Chloroplast ultrastructure and thylakoid polypeptide composition are affected by different salt concentrations in the halophytic plant Arthrocnemum macrostachyum

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosynthetic complexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied. Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found that the PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunits with a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, as well as the organization of thylakoid membranes were affected by NaCl concentration, with many grana containing few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mM NaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.     

Structural-functional state of thylakoid membranes of wheat genotypes under water stress

Plants were grown in field conditions in the wide area under normal water supply and severe water deficit. Two wheat (Triticum aestivum L.) genotypes contrasting by architectonics and differing in drought-resistance were used: Giymatli-2/17, short stature, with broad and drooping leaves, drought-sensitive, and Azamatli-95, short stature, with vertically oriented small leaves, drought-tolerant). It was found out that Giymatli-2/17 was characterized by relatively low content of Chl a-protein of PS I (CP I) and β-subunit of ATP-synthase complex, the high content of proteins in the 33-30.5 kDa region and LHC polypeptides (28-24.5 kDa), the intensive fluorescence at 740 nm and more high photochemical activity of PS II under normal irrigation compared with Azamatli-95. However, the content of CP I (M_r 115 kDa) and apoprotein of P700 with M_r 63 kDa insignificantly increases in the drought-resistant genotype Azamatli-95 under extreme water supply condition while their content decreases in drought-sensitive cv Giymatli-2/17. Intensity of synthesis α- and β-subunits of CF₁ (55 and 53.5 kDa) also decreases in Giymatli-2/17. The levels of the core antenna polypeptides of FS II with M_r 46 and 44.5 kDa (CP47 and CP43) remains stable both in normal, and stressful conditions. At the same time the significant reduction is observed in the content of polypeptides in the 33-30.5 kDa region in the more sensitive genotype Giymatli-2/17. There is an increase in the LHC II polypeptides level in tolerant genotype Azamatli-95 in contrast to Giymatli-2/17 (where the content of these subunits is observed decreasing). The intensity of short wavelength peaks at 687 and 695 nm sharply increases in the fluorescence spectra (77 K) of chloroplasts from sensitive genotype Giymatli-2/17 under water deficiency and there is a stimulation of the ratio of fluorescence band intensity F687/F740. After exposure to drought, cv Giymatli-2/17 shows a larger reduction in the actual PS II photochemical efficiency of chloroplasts than cv Azamatli-95.     

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.     

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 °C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 °C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 °C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.     

AtFtsH heterocomplex-mediated degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to stresses

Chloroplastic heterocomplex consisting of AtFtsH1, 2, 5 and 8 proteases, integrally bound to thylakoid membrane was shown to play a critical role in degradation of photodamaged PsbA molecules, inherent to photosystem II (PSII) repair cycle and in plastid development. As no one thylakoid bound apoproteins besides PsbA has been identified as target for the heterocomplex-mediated degradation we investigated the significance of this protease complex in degradation of apoproteins of the major light harvesting complex of photosystem II (LHCII) in response to various stressing conditions and in stress-related changes in overall composition of LHCII trimers of PSII-enriched membranes (BBY particles). To reach this goal a combination of approaches was applied based on immunoblotting, in vitro degradation and non-denaturing isoelectrofocusing. Exposure of Arabidopsis thaliana leaves to desiccation, cold and high irradiance led to a step-wise disappearance of Lhcb1 and Lhcb2, while Lhcb3 level remained unchanged, except for high irradiance which caused significant Lhcb3 decrease. Furthermore, it was demonstrated that stress-dependent disappearance of Lhcb1–3 is a proteolytic phenomenon for which a metalloprotease is responsible. No changes in Lhcb1–3 level were observed due to exposition of var1-1 mutant leaves to the three stresses clearly pointing to the involvement of AtFtsH heterocomplex in the desiccation, cold and high irradiance-dependent degradation of Lhcb1 and Lhcb2 and in high irradiance-dependent degradation of Lhcb3. Non-denaturing isoelectrofocusing analyses revealed that AtFtsH heterocomplex-dependent differential Lhcb1–3 disappearance behaviour following desiccation stress was accompanied by modulations in abundances of individual LHCII trimers of BBY particles and that LHCII of var1-1 resisted the modulations.     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-β-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.     

