Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.     

Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism

Plants and algae have developed multiple protective mechanisms to survive under high light conditions. Thermal dissipation of excitation energy in the membrane-bound chlorophyll-antenna of photosystem II (PSII) decreases the energy arriving at the reaction center and thus reduces the generation of toxic photo-oxidative species. This process results in a decrease of PSII-related fluorescence emission, known as non-photochemical quenching (NPQ). It has always been assumed that cyanobacteria, the progenitor of the chloroplast, lacked an equivalent photoprotective mechanism. Recently, however, evidence has been presented for the existence of at least three distinct mechanisms for dissipating excess absorbed energy in cyanobacteria. One of these mechanisms, characterized by a blue-light-induced fluorescence quenching, is related to the phycobilisomes, the extramembranal antenna of cyanobacterial PSII. In this photoprotective mechanism the soluble carotenoid-binding protein (OCP) encoded by the slr1963 gene in Synechocystis sp. PCC 6803, of previously unknown function, plays an essential role. The amount of energy transferred from the phycobilisomes to the photosystems is reduced and the OCP acts as the photoreceptor and as the mediator of this antenna-related process. These are novel roles for a soluble carotenoid protein.     

A soluble carotenoid protein involved in phycobilisome-related energy dissipation in cyanobacteria

Photosynthetic organisms have developed multiple protective mechanisms to survive under high-light conditions. In plants, one of these mechanisms is the thermal dissipation of excitation energy in the membrane-bound chlorophyll antenna of photosystem II. The question of whether or not cyanobacteria, the progenitor of the chloroplast, have an equivalent photoprotective mechanism has long been unanswered. Recently, however, evidence was presented for the possible existence of a mechanism dissipating excess absorbed energy in the phycobilisome, the extramembrane antenna of cyanobacteria. Here, we demonstrate that this photoprotective mechanism, characterized by blue light-induced fluorescence quenching, is indeed phycobilisome-related and that a soluble carotenoid binding protein, ORANGE CAROTENOID PROTEIN (OCP), encoded by the slr1963 gene in Synechocystis PCC 6803, plays an essential role in this process. Blue light is unable to quench fluorescence in the absence of phycobilisomes or OCP. The fluorescence quenching is not DeltapH-dependent, and it can be induced in the absence of the reaction center II or the chlorophyll antenna, CP43 and CP47. Our data suggest that OCP, which strongly interacts with the thylakoids, acts as both the photoreceptor and the mediator of the reduction of the amount of energy transferred from the phycobilisomes to the photosystems. These are novel roles for a soluble carotenoid protein.     

The Supramolecular Structure of Photosystem II — Phycobilisome-Complexes of Porphyridium cruentum

The structure and arrangement of phycobilisomes of the unicellular red alga Porphyridium cruentum is compared with the organization of the thylakoid freeze-fracture particles in order to determine the relationship between phycobilisomes and photosystem II. The hemi-ellipsoidal phycobilisomes, 20 nm thick, are predominantly organized into rows; their centre to centre periodicity is 30–40 nm, so that they are well separated by a gap of 10–20 nm. The phycobilisomes are cleaved by a central faint furrow, parallel to the long axis from top to base. The organization of the exoplasmic particles in rows is similar to the arrangement of the phycobilisomes so that a structural relationship between both systems, previously demonstrated in cyanobacteria, is evident. Within the rows, the 10 nm EF-particles are grouped in tetrameric complexes separated by distances similar to those observed for phycobilisomes. We propose that the tetrameric EF-particle complexes correspond to tetrameric photosystem II complexes which bind one hemi-ellipsoidal phycobilisome on the stroma exposed surface of the thylakoid. A hypothetical model of this photosystem II-phycobilisome complex is presented.     

Cyanobacterial phycobilisomes

Cyanobacterial phycobilisomes harvest light and cause energy migration usually toward photosystem II reaction centers. Energy transfer from phycobilisomes directly to photosystem I may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the four chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by complementary chromatic adaptation is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The linkers govern the assembly of the biliproteins into phycobilisomes, and, even if colorless, in certain cases they have been shown to improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Details of how linkers perform their tasks are still topics of interest. The transfer of excitation energy from bilin to bilin is considered, particularly for monomers and trimers of C-phycocyanin, phycoerythrocyanin, and allophycocyanin. Phycobilisomes are one of the ways cyanobacteria thrive in varying and sometimes extreme habitats. Various biliprotein properties perhaps not related to photosynthesis are considered: the photoreversibility of phycoviolobilin, biophysical studies, and biliproteins in evolution. Copyright 1998 Academic Press.     

