of the ligands to an inorganic Mn(4)Ca center that is essential for water oxidation. Because of its unusual redox chemistry, PSII often undergoes degradation followed by stepwise assembly. Psb27, a small luminal polypeptide, functions as an important accessory factor in this elaborate assembly pathway. However, the structural location of Psb27 within PSII assembly intermediates has remained elusive. Here we report that Psb27 binds to CP43 in such assembly intermediates. We treated purified genetically tagged PSII assembly intermediate complexes from the cyanobacterium Synechocystis 6803 with chemical cross-linkers to examine intermolecular interactions between Psb27 and various PSII proteins. First, the water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to cross-link proteins with complementary charged groups in close association to one another. In the His27△ctpAPSII preparation, a 58-kDa cross-linked species containing Psb27 and CP43 was identified. This species was not formed in the HT3△ctpA△psb27PSII complex in which Psb27 was absent. Second, the homobifunctional thiol-cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) was used to reversibly cross-link Psb27 to CP43 in His27△ctpAPSII preparations, which allowed the use of liquid chromatography/tandem MS to map the cross-linking sites as Psb27K(63)↔CP43D(321) (trypsin) and CP43K(215)↔Psb27D(58)AGGLK(63)↔CP43D(321) (chymotrypsin), respectively. Our data suggest that Psb27 acts as an important regulatory protein during PSII assembly through specific interactions with the luminal domain of CP43.

| S-EPMC3215006 | biostudies-literature

Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes.

Project description:Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.

| S-EPMC3083219 | biostudies-literature

Mass spectrometry-based footprinting reveals structural dynamics of loop E of the chlorophyll-binding protein CP43 during photosystem II assembly in the cyanobacterium Synechocystis 6803.

Project description:The PSII repair cycle is required for sustainable photosynthesis in oxygenic photosynthetic organisms. In cyanobacteria and higher plants, proteolysis of the precursor D1 protein (pD1) to expose a C-terminal carboxylate group is an essential step leading to coordination of the Mn4CaO5 cluster, the site of water oxidation. Psb27 appears to associate with both pD1- and D1-containing PSII assembly intermediates by closely interacting with CP43. Here, we report that reduced binding affinity between CP43 and Psb27 is triggered by the removal of the C-terminal extension of the pD1 protein. A mass spectrometry-based footprinting strategy was adopted to probe solvent-exposed aspartic and glutamic acid residues on the CP43 protein. By comparing the extent of footprinting between HT3ΔctpAΔ27PSII and HT3ΔctpAPSII, two genetically modified PSII assembly complexes, we found that Psb27 binds to CP43 on the side of Loop E distal to the pseudo-symmetrical D1-D2 axis. By comparing a second pair of PSII assembly complexes, we discovered that Loop E of CP43 undergoes a significant conformational rearrangement due to the removal of the pD1 C-terminal extension, altering the Psb27-CP43 binding interface. The significance of this conformational rearrangement is discussed in the context of recruitment of the PSII lumenal extrinsic proteins and Mn4CaO5 cluster assembly. In addition to CP43's previously known function as one of the core PSII antenna proteins, this work demonstrates that Loop E of CP43 plays an important role in the functional assembly of the Water Oxidizing Center (WOC) during PSII biogenesis.

| S-EPMC3656277 | biostudies-literature

The RUBISCO to Photosystem II Ratio Limits the Maximum Photosynthetic Rate in Picocyanobacteria.

Project description:Marine Synechococcus and Prochlorococcus are picocyanobacteria predominating in subtropical, oligotrophic marine environments, a niche predicted to expand with climate change. When grown under common low light conditions Synechococcus WH 8102 and Prochlorococcus MED 4 show similar Cytochrome b6f and Photosystem I contents normalized to Photosystem II content, while Prochlorococcus MIT 9313 has twice the Cytochrome b6f content and four times the Photosystem I content of the other strains. Interestingly, the Prochlorococcus strains contain only one third to one half of the RUBISCO catalytic subunits compared to the marine Synechococcus strain. The maximum Photosystem II electron transport rates were similar for the two Prochlorococcus strains but higher for the marine Synechococcus strain. Photosystem II electron transport capacity is highly correlated to the molar ratio of RUBISCO active sites to Photosystem II but not to the ratio of cytochrome b6f to Photosystem II, nor to the ratio of Photosystem I: Photosystem II. Thus, the catalytic capacity for the rate-limiting step of carbon fixation, the ultimate electron sink, appears to limit electron transport rates. The high abundance of Cytochrome b6f and Photosystem I in MIT 9313, combined with the slower flow of electrons away from Photosystem II and the relatively low level of RUBISCO, are consistent with cyclic electron flow around Photosystem I in this strain.

| S-EPMC4390859 | biostudies-other

Thylakoid protein FPB1 synergistically cooperates with PAM68 to promote CP47 biogenesis and Photosystem II assembly.

