Tünde Tóth

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes.

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.

Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium, Synechocystis PCC 6803

Despite intense research, the mechanism of Cd(2+) toxicity on photosynthesis is still elusive because of the multiplicity of the inhibitory effects and different barriers in plants. The quick Cd(2+) uptake in Synechocystis PCC 6803 permits the direct interaction of cadmium with the photosynthetic machinery and allows the distinction between primary and secondary effects. We show that the CO(2) -dependent electron transport is rapidly inhibited upon exposing the cells to 40 µm Cd(2+) (50% inhibition in ∼15 min). However, during this time we observe only symptoms of photosystem I acceptor side limitation and a build of an excitation pressure on the reaction centres, as indicated by light-induced P700 redox transients, O(2) polarography and changes in chlorophyll a fluorescence parameters. Inhibitory effects on photosystem II electron transport and the degradation of the reaction centre protein D1 can only be observed after several hours, and only in the light, as revealed by chlorophyll a fluorescence transients, thermoluminescence and immunoblotting. Despite the marked differences in the manifestations of these short- and long-term effects, they exhibit virtually the same Cd(2+) concentration dependence. These data strongly suggest a cascade mechanism of the toxic effect, with a primary effect in the dark reactions.

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering ☆ ☆☆

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts-using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023Å(-1), corresponding to spatially and statistically averaged repeat distance values of about 275-330 Å⁻¹. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D2O in dim laboratory light or transpired with D2O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230 Å⁻¹, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

Carotenoids Assist in Cyanobacterial Photosystem II Assembly and Function

Carotenoids (carotenes and xanthophylls) are ubiquitous constituents of living organisms. They are protective agents against oxidative stresses and serve as modulators of membrane microviscosity. As antioxidants they can protect photosynthetic organisms from free radicals like reactive oxygen species that originate from water splitting, the first step of photosynthesis. We summarize the structural and functional roles of carotenoids in connection with cyanobacterial Photosystem II. Although carotenoids are hydrophobic molecules, their complexes with proteins also allow cytoplasmic localization. In cyanobacterial cells such complexes are called orange carotenoid proteins, and they protect Photosystem II and Photosystem I by preventing their overexcitation through phycobilisomes (PBS). Recently it has been observed that carotenoids are not only required for the proper functioning, but also for the structural stability of PBSs.

Fingerprinting the macro-organisation of pigment-protein complexes in plant thylakoid membranes in vivo by circular-dichroism spectroscopy.

Macro-organisation of the protein complexes in plant thylakoid membranes plays important roles in the regulation and fine-tuning of photosynthetic activity. These delicate structures might, however, undergo substantial changes during isolating the thylakoid membranes or during sample preparations, e.g., for electron microscopy. Circular-dichroism (CD) spectroscopy is a non-invasive technique which can thus be used on intact samples. Via excitonic and psi-type CD bands, respectively, it carries information on short-range excitonic pigment-pigment interactions and the macro-organisation (chiral macrodomains) of pigment-protein complexes (psi, polymer or salt-induced). In order to obtain more specific information on the origin of the major psi-type CD bands, at around (+)506, (-)674 and (+)690nm, we fingerprinted detached leaves and isolated thylakoid membranes of wild-type and mutant plants and also tested the effects of different environmental conditions in vivo. We show that (i) the chiral macrodomains disassemble upon mild detergent treatments, but not after crosslinking the protein complexes; (ii) in different wild-type leaves of dicotyledonous and monocotyledonous angiosperms the CD features are quite robust, displaying very similar excitonic and psi-type bands, suggesting similar protein composition and (macro-) organisation of photosystem II (PSII) supercomplexes in the grana; (iii) the main positive psi-type bands depend on light-harvesting protein II contents of the membranes; (iv) the (+)506nm band appears only in the presence of PSII-LHCII supercomplexes and does not depend on the xanthophyll composition of the membranes. Hence, CD spectroscopy can be used to detect different macro-domains in the thylakoid membranes with different outer antenna compositions in vivo.

