[Show abstract][Hide abstract] ABSTRACT: In the last years new renewable energy sources started to be exploited to compensate exhausting fossil fuels and minimize anthropogenic factors on climate change. Microalgae have reemerged as potential next-generation feedstock for biofuels and they are considered very promising on the long term, since they have a potentially high productivity per area and they can be grown on marginal land without competing with food crops. Our work is focused on the seawater microalga Nannochloropsis gaditana, which combines a fast growth rate with a strong accumulation of lipids and therefore yields great potential for these kinds of applications. Solar radiation provides all the energy supporting algae growth and lipids production and for this reason the available radiation must be exploited with the highest possible efficiency to optimize productivity and make their cultivation on a large scale competitive. Investigation of the bases affecting light use efficiency is thus seminal to elucidate the connection between light and the lipids/biomass productivity. To this aim we investigated the influence of different illumination regimes, nutrients availability and batch/ continuous cultivation on N. gaditana cultures productivity and light use efficiency. This information will be exploited for optimization of growing systems but also to design Nannochloropsis gaditana genetic manipulations. Strains with altered composition of the photosynthetic apparatus and modified regulation of photosynthesis are being selected and characterized to increase Nannochloropsis gaditana productivity in photobioreactors.
[Show abstract][Hide abstract] ABSTRACT: Nannochloropsis gaditana belongs to Eustigmatophyceae, a class of eukaryotic algae resulting from a secondary endosymbiotic event. Species of this class have been poorly characterized thus far but are now raising increasing interest in the scientific community because of their possible application in biofuel production. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of only chlorophyll a, with violaxanthin and vaucheriaxanthin esters as the most abundant carotenoids. In this study, the photosynthetic apparatus of this species was analyzed by purifying the thylakoids and isolating the different pigment-binding complexes upon mild solubilization. The results from the biochemical and spectroscopic characterization showed that the photosystem II antenna is loosely bound to the reaction center, whereas the association is stronger in photosystem I, with the antenna-reaction center super-complexes surviving purification. Such a supramolecular organization was found to be conserved in photosystem I from several other photosynthetic eukaryotes, even though these taxa are evolutionarily distant. A hypothesis on the possible selective advantage of different associations of the antenna complexes of photosystems I and II is discussed.
[Show abstract][Hide abstract] ABSTRACT: Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel (SynCaK) in the genome of the freshwater cyanobacterium Synechocystis PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a Ca2+-dependent potassium channel isolated from M. thermoautotrophicum. Expression of SynCaK in fusion with EGFP in mammalian Chinese Hamster Ovary (CHO) cells' plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasmamembrane. In order to understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803 (ΔSynCaK) has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in wild-type, ΔSynCaK was characterized by a depolarized resting membrane potential as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress, that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor which also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.
[Show abstract][Hide abstract] ABSTRACT: Antimicrobial photodynamic therapy (PDT) is a promising tool to combat antibiotic-resistant bacterial infections. During PDT, bacteria are killed by reactive oxygen species generated by a visible light absorbing photosensitizer (PS). We used a classical proteomic approach that included two-dimensional gel electrophoresis and mass spectrometry analysis, to identify some proteins of Staphylococcus aureus that are damaged during PDT with the cationic PS meso-tetra-4-N-methyl pyridyl porphine (T4). Suspensions of S. aureus cells were incubated with selected T4 concentrations and irradiated with doses of blue light that reduced the survival to about 60% or 1%. Proteomics analyses of a membrane proteins enriched fraction revealed that these sub-lethal PDT treatments affected the expression of several functional classes of proteins, and that this damage is selective. Most of these proteins were found to be involved in metabolic activities, in oxidative stress response, in cell division and in the uptake of sugar. Subsequent analyses revealed that PDT treatments delayed the growth and considerably reduced the glucose consumption capacity of S. aureus cells. This investigation provides new insights towards the characterization of PDT induced damage and mechanism of bacterial killing using, for the first time, a proteomic approach.
