Báo cáo khoa học: J1–Photosynthetic Light Harvesting and Reaction Center Complexes ppt

J1–Photosynthetic Light Harvesting and Reaction Center Complexes J1-001 Cocrystals of photosystem I with its soluble natural electron acceptor ferredoxin at 4 A resolution R. Fromme, H. Yu, I. Grotjohann and P. Fromme Department of Chemistry and Biochemistry, Arizona State Univer- sity, Tempe, AZ, USA. E-mail: Raimund.Fromme@asu.edu We measured cocrystals of the membrane protein Photosystem I with its soluble electron acceptor ferredoxin for the first time at the ALS (Berkeley, CA). Previous data collected at our home source and at other synchrotron sources showed crystals with very high mosaicity (2–5%) and a diffraction limit to 7–8 Aresolution. This first beamtime at ALS was very successful and may represent a breakthrough for the determination of the crystal structure. 110 crystals, grown under modified crystallization conditions, were scanned and for the first time, crystals diffraction to 3.5 A were observed. These crystals have a different space group and unit cell dimensions than all previous grown PSI-ferr cocrystals. The space group has been determined to be P21 with a = 214.5, b = 235.6, c = 261.2 alpha = 90.0 beta = 100.47 gamma = 90.0. A full data set (360 images with 1.5 min exposure/ image and 0.5 rota- tion/ image) was collected from one crystal, even if the long X-ray exposure has limited the completeness of the higher resolution data, being 99% complete at 4.0 A and 63% complete at 3.5 A. The mosaicity of the crystal was with 0.84% much lower than the mosaicity of all previously measured crystals of the PSI-ferredoxin supercomplex. In addition to the native data set, two low resolu- tion datasets on the iron edge and peak for MAD were collected to 7 A resolution. These data sets were measured after the native data sets on the same crystal with decreasing diffraction quality due to X-ray damage. Therefore the data evaluation was limited to 8–9 A, which is not sufficient for MAD phasing based on the FeS clusters in PS I and ferredoxin. Preliminary phasing with Molecular Replacement revealed that there are two trimers of the PS I com- plex in the PS I-ferredoxin asymmetric unit, which corresponds to a molecular mass of 2.1 million Da. The actual resolution of the ED map shows that experimental phases and improved native data sets are essential for the unraveling of the structure of the PS I-fer- redoxin complex. We plan to collect native data sets from several crystals of the new form, which can be merged to limited the X-ray exposure of individual crystals during data collection. J1-002 Breaking biological symmetry in membrane proteins: how PsaC evolved to orient asymmetrically on the photosystem I core J. H. Golbeck 1 , M. Antonkine 2 and D. Stehlik 2 1 Biochemistry and Molecular Biology; Chemistry, The Pennsylva- nia State University, University Park, PA, USA, 2 Fachbereich Physik, Freie Universita ¨ t, Berlin, Germany. E-mail: jhg5@psu.edu Photosystem I is particularly intriguing because the reaction center core is a pseudo-C2-symmetric heterodimer that likely evolved from a C2-symmetric homodimeric precursor. This change was accompanied by the recruitment of a bacterial dicluster ferredoxin, now known as PsaC, which serves as the terminal electron accep- tor. The involvement of FA and FB in electron transfer lengthened the time of charge separation, thereby ensuring a high quantum yield. The FX region on PsaA/PsaB is highly symmetric, yet PsaC binds asymmetrically (i.e. in one of two possible orientations). For PsaC to bind asymmetrically, a number of alterations were neces- sary in the structures of both PsaC and the PsaA/PsaB heterodi- mer. We have studied the assembly of these subunits by comparing the three-dimensional NMR solution structure of unbound PsaC (1K0T) with the atomic-resolution X-ray crystal structure of PsaC bound to the PS I reaction center. This is the first instance in which bound and unbound three-dimensional structures are available for a membrane-associated protein. We found structural features, spe- cific to unbound PsaC in solution, which prevent binding of this protein to the PS I core in the incorrect orientation. We propose that during binding to PS I core PsaC undergoes step-wise struc- tural change that assures its binding in the correct orientation. Sim- ilar fundamental principles may have evolved in the assembly of membrane-associated subunits of other symmetrical complexes. J1-003 Supercomplexes of photosystem I and antenna proteins in green plants and cyanobacteria R. Kouril 1 , A. Zygadlo 2 , P. E. Jensen 2 , N. Yeremenko 3 , H. Matthijs 3 , H. D’Haene 4 , J. Dekker 4 and E. Boekema 1 