Heat- and light-induced detachment of the light harvesting complex from isolated photosystem I supercomplexes

In a previous study, using photosystem I enriched stroma thylakoid membrane vesicles, we have shown that the light harvesting complexes of this photosystem are prone to heat- and light-induced, thermo-optically driven detachment from the supercomplex [43]. We have also shown that the splitting of the supercomplex occurs in a gradual and specific manner, selectively affecting the different constituents of the antenna complexes. Here we further analyse these heat- and light-induced processes in isolated Photosystem I supercomplex using circular dichroism and 77K fluorescence emission spectroscopy and immuno blotting, and obtain further details on the sequence of events of the dissociation process as well as on the thermal stability of the different components. Our absorption and circular dichroism spectroscopy and immuno blotting data show that the dissociation of LHCI from PSI-LHCI supercomplex starts above 50°C. Also, the low temperature fluorescence emission spectra depicts decrease of maximum fluorescence emission at 730nm and an increase of the intensity at 685nm, and about 10nm blue-shifts, from 730 to 720nm and from 685 to 676nm, respectively, indicating the heat (50°C) induced detachment of LHCI from PSI core complexes. The reaction centre proteins are highly stable even at high temperatures. Lhca2 is more heat stable than the other light harvesting protein complexes of PSI, whereas Lhca4 and Lhca3 are rather labile. Combined heat and light treatments significantly enhances the disorganization of PSI-LHCI supercomplexes, indicating a thermo-optic mechanism, which might have significant role under combined heat and light stress conditions.

β-Carotene influences the phycobilisome antenna of cyanobacterium Synechocystis sp. PCC 6803

We investigated the relation between the carotenoid composition and the structure of phycobilisome (PBS) antenna of cyanobacterium Synechocystis sp. PCC 6803. PBS is a large soluble protein complex enhances the light harvesting efficiency of the cells. It is composed of a central allophycocyanin core and radial phycocyanin rods, but it does not contain carotenoids. However, the absence or low level of carotenoids were previously shown to lead the co-existence of unconnected rod units and assembled PBS with shorter peripheral rods. Here we show that the lack of β-carotene, but not of xanthophylls or the distortion of photosystem structure, evoked unconnected rods. Thus, these essential β-carotene molecules are not bound by Photosystem I or Photosystem II. Our results do not show correlation between the reactive oxygen species (ROS) and PBS distortion despite the higher singlet oxygen producing capacity and light sensitivity of the mutant cells. Reduced cellular level of those linker proteins attaching the rod units together was also observed, but the direct damage of the linkers by ROS are not supported by our data. Enzymatic PBS proteolysis induced by nitrogen starvation in carotenoid mutant cells revealed a retarded degradation of the unconnected rod units.

Zeaxanthin and echinenone modify the structure of photosystem I trimer in Synechocystis sp. PCC 6803

The function of xanthophylls in the organisation and structure of the photosynthetic complexes is not completely clarified yet. Recently, we observed a reduced level of the photosystem oligomers upon xanthophyll deficiency, although xanthophylls are not considered to be part of the photosynthetic complexes of cyanobacteria. The present study aimed at further investigating the relationship between xanthophylls and photosytem I (PSI) complex in the cyanobacterium Synechocystis sp. PCC 6803. Interestingly, we recorded the presence of echinenone and zeaxanthin in the isolated PSI trimers. These two xanthophyll species are among the most abundant xanthophylls in this cyanobacterial species. Various xanthophyll biosynthesis mutants were used to investigate the specific role of these xanthophylls. Our spectroscopic results revealed specific structural changes manifested in altered pigment-pigment or pigment-protein interactions within PSI complex in the absence of zeaxanthin and echinenone. These structural modifications of the complexes seem to destabilize the PSI trimeric complexes and eventually result in an increased propensity for monomerization. Our results clearly demonstrate that xanthophylls are important for the fine-tuning of the PSI trimer structure. These xanthophylls could be part of the complex or be embedded in the membrane in the vicinity of PSI.