Journal of proteomics 09/2012; 77:329–343. DOI:10.1016/j.jprot.2012.09.007 · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidermis of different scales in the lizard Anolis carolinensis expresses specific keratin-associated beta-proteins (beta-keratins). In order to localize the sites of accumulation of different beta-proteins, we have utilized antibodies directed against representative members of the main families of beta-proteins, the glycine-rich (HgG5), glycine-cysteine rich (HgGC3), glycine-cysteine medium-rich (HgGC10), and cysteine-rich (HgC1) beta-proteins. Immunoblotting and immunocytochemical controls confirm the specificity of the antibodies made against these proteins. Light and ultrastructural immunocytochemistry shows that the glycine-rich protein HgG5 is present in beta-layers of different body scales but is scarce in the oberhautchen and claws, and is absent in alpha-layers and adhesive setae. The cysteine-glycine-rich protein HgGC3 is low to absent in the oberhautchen, beta-layer, and mesos-layer but increases in alpha-layers. This beta-protein is low in claws where it is likely associated with the hard alpha-keratins previously studied in this lizard. The glycine-cysteine medium-rich HgGC10 protein is low in the beta-layer, higher in alpha-layers, and in the oberhautchen. This protein forms a major component of setal proteins including those of the adhesive spatula that allow this lizard to stick on vertical surfaces. HgC1 is poorly localized in most epidermis analyzed including adhesive setae and claws and appears as a minor component of the alpha-layers. In conclusion, the present study suggests that beta- and alpha-layers of lizard epidermis represent regions with different accumulation of glycine-rich proteins (mainly for mechanical resistance and hydrophobicity in the beta-layer) or cysteine-glycine-rich proteins (for both resistance and elasticity in both alpha- and beta-layers).
Journal of Experimental Zoology Part B Molecular and Developmental Evolution 07/2012; 318(5):388-403. DOI:10.1002/jez.b.22454 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ΔpH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.
Proceedings of the National Academy of Sciences 06/2012; 109(27):11043-8. DOI:10.1073/pnas.1205960109 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bioinformatic approaches have allowed the identification in Arabidopsis thaliana of twenty genes encoding for homologues of animal ionotropic glutamate receptors (iGLRs). Some of these putative receptor proteins, grouped into three subfamilies, have been located to the plasmamembrane, but their possible location in organelles has not been investigated so far. In the present work we provide multiple evidence for the plastid localization of a glutamate receptor, AtGLR3.4, in Arabidopsis and tobacco. Biochemical analysis was performed using an antibody shown to specifically recognize both the native protein in Arabidopsis and the recombinant AtGLR3.4 fused to YFP expressed in tobacco. Western blots indicate the presence of AtGLR3.4 in both the plasmamembrane and in chloroplasts. In agreement, in transformed Arabidopsis cultured cells as well as in agroinfiltrated tobacco leaves, AtGLR3.4::YFP is detected both at the plasmamembrane and at the plastid level by confocal microscopy. The photosynthetic phenotype of mutant plants lacking AtGLR3.4 was also investigated. These results identify for the first time a dual localization of a glutamate receptor, revealing its presence in plastids and chloroplasts and opening the way to functional studies.
[Show abstract][Hide abstract] ABSTRACT: Thylakoid membranes in higher plant chloroplasts are composed by two distinct domains: stacked grana and stroma lamellae. We developed a procedure for biochemical isolation of grana membranes using mild detergent to maintain membrane structure. Pigment and polypeptide analyses of membrane preparation showed the preparations were indeed enriched in grana membranes. The method was shown to be effective in four different plant species, although with small changes in detergent concentration. Electron microscopy analyses also showed that the preparation consisted of large membrane patches with roughly round shape and diameter comparable with grana membranes in vivo. Furthermore, protein complexes distribution was shown to be maintained with respect to freeze fracture studies, demonstrating that the protocol was successful in isolating membranes close to their in vivo state.
Journal of Bioenergetics 02/2010; 42(1):37-45. DOI:10.1007/s10863-009-9261-3 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bioinformatic approaches have allowed the identification of twenty genes, grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors (iGLRs) in the Arabidopsis thaliana model plant. Indirect evidence suggests that plant iGLRs function as non-selective cation channels. In the present work we provide biochemical and electrophysiological evidences for the chloroplast localization of glutamate receptor(s) of family 3 (iGLR3) in spinach. A specific antibody, recognizing putative receptors of family 3 locates iGLR3 to the inner envelope membrane of chloroplasts. In planar lipid bilayer experiments, purified inner envelope vesicles from spinach display a cation-selective electrophysiological activity which is inhibited by DNQX (6,7-dinitroquinoxaline-2,3-dione), considered to act as an inhibitor on both animal and plant iGLRs. These results identify for the first time the intracellular localization of plant glutamate receptor(s) and a DNQX-sensitive, glutamate-gated activity at single channel level in native membrane with properties compatible with those predicted for plant glutamate receptors.