1 Department of Biophysical Chemistry, Groningen University, Groningen, The Netherlands, 2 Agricultural University, Copenhagen, Denmark, 3 University of Amsterdam, Amsterdam, The Nether- lands, 4 Vrije Universiteit Amsterdam, Amsterdam, The Nether- lands. E-mail: e.j.boekema@rug.nl Photosystem I (PSI) and Ph otosystem II ( PSII) can form supercom- plexes with various types of antenna proteins. One such protein is Light-harvesting complex I I ( LHCII). In plants it is bound in its tri- meric form to dimeric PSII. In spinach and Arabidopsis thaliana most dimers bind 2–4 LHCII trimers. We have characterized the P SI- LHCII complex from Arabidopsis membranes in state two by single particle el ectron microscopy at about 1 6 A ˚ . PSI binds one trimer at the site o f the PsaL and PsaK subunits. Cyanoba cteria e xpress large quantities of the Iron Stress Inducible protein IsiA under iron defici- ency. IsiA can assemble into numerous types of single or double rings surrounding PS I. These supercomplexes are functional in light-har- vesting, but empty IsiA rings are effective energy diss ipators. Electron microscopy studies on over 130 0 00 p article projections of th ese supercomplexes show th at photo system I trimers find 18 IsiA copies in a single ring; whereas monomers may bind up to 35 copies in two rings. The double rings are formed by inner rings of 12, 13 or 14 cop- ies and the outer ones of 19, 20 and 21 copies, respectively. This shows that IsiA c a n form a remarkable large variety of ring-like structures. Work on particles purified from mutants indicates that the PsaF and PsaL subunits facilitate the formation of closed rings around PSI monomers. But these subunits are not obl igatory compo- nents in the formation of P SI-IsiA supercomplexes. J1-004 Initial charge separation in photosystem II reaction centers identified with femtosecond mid-infrared spectroscopy M L. Groot 1 , N. P. Pawlowicz 1 , L. J. van Wilderen 1 , J. Breton 2 , I. H. van Stokkum 1 and R. van Grondelle 1 1 Department of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands, 2 CEA, Saclay, France. E-mail: rienk@few.vu.nl In spite of the apparent similarity between the plant Photosystem II reaction center and its purple bacterial counterpart, we show Abstracts 449 that the mechanism of charge separation is very different for the two photosynthetic reaction centers. Using femtosecond visible- pump-mid-infrared probe spectroscopy in the region of the chlo- rophyll ester and keto modes, between 1775 and 1585 cm )1 , with 150 fs time resolution we show that the reduction of pheophytin occurs on a 0.6–0.8 ps time scale, whereas P+, the precursor state for water oxidation, is formed after ~6 ps. We conclude therefore that in the PS II RC the primary charge separation occurs between the ‘‘accessory chlorophyll’’, ChlD1 and pheo- phytin on the so-called active branch. J1-005 The primary light energy conversion in bacterial reaction centers: coupling electron transfer and nuclear motions V. A. Shuvalov Primary Processes of Photosynthesis, Institute of Basic Biological Problems, Russian Academy of Sciences, Pushchino, Moscow region Russian Federation. E-mail: shuvalov@issp.serpukhov.su The primary light energy conversion at photosynthesis occurs in photoreaction center (RC) of photosynthetic bacteria and green plants (photosystem I and II). In all known reaction centers the primary electron donor is a (bacterio)chlorophyll dimer, P, and the primary acceptor is a (bacterio)chlorophyll monomer (bacter- ial and PSII RCs), BA, or dimer (PSI RCs). The primary charge separation is observed within 2–10 ps and accompanied by elec- tron transfer to the secondary electron acceptors (pheophytin, qui- none molecules and iron-sulfur centers). The nuclear wave packet formed by 18-fs excitation on the P* potential energy surface in native and mutant reaction centers (RCs) of Rhodobacter (Rb) sphaeroides was found to be accompanied by the nuclear motions with frequency of 130–150 cm )1 inside of P* with the charge transfer from PA to PB molecule. The negative charge on PB - is reversibly transferred to the P+BA- surface at 120, 380, etc. fem- tosecond (fs) delays (monitored by measurements of BA- absorp- tion band at 1020–1028 nm) via a molecular pathway including the water molecule at the position 55 (Rb sphaeroides). In the absence of tyrosine M210 replaced by W or L the most simple pattern of fs oscillations with seven periods of 230 fs in stimulated emission from P* is observed. Identical reversible oscillations are observed in the 1020-nm band of BA- of the mutants showing the absence of the stabilization of the state P+BA The obtained results are discussed in terms of electron