Light-independent pathway of STN7 kinase activation under low temperature stress in runner bean (Phaseolus coccineus L.).

BACKGROUND: The phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet. RESULTS: In this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation. CONCLUSIONS: Our results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling.

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana.

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery.

SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals-The Etiological Factor of Chondrocalcinosis.

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg2+-dependent and also works against soluble species of pyrophosphate.

In vivo creation of plasmid pCRT01 and its use for the construction of carotenoid-producing Paracoccus spp. strains that grow efficiently on industrial wastes.

BACKGROUND: Carotenoids are natural tetraterpene pigments widely utilized in the food, pharmaceutical and cosmetic industries. Currently, chemical synthesis of these compounds outperforms their production in Escherichia coli or yeast due to the limited efficiency of the latter. The use of natural microbial carotenoid producers, such as bacteria of the genus Paracoccus (Alphaproteobacteria), may help to optimize this process. In order to couple the ability to synthesize these pigments with the metabolic versatility of this genus, we explored the possibility of introducing carotenoid synthesis genes into strains capable of efficient growth on simple low-cost media. RESULTS: We constructed two carotenoid-producing strains of Paracoccus carrying a new plasmid, pCRT01, which contains the carotenoid synthesis gene locus crt from Paracoccus marcusii OS22. The plasmid was created in vivo via illegitimate recombination between crt-carrying vector pABW1 and a natural "paracoccal" plasmid pAMI2. Consequently, the obtained fusion replicon is stably maintained in the bacterial population without the need for antibiotic selection. The introduction of pCRT01 into fast-growing "colorless" strains of Paracoccus aminophilus and Paracoccus kondratievae converted them into efficient producers of a range of both carotenes and xanthophylls. The exact profile of the produced pigments was dependent on the strain genetic background. To reduce the cost of carotenoid production in this system, we tested the growth and pigment synthesis efficiency of the two strains on various simple media, including raw industrial effluent (coal-fired power plant flue gas desulfurization wastewater) supplemented with molasses, an industrial by-product rich in sucrose. CONCLUSIONS: We demonstrated a new approach for the construction of carotenoid-producing bacterial strains which relies on a single plasmid-mediated transfer of a pigment synthesis gene locus between Paracoccus strains. This strategy facilitates screening for producer strains in terms of synthesis efficiency, pigment profile and ability to grow on low-cost industrial waste-based media, which should increase the cost-effectiveness of microbial production of carotenoids.

Genome-Based Insights into the Production of Carotenoids by Antarctic Bacteria, Planococcus sp. ANT_H30 and Rhodococcus sp. ANT_H53B.

Antarctic regions are characterized by low temperatures and strong UV radiation. This harsh environment is inhabited by psychrophilic and psychrotolerant organisms, which have developed several adaptive features. In this study, we analyzed two Antarctic bacterial strains, Planococcus sp. ANT_H30 and Rhodococcus sp. ANT_H53B. The physiological analysis of these strains revealed their potential to produce various biotechnologically valuable secondary metabolites, including surfactants, siderophores, and orange pigments. The genomic characterization of ANT_H30 and ANT_H53B allowed the identification of genes responsible for the production of carotenoids and the in silico reconstruction of the pigment biosynthesis pathways. The complex manual annotation of the bacterial genomes revealed the metabolic potential to degrade a wide variety of compounds, including xenobiotics and waste materials. Carotenoids produced by these bacteria were analyzed chromatographically, and we proved their activity as scavengers of free radicals. The quantity of crude carotenoid extracts produced at two temperatures using various media was also determined. This was a step toward the optimization of carotenoid production by Antarctic bacteria on a larger scale.

Galactolipid deficiency disturbs spatial arrangement of the thylakoid network in Arabidopsis thaliana plants.