ACCLIMATION OF THE PHOTOSYNTHETIC APPARATUS OF PALMARIA PALMATA (RHODOPHYTA) TO LIGHT QUALITIES THAT PREFERENTIALLY EXCITE PHOTOSYSTEM I OR PHOTOSYSTEM II1

Acclimation of the photosynthetic apparatus to light absorbed primarily by phycobilisomes (which transfer energy predominantly to photosystem II) or absorbed by chlorophyll a (mainly present in the antenna of photosystem I) was studied in the macroalga Palmaria palmata L. In addition, the influence of blue and yellow light, exciting chlorophyll a and phycobilisomes, respectively, ivas investigated. All results were compared to a white light control. Complementary chromatic adaptation in terms of an enhanced ratio of phycoerythrin to phycocyanin under green light conditions was observed. Red light (mainly absorbed by chlorophyll a) and green light (mainly absorbed by phycobilisomes) caused an increase of the antenna system, which was not preferentially excited. Yellow and blue light led to intermediate states comparable to each other and white light. Growth was reduced under all light qualities in comparison to white light, especially under conditions preferably exciting phycobilisomes (green light-adapted algae had a 58% lower growth rate compared to white light-adapted algae). Red and blue light-adapted algae showed maximal photosynthetic capacity with white light excitation and significantly lower values with green light excitation. In contrast, green and yellow light-adapted algae exhibited comparable photosynthetic capacities at all excitation wavelengths. Low-temperature fluorescence emission analysis showed an increase of photosystem II emission in red light-adapted algae and a decrease in green light-adapted algae. A small increase of photosystem I emission teas also found in green light-adapted algae, but this was much less than the photosystem II emission increase observed in red light-adapted algae (both compared to phycobilisome emission). Efficiency of energy transfer from phycobilisomes to photosystem II was higher in red than in green light-adapted algae. The opposite was found for the energy transfer efficiency from phycobilisomes to photosystem I. Zeaxanthin content increased in green and blue light-adapted algae compared to red, white, and yellow light-adapted algae. Results are discussed in comparison to published data on unicellular red algae and cyanobacteria.     

A novel protein involved in the functional assembly of the oxygen-evolving complex of photosystem II in Synechocystis sp. PCC 6803

Mutation of Glu69 to Gln in the D2 protein of photosystem II is known to lead to a loss of photoautotrophic growth in Synechocystis sp. PCC 6803. However, second-site mutants (pseudorevertants) with restored photoautotrophic growth but still maintaining the E69Q mutation in D2 are easily obtained. Using a genomic mapping technique involving functional complementation, the secondary mutation was mapped to slr0286 in two independent mutants. The mutations in Slr0286 were R42M or R394H. To study the function of Slr0286, mutants of E69Q and of the wild-type strain were made that lacked slr0286. Deletion of slr0286 did not affect photoautotrophic capacity in wild type but led to a marked decrease in the apparent affinity of Ca(2+) to its binding site at the water-splitting system of photosystem II and to a reduced heat tolerance of the oxygen-evolving system, particularly in E69Q. Moreover, a small increase in the half-time for photoactivation of the oxygen-evolving complex of photosystem II for both wild type and the E69Q mutant was observed in the absence of Slr0286. The accumulation of photosystem II reaction centers, dark stability of the oxygen-evolving apparatus, stability of oxygen evolution, and the kinetics of charge recombination between Q(A)(-) and the donor side were not affected by deletion of slr0286. Slr0286 lacks clear functional motifs, and no homologues are apparent in other organisms, even not in other cyanobacteria. In any case, Slr0286 appears to help the functional assembly and stability of the water-splitting system of photosystem II.     