transfer processes coupled to the nuclear wave packet motions on the P* poten- tial energy surface which are transferred to the product state (P+BA-). The stabilization of P+BA- is suggested to occur due to the motion of H+ in tyrosine O-H+ group. These processes seem to play a key role in the primary charge separation. J1-006 Structure of the purple bacterial LH1/RC complex and its role within the photosynthetic unit A. T. Gardiner 1 , A. W. Roszak 2 , J. Southall 1 , C. J. Law 3 , R. J. Codgell 1 and N. W. Isaacs 2 1 Laboratory of Microbial Photosynthesis, Department of Biochem- istry and Molecular Biology, University of Glasgow, Glasgow, UK, 2 Laboratory of Protein Crystallography, Department of Chemistry, University of Glasgow, Glasgow, UK, 3 L2-009, Department of Chemistry and Environmental Science, University of Limerick, Limerick, Ireland. E-mail: atg3v@udcf.gla.ac.uk In purple non-sulphur photosynthetic bacteria each LH1 is intimately associated with a reaction centre (RC), in a fixed 1:1 stoichiometry, forming the so-called ‘‘core’’ complex. If the RC is surrounded by a palisade of rigid a-helices from the LH1 com- plex, then how is the ubiquinol able to ‘‘escape’’ and diffuse to the cytochrome b/c1? This question has been answered, in part, by the 4.8A ˚ crystal structure LH1/RC ‘‘core’’ complex from Rhodopseudomonas palustris. The RC is surrounded by an oval, rather than circular, LH1 complex consisting of 15 ba-pairs and their associated pigments. The orientation of the long axis of the ellipse coincides with the long axis of the RC and allows the LH1 complex to wrap tightly around the RC. The LH1 complex is prevented from completely encircling the RC by a single trans- membrane helix (called W). Protein W replaces a ba-pair and is located out of register with the other 15 ba pairs. Little is known about W but it is thought to be structurally equivalent to the Puf X protein present in Rhodobacter (Rb.) sphaeroides and Rb. cap- sulatus and appears to be associated with the LH1 complex facili- tating ubiquinol/ubiquinone exchange between the RC and the cytochrome b/c 1 complex. W is located directly in front of the Q b binding site in the RC and is therefore ideally placed to enable ubiquinol to exit. This presentation will provide details of our current understanding of the structure and function of the purple bacterial antenna LH1/RC ‘‘core’’ complex. J1-007P Light stress-induced one-helix protein of the chlorophyll a/b-binding family associated with photosystem I U. B. Andersson 1,2 , M. Heddad 1 and I. Adamska 1,2 1 Department of Biochemistry and Biophysics, Stockholm Univer- sity, Stockholm, Sweden, 2 Department of Biology, University of Konstanz, Konstanz, Germany. E-mail: ulrica@dbb.su.se The chlorophyll a/b-binding (Cab) protein family contains not only members composing the light-harvesting antennae but also several proteins associated with the photosystems that have other non-light-harvesting functions. All Cab superfamily proteins are intrinsic membrane proteins consisting of one to four transmem- brane helices and they share a conserved motif in the first and third helix. One subfamily of the Cab proteins constitutes the early light-induced proteins (Elips) that are proposed to partici- pate in protection against excessive light. One member of the Elips family in Arabidopsis thaliana is the One-helix protein 2 (Ohp2), a 14 kDa protein with one predicted transmembrane helix. The transcripts and protein of Ohp2 accumulate in higher light intensity but not in response to other stress conditions, such as salt, oxidative, cold or heat stress. UV-A irradiation affected the transcript level but not the protein level, suggesting that the amount of Ohp2 is controlled on both RNA and protein level. Localization studies showed that Ohp2 is found exclusively in photosystem I (PSI) under low as well as high light conditions. This is in contrast to other Elips investigated, which are associ- ated with photosystem II. PSI was long believed not to suffer from photoinhibition, but recently it was found that high light in combination with cold stress caused severe damage to PSI. We therefore suggest that Ohp2 has a protective role for PSI during light stress conditions. Abstracts 450 J1-008P Assembly of the heterodimeric light- harvesting complex LHCI-730 depends on amino acids in the second transmembrane helix of the Lhca4 subunit D. Corbet and V. H. Schmid Institute of General Botany, Johannes Gutenberg University, Mainz, Germany. E-mail: corbet@uni-mainz.de Alignments of apoproteins of light-harvesting complexes (Lhc) of photosystem (PS) I and PSII show considerable amino acid sequence conservation in several