3-[18F]Acetylcyclofoxy: a useful probe for the visualization of opiate receptors in living animals

A fluoro-analogue of the potent narcotic antagonist, naltrexone, was synthesized and shown to bind with high affinity to opiate receptors in vitro. 3-[18F]acetylcyclofoxy was prepared via a one-step triflate displacement reaction with the positron emitting 18F ion from tetraethylammonium [18F] fluoride. 3-[18F]acetylcyclofoxy accumulation in opiate receptor rich brain regions of both rat and baboon is shown to be completely displaced by the active enantiomer of naloxone [-)-naloxone) while the identical dose of the pharmacologically inert (+)-naloxone has no detectable effect. Moreover, both rat and baboon brain showed the well documented, typical opiate receptor distribution so that basal ganglia and thalamus are clearly visible in the living baboon brain up to 95 min after intravenous injection of 3-[18F] acetylcyclofoxy. We expect that 3-[18F )acetylcyclofoxy will be a useful probe for visualizing opiate receptors in living humans.     

Unique constitution of photosystem I with a novel subunit in the cyanobacterium Gloeobacter violaceus PCC 7421

Constitution of the photosystem I complex isolated from the cyanobacterium Gloeobacter violaceus PCC 7421 was investigated by tricine-urea-SDS-PAGE, followed by peptide mass fingerprinting or N-terminal sequencing. Eight subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL and PsaM) were identified as predicted from the genome sequence. A novel subunit (PsaZ) was discovered, but PsaI, PsaJ, PsaK and PsaX were absent. PsaB has a C-terminal extension with 155 amino acids in addition to the conserved region and this domain is similar to the peptidoglycan-binding domain. These results suggest that PS I complexes of G. violaceus have unique structural properties.     

C-terminal amidation of neuropeptides. Gly-Lys-Arg extension an efficient precursor of C-terminal amide

Biosynthesis of the C-terminal carboxamide group of peptide hormones was studied using comparatively pGlu-His-Pro-Gly and Glu-His-Pro-Gly-Lys-Arg as putative precursors of the tripeptide, thyroliberin (TRH). Rat hypothalamus granules were found to contain an amide group forming activity which converts both peptide substrates into TRH. Comparison of the rate of conversion of the two substrates indicated that the C-terminal dibasic extension favored a 10-fold increase in the production of amidated peptide. It is suggested that this type of structure may be present in the putative biosynthetic precursor of TRH and that it may provide a better substrate for the enzyme(s) involved in C-terminal amidation.     

Spermidine levels and its relationship to DNA synthesis in outgrowing spores and vegetative cells of Bacillus subtilis

It is now established that the light-harvesting chlorophyll—protein complex (LHCP) of chloroplasts becomes phosphorylated in the light. In this study subfractionation of phosphorylated intact chloroplasts has been carried out to compare the phosphorylation of LHCP in non-appressed and appressed thylakoid regions. The results show around 10-times higher relative phosphorylation in the non-appressed regions than in the appressed ones. Since the non-appressed thylakoids also contain almost all photosystem 1, this region is likely to be the site for energy transfer from LHCP to photosystem 1 under phosphorylated conditions.     

EDTA-induced release of manganese and proteins from inside-out thylakoid vesicles and the inhibition of oxygen evolution

Washing of inside-out, but not right-way-round, pea chloroplast thylakoid vesicles with 2 mM EDTA inhibits O2 evolution. Artificial electron donor/acceptor studies indicate that the site of inhibition is on the oxidising side of photosystem two (PS2), a conclusion reinforced by chlorophyll fluorescence measurements. Evidence is presented that the EDTA inhibition of O2 evolution is linked partly to the removal of one Mn atom per PS2 reaction centre and partly to the removal of extrinsic membrane proteins having apparent molecular weights between 58 and 70 kdaltons.     

Comparative analysis of P2Y4 and P2Y6 receptor architecture in native and transfected neuronal systems

Although extensive studies provided molecular and pharmacological characterization of metabotropic P2Y receptors for extracellular nucleotides, little is still known about their quaternary structure. By the use of transfected cellular systems and SDS-PAGE, in our previous work we established the propensity of P2Y₄ receptor to form dimeric interactions. Here we focused on endogenously expressed P2Y₄ and P2Y₆ subtypes, comparing their oligomeric complexes under Blue Native (BN) gel electrophoresis. We provided evidence that P2Y₄ and P2Y₆ receptors form high order complexes in native neuronal phenotypes and that the oligomers can be disaggregated down to the dimeric P2Y₄ or to the dimeric and monomeric P2Y₆ receptor. Moreover, dimeric P2Y₄ and monomeric P2Y₆ proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Ligand activation by UTP shifted the oligomerization of P2Y₆ but not of P2Y₄ receptor, as analysed by BN electrophoresis. Finally, whereas transfected P2Y₄ and P2Y₆ proteins homo-interact and posses the appropriate domains to associate with all P2Y_1,2,4,6,11 subtypes, in naive PC12 cells the endogenous P2Y₄ forms hetero-oligomers only with the P2Y₆ subunit. In conclusion, our results indicate that quaternary structure distinguishing P2Y₄ from P2Y₆ receptors might be crucial for specific ligand activation, membrane partitioning and consequent functional regulation.     

Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803

The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA⁺, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.     

Orientation of pigments in the thylakoid membrane and in the isolated chlorophyll-protein complexes of higher plants. II. Linear dichroism spectra of isolated pigment-protein complexes oriented in polyacrylamide gels at 300 and 100 K

The absorption and linear dichroism (LD) spectra (380–780 nm) of isolated light-harvesting complex (LHC), Photosystem I (PS I), Photosystem II (PS II), as well as intact thylakoids have been determined at 300 and 100 K. The samples were oriented in squeezed polyacrylamide gel. The low-temperature spectra of LHC and PS I present LD signals which are characteristic enough to be recognized in the LD spectrum of thylakoids. Tentative assignments of the various features of the LD spectra to the major photosynthetic pigments are discussed. A shoulder in the low-temperature absorption spectra is observed at about 673 nm in all the systems under investigation. The absence of an associated LD signal suggests that this ubiquitous chlorophyll (Chl) a form is non-dichroic. Furthermore, in the three isolated chlorophyll-protein complexes described in this study the sign of the LD signal indicates that both the Q_y transition of the Chl a and the carotenoid molecules are preferentially oriented parallel to the largest dimension(s) of the particles.     

Properties of thylakoids and thylakoid particles derived from structurally different chloroplasts

Structurally and functionally different tobacco chloroplasts were subjected to digitonin treatment and subsequent fractional centrifugation. The light-harvesting $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b-}\text{protein}$ complex was found to be enriched in the most dense fraction regardless of the presence of grana in the original preparation. It is suggested that isolated thylakoid membranes and fragments thereof which contain sufficient light-harvesting protein may, under appropriate ionic conditions, form aggregates even when they originate from unstacked thylakoid systems. Comparative studies of fluorescence properties and polypeptide composition of the thylakoids suggest that the light-harvesting protein does not contribute significantly to the fluorescence spectrum of isolated chloroplasts as long as this protein is intimately associated with the Photosystem II (PS II) pigment-protein complex responsible for the 685 nm emission. While the PS II-deficient mutant chloroplasts of the variegated tobacco variety NC 95 lacked both the 685 nm fluorescence component and two or three PS II proteins, one of these proteins was found to be very prominent in our chlorophyll b-deficient mutant thylakoids which also displayed an intense 685 nm fluorescence peak. This correlation supports the contention that a 45 kdalton polypeptide is an apoprotein of pigments associated with the PS II reaction center.     

Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and X-ray crystallography

PsbY is one of the low molecular mass subunits of oxygen-evolving photosystem II (PSII). Its location, however, has not been identified in the current crystal structure of PSII. We constructed a PsbY-deletion mutant of Thermosynechococcus elongatus, crystallized, and analyzed the crystal structure of the mutant PSII dimer. The results obtained showed that PsbY is located in the periphery of PSII close to the alpha- and beta-subunits of cytochrome b559, which corresponded to an unassigned helix in the 3.7A structure of T. vulcanus or helix X2 in the 3.0A structure of T. elongatus. Our results also indicated that the C-terminal loop of PsbY is protruded toward the stromal side, instead of the lumenal side predicted previously.     

Characterization of a novel Photosystem I-LHCI supercomplex isolated from Chlamydomonas reinhardtii under anaerobic (State II) conditions

A novel supercomplex of Photosystem I (PSI) with light harvesting complex I (LHCI) was isolated from the green alga Chlamydomonas reinhardtii. This novel supercomplex is unique as it is the first stable supercomplex of PSI together with its external antenna. The supercomplex contains 256 chlorophylls per reaction center. The supercomplex was isolated under anaerobic conditions and may represent the State II form of the photosynthetic unit. In contrast to previously reported supercomplexes isolated in State I, which contain only 4 LHC I proteins, this supercomplex contains 10-11 LHC I proteins tightly bound to the PSI core. In contrast to plants, no LHC II is tightly bound to the PSI-LHCI supercomplex in State II. Investigation of the energy transfer from the antenna system to the reaction center core shows that the LHC supercomplexes are tightly coupled to the PSI core, not only structurally but also energetically. The excitation energy transfer kinetics are completely dominated by the fast phase, with a near-complete lack of long-lived fluorescence. This tight coupling is in contrast to all reports of energy transfer in PSI-LHCI supercomplexes (in State I), which have so far been described as weakly coupled supercomplexes with low efficiency for excitation energy transfer. These results indicate that there are large and dynamic changes of the PSI-LHCI supercomplex during the acclimation from aerobic (State I) to anaerobic (State II) conditions in Chlamydomonas.     

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.     

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.