Project description:In chloroplasts, insertion of proteins with multiple transmembrane domains (TMDs) into thylakoid membranes usually occurs in a co-translational manner. Here, we have characterized a thylakoid protein designated FPB1 (Facilitator of PsbB biogenesis1) which together with a previously reported factor PAM68 (Photosynthesis Affected Mutant68) is involved in assisting the biogenesis of CP47, a subunit of the Photosystem II (PSII) core. Analysis by ribosome profiling reveals increased ribosome stalling when the last TMD segment of CP47 emerges from the ribosomal tunnel in fpb1 and pam68. FPB1 interacts with PAM68 and both proteins coimmunoprecipitate with SecY/E and Alb3 as well as with some ribosomal components. Thus, our data indicate that, in coordination with the SecY/E translocon and the Alb3 integrase, FPB1 synergistically cooperates with PAM68 to facilitate the co-translational integration of the last two CP47 TMDs and the large loop between them into thylakoids and the PSII core complex.

| S-EPMC11006888 | biostudies-literature

Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins.

Project description:Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls (Chls), pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound, Chl was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the Chls and both pheophytins. We suggest that Chl can be delivered to RCII from either monomeric Photosystem I or Ycf39/Hlips complexes.

| S-EPMC9157124 | biostudies-literature

Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II.

Project description:The Photosystem II reaction center is vulnerable to photoinhibition. The D1 and D2 proteins, lying at the core of the photosystem, are susceptible to oxidative modification by reactive oxygen species that are formed by the photosystem during illumination. Using spin probes and EPR spectroscopy, we have determined that both O2•- and HO• are involved in the photoinhibitory process. Using tandem mass spectroscopy, we have identified a number of oxidatively modified D1 and D2 residues. Our analysis indicates that these oxidative modifications are associated with formation of HO• at both the Mn4O5Ca cluster and the nonheme iron. Additionally, O2•- appears to be formed by the reduction of O2 at either PheoD1 or QA Early oxidation of D1:332H, which is coordinated with the Mn1 of the Mn4O5Ca cluster, appears to initiate a cascade of oxidative events that lead to the oxidative modification of numerous residues in the C termini of the D1 and D2 proteins on the donor side of the photosystem. Oxidation of D2:244Y, which is a bicarbonate ligand for the nonheme iron, induces the propagation of oxidative reactions in residues of the D-de loop of the D2 protein on the electron acceptor side of the photosystem. Finally, D1:130E and D2:246M are oxidatively modified by O2•- formed by the reduction of O2 either by PheoD1•- or QA•- The identification of specific amino acid residues oxidized by reactive oxygen species provides insights into the mechanism of damage to the D1 and D2 proteins under light stress.

| S-EPMC5358366 | biostudies-literature

An intermediate membrane subfraction in cyanobacteria is involved in an assembly network for Photosystem II biogenesis.

Project description:Early steps in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 are thought to occur in a specialized membrane fraction that is characterized by the specific accumulation of the PSII assembly factor PratA and its interaction partner pD1, the precursor of the D1 protein of PSII. Here, we report the molecular characterization of this membrane fraction, called the PratA-defined membrane (PDM), with regard to its lipid and pigment composition and its association with PSII assembly factors, including YCF48, Slr1471, Sll0933, and Pitt. We demonstrate that YCF48 and Slr1471 are present and that the chlorophyll precursor chlorophyllide a accumulates in the PDM. Analysis of PDMs from various mutant lines suggests a central role for PratA in the spatial organization of PSII biogenesis. Moreover, quantitative immunoblot analyses revealed a network of interdependences between several PSII assembly factors and chlorophyll synthesis. In addition, formation of complexes containing both YCF48 and Sll0933 was substantiated by co-immunoprecipitation experiments. The findings are integrated into a refined model for PSII biogenesis in Synechocystis 6803.

| S-EPMC3122249 | biostudies-literature

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