Research - Institute of Plant Biology - Laboratory of Photosynthetic Membranes

Petar LAMBREV
senior research associate

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Győző GARAB Professor Emeritus
Bettina UGHY research associate
Márta DOROGI research associate
Melinda MAGYAR research associate
Ottó ZSIROS scientific administrator
Gábor SIPKA predoctoral Ph.D. student
Parveen AKHTAR junior research associate
Mónika LINGVAY PhD student
Sai Divya KANNA PhD student
Ágnes RÉDAI laboratory assistant

LIGHT ENERGY CONVERTING BIOLOGICAL STRUCTURES, PHYSIOLOGICAL PROCESSES, PHYSICAL MECHANISMS

The main goal of our research is to understand the structure and function of photosynthetic membranes with special attention to the interplay between the dynamic macro-organization of the membranes and the regulation of excitation energy and electron flow. We also design and construct innovative scientific instruments.


Macro-organization and flexibility of the photosynthetic membranes

The main light harvesting antenna complexes (LHCII) and photosystem II (PSII) in plants are assembled into highly organized but structurally flexible macrodomains with long-range chiral order, which give rise to giant, so-called psi-type circular dichroism (CD) bands (Figure 1). The macrodomain formation explains the sorting of LHCII:PSII and LHCI:PSI supercomplexes and the stacking of membranes, the basis of the self-assembly of granal ultrastructure (Mustárdy and Garab 2003). Using electron tomography, we revealed the quasi-helical organization of the granum-stroma thylakoid membrane assembly (Mustárdy et al. 2008).



Figure 1. A. Thin-section electron micrograph of higher-plant chloroplast. B. 3D computer model of the helically organized granal thylakoid membrane, based on electron micrographs from serial sectioning of granum-stroma assemblies. C. CD spectra of thylakoid membranes at different levels of sctructural complexity: intrinsic CD due to weak chirality (multiplied by 5), measured in acetone; excitonic CD due to short-range dipole interactions in the pigment-protein complexes, measured in hypotonic, law-salt buffer; psi-type CD dependent on the long-range chiral order of chromophores in the stacked membranes suspended in isotonic buffer in the presence of Mg2+ ions.


Small-angle neutron scattering (SANS) is a non-invasive technique that can monitor dynamic structural parameters of the membrane system, such as the repeat distance, or the periodicity of thylakoid membranes. SANS studies on a large variety of photosynthetic organisms in vivo and isolated thylakoid membranes under a broad range of experimental conditions revealed unexpectedly high structural flexibility of the membrane system. Small (~10 Å) but well discernible light-induced reversible changes in the periodicity of the thylakoid membranes were discovered in living algal cells and plant leaves (Nagy et al. 2012, 2014, Ünnep et al. 2014, Herdean et al. 2016).



Figure 2. Small-angle neutron scattering (SANS) profiles of isolated thylakoid membranes in the dark or after illumination with white light in the presence of DCMU and PMS.



Molecular interactions in the photosynthetic membranes

The light-harvesting functions of the thylakoid membrane depend on the molecular organization of LHCII – changes in the LHCII conformation and intermolecular interactions balance the amount of energy directed to the PSII reaction centers or dissipated as heat. The high degree of connectivity, i.e. the large domain size (Lambrev et al. 2011), enable long-range migration of excitation energy under normal conditions. Dissociation of LHCII from PSII and non-photochemical quenching of excited states (for reviews, see Demmig-Adamas et al. 2014) in LHCII are two mechanisms protecting the reaction centers from photodamage (Miloslavina et al. 2008, Lambrev et al. 2012). Similar mechanisms seem to operate in diatoms, which have very different antenna organization (Miloslavina et al. 2009, Ghazaryan et al. 2016). These processes are tightly regulated by interactions between the pigment-protein complexes in the membranes. By CD spectroscopy we have revealed that the pigment-pigment interactions in LHCII are sensitive to its molecular environment (Lambrev et al. 2007, Akhtar et al. 2015). Extracting of the complexes from the membrane and solubilization with detergents perturbs the native structure.

