Association of fucoxanthin chlorophyll a/c-binding polypeptides with photosystems and phosphorylation in the centric diatom Cyclotella cryptica

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.     

Lectin binding in Cryptomonas and Chroomonas (Cryptophyceae)

The cell envelopes of Cryptomonas and Chroomonas exhibited significant fluorescence using FITC-labelled concanavalin A and wheat germ agglutinin when the cells were fixed prior to lectin binding. The periplast became intensely labelled in Chroomonas whereas Cryptomonas showed fluorescing granula in its gullet/furrow region and on the cell surface. Lectin labelling followed by fixation showed only label of periplast remnants of lysed cells and of the flagella of Chroomonas. Isolated periplasts of Cryptomonas and Chroomonas were intensively labelled with both concanavalin A and wheat germ agglutinin. Glycostaining of gels, onto which total cell protein extracts were loaded, showed a glycoprotein of high molecular weight for Cryptomonas and Chroomonas and an additional glycoprotein for Cryptomonas species.     

Biochemical characterization of photosystem I complexes having different subunit compositions of fucoxanthin chlorophyll a/c-binding proteins in the diatom Chaetoceros gracilis

Photosynthesis Research - Diatoms are dominant phytoplankton in aquatic environments and have unique light-harvesting apparatus, fucoxanthin chlorophyll a/c-binding protein (FCP). Diatom...     

Association of chlorophyll a/c(2) complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17x24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy

Antisera were raised against the C termini of three fucoxanthin chlorophyll a/c-binding polypeptides, Fcp2, Fcp4, and Fcp6, of the centric diatom Cyclotella cryptica. Immunogold electron microscopy of ultrathin-sectioned cells indicated that Fcp2 and Fcp4 are present in almost the same amounts, whereas approximately 8- to 10-fold less gold label was registered for Fcp6. Immunogold electron microscopy of freeze-fracture replicas of thylakoid membranes showed that the C termini of at least Fcp2 and Fcp4 were located in the thylakoid lumen, thus demonstrating a 3-dimensional structure similar to that already described for the chlorophyll a/b-binding light-harvesting polypeptides of higher plants.     

Specificity of Chroomonas (Cryptophyceae) as a source of kleptochloroplast for Nusuttodinium aeruginosum (Dinophyceae)

The unarmoured dinoflagellate Nusuttodinium aeruginosum retains a kleptochloroplast, which is a transient chloroplast stolen from members of the cryptomonad genus, Chroomonas. Both N. aeruginosum and the closely related N. acidotum have been shown to restrict their diet to a limited number of species of this blue‐green genus of cryptophyte. However, it is still unclear how flexible the predators are with regard to the ingestion and utilization of Chroomonas spp. as a source of kleptochloroplast. To address specificity of cryptomonad in N. aeruginosum, we collected the cells of N. aeruginosum from several ponds in Japan, and analysed the phylogeny of the kleptochloroplasts based on their plastidial 16S rDNA sequences. All sequences obtained in this study were restricted to only one (the subclade 4) of four subclades known to comprise the Chroomonas/Hemiselmis clade. Therefore, N. aeruginosum is specific in its dietary requirements, selecting their prey within the subclade level.     

Evidence for variable chlorophyll fluorescence of photosystem I in vivo

Photosynthesis Research - Room temperature fluorescence in vivo and its light-induced changes are dominated by chlorophyll a fluorescence excited in photosystem II, F(II), peaking around...     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes

A photosystem I (PSI)-fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI-FCP sample revealed a monomeric complex comparable in size and shape to the PSI-LHCI complex of green algae.     

Observations on the ultrastructure of representatives of the genera Hemiselmis and Chroomonas (Cryptophyceae)

Hemiselmis rufescens Parke and three species of the genus Chroomonas have been examined by electron microscopy. They demonstrate certain characteristic features of the Cryptophyceae, such as the presence of trichocysts and the arrangement of thylakoids in pairs in the plastid. The prominent pyrenoid of the genus Chroomonas is penetrated longitudinally by a tongue of cytoplasmic matrix which originates from between the two pairs of plastid membranes. In the genus Hemiselmis, however, the pyrenoid is traversed by a pair of thylakoids. The nucleus, Golgi apparatus and Corps de Maupas also occupy characteristic positions. These features indicate a close relationship between these two genera and the other major genus Cryptomonas, and support the suggestion that the Cryptophyceae is a discrete taxonomic group.     

