Biogenesis of chlorophyll-binding proteins under iron stress in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The biogenesis of chlorophyll-binding proteins under iron stress has been investigated in vivo in a chlN deletion mutant of Synechocystis sp. PCC 6803. The chlN gene encodes one subunit of the light-independent protochlorophyllide reductase. The mutant is unable to synthesize chlorophyll in darkness, causing chlorophyll biosynthesis to become light dependent. When the mutant was propagated in darkness, essentially no chlorophyll and photosystems were detected. Upon return of the chlN deletion mutant to light, 77 K fluorescence emission spectra and oxygen evolution of greening cells under iron-sufficient or-deficient conditions were measured. The gradual blue shift of the photosystem I (PS I) peak upon greening under iron stress suggested the structural alteration of newly synthesized PS I. Furthermore, the rate of biogenesis of PS II was delayed under iron stress, which might be due to the presence of IsiA.     

Biogenesis of chlorophyll-binding proteins under iron stress in Synechocystis sp. PCC 6803

The biogenesis of chlorophyll-binding proteins under iron stress has been investigated in vivo in a chlN deletion mutant of Synechocystis sp. PCC 6803. The chlN gene encodes one subunit of the light-independent protochlorophyllide reductase. The mutant is unable to synthesis chlorophyll in darkness, causing chlorophyll biosynthesis to become light dependent. When the mutant was propagated in darkness, essentially no chlorophyll and photosystems were detected. Upon return of the chlN deletion mutant to light, 77 K fluorescence emission spectra and oxygen evolution of greening cells under iron-sufficient or -deficient conditions were measured. The gradual blue shift of the photosystem I (PS I) peak upon greening under iron stress suggested the structural alteration of newly synthesized PS I. Furthermore, the rate of biogenesis of PS II was delayed under iron stress, which might be due to the presence of IsiA.     

Functional characteristics of green alga Scenedesmus obliquus (Chlorophyceae): 276–6 wild type and its two photosystems deficient mutants cultured under photoautotrophic,mixotrophic and heterotrophic conditions

Functional features of Scenedesmus obliquus: wild type 276–6 strain (WT) and its two mutants reported as photosystem I‐deficient (mutant 56.80) and photosystem II‐deficient (mutant 57.80) were characterized. Algae were cultured aseptically under continuous light or in darkness on mineral bold basal medium (BBM), yeast extract‐enriched BBM and yeast extract to evaluate the physiology of algal cells under photoautotrophic, mixotrophic and heterotrophic conditions. Growth, superoxide dismutase activity and photosynthetic parameters, including polyphasic fluorescence rise during the first seconds of chlorophyll a illumination (OJIP), were analyzed to find relationships between the photosynthetic/respiratory activity of the cells, occurrence of oxidative stress and trophic conditions applied to PSs‐deficient algae. Despite the highest superoxide dismutase activity, indicating the presence of oxidative stress, mixotrophic conditions appeared to be optimal for S. obliquus WT and mutant strains kept in non‐aerated cultures. OJIP analysis indicated that in mutant 56.80 part of photosystem (PS) I was functional and in mutant 57.80 residual PS II activity was found.     

A comparative fluorescence kinetics study of Photosystem I monomers and trimers from Synechocystis PCC 6803

Picosecond time-resolved fluorescence measurements have been performed as a function of emission wavelengths in order to investigate the possible functional differences between monomeric and trimeric Photosystem I (PS I) particles from a cyanobacterium Synechocystis. Applying global analysis, four kinetic components were found necessary to describe the fluorescecne decay for both monomers and trimers of PS I. The lifetimes and spectra of the respective components are quite similar, indicating that they can be attributed to identical processes in both the monomers and trimers. It is concluded that both forms of PS I are capable of efficient energy transfer and charge separation, in agreement with a physiological role of both forms. Small differences in the fluorescence decays are discussed in terms of a slightly higher ratio of red emitting pigments per reaction centre in trimers of PS I. A comparison to Synechococcus PS I particles reveals the higher red chlorophyll content of the latter.Abbreviations -DM- -dodecyl-maltoside - Chl- chlorophyll - CMC- critical micellar concentration - DAS- decay-associated spectrum - DCM- 4-dicyano-methylene-2-methyl-6-(-dimethyl-aminostyryl)-4h-pyran - FWHM- full-width at half-maximum - P700- primary electron donor of Photosystem I - PS- photosystem - RC- reaction centre     

In vitro oligomerization of a membrane protein complex. liposome-based reconstitution of trimeric photosystem I from isolated monomers.

Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacterium Spirulina platensis, trimers in contrast to monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platensis with n-dodecyl beta-D-maltoside and further purified by perfusion chromatography steps. The isolated complexes had the same polypeptide composition as other cyanobacteria (PsaA-PsaF and PsaI-PsaM), as determined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emission at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high performance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the formation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.     