The anisotropic CD (ACD) of macroscopically aligned samples – an extension of the CD spectroscopy – provides additional information about the molecular orientation of the participating pigment dipoles. For example, we have applied ACD to validate the structural model of the bacterial chlorosome baseplate protein CsmA (Nielsen et al. 2016). Likewise, LHCII in isolated form or in the native membrane, as well as LHCII microcrystals have a distinct ACD signature (Miloslavina et al. 2011) showing the relative orientation of the transition dipole moments participating in the pigment interactions with respect to the membrane.

Electrostatic interactions between LHCIIs (mediated by charge-screening cations), are partly responsible for the stacking of thylakoid membranes and the formation of grana. We have shown that LHCII has an in-built light switch that dynamically controls the membrane stacking (Hind et al. 2014). This effect is attributed to the so-called thermo-optic effect, a novel mechanism, discovered in our laboratory (for review, see Garab 2014) (Figure 3) – elementary structural changes in light-harvesting antenna complexes, elicited by local heat transients due to the dissipation of photon energy (Gulbinas et al. 2006). In general, our knowledge about the mechanisms and effects of the dissipation of excess excitation energy in photosynthesis is quite rudimentary. A reconstituted Ni-bacterochlorophyll-containing bacterial antenna complex, with well-defined dissipation site and time, has thus been constructed (Lambrev et al. 2013), which will enable us to monitor the ultrafast kinetics of molecular vibrations due to the appearance of dissipation-induced heat package.



Figure 3. Thermo-optic effect in thylakoid membranes. A. Light-induced dark-reversible structural changes in the macroorganization of isolated thylakoid membranes monitored by amplitude variations of the psi-type CD band at 510 nm. B. Schematic representation of the proposed three-state model for explaining the thermo-optic mechanism and the temperature-dependence of thermo-optically induced reorganizations. C. Crystal structure of LHCII (monomer) illustrating the proximity of the site of dissipation (red chlorophylls) to the N-terminal region of the protein, possessing high flexibility and the ability to bind cations. D. Schematic model of the cation-assisted stacking of membrane layers in dark conditions and the thermo-optically induced cation release and concomitant membrane unstacking under light irradiation.



Reconstituted models of the photosynthetic membranes

As experimental systems of intermediate complexity bridging the gap between the full thylakoid membrane and isolated complexes, we use artificially reconstituted membrane models –proteoliposomes and supported lipid bilayers – containing predefined composition of lipids, protein complexes and cofactors. With the help of these models we can mimic and understand various functions of the native photosynthetic membranes. LHCII-lipid membranes show the propensity of LHCII to self-aggregate creating domains with high degree of energetic connectivity (Akhtar et al. 2015). By picosecond time-resolved fluorescence spectroscopy of reconstituted membranes with LHCII and Photosystem I (PSI) we could show that LHCII can transfer excitations efficiently to PSI, substantially increasing the total absorption cross-section of PSI with a minor loss in the quantum yield of photochemistry (Akhtar et al. 2016).



Figure 4. Reconstituted thylakoid membrane models. A – freeze-fracture EM image of a proteoliposome containing LHCII and Photosystem I; B – species-associated emission spectra of LHCII and Photosystem I (bulk and “red” Chls) from time-resolved fluorescence analysis; C – kinetic scheme of the connectivity of LHCII and Photosystem I with rate constants in ns-1.


We proposed that non-bilayer lipids (such as MGDG, which constitutes ~50% of the lipid content of bilayer thylakoid membranes) play key role in self-regulating the protein content of membranes and hypothesized the existence of a non-bilayer lipid phase associated and exchanging with the bilayer membrane (Garab et al. 2000). In the past years, using P31-NMR and fluorescence spectroscopy, we provided experimental evidence supporting this hypothesis (Krumova et al. 2008, Garab et al. 2016).

Technical development projects and applications

We transform confocal laser scanning microscopes (LSMs) into differential-polarization (DP-)LSMs, which, via measuring pixel-by-pixel different physical quantities allow the mapping of anisotropic molecular organization of biological samples. For papers, exhibitions and prizes, and patents see www.dp-lsm.com.

