ChloroSpec: A new in vivo chlorophyll fluorescence spectrometer for simultaneous wavelength- and time-resolved detection.

Chlorophyll fluorescence is a ubiquitous tool in basic and applied plant science research. Various standard commercial instruments are available for characterization of photosynthetic material like leaves or microalgae, most of which integrate the overall fluorescence signals above a certain cut-off wavelength. However, wavelength-resolved (fluorescence signals appearing at different wavelengths having different time dependent decay) signals contain vast information required to decompose complex signals and processes into their underlying components that can untangle the photo-physiological process of photosynthesis.

Flavodiiron-mediated O photoreduction at photosystem I acceptor-side provides photoprotection to conifer thylakoids in early spring.

Green organisms evolve oxygen (O) via photosynthesis and consume it by respiration. Generally, net O consumption only becomes dominant when photosynthesis is suppressed at night. Here, we show that green thylakoid membranes of Scots pine (Pinus sylvestris L) and Norway spruce (Picea abies) needles display strong O consumption even in the presence of light when extremely low temperatures coincide with high solar irradiation during early spring (ES).

Direct energy transfer from photosystem II to photosystem I confers winter sustainability in Scots Pine.

Evergreen conifers in boreal forests can survive extremely cold (freezing) temperatures during long dark winter and fully recover during summer. A phenomenon called "sustained quenching" putatively provides photoprotection and enables their survival, but its precise molecular and physiological mechanisms are not understood. To unveil them, here we have analyzed seasonal adjustment of the photosynthetic machinery of Scots pine (Pinus sylvestris) trees by monitoring multi-year changes in weather, chlorophyll fluorescence, chloroplast ultrastructure, and changes in pigment-protein composition.

Dynamic pH-induced conformational changes of the PsbO protein in the fluctuating acidity of the thylakoid lumen.

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown.

Crystal structure and functional characterization of photosystem II-associated carbonic anhydrase CAH3 in Chlamydomonas reinhardtii.

In oxygenic photosynthesis, light energy is stored in the form of chemical energy by converting CO2 and water into carbohydrates. The light-driven oxidation of water that provides the electrons and protons for the subsequent CO2 fixation takes place in photosystem II (PSII). Recent studies show that in higher plants, HCO3 (-) increases PSII activity by acting as a mobile acceptor of the protons produced by PSII.

Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation.

Cyanobacteria, algae, and plants oxidize water to the O2 we breathe, and consume CO2 during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO2/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast's lumen, resulting in a light-driven production of O2 and CO2.

Efficiency of photosynthetic water oxidation at ambient and depleted levels of inorganic carbon.

Over 40 years ago, Joliot et al. (Photochem Photobiol 10:309-329, 1969) designed and employed an elegant and highly sensitive electrochemical technique capable of measuring O2 evolved by photosystem II (PSII) in response to trains of single turn-over light flashes. The measurement and analysis of flash-induced oxygen evolution patterns (FIOPs) has since proven to be a powerful method for probing the turnover efficiency of PSII.

Phosphorylation controls the localization and activation of the lumenal carbonic anhydrase in Chlamydomonas reinhardtii.

Background: Cah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO(2) fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO(2) conditions.

Importance of post-translational modifications for functionality of a chloroplast-localized carbonic anhydrase (CAH1) in Arabidopsis thaliana.

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known.

The mechanism of anthracene interaction with photosynthetic apparatus: a study using intact cells, thylakoid membranes and PS II complexes isolated from Chlamydomonas reinhardtii.

Intact cells of Chlamydomonas reinhardtii as well as isolated thylakoid membranes and photosystem II complexes were used to examine a possible mechanism of anthracene (ANT) interaction with the photosynthetic apparatus. Since ANT concentrations above 1 mM were required to significantly inhibit the rate of oxygen evolution in PS II membrane fragments it may indicate that the toxicant did not directly interact with this photosystem. On the other hand, stimulation of oxygen uptake by ANT-treated thylakoids suggested that ANT could either act as an artificial electron acceptor in the photosynthetic electron transport chain or function as an uncoupler.

A carbonic anhydrase inhibitor induces bicarbonate-reversible suppression of electron transfer in pea photosystem 2 membrane fragments.

The effects of suppression of the carbonic anhydrase (CA) activity by a CA-inhibitor, acetazolamide (AA), on the photosynthetic activities of photosystem II (PS II) particles from higher plants were investigated. AA along with CA-activity inhibits the PS II photosynthetic electron transfer and the AA-induced suppression is totally reversed by the addition of bicarbonate (3-5 mM). Similar effect of recovery in the PS II photosynthetic activity was also revealed upon the addition of known artificial electron donors (potassium ferrocyanide and TMPD).

A metabolomic approach to study major metabolite changes during acclimation to limiting CO2 in Chlamydomonas reinhardtii.