Late assembly steps and dynamics of the cyanobacterial photosystem I

The dynamics of photosystem I assembly in cyanobacteria have been addressed using in vivo pulse-chase labeling of Synechocystis sp. PCC 6803 proteins in combination with blue native polyacrylamide gel electrophoresis. The analyses indicate the existence of three different monomeric photosystem I complexes and also the high stability of photosystem I trimers. We show that in addition to a complete photosystem I monomer, containing all 11 subunits, we detected a PsaK-less monomer and a short-lived PsaL/PsaK-less complex. The latter two monomers were missing in the ycf37 mutant of Synechocystis sp. PCC 6803 that accumulates also less trimers. Pulse-chase experiments suggest that the three monomeric complexes have different functions in the biogenesis of the trimer. Based on these findings we propose a model where PsaK is incorporated in the latest step of photosystem I assembly. The PsaK-less photosystem I monomer may represent an intermediate complex that is important for the exchange of the two PsaK variants during high light acclimation. Implications of the presented data with respect to Ycf37 function are discussed.     

Organization and role of the long-wave chlorophylls in the photosystem I of the Cyanobacterium spirulina.

The data on the organization and function of the photosystem I pigment-protein complexes of the cyanobacterium Spirulina and the characteristics of pigment antenna of the photosystem I monomeric and trimeric core complexes are presented and discussed. We proved that the photosystem I complexes in the cyanobacterial membrane pre-exist mainly as trimers, though both types of complexes contribute to the photosynthetic electron transport. In contrast to monomers, the antenna of the photosystem I trimeric complexes of Spirulina contains the extreme long-wave chlorophyll form absorbing at 735 nm and emitting at 760 nm (77 K). The intensity of fluorescence at 760 nm depends strongly on the P700 redox state: it is maximum with the reduced P700 and strongly decreased with the oxidized P700 which is the most efficient quencher of fluorescence at 760 nm. The energy absorbed by the extreme long-wave chlorophyll form is active in the photooxidation of P700 in the trimeric complex. The data obtained indicate that the long-wave form of chlorophyll originates from interaction of the chlorophyll molecules localized on monomeric subunits forming the photosystem I trimer. Kinetic analysis of the P700 photooxidation and light-induced quenching of fluorescence at 760 nm (77 K) allows the suggestion that the excess energy absorbed by the antenna monomeric subunits within the trimer migrates via the extreme long-wave chlorophyll to the P700 cation radical and is quenched, which prevents the photodestruction of the pigment-protein complex.     

Recruitment of a foreign quinone into the A(1) site of photosystem I. I. Genetic and physiological characterization of phylloquinone biosynthetic pathway mutants in Synechocystis sp. pcc 6803

Genes encoding enzymes of the biosynthetic pathway leading to phylloquinone, the secondary electron acceptor of photosystem (PS) I, were identified in Synechocystis sp. PCC 6803 by comparison with genes encoding enzymes of the menaquinone biosynthetic pathway in Escherichia coli. Targeted inactivation of the menA and menB genes, which code for phytyl transferase and 1,4-dihydroxy-2-naphthoate synthase, respectively, prevented the synthesis of phylloquinone, thereby confirming the participation of these two gene products in the biosynthetic pathway. The menA and menB mutants grow photoautotrophically under low light conditions (20 microE m(-2) s(-1)), with doubling times twice that of the wild type, but they are unable to grow under high light conditions (120 microE m(-2) s(-1)). The menA and menB mutants grow photoheterotrophically on media supplemented with glucose under low light conditions, with doubling times similar to that of the wild type, but they are unable to grow under high light conditions unless atrazine is present to inhibit PS II activity. The level of active PS II per cell in the menA and menB mutant strains is identical to that of the wild type, but the level of active PS I is about 50-60% that of the wild type as assayed by low temperature fluorescence, P700 photoactivity, and electron transfer rates. PS I complexes isolated from the menA and menB mutant strains contain the full complement of polypeptides, show photoreduction of F(A) and F(B) at 15 K, and support 82-84% of the wild type rate of electron transfer from cytochrome c(6) to flavodoxin. HPLC analyses show high levels of plastoquinone-9 in PS I complexes from the menA and menB mutants but not from the wild type. We propose that in the absence of phylloquinone, PS I recruits plastoquinone-9 into the A(1) site, where it functions as an efficient cofactor in electron transfer from A(0) to the iron-sulfur clusters.     