Investigation of the structure of trimeric and monomeric photosystem I reaction centre complexes

Electron microscopy of monomeric and trimeric forms of the reaction centre of photosystem I from the thermophilic cyanobacterium Phormidium laminosum has allowed the construction of a three-dimensional model describing the shape of the complex. The trimeric form of the Photosystem I reaction centre complex was found to have a very regular shape corresponding to a rounded equilateral triangle with edges ˜18 nm long and a thickness of ˜6 nm. A distinctive chiral arrangement of the three reaction centres in the trimer could be observed on one face of the complex, whereas the opposing face appeared to be smooth with no distinctive internal features. The monomeric reaction centre is roughly pearshaped, with a length of ˜15 nm and a width of ˜9 nm. A thickness of 6 nm is assumed from comparison with the trimer. It is predicted to lie with its shortest axis spanning the membrane. A double-lobed structure, with one lobe larger than the other, was occasionally observed for the monomeric reaction centre. No experimental evidence could be obtained for the existence of the trimeric form in the membrane. The formation of the trimeric form after detergent extraction is suggested. The trimeric form was found to be more stable than the monomeric form in solutions containing anionic and non-ionic detergents.     

Evidence for hydrogen bond formation to the PsaB chlorophyll of P700 in photosystem I mutants of Synechocystis sp. PCC 6803

P700, the primary electron donor of photosystem I, is an asymmetric dimer made of one molecule of chlorophyll a' (P(A)) and one of chlorophyll a (P(B)) that are bound to the homologous PsaA and PsaB polypeptides. While the carbonyl groups of P(A) are involved in hydrogen-bonding interactions with several surrounding amino acid side chains and a water molecule, P(B) does not engage hydrogen bonds with the protein. Notably, the residue Thr A739 is donating a strong hydrogen bond to the 9-keto C=O group of P(A) and the homologous residue Tyr B718 is free from interaction with P(B). Light-induced FTIR difference spectroscopy of the photooxidation of P700 has been combined with a site-directed mutagenesis attempt to introduce hydrogen bonds to the carbonyl groups of P(B) in Synechocystis sp. PCC 6803. The FTIR study of the Y(B718)T mutant provides evidence that the 9-keto C=O group of P(B) and P(B)(+) engages a relatively strong hydrogen-bonding interaction with the surroundings in a significant fraction (40 +/-10%) of the reaction centers. Additional mutations on the two PsaB residues homologous to those involved in the main interactions between the PsaA polypeptide and the 10a-carbomethoxy groups of P(A) affect only marginally the vibrational frequency of the 10a-ester C=O group of P(B). The FTIR data on single, double, and triple mutants at these PsaB sites indicate a plasticity of the interactions of the carbonyl groups of P(B) with the surrounding protein. However, these mutations do not perturb the hydrogen-bonding interactions assumed by the 9-keto and 10a-ester C=O groups of P(A) and P(A)(+) with the protein and have only a limited effect on the relative charge distribution between P(A)(+) and P(B)(+).     

Characterization of chlorophyll a/b proteins of photosystem I from Chlamydomonas reinhardtii.

In this study we have isolated the chlorophyll a/b-binding proteins from a photosystem I preparation of the green alga Chlamydomonas reinhardtii and characterized them by N-terminal sequencing, fluorescence, and absorption spectroscopy and by immunochemical means. The results indicate that in this organism, the light-harvesting complex of photosystem I (LHCI) is composed of at least seven distinct polypeptides of which a minimum number of three are shown to bind chlorophyll a and b. Both sequence homology and immunological cross-reactivity with other chlorophyll-binding proteins suggest that all of the LHCI polypeptides bind pigments. Fractionation of LHCI by mildly denaturing methods showed that, in contrast to higher plants, the long wavelength fluorescence emission typical of LHCI (705 nm in C. reinhardtii) cannot be correlated with the presence of specific polypeptides, but rather with changes in the aggregation state of the LHCI components. Reconstitution of both high aggregation state and long wavelength fluorescence emission from components that do not show these characteristics confirm this hypothesis.     

Identification of the photosystem I antenna polypeptides in barley. Isolation of three pigment-binding antenna complexes.

The light-harvesting antenna of barley photosystem I (LHCI) was isolated from native photosystem I (PSI) complexes and fractionated into three pigment-protein subcomplexes using two consecutive rounds of green gel electrophoresis. Each complex showed a characteristic polypeptide composition and low-temperature fluorescence emission spectrum; they were designated as LHCI-730, LHCI-680A and LHCI-680B. Their four apoproteins of 21, 22, 23 and 25 kDa were purified and NH2-terminal sequences were determined; in the case of the NH2-terminally blocked 25-kDa protein, an internal sequence was obtained after cleavage with endoproteinase Lys-C. This made possible an assignment of the four proteins to the four types (I-IV) of genes coding for chlorophyll a/b proteins of PSI (cab or lha genes). The LHCI-730 complex was isolated as a heterodimer composed of the 21-kDa (LHCI type IV) and the 22-kDa (LHCI type I) polypeptides. Each LHCI-680 complex had a single apoprotein. LHCI-680A consisted of the 25-kDa (LHCI type III) and LHCI-680B of the 23-kDa (LHCI type II) polypeptides. LHCI-680B was associated with the non-pigmented PSI-E subunit, indicating that this protein may function in the binding of this antenna to the reaction centre.     