Selected publications

Akhtar, P., Dorogi, M., Pawlak, K., Kovács, L., Bóta, A., Kiss, T., Garab, G. & Lambrev, P.H. Pigment interactions in light-harvesting complex II in different molecular environments, J. Biol. Chem. 290: 4877-4886 (2015)

Akhtar, P., Lingvay, M., Kiss, T., Deák, R., Bóta, A., Ughy, B., Garab, G., Lambrev, P.H. Excitation energy transfer between light-harvesting complex II and Photosystem I in reconstituted membranes, Biochim. Biophys. Acta 1857:462-72 (2016)

Akhtar, P., Zhang, C., Do, T.N., Garab, G., Lambrev, P.H., Tan, H.-S. Two-dimensional spectroscopy of chlorophyll a excited-state equilibration in light-harvesting complex II. J. Phys. Chem. Lett. 8: 257-263 (2017)

Demmig-Adams, B., Garab, G. & Adams III, W. (Eds) Non-Photochemical Quenching and Energy Dissipation in Plants, Algae and Cyanobacteria. Adv. Photosynth. Respir., Vol. 40, Springer Science+Business Media, Dordrecht (2014)

Garab, G. & Mustárdy, L. Role of LHCII-containing macrodomains in the structure, function and dynamics of grana. Funct. Plant Biol. 27: 279-279 (2000)

Garab, G. Hierarchical organization and structural flexibility of thylakoid membranes. Biochim. Biophys. Acta 1837: 481-494 (2014)

Garab, G., Ughy, B. & Goss, R. Role of MGDG and non-bilayer lipid phases in the structure and dynamics of chloroplast thylakoid membranes. Lipids in Plant and Algae Dev., Springer International Publishing, Vol. 86: 127-157 (2016)

Ghazaryan, A., Akhtar, P., Garab, G., Lambrev, P.H. & Büchel C. Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes. Biochim. Biophys. Acta 1857: 1373-1379 (2016)

Gulbinas, V., Karpicz, R., Garab, G. & Valkunas, L. Nonequilibrium heating in LHCII complexes monitored by ultrafast absorbance transients. Biochemistry 45: 9559-9565 (2006)

Herdean, A., Teardo, E., Nilsson, A.K., Pfeil, B.E., Johansson, O.N., Ünnep, R., Nagy, G., Zsiros, O., Dana, S., Solymosi, K., Garab, G., Szabó, I., Spetea, C. & Lundin, B. A voltage-dependent chloride channel fine-tunes photosynthesis in plants, Nat. Commun. 7: 11654 (2016)

Hind, G., Wall, J.S., Várkonyi, Z., Istokovics, A., Lambrev, P.H. & Garab, G. Membrane crystals of plant light-harvesting complex II disassemble reversibly in light. Plant Cell Physiol. 55: 1296-303 (2014)

Krumova, S.B., Koehorst, R.B.M., Bóta, A., Páli, T., van Hoek, A., Garab, G. & van Amerongen, H. Temperature dependence of the lipid packing in thylakoid membranes studied by time-and spectrally resolved fluorescence of Merocyanine 540. Biochim. Biophys. Acta 1778: 2823-2833 (2008)

Krumova, S.B., Laptenok, S.P., Kovács, L., Tóth, T., van Hoek, A., Garab, G. & van Amerongen, H. Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes. Photosynth. Res. 105: 229-242 (2010)

Lambrev, P.H. & Miloslavina, Y, On the relationship between non-photochemical quenching and photoprotection of Photosystem II. Biochim. Biophys. Acta 1817: 760–769 (2012)

Lambrev, P.H., Miloslavina, Y., Van Stokkum, I.H.M., Stahl, A.D., Michalik, M., Susz, A., Tworzydło, J., Fiedor, J., Huhn, G., Groot, M.L., Van Grondelle, R., Garab, G. & Fiedor, L. Excitation energy trapping and dissipation by Ni-substituted bacteriochlorophyll a in reconstituted LH1 complexes from Rhodospirillum rubrum. J. Phys. Chem. B 117: 11260-11271 (2013)