State of the phycobilisome determines effective absorption cross-section of Photosystem II in Synechocystis sp. PCC 6803

Quenching of excess excitation energy is necessary for the photoprotection of light-harvesting complexes. In cyanobacteria, quenching of phycobilisome (PBS) excitation energy is induced by the Orange Carotenoid Protein (OCP), which becomes photoactivated under high light conditions. A decrease in energy transfer efficiency from the PBSs to the reaction centers decreases photosystem II (PS II) activity. However, quantitative analysis of OCP-induced photoprotection in vivo is complicated by similar effects of both photochemical and non-photochemical quenching on the quantum yield of the PBS fluorescence overlapping with the emission of chlorophyll. In the present study, we have analyzed chlorophyll a fluorescence induction to estimate the effective cross-section of PS II and compared the effects of reversible OCP-dependent quenching of PBS fluorescence with reduction of PBS content upon nitrogen starvation or mutations of key PBS components. This approach allowed us to estimate the dependency of the rate constant of PS II primary electron acceptor reduction on the amount of PBSs in the cell. We found that OCP-dependent quenching triggered by blue light affects approximately half of PBSs coupled to PS II, indicating that under normal conditions, the concentration of OCP is not sufficient for quenching of all PBSs coupled to PS II.     

Interaction of phycobilisomes with photosystem II dimers and photosystem I monomers and trimers in the cyanobacterium Spirulina platensis.

Distribution of phycobilisomes between photosystem I (PSI) and photosystem II (PSII) complexes in the cyanobacterium Spirulina platensis has been studied by analysis of the action spectra of H2 and O2 photoevolution and by analysis of the 77 K fluorescence excitation and emission spectra of the photosystems. PSI monomers and trimers were spectrally discriminated in the cell by the unique 760 nm low-temperature fluorescence, emitted by the trimers under reductive conditions. The phycobilisome-specific 625 nm peak was observed in the action spectra of both PSI and PSII, as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 695 nm (PSII), 730 nm (PSI monomers), and 760 nm (PSI trimers). The contributions of phycobilisomes to the absorption, action, and excitation spectra were derived from the in vivo absorption coefficients of phycobiliproteins and of chlorophyll. Analyzing the sum of PSI and PSII action spectra against the absorption spectrum and estimating the P700:P680 reaction center ratio of 5.7 in Spirulina, we calculated that PSII contained only 5% of the total chlorophyll, while PSI carried the greatest part, about 95%. Quantitative analysis of the obtained data showed that about 20% of phycobilisomes in Spirulina cells are bound to PSII, while 60% of phycobilisomes transfer the energy to PSI trimers, and the remaining 20% are associated with PSI monomers. A relevant model of organization of phycobilisomes and chlorophyll pigment-protein complexes in Spirulina is proposed. It is suggested that phycobilisomes are connected with PSII dimers, PSI trimers, and coupled PSI monomers.     

Isolation of state transition mutants of Chlamydomonas reinhardtii by fluorescence video imaging

Oxygenic photosynthetic organisms adapt to varying light conditions by changing the distribution of light energy between Photosystem II (PS II) and photosystem I (PS I) during so-called state transitions. To identify the genes involved in this process, we have exploited a simple chlorophyll fluorescence video-imaging technique to screen a library of nuclear mutants of Chlamydomonas reinhardtii for colonies grown on agar plates that are disturbed in their ability to regulate light energy distribution between PS I and PS II. Subsequent modulated fluorescence measurements at room temperature and 77 K fluorescence emission spectra confirmed that 5 mutants (0.025% of total number screened) were defective in state transitions. [³²P]orthophosphate phosphorylation experiments in vivo revealed that in one of these mutants, designated stm1, the level of LHC II polypeptide phosphorylation was drastically reduced compared with wild type. Despite WT levels of PS I and PS II, stm1 grew photoautotrophically at reduced rates, compared with WT especially under low light conditions, which is consistent with an important physiological role for state transitions. Our results highlight the feasibility of video imaging in tandem with mutagenesis as a means of identifying the genes involved in controlling state transitions in eukaryotic photosynthetic organisms.     

Spectral properties of the CP43-deletion mutant of Synechocystis sp. PCC 6803

Spectral properties, particularly fluorescence spectra and their time-dependent behavior, were investigated for a mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the 43 kDa chlorophyll-protein (CP43, PsbC). Lack of CP43 was confirmed by a size shift of the corresponding gene and by Western blotting. The CP43-deletion mutant grown under heterotrophic conditions accumulated a small amount of photosystem (PS) II, but virtually no PS II fluorescence was observed. A 686-nm fluorescence band was clearly observed by phycocyanin excitation, coming from the terminal pigments of phycobilisomes. In contrast, no PS I fluorescence was detected by phycocyanin excitation when accumulation of PS II components was not proved by a fluorescence excitation spectrum, indicating that energy transfer to PS I chlorophyll a was mediated by PS II chlorophyll a. Direct connection of phycobilisomes with PS I was not suggested. Based on these fluorescence properties, the energy flow in the CP43-deletion mutant cells is discussed.     