regions. Despite this similarity, Lhc proteins of PSII form either monomers or trimers, and Lhc proteins of PSI dimers. To get insight into the amino acids involved in formation of the heterodimeric LHCI-730 we used mutated Lhca1 and Lhca4 apoproteins for in vitro reconstitu- tions. By this approach, we earlier identified tryptophan residues at the N- and C-terminus of Lhca1 that are important for dimer formation. In order to analyze the involvement of the 2nd helix of Lhca1 and Lhca4 in LHCI-730 assembly we produced chimers of Lhca1 and Lhca4 that contained the 2nd helix of Lhca3, which does not form dimers with Lhca1 or Lhca4. The Lhca4/ Lhca3 chimer did not form dimers with the Lhca1 wild type. By contrast, exchange of the 2nd helix in Lhca1 did not affect dime- risation. To identify the amino acids in the 2nd helix of Lhca4 that interact with Lhca1, point mutated Lhca4 proteins were pro- duced. Two groups of amino acids (86–88, 99–103) within helix 2 of Lhca4 were detected, that are involved in interaction with Lhca1. Mutation of H99G resulted in a strongly reduced dimer yield. Additional mutation of three serine residues at the begin- ning of the 2nd helix completely abolished dimerisation. Reintro- duction of those amino acids into the Lhca4/Lhca3 chimer, that are present in the original Lhca4 sequence, resulted in recovery of dimer formation capability. This demonstrates that H99 together with serine residues at positions 86–88 is involved in LHCI-730 assembly. Additional mutants with impaired dimerisa- tion will be presented and a model summarizing the current knowledge of the subunit interaction in LHCI-730 will be shown. J1-009P Mathematical modelling of electron transport reactions in photosystem II P. Chernev 1 , I. Zaharieva 1 , V. Goltsev 1 and R. J. Strasser 2 1 Department of Biophysics and Radiobiology, Faculty of Biology, University of Sofia, Sofia, Bulgaria, 2 Bioenergetics Laboratory, Department of Plant Biology, University of Geneva, Geneva, Switzerland. E-mail: pchcher@yahoo.com A mathematical model of electron transfer reactions in the Phot- osystem II supramolecular complex is designed. The model includes the electron carriers between the oxygen-evolving com- plex and the plastoquinone pool. Specialized computer software is developed that allows the automatic construction of the differ- ential equations describing the transitions between the redox states of the Photosystem II complex. The model is tested using the luminescent characteristics of Photosystem II – prompt and delayed chlorophyll a fluorescence transients from dark to light- adapted state. By fitting the model curves to experimental ones by the same software, the values of the rate constants of the par- ticular electron transfer reactions are assessed. An approach to fitting is proposed that avoids over-parameterization and allows the acquisition of correct values of the rate constants by the sim- ultaneous fitting of several types of experimental curves (prompt and delayed chlorophyll a fluorescence) and curves obtained at different experimental conditions. The method allows the evalua- tion of the rate constants in native plants and in plants under the influence of different environmental factors in in vivo and in situ measurements. For example, as it is expected, the growing of bar- ley plants at different light intensities causes the change of the parameter that describes the Photosystem II antenna size. J1-010P New Insigths into the structure and function of photosystem I and II P. Fromme, H. Yu, Y. Bukman, D. Ni, B. Varco-Merth, D. Chauhan, C. Vanselow, C. Jolley, R. Fromme and I. Grotjohann Department of Chemistry and Biochemsitry, Arizona State University, Tempe, AZ, USA. E-mail: pfromme@asu.edu Oxygenic photosynthesis converts the light energy from the sun into chemical energy. The primary step in this energy conversion, the light induced charge separation, is catalyzed by Photosystem I and II. Photosystem I of cyanobacteria consists of 12 protein subunits, to which more than 100 cofactors are non-covalently bound. The X-ray structure at 2.5 A ˚ [1] showed the location of the individual subunits and cofactors and provided new informa- tion on the protein-cofactor interactions. The structural model of plant PS I was determined at 4.4 A ˚ by Ben-Shem et al [2]. Recent computations provided an atomic model of plant Photosystem I, shining light into the similarities and differences between both systems. Photosystem II consists of 17 protein subunits to which about 50 cofactors are non-covalently bound. The X-ray struc- tural model of the intact PS II complex at 3.8 A ˚ resolution [3] thereby providing the first insight into the structure of the water splitting Photosystem II. In the meantime, more structures from PS II has been published at 3.7–3.2 A ˚ , revealing more details of the structures [4, 5, 6, 7]. The different models and the many open questions which still remain will be discussed in respect to the functional aspects of the mechanism of water oxidation, elec- tron transfer and the process of light capturing. Acknowledgment: This work is supported by NIH (1 R01 GM71619-01), NSF MCB-0417142 and USDA (2003-35318- 13573 References 1. Jordan et al. Nature 2001; 411: 909 2. Ben-Shem et al. Nature 2003; 426: 630 3. Zouni et al. Nature 2001; 409: 739. 