Lambrev, P.H., Schmitt, F.J., Kussin, S., Schoengen, M., Várkonyi, Zs., Eichler, H.J., Garab, G. & Renger G. Functional domain size in aggregates of light-harvesting complex II and thylakoid membranes. Biochim. Biophys. Acta 1807: 1022-1031 (2011)

Lambrev, P.H., Várkonyi, Zs., Krumova, S., Kovács, L., Miloslavina, Y., Holzwarth, A.R. & Garab, G. Importance of trimer–trimer interactions for the native state of the plant light-harvesting complex II. Biochim. Biophys. Acta 1767: 847-853 (2007)

Miloslavina, Y., Grouneva, I., Lambrev, P.H., Lepetit, B., Goss, R., Wilhelm, C. & Holzwarth, A.R. Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms. Biochim. Biophys. Acta 1787: 1189-1197 (2009)

Miloslavina, Y., Lambrev, P.H., Jávorfi, T., Várkonyi, Zs., Karlický, V., Wall, J.S., Hind, G. & Garab, G. Anisotropic circular dichroism signatures of oriented thylakoid membranes and lamellar aggregates of LHCII. Photosynth. Res. 111: 29-39 (2011)

Miloslavina, Y., Wehner, A., Lambrev, P.H., Wientjes, E., Reus, M., Garab, G., Croce, R. & Holzwarth, A.R. Far-red fluorescence: a direct spectroscopic marker for LHCII oligomer formation in non-photochemical quenching. FEBS Lett. 582: 3625-3631 (2008)

Mustárdy, L. & Garab, G. Granum revisited. A three-dimensional model–where things fall into place. Trends Plant Sci. 8: 117-122 (2003)

Mustárdy, L., Buttle, K., Steinbach, G. & Garab, G. The three-dimensional network of the thylakoid membranes in plants: quasihelical model of the granum-stroma assembly. Plant Cell 20: 2552-2557 (2008)

Nagy, G., Szabó, M., Ünnep, R., Káli, G., Miloslavina, Y., Lambrev, P.H., Zsiros, O., Porcar, L., Timmins, P., Rosta, L. & Garab, G. Modulation of the multilamellar membrane organization and of the chiral macrodomains in the diatom revealed by small-angle neutron scattering and circular dichroism spectroscopy. Photosynth. Res. 111: 71-79 (2012)

Nagy, G., Ünnep, R., Zsiros, O., Tokutsu, R., Takizawa, K., Porcar, L., Moyet, L., Petroutsos, D., Garab, G., Finazzi, G. & Minagawa, J. Chloroplast remodeling during state transitions in Chlamydomonas reinhardtii as revealed by non-invasive techniques in vivo, Proc. Natl. Acad. Sci. USA, 111: 5042-5047 (2014)

Nielsen, J.T., Kulminskaya, N.V., Bjerring, M., Linnanto, J.M., Rätsep, M., Pedersen, M.Ø., Lambrev, P.H., Dorogi, M., Garab, G., Thomsen, K., Jegerschöld, C., Frigaard, N.U., Lindahl, M., Nielsen, N.Chr. In situ high-resolution structure of the baseplate antenna complex in Chlorobaculum tepidum. Nat. Commun. 7: 12454 (2016)

Schansker, G., Tóth, S.Z., Kovács, L., Holzwarth, A.R. & Garab, G. Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise. Biochim. Biophys. Acta 1807: 1032-1043 (2011)

Ünnep, R., Zsiros, O., Solymosi, K., Kovács, L., Lambrev, P.H., Tóth, T., Schweins, R., Posselt, D., Székely, N.K., Rosta, L., Nagy, G. & Garab G. The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering. Biochim. Biophys. Acta 1837: 1572-1580 (2014)

Wells, K.L., Lambrev, P.H., Zhang, Z., Garab, G. & Tan, H.S. Pathways of energy transfer in LHCII revealed by room-temperature 2D electronic spectroscopy, Phys. Chem. Chem. Phys. 16: 1463-9076 (2014) Zhang, Z., Lambrev, P.H., Wells, K.L., Garab, G. & Tan, H.S. Direct observation of multistep energy transfer in LHCII with fifth-order 3D electronic spectroscopy. Nat. Commun. 6: 7914 (2015)