State transitions,photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F relative fluorescence intensity at emission wavelength nm - F _o fluorescence intensity when all PS II traps are open - light 1 light absorbed preferentially by PS I - light 2 light absorbed preferentially by PS II - PBS phycobilisome - PS photosystem     

Xanthophyll cycle mutants from Chlamydomonas reinhardtii indicate a role for zeaxanthin in the D1 protein turnover

Photosynthetic activity, pigment conversion and D1 protein degradation under high light stress has been investigated in a wild type strain and two xanthophyll cycle mutants (npq1 and npq2) of Chlamydomonas reinhardtii. Wild type cells exhibited the well-known inactivation of photosystem II in high light, which was accompanied by the loss of β-carotene and a concomitant increase of zeaxanthin. Complete degradation of D1 protein was found after 2 h of illumination in the presence of chloramphenicol, an inhibitor of chloroplast protein synthesis. The npq1 mutant, which is unable to convert violaxanthin to zeaxanthin, showed a very similar behaviour. For the npq2 mutant, however, which is unable to form violaxanthin from zeaxanthin and thus contains high amounts of zeaxanthin even in low light, photosystem II inactivation was less pronounced. This was paralleled by a much slower D1 protein degradation in chloramphenicol treated cells. Our results support a protective role for zeaxanthin against high light-induced photosystem II inactivation resulting in a slowed-down D1 protein turnover.     

Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.

We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700-chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light-activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild-type cells showed a 4-fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700-apoprotein; greatly reduced whole-chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA-C and PSA-D were not detected in this mutant. ADK9 does assemble near wild-type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6-dichloro-p-benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site-directed mutagenesis of the PS I core.     

The coleoptile chloroplast: Distinct distribution of xanthophyll cycle pigments,and enrichment in Photosystem II

Recent studies have shown that coleoptile chloroplasts operate the xanthophyll cycle, and that their zeaxanthin concentration co-varies with their sensitivity to blue light. The present study characterized the distribution of photosynthetic pigments in thylakoid pigment–protein complexes from dark-adapted and light-treated coleoptile and mesophyll chloroplasts, the low temperature fluorescence emission spectra, and the rates of PS I and PS II electron transport in both types of chloroplasts from 5-day-old corn seedlings. Pigments were extracted from isolated PS I holocomplex, LHC IIb trimeric and LHC II monomeric complexes and analyzed by HPLC. Chlorophyll distribution in coleoptile thylakoids showed 31% of the total collected Chl in PS I and 65% in the light harvesting complexes of PS II. In mesophyll thylakoids, the values were 44% and 54%, respectively. Mesophyll and coleoptile PS I holocomplexes differed in their Chl t a/Chl t b ratios (8.1 and 6.1, respectively) and -carotene content. In contrast, mesophyll and coleoptile LHC IIb trimers and LHC II monomers had similar Chl t a/Chl t b ratios and -carotene content. The three analyzed pigment–protein complexes from dark-adapted coleoptile chloroplasts contained zeaxanthin, whereas there was no detectable zeaxanthin in the complexes from dark-adapted mesophyll chloroplasts. In both chloroplast types, zeaxanthin and antheraxanthin increased markedly in the three pigment–protein complexes upon illumination, while violaxanthin decreased. In mesophyll thylakoids, zeaxanthin distribution as a percentage of the xanthophyll cycle pool was: LHC II monomers > LHC IIb trimers > PS I holocomplex, and in coleoptile thylakoids, it was: LHC IIb trimers > LHC II monomers = PS I holocomplex. Low temperature (77 K) fluorescence emission spectra showed that the 686 nm emission of coleoptile chloroplasts was approximately 50% larger than that of mesophyll chloroplasts when normalized at 734 nm. The pigment and fluorescence analysis data suggest that there is relatively more PS II per PS I and more LHC I per CC I in coleoptile chloroplasts than in mesophyll chloroplasts. Measurements of t in vitro uncoupled photosynthetic electron transport showed approximately 60% higher rates of electron flow through PS II in coleoptile chloroplasts than in mesophyll chloroplasts. Electron transport rates through PS I were similar in both chloroplast types. Thus, when compared to mesophyll chloroplasts, coleoptile chloroplasts have a distinct PS I pigment composition, a distinct chlorophyll distribution between PS I and PS II, a distinct zeaxanthin percentage distribution among thylakoid pigment–protein complexes, a higher PS II-related fluorescence emission, and higher PS II electron transport capacity. These characteristics may be associated with a sensory transducing role of coleoptile chloroplasts.