4. Fromme et al. Philos T Roy Soc B 2002; 357: 1337. 5. Kamiya. and Shen, Proc Natl Acad Sci U S A 2003; 100: 98 6. Ferreira et al. Science 2004; 303: 1831. 7. Biesiadka Phys Chem Chem Phys 2004; 6: 4733 J1-011P Kinetics of milliseconds delayed chlorophyll a fluorescence in whole leaves V. Goltsev 1 , I. Zaharieva 1 , P. Chernev 1 and R. J. Strasser 2 1 Department of Biophysics and Radiobiology, Faculty of Biology , St. Kliment Ohridski University of Sofia, Sofia, Bulgaria, 2 Bioener- getics Laboratory, Department of Plant Biology, University of Geneva, Geneva, Switzerland. E-mail: goltsev@biofac.uni-sofia.bg Delayed fluorescence dark decays in time interval from 0.35 to 5.5 ms are measured during dark to light adaptation in whole bar- ley leaves using a disc phosphoroscope. The changes of delayed fluorescence features are compared with variable chlorophyll fluor- escence simultaneously registered with the same apparatus as well as in parallel by Handy PEA (Hansatech Instruments Ltd.) and absorbance changes at 820 nm. The registered delayed fluorescence signal is a sum of three components – sub-millisecond with life- time about 0.6 ms, millisecond decayed 2–4 ms and slow com- ponent with life-time >> 5.5 ms. The sub-millisecond delayed Abstracts 451 fluorescence component is proposed to be a result of radiative charge recombination in Photosystem II reaction centers in state Z + PQ A - Q B - , and its lifetime is determined by the rate of electron transfer from Q A - to Q B - . The millisecond delayed fluorescence component is associated with recombination in Z + PQ A - Q B = cen- ters with a lifetime determined by the sum of the rate constants of electron transfer from the oxygen-evolving complex to Z + and of the exchange between the reduced and oxidized plastoquinone pool in the Q B - site. On the basis of these assumptions and of the differ- ent share of the three components in the integral delayed fluores- cence during induction, an attempt is made to interpret the changes in the delayed fluorescence intensity during the transition of the photosynthetic apparatus from dark to light adapted state. J1-012P Thermo-optically induced monomeriza tion of trimers of the main light harvesting antenna complexes of plants in vivo and in vitro. Quantum yield and dependencies on the phosophorylation and zeaxanthin content of the membranes P. H. Lambrev,Z.Va ´ rkonyi, T. Ja ´ vorfi, A. Kiss, B A. Namkhainyambuu, M. Szabo ´ and G. Garab Institute of Plant Biology, Biological Research Center, Szeged, Hungary. E-mail: lambrev@brc.hu Earlier we have shown that the macro-organization and the olig- omerization state of LHCII, the main chlorophyll a/b light harvest- ing antenna complex of plants, possesses a remarkable and unexpected structural flexibility: they are capable of undergoing light-induced reversible structural reorganizations that are largely independent of the photochemical activity of thylakoids. The reorganizations are approximately linearly proportional to the light intensity above the saturation of photosynthesis – a poten- tially very important, unique feature with respect to adaptation and protection of plants against excess excitation. This structural flexibility is also carried by and probably ‘borrowed’ from LHCII, and have been shown to be driven by a novel, biological thermo- optic mechanism. Fast thermal transients arising from dissipated excitation energy can lead to elementary structural transitions because of the existence of ‘‘built-in’’ thermal instabilities in the close vicinity (<2 nm) of the sites of dissipation. We have identi- fied three well discernible thermo-optically induced structural changes in LHCII-containing systems in vivo (in whole plants, chloroplasts and sub-chloroplasts membrane preparations) and in vitro (isolated trimers and lamellar aggregates of LHCII), including monomerization of the trimers [1, 2, 3]. In this work, we present data on the quantum yield of this last step and its dependences on the phosphorylation and zeaxanthin content of the membranes. Acknowledgment: This work was supported by EU-FP6 MCRTN INTRO2. References 1. Cseh et al. Biochemistry 2000; 39: 15250. 