集胞藻PCC6803 EGY同源基因破坏突变体的构建及表型分析

摘要：拟南芥中近来发现的定位于叶绿体的膜嵌合金属蛋白酶EGY1影响叶绿体发育与脂肪酸合成,经生物信息学分析,集胞藻PCC6803 (Synechocystis sp. PCC6803)中slr0643、sll0862基因编码同源蛋白。【目的】为了鉴定这两个基因的功能,【方法】本文通过同源重组插入卡那霉素抗性基因、切断目的基因,分别构建了slr0643::km和sll0862::km两种突变体,检测突变体的生理生化表型。【结果】在30℃,20 μE/m2s自养培养下,slr0643::km与野生型相比,早期     

Functional linkage between phycobilisome and reaction center in two phycobilisome oxygen-evolving photosystem II preparations isolated from the thermophilic cyanobacterium Synechococcus sp

Photosystem II oxygen-evolving preparations with attached phycobilisomes were isolated from the thermophilic cyanobacterium Synechococcus sp. with beta-octylglucoside or digitonin. Fluorescence emission spectra of the two preparations determined at 77 K largely lacked a far red band which originates from photosystem I. The spectrum of the digitonin preparation was otherwise similar to that of intact cells, whereas the beta-octylglucoside preparation showed a pronounced band at 687 nm, which is considered to be emitted from phycobilisomes. The relative yield of phycobilin fluorescence was similar between the digitonin preparations and the cells but was considerably larger in the beta-octylglucoside preparations at room temperature. The quantum yield of ferricyanide photoreduction determined with light which is absorbed mainly by phycobiliproteins was 0.85 for the digitonin preparation and 0.57 for the beta-octylglucoside preparation. The results indicate that excitation energy is transferred from phycobilisomes to photosystem II reaction centers in the digitonin preparation as efficiently as in intact cells, while a significant portion of light energy harvested by phycobilisomes is not utilized by the primary photochemistry in the beta-octylglucoside preparation. Digitonin and beta-octylglucoside preparations had 65 and 48 chlorophyll a molecules per photosystem II reaction center, respectively. The beta-octylglucoside preparation contained twice as much phycocyanin and allophycocyanin per photosystem II reaction center as the digitonin preparation, which has a phycobiliprotein-to-photosystem II reaction center ratio very similar to that of cells. It is concluded that whereas the beta-octylglucoside preparation contains a considerable amount of free phycobilisomes, all phycobilisomes present in the digitonin preparation are physically and functionally linked to photosystem II reaction center complexes.     

Protective dissipation of excess absorbed energy by photosynthetic apparatus of cyanobacteria: role of antenna terminal emitters

Two mechanisms of photoprotective dissipation of the excessively absorbed energy by photosynthetic apparatus of cyanobacteria are described that divert energy from reaction centers. Energy dissipation, monitored as nonphotochemical fluorescence quenching, occurs at different steps of energy transfer within the phycobilisomes or core antenna of photosystem I. Although these mechanisms differ significantly, in both cases, energy dissipates mainly from terminal emitters: allophycocyanin B or core membrane linker protein (L_CM) in phycobilisomes, or the longest-wavelength chlorophylls in photosystem I antenna. It is supposed that carotenoid-induced energy dissipation in phycobilisomes is triggered by light-induced transformation of the nonquenched state of antenna into quenched state due to conformation changes caused by orange carotinoid-binding protein (OCP)–phycobilisome interaction. Fluorescence of the longest-wavelength chlorophylls of photosystem I antenna is strongly quenched by P700 cation radical or by P700 triplet state, dependent on redox state of the acceptor side cofactors of photosystem I.     

Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts

Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes. Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F. diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A. nidulans or C. reinhardtii; and complexes containing reaction center I of F. diplosiphon or C. reinhardtii. Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A. nidulans or C. reinhardtii. Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers. We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids. This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.     

Inactivation of the open reading frame slr0399 in Synechocystis sp. PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II. A possible role of Slr0399 as a chaperone for quinone binding.