Akridon és akridin egységet tartalmazó koronaéter alapú szenzor- és szelektormolekulák szintézise, kation- és enantiomerfelismerése

Napjainkban széleskörû tudományos érdeklõdés irányul a különbözõ fémionok, illetve királis vegyületek enantiomerjeinek egyre szelektívebb felismerését biztosító szenzormolekulák, valamint az ezek elválasztását is lehetõvé tevõ szelektormolekulák kifejlesztésére, mivel alkalmazásukra a gyógyszeriparban, az élelmiszeriparban és a környezetvédelemben is lehetõség nyílik. Ezen vegyületek szelektív komplexképzõ tulajdonságainak alapja a molekuláris felismerés jelensége, amely a legjobban két vagy több molekula közötti szelektív megkülönböztetésként jellemezhetõ, amely során másodlagos (nem kovalens) kölcsönhatások kialakulása révén rendezett szerkezetek, komplexek keletkeznek. A molekuláris felismerés fogalmát Emil Fischer dolgozta ki 1894-ben, mikor egy adott enzim adott szubsztráthoz való specifikus kötõdését a „kulcs-zár” elmélettel magyarázta. A szupramolekuláris kémia fogalmának bevezetése pedig Jean-Marie Lehn nevéhez fûzõdik: véleménye szerint ez úgy határozható meg, mint “kémia a molekulán túl”1. Az egyre szélesebb körben terjedõ szupramolekuláris kémia másodlagos kötõerõkkel összetartott molekuláris asszociátumok keletkezésével, tulajdonságainak és alkalmazási lehetõségeinek vizsgálatával foglalkozik. Mióta az egyik úttörõje az ilyen irányú kutatásoknak, Charles J. Pedersen, publikálta az elsõ szintetikus gazdamolekulák, a koronaéterek szintézisét és vizsgálatát2,3, ez a kutatási terület jelentõs érdeklõdésre tett szert4. E terület elismerését jelzi, hogy a nagy szelektivitással rendelkezõ szerkezet-specifikus kölcsönhatások kialakítására képes molekulák kifejlesztésért Charles J. Pedersennek, Donald J. Cramnek és Jean-Marie Lehnnek ítélték oda az 1987. évi kémiai Nobel-díjat. A 2016. évi kémiai Nobel-díjat Jean-Pierre Sauvage, Sir J. Fraser Stoddart és Bernard L. Feringa kapta molekuláris gépek fejlesztéséért, amely azt mutatja, hogy a kémia e területe most is nagy érdeklõdésre tart számot.

Critical evaluation of rate coefficients for hydroxyl radical reactions with antibiotics: A review

Abstract Rate coefficients (k•OH) of hydroxyl radical + antibiotic molecule reactions (c.a. 200 k•OH values for 70 molecules) were collected from published studies. Data measured in different laboratories, occasionally by different methods, were compared. Excluding the rate coefficients with values above the diffusion controlled limit and adjusting rate coefficients determined by competition kinetics, the data were averaged and the most probable values were selected. Rate coefficients for compounds with aromatic rings (e.g., amoxicillin) or conjugated double bonds (e.g., tylosin) are in the (6 – 8) × 109 mol−1 dm3 s−1 range. The basic reaction is radical addition. The value may be smaller when strong electron withdrawing substituent, like –NO2 is attached to the ring, and N-atoms in the aromatic ring also reduce the rate coefficient. The rate coefficients for molecules without aromatic rings are smaller with values in the (3 – 6) × 109 mol−1 dm3 s−1 range, where hydrogen abstraction reactions dominate. Carefully executed pulse radiolysis and steady-state experiments (with correct reference values) give similar rate coefficients.