The chloroplast thylakoid network is a dynamic structure which, through possible rearrangements, plays a crucial role in regulation of photosynthesis. Although the importance of the main components of the thylakoid membrane matrix, galactolipids, in the formation of the network of internal plastid membrane was found before, the structural role of monogalactosyldiacylglycerol (MGDG) and digalactosylidacylglycerol (DGDG) is still largely unknown. We elucidated detailed structural modifications of the thylakoid membrane system in Arabidopsis thaliana MGDG- and DGDG-deficient mutants. An altered MGDG/DGDG ratio was structurally reflected by formation of smaller grana, local changes in grana stacking repeat distance, and significant changes in the spatial organization of the thylakoid network compared with wild-type plants. The decrease of the MGDG level impaired the formation of the typical helical grana structure and resulted in a 'helical-dichotomic' arrangement. DGDG deficiency did not affect spatial grana organization but changed the shape of the thylakoid membrane network in situ from lens like into a flattened shape. Such structural disturbances were accompanied by altered composition of carotenoid and chlorophyll-protein complexes, which eventually led to the decreased photosynthetic efficiency of MGDG- and DGDG-deficient plants.

Three-Dimensional Visualization of the Tubular-Lamellar Transformation of the Internal Plastid Membrane Network during Runner Bean Chloroplast Biogenesis.

Chloroplast biogenesis is a complex process that is integrated with plant development, leading to fully differentiated and functionally mature plastids. In this work, we used electron tomography and confocal microscopy to reconstruct the process of structural membrane transformation during the etioplast-to-chloroplast transition in runner bean (Phaseolus coccineus). During chloroplast development, the regular tubular network of paracrystalline prolamellar bodies (PLBs) and the flattened porous membranes of prothylakoids develop into the chloroplast thylakoids. Three-dimensional reconstruction is required to provide us with a more complete understanding of this transformation. We provide spatial models of the bean chloroplast biogenesis that allow such reconstruction of the internal membranes of the developing chloroplast and visualize the transformation from the tubular arrangement to the linear system of parallel lamellae. We prove that the tubular structure of the PLB transforms directly to flat slats, without dispersion to vesicles. We demonstrate that the grana/stroma thylakoid connections have a helical character starting from the early stages of appressed membrane formation. Moreover, we point out the importance of particular chlorophyll-protein complex components in the membrane stacking during the biogenesis. The main stages of chloroplast internal membrane biogenesis are presented in a movie that shows the time development of the chloroplast biogenesis as a dynamic model of this process.

Overlapping toxic effect of long term thallium exposure on white mustard (Sinapis alba L.) photosynthetic activity.

BACKGROUND: Heavy metal exposure affect plant productivity by interfering, directly and indirectly, with photosynthetic reactions. The toxic effect of heavy metals on photosynthetic reactions has been reported in wide-ranging studies, however there is paucity of data in the literature concerning thallium (Tl) toxicity. Thallium is ubiquitous natural trace element and is considered the most toxic of heavy metals; however, some plant species, such as white mustard (Sinapis alba L.) are able to accumulate thallium at very high concentrations. In this study we identified the main sites of the photosynthetic process inhibited either directly or indirectly by thallium, and elucidated possible detoxification mechanisms in S. alba. RESULTS: We studied the toxicity of thallium in white mustard (S. alba) growing plants and demonstrated that tolerance of plants to thallium (the root test) decreased with the increasing Tl(I) ions concentration in culture media. The root growth of plants exposed to Tl at 100 µg L(-1) for 4 weeks was similar to that in control plants, while in plants grown with Tl at 1,000 µg L(-1) root growth was strongly inhibited. In leaves, toxic effect became gradually visible in response to increasing concentration of Tl (100 - 1,000 µg L(-1)) with discoloration spreading around main vascular bundles of the leaf blade; whereas leaf margins remained green. Subsequent structural analyses using chlorophyll fluorescence, microscopy, and pigment and protein analysis have revealed different effects of varying Tl concentrations on leaf tissue. At lower concentration partial rearrangement of the photosynthetic complexes was observed without significant changes in the chloroplast structure and the pigment and protein levels. At higher concentrations, the decrease of PSI and PSII quantum yields and massive oxidation of pigments was observed in discolored leaf areas, which contained high amount of Tl. Substantial decline of the photosystem core proteins and disorder of the photosynthetic complexes were responsible for disappearance of the chloroplast grana. CONCLUSIONS: Based on the presented results we postulate two phases of thallium toxicity on photosynthesis: the non-destructive phase at early stages of toxicant accumulation and the destructive phase that is restricted to the discolored leaf areas containing high toxicant content. There was no distinct border between the two phases of thallium toxicity in leaves and the degree of toxicity was proportional to the migration rate of the toxicant outside the vascular bundles. The three-fold (nearly linear) increase of Tl(I) concentration was observed in damaged tissue and the damage appears to be associated with the presence of the oxidized form of thallium - Tl(III).