Antibodies to the photosystem I chlorophyll a + b antenna cross-react with polypeptides of CP29 and LHCII

A chlorophyll (a + b)--protein complex associated with photosystem I (PSI) was isolated from a larger PSI complex (CPIa) produced by electrophoresis of barley thylakoids solubilized with 300 mM octyl glucoside. It had an apparent Mr of 35,000-43,000 on 7.5% and 10% acrylamide gels respectively, and a chlorophyll a/b ratio of 2.5 +/- 1.5. Denaturation released four polypeptides migrating between 21-24 kDa. They were well separated from the polypeptides of the two photosystem II chlorophyll a + b antenna complexes: LHCII (25-27 kDa) and CP29 (28-29 kDa). In order to study the PSI antenna complex, antibodies were raised against highly purified CPIa. The antigen appeared to be pure when electrophoresed, blotted and reacted with its antiserum, i.e. anti-CPIa detected only the 64-66-kDa CPI apoprotein and the four 21-24 kDa antenna polypeptides. However, when blotted against the whole spectrum of thylakoid proteins, it cross-reacted with both LHCII and CP29 apoproteins. Removal of anti-CPI activity from the anti-CPIa did not affect these cross-reactions, showing that they were not due to antibodies directed against CPI. To show that the same antibody population was reacting with both the photosystem I and photosystem II antenna polypeptides, anti-CPIa was adsorbed onto highly purified CPIa on nitrocellulose. The bound antibody was eluted and used again in a Western blot against whole thylakoid proteins. This selected antibody population showed the same relative strength of reaction with photosystem I and photosystem II antenna polypeptides as the original antibody population had. Similar observations have been made with antibodies to the two photosystem II antenna complexes. We therefore conclude that there are antigenic determinants in common among the chlorophyll a + b binding polypeptides, and predict that there could be amino acid sequence similarities.     

Characterization of a Synechococcus sp. strain PCC 7002 spontaneous mutant strain defective in accumulation of photosystem II core chlorophyll-protein complexes.

Two photosystem II-associated chlorophyll-protein complexes of Synechococcus sp. strain PCC 7002 were identified. Their polypeptide compositions were similar to those of chlorophyll-containing antenna complexes of other cyanobacteria. Strain GT8B did not possess the complex responsible for 695-nm fluorescence and was unable to grow photoautotrophically; hence, this complex is necessary for photosystem II function in vivo.     

Comparison of oligomeric states and polypeptide compositions of fucoxanthin chlorophyll a/c-binding protein complexes among various diatom species

Fucoxanthin chlorophyll a/c-binding protein (FCP) is a unique light-harvesting apparatus in diatoms. Several biochemical characteristics of FCP oligomer and trimer from different diatom species have been reported previously. However, the integration of information about molecular organizations and polypeptides of FCP through a comparison among diatoms has not been published. In this study, we used two-dimensional clear-native/SDS-PAGE to compare the oligomeric states and polypeptide compositions of FCP complexes from four diatoms: Chaetoceros gracilis, Thalassiosira pseudonana, Cyclotella meneghiniana, and Phaeodactylum tricornutum. FCP oligomer was found in C. gracilis, T. pseudonana, and C. meneghiniana, but not in P. tricornutum. The oligomerization varied among the three diatoms, although a predominant subunit having similar molecular weight was recovered in each FCP oligomer. These results suggest that the predominant subunit is involved in the formation of high FCP oligomerization in each diatom. In contrast, FCP trimer was found in all the diatoms. The trimerizations were quite similar, whereas the polypeptide compositions were markedly different. On the basis of this information and that from mass spectrometric analyses, the gene products in each FCP complex were identified in T. pseudonana and P. tricornutum. Based on these results, we discuss the role of FCP oligomer and trimer from the four diatoms.     

Association of chlorophyll a/c2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c₂ proteins were found. The most common complexes have Chl a/c₂ complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c₂ light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c₂ proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c₂ proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Composition of a photosystem I chlorophyll protein complex from Anabaena flos-aquae.

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.