The growth of plants, algae, and cyanobacteria relies on the catalytic activity of the oxygen-evolving PSII complex, which uses solar energy to extract electrons from water to feed into the photosynthetic electron transport chain. PSII is proving to be an excellent system to study how large multi-subunit membrane-protein complexes are assembled in the thylakoid membrane and subsequently repaired in response to photooxidative damage. Here we summarize recent developments in understanding the biogenesis of PSII, with an emphasis on recent insights obtained from biochemical and structural analysis of cyanobacterial PSII assembly/repair intermediates. We also discuss how chlorophyll synthesis is synchronized with protein synthesis and suggest a possible role for PSI in PSII assembly. Special attention is paid to unresolved and controversial issues that could be addressed in future research.

• MeSH
• Chlorophyll metabolism   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Cyanobacteria * metabolism   MeSH
• Thylakoids metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Chlorophyll MeSH
• Photosystem II Protein Complex * MeSH

In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.

• Keywords
• CP: Plants, Synechocystis, arginine metabolism, bilins, chlorophyll, genome-uncoupled-4, nitrogen homeostasis, tetrapyrrole biosynthesis,
• MeSH
• Arginine metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Nitrogen MeSH
• Ornithine MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arginine MeSH
• Chlorophyll MeSH
• Nitrogen MeSH
• Ornithine MeSH

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

• Keywords
• chlorophyll, cyanobacteria, cyclase, microevolution, photosynthesis,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bacteriochlorophylls biosynthesis   genetics   MeSH
• Gene Deletion * MeSH
• Oxygenases genetics   metabolism   MeSH
• Prochlorococcus genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Bacteriochlorophylls MeSH
• Oxygenases MeSH

Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.

• Keywords
• Synechocystis, carotenoid, chlorophyll, chloroplast, ferrochelatase, heme, light harvesting complex (LHC)-like proteins, membrane protein, photosynthesis, photosynthetic pigment, pigment binding, plant biochemistry,
• MeSH
• Chlorophyll A metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Dimerization MeSH
• Ferrochelatase chemistry   metabolism   MeSH
• Phylogeny MeSH
• Carotenoids metabolism   MeSH
• Protein Conformation MeSH
• Light-Harvesting Protein Complexes metabolism   MeSH
• Synechocystis enzymology   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chlorophyll A MeSH
• Chlorophyll MeSH
• chlorophyll b MeSH Browser
• Ferrochelatase MeSH
• Carotenoids MeSH
• Light-Harvesting Protein Complexes MeSH

Stress-induced senescence is a global agro-economic problem. Cytokinins are considered to be key plant anti-senescence hormones, but despite this practical function their use in agriculture is limited because cytokinins also inhibit root growth and development. We explored new cytokinin analogs by synthesizing a series of 1,2,3-thiadiazol-5-yl urea derivatives. The most potent compound, 1-(2-methoxy-ethyl)-3-1,2,3-thiadiazol-5-yl urea (ASES - Anti-Senescence Substance), strongly inhibited dark-induced senescence in leaves of wheat (Triticum aestivum L.) and Arabidopsis thaliana. The inhibitory effect of ASES on wheat leaf senescence was, to the best of our knowledge, the strongest of any known natural or synthetic compound. In vivo, ASES also improved the salt tolerance of young wheat plants. Interestingly, ASES did not affect root development of wheat and Arabidopsis, and molecular and classical cytokinin bioassays demonstrated that ASES exhibits very low cytokinin activity. A proteomic analysis of the ASES-treated leaves further revealed that the senescence-induced degradation of photosystem II had been very effectively blocked. Taken together, our results including data from cytokinin content analysis demonstrate that ASES delays leaf senescence by mechanism(s) different from those of cytokinins and, more effectively. No such substance has yet been described in the literature, which makes ASES an interesting tool for research of photosynthesis regulation. Its simple synthesis and high efficiency predetermine ASES to become also a potent plant stress protectant in biotechnology and agricultural industries.

• Keywords
• ASES, cytokinin, photosystem II, senescence, stress, thidiazuron, wheat,
• Publication type
• Journal Article MeSH

In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light-inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co-ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG-YidC/Alb3 complex may be evolutionarily conserved in algae and plants.