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.

A reaction center-dependent photoprotection mechanism in a highly robust photosystem II from an extremophilic red alga, Cyanidioschyzon merolae.

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extremely low pH levels and moderately high temperatures. The photosynthetic apparatus of the red alga Cyanidioschyzon merolae represents an intermediate type between cyanobacteria and higher plants, suggesting that this alga may provide the evolutionary link between prokaryotic and eukaryotic phototrophs. Although we now have a detailed structural model of photosystem II (PSII) from cyanobacteria at an atomic resolution, no corresponding structure of the eukaryotic PSII complex has been published to date. Here we report the isolation and characterization of a highly active and robust dimeric PSII complex from C. merolae. We show that this complex is highly stable across a range of extreme light, temperature, and pH conditions. By measuring fluorescence quenching properties of the isolated C. merolae PSII complex, we provide the first direct evidence of pH-dependent non-photochemical quenching in the red algal PSII reaction center. This type of quenching, together with high zeaxanthin content, appears to underlie photoprotection mechanisms that are efficiently employed by this robust natural water-splitting complex under excess irradiance. In order to provide structural details of this eukaryotic form of PSII, we have employed electron microscopy and single particle analyses to obtain a 17 Å map of the C. merolae PSII dimer in which we locate the position of the protein mass corresponding to the additional extrinsic protein stabilizing the oxygen-evolving complex, PsbQ'. We conclude that this lumenal subunit is present in the vicinity of the CP43 protein, close to the membrane plane.

Chloroplast biogenesis - correlation between structure and function.

Chloroplast biogenesis is a multistage process leading to fully differentiated and functionally mature plastids. Complex analysis of chloroplast biogenesis was performed on the structural and functional level of its organization during the photoperiodic plant growth after initial growth of seedlings in the darkness. We correlated, at the same time intervals, the structure of etioplasts transforming into mature chloroplasts with the changes in the photosynthetic protein levels (selected core and antenna proteins of PSI and PSII) and with the function of the photosynthetic apparatus in two plant species: bean (Phaseolus vulgaris L.) and pea (Pisum sativum L). We selected these plant species since we demonstrated previously that the mature chloroplasts differ in the thylakoid organization. We showed that the protein biosynthesis as well as photosynthetic complexes formation proceeds gradually in both plants in spite of periods of darkness. We found that both steady structural differentiation of the bean chloroplast and reformation of prolamellar bodies in pea were accompanied by a gradual increase of the photochemical activity in both species. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.

The coupled photocycle of phenyl-p-benzoquinone and Light-Harvesting Complex II (LHCII) within the biohybrid system.