2. Garab et al. Biochemistry 2002; 41: 15121. 3. Dobrikova et al. Biochemistry 2003; 42: 11272. J1-013P Damage and protection of photosystem II pigment-protein complexes under heat stress N. L. Pshybytko, L. N. Kalituho and L. F. Kabashnikova Institute of Biophysics and Cellular Engineering, National Academy of Sciences of Belarus, Minsk, Belarus. E-mail: pshybytko@rambler.ru The seedlings of different ages are characterized by various rates of the photosynthetic reactions, therefore it is possible to suppose, that they could possess different stability to the stress impact. In this connection the effects of heat shock (40°C, 3 h) on the photo- synthetic activity of 4, 7 and 11-day-old barley seedlings were stud- ied. The rate of CO 2 gas exchange in young leaves was not changed under heat shock while in 11-day-old seedlings the high tempera- ture affected both quantum yield and maximum rate of CO 2 fix- ation. The thermostability of photosynthetic apparatus in young leaves can be caused by protective role of heat shock proteins (HSP). The appearance of 30 kDa HSP in young leaves and in less degree in old leaves was illustrated by means of Western blot ana- lysis. This HSP is known to protect photosystem II (PSII).The pho- tochemical activity of PSII in both young and old barley leaves was decreased under high temperature. However the causes of PSII thermoinactivation were different. In 4-day-old leaves the effective quantum yield of PSII photochemistry was reduced while in old leaves high temperature decreased amount of active PSII com- plexes. The causes of PSII disturbance in old leaves were the increase of the proton gradient on the thylakoid membrane and the increase of plastoquinone pool reduction. The increase of plasto- quinone pool reduction was caused by the diminution of oxidizing ability of cytochrome b6/f complex. The increased pH can induced the damage of electron transport chain and the degradation of pig- ment-protein complex of PS II. The breaking of D1 and D2 protein was shown in 11-day-old leaves under high temperature. Similar events were not obtained in young leaves, in which HSPs protect PSII from damage. J1-014P Structure and dynamics of the photosystem II reaction center pigment-protein complex P. Palencar 1 , F. Vacha 2,3 and M. Kuty 1 1 Laboratory of High Performance Computing, Institute of Physical Biology and Institute of Landscape Ecology, University of South Bohemia in Ceske Budejovice, AS CR, Nove Hrady, Czech Repub- lic, 2 Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic, 3 Institute of Physical Biology, University of South Bohemia in Ceske Budejo- vice, Nove Hrady, Czech Republic. E-mail: palencar@greentech.cz Photosystem II (PSII) is a light-absorbing pigment-protein com- plex located in thylakoid membrane of cyanobacteria, algae and higher plants. Changes in excitonic interactions in PSII reaction center (RC) pigments upon light-induced oxidation of primary donor (P680) or reduction of primary acceptor pheophytin a (Phe a) were analyzed using absorption and circular dichroism (CD) spectra [1]. In contrast to the oxidation of primary donor, the light-induced change in the CD spectrum upon primary acceptor reduction was temperature-dependent. This suggests a hypothesis that at room temperature the reduced Phe a induces conformation- al changes of the RC protein environment, which affects the exci- tonic interaction of the RC chlorophylls. For better understanding and interpreting measured optical spectra [2], molecular dynamics (MD) coupled with ab initio calculations are appropriate methods to be applied on PSII RC pigment-protein environment. Having chemically well defined 3D molecular structure [3] and so-called force field (FF) parameters, conformational study of the PSII RC can be performed consequently by using MD technique. Force field parameters (charge distribution and force constants) of the chlorophyll a, Phe a, heme, plastoquinone and surrounding amino acids of the PSII RC structure were calculated quantum chemic- ally and partially transferred from various studies concerning bac- terial pigments [4, 5] and heme prosthetic group [6]. Development of all missing FF parameters of pigments from our truncated PSII RC and calculation of charge distribution on reduced Phe a mole- cule and surrounding protein environment were necessary steps to run appropriate MD simulation and subsequently better under- stand processes in PSII RC. Abstracts 452 Acknowledgment: This work was supported by MSMT (MSM6007665808, GACR206/02/D177) and by AV CR (AVOZ60870520). References 1. Vacha F, Durchan M and Siffel P. Biochim Biophys Acta. 2002; 147: 1554. 