[Show abstract][Hide abstract] ABSTRACT: Various ion channel activities can be recorded by electrophysiological methods in the outer and inner envelope membranes of chloroplasts as well as in the thylakoid membrane. However, most of these channels are poorly characterized from a pharmacological point of view. Furthermore, the molecular identity has been determined only for a few of them, preventing an understanding of their role in plant physiology. By allowing specific ion fluxes across plastidial membranes, these ion channels may either directly or indirectly regulate photosynthesis, as has been hypothesized earlier. We have determined the effect of various ion channel modulators [indole-3-acetic acid, 5-nitro-2-(3-phenylpropylamino)-benzoate, (-)-epigallocatechin-3-gallate, p-chlorophenoxyacetic acid, Konig's polyanion, Cs+, Gd3+, 4-aminopyridine, tetraethylammonium chloride, charybdotoxin, nimodipine, and cyclosporin A] on the efficiency of photosynthetic oxygen evolution in intact chloroplasts, broken chloroplasts, and isolated thylakoids. The data may improve our understanding of chloroplast ion channels and identifies inhibitors which may be exploited for electrophysiological studies.
Journal of Chemical Information and Modeling 11/2005; 45(6):1691-700. DOI:10.1021/ci0501802 · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Seven genes seem to encode for putative ClC chloride channels (AtClC-a to AtClC-g) in Arabidopsis thaliana. Their function and localization is still largely unknown. AtClC-f shares considerable sequence similarity with putative ClC channel proteins from Synechocystis, considered to represent the precursor of chloroplasts. We show by biochemical and mass spectrometry analysis that ClC-f is located in the outer envelope membrane of spinach chloroplasts. Consistent with the plastidial localization of ClC-f, p-chlorophenoxy-acetic acid (CPA) reduces photosynthetic activity and the protein is expressed in etioplasts and chloroplasts but not in root tissue. These findings may represent a step toward the molecular identification of ion channel activities in chloroplast membranes.
[Show abstract][Hide abstract] ABSTRACT: As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll-protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.
[Show abstract][Hide abstract] ABSTRACT: In higher plants, the PsbS subunit of photosystem II (PSII) plays a crucial role in pH- and xanthophyll-dependent nonphotochemical quenching of excess absorbed light energy, thus contributing to the defense mechanism against photoinhibition. We determined the amino acid sequence of Zea mays PsbS and produced an antibody that recognizes with high specificity a region of the protein located in the stroma-exposed loop between the second and third putative helices. By means of this antiserum, the thylakoid membranes of various higher plant species revealed the presence of a 42-kDa protein band, indicating the formation of a dimer of the 21-kDa PsbS protein. Crosslinking experiments and immunoblotting with other antisera seem to exclude the formation of a heterodimer with other PSII protein components. The PsbS monomer/dimer ratio in isolated thylakoid membranes was found to vary with luminal pH in a reversible manner, the monomer being the prevalent form at acidic and the dimer at alkaline pH. In intact chloroplasts and whole plants, dimer-to-monomer conversion is reversibly induced by light, known to cause luminal acidification. Sucrose-gradient centrifugation revealed a prevalent association of the PsbS monomer and dimer with light-harvesting complex and PSII core complexes, respectively. The finding of the existence of a light-induced change in the quaternary structure of the PsbS subunit may contribute to understanding the mechanism of PsbS action during nonphotochemical quenching.