• Keywords
• Arabidopsis, YidC/Alb3/OxaI, chlorophyll, chlorophyll synthase, cyanobacteria, high light-inducible proteins,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Photosynthesis radiation effects   MeSH
• Photosystem II Protein Complex genetics   metabolism   MeSH
• Phylogeny MeSH
• Carbon-Oxygen Ligases classification   genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Light MeSH
• Synechocystis genetics   metabolism   MeSH
• Thylakoids metabolism   radiation effects   MeSH
• Protein Binding radiation effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ALBINO 3 protein, Arabidopsis MeSH Browser
• Bacterial Proteins MeSH
• chlorophyll synthetase MeSH Browser
• Photosystem II Protein Complex MeSH
• Carbon-Oxygen Ligases MeSH
• Arabidopsis Proteins MeSH

Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and β-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and β-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound β-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two β-carotenes.

• Keywords
• Chlorophyll, HLIPs, HliC, Raman spectroscopy, Synechocystis, β-Carotene,
• MeSH
• Bacterial Proteins chemistry   genetics   isolation & purification   metabolism   MeSH
• beta Carotene metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Protein Multimerization MeSH
• Spectrum Analysis, Raman MeSH
• Recombinant Proteins genetics   isolation & purification   metabolism   MeSH
• Light-Harvesting Protein Complexes genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   physiology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• beta Carotene MeSH
• Chlorophyll MeSH
• high light-inducible protein, cyanobacteria MeSH Browser
• Recombinant Proteins MeSH
• Light-Harvesting Protein Complexes MeSH

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Photosystem II Protein Complex genetics   metabolism   MeSH
• Mutation MeSH
• Ribosomes metabolism   MeSH
• Light-Harvesting Protein Complexes genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Chlorophyll MeSH
• Phosphoproteins MeSH
• Photosystem II Protein Complex MeSH
• photosystem II, chlorophyll-binding protein, CP-47 MeSH Browser
• photosystem II, psbH subunit MeSH Browser
• Light-Harvesting Protein Complexes MeSH

Cyanobacterium Synechocystis PCC 6803 represents a favored model organism for photosynthetic studies. Its easy transformability allowed construction of a vast number of Synechocystis mutants including many photosynthetically incompetent ones. However, it became clear that there is already a spectrum of Synechocystis "wild-type" substrains with apparently different phenotypes. Here, we analyzed organization of photosynthetic membrane complexes in a standard motile Pasteur collection strain termed PCC and two non-motile glucose-tolerant substrains (named here GT-P and GT-W) previously used as genetic backgrounds for construction of many photosynthetic site directed mutants. Although, both the GT-P and GT-W strains were derived from the same strain constructed and described by Williams in 1988, only GT-P was similar in pigmentation and in the compositions of Photosystem II (PSII) and Photosystem I (PSI) complexes to PCC. In contrast, GT-W contained much more carotenoids but significantly less chlorophyll (Chl), which was reflected by lower level of dimeric PSII and especially trimeric PSI. We found that GT-W was deficient in Chl biosynthesis and contained unusually high level of unassembled D1-D2 reaction center, CP47 and especially CP43. Another specific feature of GT-W was a several fold increase in the level of the Ycf39-Hlip complex previously postulated to participate in the recycling of Chl molecules. Genome re-sequencing revealed that the phenotype of GT-W is related to the tandem duplication of a large region of the chromosome that contains 100 genes including ones encoding D1, Psb28, and other PSII-related proteins as well as Mg-protoporphyrin methylester cyclase (Cycl). Interestingly, the duplication was completely eliminated after keeping GT-W cells on agar plates under photoautotrophic conditions for several months. The GT-W strain without a duplication showed no obvious defects in PSII assembly and resembled the GT-P substrain. Although, we do not exactly know how the duplication affected the GT-W phenotype, we hypothesize that changed stoichiometry of protein components of PSII and Chl biosynthetic machinery encoded by the duplicated region impaired proper assembly and functioning of these multi-subunit complexes. The study also emphasizes the crucial importance of a proper control strain for evaluating Synechocystis mutants.

• Keywords
• Synechocystis 6803, chlorophyll, large tandem duplication, photosystem I, photosystem II assembly,
• Publication type
• Journal Article MeSH

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

• Keywords
• Gun4, Mgprotoporphyrin IX, Synechocystis 6803, chlorophyll, cyanobacteria, docking, molecular modeling, porphyrin,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Chlorophyll biosynthesis   MeSH
• Circular Dichroism MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Mutation MeSH
• Porphyrins metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Synechocystis genetics   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Chlorophyll MeSH
• Porphyrins MeSH