Using a gas chromatography-mass spectrometry-time of flight technique, we determined major metabolite changes during induction of the carbon-concentrating mechanism in the unicellular green alga Chlamydomonas reinhardtii. In total, 128 metabolites with significant differences between high- and low-CO(2)-grown cells were detected, of which 82 were wholly or partially identified, including amino acids, lipids, and carbohydrates. In a 24-h time course experiment, we show that the amino acids serine and phenylalanine increase transiently while aspartate and glutamate decrease after transfer to low CO(2).

Importance of a single disulfide bond for the PsbO protein of photosystem II: protein structure stability and soluble overexpression in Escherichia coli.

PsbO protein is an important constituent of the water-oxidizing complex, located on the lumenal side of photosystem II. We report here the efficient expression of the spinach PsbO in E. coli where the solubility depends entirely on the formation of the disulfide bond.

The photosystem II-associated Cah3 in Chlamydomonas enhances the O2 evolution rate by proton removal.

Water oxidation in photosystem II (PSII) is still insufficiently understood and is assumed to involve HCO(3)(-). A Chlamydomonas mutant lacking a carbonic anhydrase associated with the PSII donor side shows impaired O(2) evolution in the absence of HCO(3)(-). The O(2) evolution for saturating, continuous illumination (R(O2)) was slower than in the wild type, but was elevated by HCO(3)(-) and increased further by Cah3.

A cluster of carboxylic groups in PsbO protein is involved in proton transfer from the water oxidizing complex of Photosystem II.

The hypothesis presented here for proton transfer away from the water oxidation complex of Photosystem II (PSII) is supported by biochemical experiments on the isolated PsbO protein in solution, theoretical analyses of better understood proton transfer systems like bacteriorhodopsin and cytochrome oxidase, and the recently published 3D structure of PS II (Pdb entry 1S5L). We propose that a cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as a buffering network providing efficient acceptors of protons derived from substrate water molecules. The charge delocalization of the cluster ensures readiness to promptly accept the protons liberated from substrate water.

Subsequent events to GTP binding by the plant PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem II complex.

Besides an essential role in optimizing water oxidation in photosystem II (PSII), it has been reported that the spinach PsbO protein binds GTP [C. Spetea, T. Hundal, B.

[Lipid metabolism characteristics in disabled persons as a consequence of cerebrovascular diseases].

Lipid metabolism condition in young, mature (till 35 y.o) and elderly disabled persons as a consequence of cerebral diseases has been investigated. It was established that all three groups differ in clinical presentation and various kinds of lipid metabolism abnormalities.

Structural dynamics of the manganese-stabilizing protein-effect of pH, calcium, and manganese.

The photosystem-II-associated 33-kDa extrinsic manganese-stabilizing protein is found in all oxygen-evolving organisms. In this paper, we show that this protein undergoes pH-induced conformational changes in the physiological pH range. At a neutral pH of 7.

Carbonic anhydrase activities in pea thylakoids.

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 micromol H(+) (mg Chl)(-1) h(-1) at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal beta-CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA.

Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae.

Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae.

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus.

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp(241) is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310-395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.

Quantitative spin trapping and triplet quenching by radicals for in vivo studies. I. The chemical model.

In order to determine quantitatively the free radical content and its changes affected by additives using spin trapping under in vivo conditions, an approach is suggested carrying out experiments in a completely mixed open system (CMOS). Measurements have been carried out for a chemical oxidation process as a model system, and analysis of products and of the spin trap was extended by kinetic ESR spectrometry of the spin adducts. Since in a CMOS differential equations of accumulation of all species can be transformed into algebraic expressions using available rate constants for the formation of the spin adducts, corresponding concentrations of free radicals have been calculated.

A photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution.

We show for the first time that Cah3, a carbonic anhydrase associated with the photosystem II (PSII) donor side in Chlamydomonas reinhardtii, regulates the water oxidation reaction. The mutant cia3, lacking Cah3 activity, has an impaired water splitting capacity, as shown for intact cells, thylakoids and PSII particles. To compensate this impairment, the mutant overproduces PSII reaction centres (1.

Origin and properties of fluorescence emission from the extrinsic 33 kDa manganese stabilizing protein of higher plant water oxidizing complex.

The fluorescence properties of the isolated extrinsic 33 kDa subunit acting as 'manganese stabilizing protein' (MSP) of the water oxidizing complex in photosynthesis was analyzed in buffer solution. Measurements of the emission spectra as a function of excitation wavelength, pH and temperature led to the following results: (a) under all experimental conditions the spectra monitored were found to be the composite of two contributions referred to as '306 nm band' and 'long-wavelength band', (b) the excitation spectra of these two bands closely resemble those of tyrosine and tryptophan in solution, respectively, (c) the spectral shape of the '306 nm band' is virtually independent on pH but its amplitude drastically decreases in the alkaline with a pK of 11.7, (d) the amplitude of the 'long-wavelength' emission band at alkaline pH slightly increases when the pH rises from 7.