Proceedings of the National Academy of Sciences 01/2004; 100(25):15265-70. DOI:10.1073/pnas.2533072100 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Photosystem I-less Synechocystis 6803 mutants carrying modified PsbH proteins, derived from different combinations of wild-type cyanobacterial and maize genes, were constructed. The mutants were analyzed in order to determine the relative importance of the intra- and extramembrane domains of the PsbH subunit in the functioning of photosystem (PS) II, by a combination of biochemical, biophysical, and physiological approaches. The results confirmed and extended previously published data showing that, besides D1, the whole PsbH protein is necessary to determine the correct structure of a QB/herbicide-binding site. The different turnover of the D1 protein and chlorophyll photobleaching displayed by mutant cells in response to photoinhibitory treatment revealed for the first time the actual role of the PsbH subunit in photoprotection. A functional PsbH protein is necessary for (i) rapid degradation of photodamaged D1 molecules, which is essential to avoid further oxidative damage to the PSII core, and (ii) insertion of newly synthesized D1 molecules into the thylakoid membrane. PsbH is thus required for both initiation and completion of the repair cycle of the PSII complex in cyanobacteria.
[Show abstract][Hide abstract] ABSTRACT: The effect of visible light on photosystem II reaction centre D1 protein in plants treated with ultraviolet-B light was studied. It was found that a 20 kDa C-terminal fragment of D1 protein generated during irradiation with ultraviolet-B light was stable when plants were incubated in the dark, but was degraded when plants were incubated in visible light. In this condition the recovery of photosynthetic activity was also observed. Even a low level of white light was sufficient to promote both further degradation of the fragment and recovery of activity. During this phase, the D1 protein is the main synthesized thylakoid polypeptide, indicating that other photosystem II proteins are recycled in the recovery process. Although both degradation of the 20 kDa fragment and resynthesis of D1 are light-dependent phenomena, they are not closely related, as degradation of the 20 kDa fragment may occur even in the absence of D1 synthesis. Comparing chemical and physical factors affecting the formation of the fragment in ultraviolet-B light and its degradation in white light, it was concluded that the formation of the fragment in ultraviolet-B light is a photochemical process, whereas the degradation of the fragment in white light is a protease-mediated process.
[Show abstract][Hide abstract] ABSTRACT: A novel Zn(II)-phthalocyanine (1). peripherally substituted with four bis(N,N,N-trimethyl)amino-2-propyloxy groups prepared by chemical synthesis is shown to be an efficient photodynamic sensitizer with a quantum yield of 0.6 for singlet oxygen generation in neat water, which is reduced to about 0.3 in phosphate-buffered saline. The physicochemical properties of 1 in both the ground and the electronically excited states strongly depend on the nature of the medium; in particular, aggregation of 1 was favoured by polar media of high ionic strength. Compound 1 exhibited an appreciable affinity for a typical Gram-positive bacterium (Staphylococcus aureus) and a typical Gram-negative bacterium (Escherichia coli). Both bacterial strains were extensively inactivated upon 5 min-irradiation with 675 nm light in the presence of 1 microM photosensitizer, even though the binding of 1 to the two bacterial cells appears to occur according to different pathways. In particular, E. coli cells underwent initial photodamage at the level of specific proteins in the outer wall, thus promoting the penetration of the photosensitizer to the cytoplasmic membrane where some enzymes critical for cell survival were inactivated.
[Show abstract][Hide abstract] ABSTRACT: Four mutants of the cyanobacterium Synechocystis sp. PCC 6803, carrying a modified PsbH subunit on a PSI-less background, were characterized by optically-detected magnetic resonance (ODMR), electron transport kinetics, and oxygen-evolving activity. Their relative tolerance to light stress was measured. Results indicate that: (i) the PsbH protein is deeply involved in determining structural and functional properties of the QB site on the D1 protein, whereas the environment of the primary donor P680 and its acceptors pheophytin and QA are not significantly affected by modifications of this subunit or its deletion; (ii) the charge recombination rate, in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), is reduced by a factor of 2, independently of the particular modification. The same result is found with the strain in which the subunit has been deleted. This result is taken as an indication that PsbH is important in regulating protein dynamics of the entire PSII core complex; (iii) all investigated mutants display reduced tolerance to light stress, the extent of which depends on the particular modification. In this respect, mutations introduced in the transmembrane portion of the polypeptide are more effective than those involving the extramembrane N-terminal extension.
[Show abstract][Hide abstract] ABSTRACT: A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.
European Journal of Biochemistry 11/2001; 268(19):5129-34. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47–RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47–RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47–RC in studying primary reactions of oxygenic photosynthesis.