The combination of trimeric form of the light-harvesting complex II (LHCII3), a porous graphite electrode (GE), and the application of phenyl-p-benzoquinone (PPBQ), the quinone derivative, allow the construction of a new type of biohybrid photoactive system. The Chl fluorescence decay and voltammetric analyzes revealed that PPBQ impacts LHCII3 proportionally to accessible quenching sites and that PPBQ forms redox complexes with Chl in both ground and excited states. As a result, photocurrent generation is directly dependent on PPBQ-induced quenching of Chl fluorescence. Since PPBQ also undergoes photoactivation, the action of GE-LHCII3-PPBQ depends on the mutual coupling of LHCII3 and PPBQ photocycles. The GE-LHCII3-PPBQ generates a photocurrent of up to 4.5 µA and exhibits considerable stability during operation. The three-dimensional arrangement of graphite scraps in GE builds an active electrode surface and stabilizes LHCII3 in its native form in low-density multilayers. The results indicate the future usability of such designed photoactive device.

3-D modelling of chloroplast structure under (Mg2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking.

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.

Structural and functional modifications of the major light-harvesting complex II in cadmium- or copper-treated Secale cereale.

The effects of 50 microM cadmium (Cd) or copper (Cu) ions on the supramolecular conformation of the light-harvesting pigment-protein complex of PSII (LHCII) isolated from rye seedlings were studied. It was found that the action of these two metal ions on the LHCII structure and organization is dissimilar. The Fourier transform infrared (FTIR) measurements indicated inhibition or stimulation of formation of parallel beta-structures and aggregates in the presence of Cd or Cu ions, respectively. The Chl a fluorescence excitation spectra of LHCII extracted from Cd-treated plants showed that the decreased aggregation of complexes was correlated with a decline in efficiency of quenching of excitation energy. From the results of mass spectrometry, changes in LHCII aggregation in the presence of Cd ions might be based on decreases in the molecular mass of Lhcb1 and Lhcb2 proteins. An increase in the content of LHCII aggregates under Cu ion excess was associated with changes in the LHCII xanthophyll pigment pool. In the complexes isolated from Cu-treated plants, all-trans violaxanthin and 9'-cis neoxanthin content declined and the simultaneous appearance of the fraction of 9-cis violaxanthin was observed. 9-cis violaxanthin formation under Cu ion excess might facilitate LHCII inter-trimer interaction and, therefore, aggregation of complexes. RLS (resonance light scattering) spectra indicated that the excitonic interaction between Chl molecules and between Chls and xanthophylls was responsible for the effective dissipation of excitation energy in LHCII isolated from Cu-treated plants. Also, changes in singlet excitation energy transfer between carotenoids and Chls under the action of heavy metals were observed.

Small Cyclic Peptide for Pyrophosphate Dependent Ligation in Prebiotic Environments.

All life on Earth uses one universal biochemistry stemming from one universal common ancestor of all known living organisms. One of the most striking features of this universal biochemistry is its utter dependence on phosphate group transfer between biochemical molecules. Both nucleic acid and peptide biological synthesis relies heavily on phosphate group transfer. Such dependents strongly indicate very early incorporation of phosphate chemistry in the origin of life. Perhaps as early as prebiotic soup stage. We report here on a short cyclic peptide, c(RPDDHR), designed rationally for pyrophosphate interaction, which is able to create a new amide bond dependent on the presence of pyrophosphate. We believe this result to be a first step in the exploration of Phosphate Transfer Catalysts that must have been present and active in prebiotic soup and must have laid down foundations for the universal bioenergetics.

Specific Composition of Lipid Phases Allows Retaining an Optimal Thylakoid Membrane Fluidity in Plant Response to Low-Temperature Treatment.