The Synechocystis sp. PCC 6803 triple mutant D2R8 with V247M/A249T/M329I mutations in the D2 subunit of the photosystem II is impaired in Q(A) function, has an apparently mobile Q(A), and is unable to grow photoautotrophically. Several photoautotrophic pseudorevertants of this mutant have been isolated, each of which retained the original psbDI mutations of D2R8. Using a newly developed mapping technique, the site of the secondary mutations has been located in the open reading frame slr0399. Two different nucleotide substitutions and a deletion of about 60% of slr0399 were each shown to restore photoautotrophy in different pseudorevertants of the mutant D2R8, suggesting that inactivation of Slr0399 leads to photoautotrophic growth in D2R8. Indeed, a targeted deletion of slr0399 restores photoautotrophy in D2R8 and in other psbDI mutants impaired in Q(A) function. Slr0399 is similar to the hypothetical protein Ycf39, which is encoded in the cyanelle genome of Cyanophora paradoxa; in the chloroplast genomes of diatoms, dinoflagellates, and red algae; and in the nuclear genome of Arabidopsis thaliana. Slr0399 and Ycf39 have a NAD(P)H binding motif near their N terminus and have some similarity to isoflavone reductase-like proteins and to a subunit of the eukaryotic NADH dehydrogenase complex I. Deletion of slr0399 in wild type Synechocystis sp. PCC 6803 has no significant phenotypic effects other than a decrease in thermotolerance under both photoautotrophic and photomixotrophic conditions. We suggest that Slr0399 is a chaperone-like protein that aids in, but is not essential for, quinone insertion and protein folding around Q(A) in photosystem II. Moreover, as the effects of Slr0399 are not limited to photosystem II, this protein may also be involved in assembly of quinones in other photosynthetic and respiratory complexes.     

Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.     

The FtsH protease slr0228 is important for quality control of photosystem II in the thylakoid membrane of Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 contains four members of the FtsH protease family. One of these, FtsH (slr0228), has been implicated recently in the repair of photodamaged photosystem II (PSII) complexes. We have demonstrated here, using a combination of blue native PAGE, radiolabeling, and immunoblotting, that FtsH (slr0228) is required for selective replacement of the D1 reaction center subunit in both wild type PSII complexes and in PSII subcomplexes lacking the PSII chlorophyll a-binding subunit CP43. To test whether FtsH (slr0228) has a more general role in protein quality control in vivo, we have studied the synthesis and degradation of PSII subunits in wild type and in defined insertion and missense mutants incapable of proper assembly of the PSII holoenzyme. We discovered that, when the gene encoding FtsH (slr0228) was disrupted in these strains, the overall level of assembly intermediates and unassembled PSII proteins markedly increased. Pulse-chase experiments showed that this was due to reduced rates of degradation in vivo. Importantly, analysis of epitope-tagged and green fluorescent protein-tagged strains revealed that slr0228 was present in the thylakoid and not the cytoplasmic membrane. Overall, our results show that FtsH (slr0228) plays an important role in controlling the removal of PSII subunits from the thylakoid membrane and is not restricted to selective D1 turnover.     

Time-resolved fluorescence study of excitation energy transfer in the cyanobacterium Anabaena PCC 7120

Photosynthesis Research - Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been...     

The role of the FtsH and Deg proteases in the repair of UV-B radiation-damaged Photosystem II in the cyanobacterium Synechocystis PCC 6803

The photosystem two (PSII) complex found in oxygenic photosynthetic organisms is susceptible to damage by UV-B irradiation and undergoes repair in vivo to maintain activity. Until now there has been little information on the identity of the enzymes involved in repair. In the present study we have investigated the involvement of the FtsH and Deg protease families in the degradation of UV-B-damaged PSII reaction center subunits, D1 and D2, in the cyanobacterium Synechocystis 6803. PSII activity in a ΔFtsH (slr0228) strain, with an inactivated slr0228 gene, showed increased sensitivity to UV-B radiation and impaired recovery of activity in visible light after UV-B exposure. In contrast, in ΔDeg-G cells, in which all the three deg genes were inactivated, the damage and recovery kinetics were the same as in the WT. Immunoblotting showed that the loss of both the D1 and D2 proteins was retarded in ΔFtsH (slr0228) during UV-B exposure, and the extent of their restoration during the recovery period was decreased relative to the WT. However, in the ΔDeg-G cells the damage and recovery kinetics of D1 and D2 were the same as in the WT. These data demonstrate a key role of FtsH (slr0228), but not the Deg proteases, for the repair of PS II during and following UV-B radiation at the step of degrading both of the UV-B damaged D1 and D2 reaction center subunits.