2. Vacha F, Psencik J, Kuty M, Durchan M, Siffel P. Photosynth Res 2005; In press. 3. Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S. Science 2004; 303: 1831. 4. Foloppe N, Ferrand M, Breton J, Smith JC. Proteins 1995; 22: 226. 5. Ceccarelli M, Procacci P, Marchi MJ. Comput Chem. 2003; 24: 129. 6. Autenrieth F, Tajkhorshid E, Baudry J, Luthey-Schulten ZJ. Comput Chem. 2004; 25: 1613. J1-015P LH1 antenna complexes of Rhodospirillum rubrum: a model for studying polypeptides interactions in membrane J. Seguin 1 , G. Ajlani 1 , J M. Verbavatz 1 , R. Gobin 1 , A. Gall 1,2 , M. Paternostre 1 and B. Robert 1 1 Service de Biophysique des Fonctions Membranaires, DBJC/CEA - URA 2096/CNRS, Gif-sur-Yvette, France, 2 IBLS/Biochemisry and Molecular Biology, University of Glasgow, Glasgow, UK. E-mail: jseguin@cea.fr From the point of view of polypeptides association within mem- brane, one of the best characterized membrane proteins is the core antenna protein LH1 from the Rhodospirillum (Rsp.) rubrum purple bacteria. This protein ensures the capture of the solar photons and the efficient funnelling of the resulting excita- tion energy toward the photochemical reaction center (RC). LH1 are large oligomers of a basic structural unit composed of a het- erodimer of two small integral membrane polypeptide (alpha and beta, ca. 50 amino acids) associated with bacteriochlorophyll and carotenoid molecules. The electronic properties of LH1 intimately depend on the association state of the polypeptides of which they are composed. In our study, the genes pufL and pufM in the puf- BALM operon encoding the L and M subunits of the RC was inactivated. This mutant was only capable to growth under aero- bic chemoheterotrophic conditions. The electron microscopy of the mutant compared to wild type show that this bacteria has a tendency to die as soon as it reaches its growing stationary phase. The intracytoplasmic membranes purified from the mutant and containing the LH1 complexes have been characterized regarding their spectroscopic properties and systematically com- pared to the spectra obtained on the intracytoplasmic membranes extracted from the wild-type S1 strain of Rps. rubrum. This char- acterization shows that the LH1 complexes formed in the intracy- toplasmic membranes of the mutant are fully comparable to the ones found in the wild type. J1-016P Does backflow of electrons from the PQ-pool contribute to the reduction of QA in heat- treated leaves? S. Z. Toth 1 , G. Schansker 1 , G. Garab 2 and R. J. Strasser 1 1 Laboratory of Bioenergetics, Department of Plant Biology, Uni- versity of Geneva, Geneva, Switzerland, 2 Institute of Plant Biology, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary. E-mail: Toth1@etu.unige.ch Photosynthetic electron transport processes, particularly within photosystem II were studied in barley leaves during recovery from a heat treatment. In heat-treated leaves photosystem II (with a destroyed oxygen-evolving complex) can produce a sin- gle stable charge separation in continuous light leading to some QA (the primary electron acceptor quinone of photosystem II) reduction and the emergence of the K-step in the chlorophyll a fluorescence rise (OKJIP). After the K-step (F~300 ls), addi- tional QA- accumulation occurs that is related to the heat- induced stimulation of dark reduction of the plastoquinone pool by a stroma factor [To ´ th et al. J. Plant Physiol, in press]. Using chlorophyll a fluorescence and 820 nm transmission measurements the interaction between the plastoquinone pool and QA was further studied. We try to answer the question if the additional QA- accumulation is possible because of slow re-reduction of TyrZ by external donors in the presence of a reduced plastoquinone pool (to study this, sequences of short light pulses (~300 ls) were used) or back flow from the plasto- quinone pool to QA. J1-017P New concept in photosynthesis: rapid reduction of carbon dioxide to formic acid and to formaldehyde by glutathione and acetyl cystein via carbamate intermediate L. Tre ´ zl 1 ,Z.Ja ´ szay 2 , L. Hulla ´ n 3 , T. Szarvas 4 , I. Petneha ´ zy 1 , A. Csiba 5 , L. Sarkadi 6 and L. To ˜ ke 1 1 Department of Organic Chemical Technology, Budapest Univer- sity of Technology and Economics, Budapest, Hungary, 2 Organic Chemical Technology Research Group, Hungarian Academy of Sci- ences, Budapest, Hungary, 3 Department of Biochemistry, National Oncological Institute, Budapest, Hungary, 4 Institute of Isotopes Co. Ltd., Budapest, Hungary, 5 Veterinary and Food Control Sta- tion, Budapest, Hungary, 6 Department of Biochemistry and Food Technology, Budapest University of Technology