Thylakoid membranes isolated from leaves of two plant species, the chilling tolerant (CT) pea and chilling sensitive (CS) runner bean, were assessed for the composition of lipids, carotenoids as well as for the arrangement of photosynthetic complexes. The response to stress conditions was investigated in dark-chilled and subsequently photo-activated detached leaves of pea and bean. Thylakoids of both species have a similar level of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), but different sulfoquinovosyldiacylglycerol to phosphatidylglycerol (PG) ratio. In pea thylakoid fraction, the MGDG, DGDG and PG, have a higher double bond index (DBI), whereas bean thylakoids contain higher levels of high melting point PG. Furthermore, the lutein to the ß-carotene ratio is higher in bean thylakoids. Smaller protein/lipid ratio in pea than in bean thylakoids suggests different lipid-protein interactions in both species. The differences between species are also reflected by the course of temperature-dependent plots of chlorophyll fluorescence pointing various temperatures of the lipid phase transitions of pea and bean thylakoids. Our results showed higher fluidity of the thylakoid membrane network in pea than in bean in optimal temperature conditions. Dark-chilling decreases the photochemical activity and induces significant degradation of MGDG in bean but not in pea leaves. Similarly, substantial changes in the arrangement of photosynthetic complexes with increase in LHCII phosphorylation and disturbances of the thylakoid structure take place in bean thylakoids only. Changes in the physical properties of bean thylakoids are manifested by the conversion of a three-phase temperature-dependent plot to a one-phase plot. Subsequent photo-activation of chilled bean leaves caused a partial restoration of the photochemistry and of membrane physical properties, but not of the photosynthetic complexes arrangement nor the thylakoid network structure. Summarizing, the composition of the thylakoid lipid matrix of CT pea allows retaining the optimal fluidity of its chloroplast membranes under low temperatures. In contrast, the fluidity of CS bean thylakoids is drastically changed, leading to the reorganization of the supramolecular structure of the photosynthetic complexes and finally results in structural remodeling of the CS bean thylakoid network.

Light-induced change of configuration of the LHCII-bound xanthophyll (tentatively assigned to violaxanthin): a resonance Raman study.

Raman scattering spectra of light-harvesting complex LHCII isolated from spinach were recorded with an argon laser, tuned to excite the most red-absorbing LHCII-bound xanthophylls (514.5 nm). The intensity of the nu(4) band (at ca. 950 cm-1) corresponding to the out-of-plane wagging modes of the C-H groups in the resonance Raman spectra of carotenoids appears to be inversely dependent on the probing laser power density. This observation can be interpreted in terms of excitation-induced change of configuration of the protein-bound xanthophyll owing to the fact that the intensity of this particular band is diagnostic of a chromophore twisting resulting from its binding to the protein environment. The comparison of the shape of the nu(4) band of a xanthophyll involved in the light-induced spectral changes with the shape of the nu(4) band of the xanthophylls present in LHCII, reported in the literature, lets us conclude that, most probably, violaxanthin is a pigment that undergoes light-driven changes of molecular configuration but also the involvement of lutein may not be excluded. Possible physical mechanisms responsible for the configuration changes and physiological importance of the effect observed are discussed.

Electrochemical characterization of LHCII on graphite electrodes - Potential-dependent photoactivation and arrangement of complexes.

Light-dependent electrochemical properties of the light harvesting complexes of Photosystem II (LHCII) and the corresponding interactions with screen-printed graphite electrodes (GEs) are determined. No exogenous soluble redox mediators are used. LHCII isolated from spinach leaves are immobilized on GE by physical adsorption and through interactions with glutaraldehyde. Importantly, the insertion of LHCII into the pores of a GE is achieved by subjecting the electrode to specific potentials. Both trimeric and aggregated forms of LHCII located within the graphite layer retain their native structures. Voltammetric current peaks centred at ca. -230 and + 50 mV vs. Ag/AgCl (+94 and + 374 mV vs. NHE) limit the investigation of the reduction and oxidation processes of immobilized LHCII. An anodic photocurrent is generated in the LHCII-GE proportional to light intensity and can reach a value of 150 nA/cm2. Light-dependent charge separation in LHCII followed by electron transfer to the GE occurs only at potentials of above -200 mV vs. Ag/AgCl (+124 mV vs. NHE). Our results illustrate the importance of the structural proximity of LHCII and GE for photocurrent generation.