and Economics, Budapest, Hungary. E-mail: trezl@oncol.hu The generally accepted photosynthetic CO 2 fixation path involves the carboxylation of ribulose 1,5-biphosphate (RuBP). Here we report a new chemical reaction, which is of general biochemical interest in plants, and may be an alternative of the Calvin cycle. Our experimental data fully support the formation of formic acid and formaldehyde from CO 2 via carbamates in a reduction process. Though the presence of formic acid and formaldehyde (free and bound) was proved in plant leaves by us and by others, too, the way of their formation and their role was not clear. We monitored the formation of the carba- mate in a fast reaction of NaH 13 CO 3 with certain proteinogenic amino acids (l-lysine, l-arginine, l-glutamine and l-asparagine) on physiological pH by 13 C-NMR and HPLC measurements. We also detected that thiol group containing reducing factors (glutathione (GSH), N-acetyl cystein (NAC)) are able to reduce the carbamino group of the amino acids to formic acid and to formaldehyde (or to its equivalents). We also showed the increased amount of formic acid in the kohlrabi leaves extract treated its NaHCO 3 solution by GSH and Arg+GSH.To get further evidence for the reduction of CO 2 to formic acid via carbamates in the plant leaves we applied photosynthetic 14 CO 2 fixation on bean leaves without treatment and pretreated with arginine. The radio thin layer chromatography of the extract of the leaves showed the presence of arginine formiate in both cases, but higher amount in the case of the leaves pooled by arginine. As we described earlier formaldehyde reacts with RuBP, providing its 2-hydroxymethyl adduct a substrate of the Rubisco enzyme, a key intermediate in photosynthesis. Abstracts 453 J1-018P Structural studies of a cryptophyte light harvesting phycocyanin PC645 K. E. Wilk, S. J. Harrop, D. Edler and P. M. Curmi Protein Structure Laboratory, School of Physics, University of NSW, Sydney, NSW Australia. E-mail: k.wilk@unsw.edu.au The photosynthesis process is essential for maintaining all forms of life on the Earth. Photosynthesis is made possible by the cooperation of many different proteins in the process of turning sunlight into useful chemical energy. In marine systems, protein pigments harvest solar energy photons and transfer excited state energy to the reaction centre protein, where charge separation takes place across a membrane. In order to utilize energy avail- able at different depths, marine photosynthesis relies on efficient light harvesting in the visible region of the spectrum. In addition to commonly present chlorophylls cyanobacteria and algae have pigments called phycobilins that are red or blue and absorb energy in the corresponding visible region of light spectrum. To maintain efficiency and a high rate of electron transfer in the reaction centre, the phycobilins are usually organized in antenna. The cryptophyte Chroomonas CCMP270 used in this project is a unicellular photosynthetic alga. The light harvesting system in the cryptophytes is distinct from all other algae and cyanobacte- rial. A peripheral antenna consists of water-soluble protein, phyc- ocyanin 645 (PC645) and a core antenna consists of protein bound chlorophylls. The chlorophylls absorb solar energy at 670 and 440 nm. The fact that phycocyanin absorbs at 645 nm, allows this algae to increase its efficiency and live at lower light- regimes and greater depths than most other algae. The pathways for energy transfer between the light harvesting systems and the reaction centre are still unknown. So far, the only structure of a cryptophyte light harvesting protein to be determined is that of PE545, which was determined by our group [Wilk et al., 1999]. This protein has a novel arrangement of protein subunits and tetrapyrrole pigments. In the current project, I will study a rela- ted cryptophyte light harvesting protein, PC645. This protein harvests longer wavelength light than PE545. Its structure should be similar to PE545 on a gross scale, but the full atomic structure will reveal differences that are important to its distinct absorb- ance characteristics. J1-019P Characterization of photosystem II by delayed chlorophyll a fluorescence I. S. Zaharieva 1 , P. C. Chernev 1 , V. N. Goltsev 1 and R. J. Strasser 2 1 Department of Biophysics and Radiobiology, Biological Faculty, University of Sofia, ’St. Kliment Ohridski’, Sofia, Bulgaria, 2 Laboratory of Bioenergetics, Department of Plant Biology, Section of Biology, University of Geneva, Geneva, Switzerland. E-mail: iva@biofac.uni-sofia.bg An approach to the investigation of structural and functional properties of Photosystem II based on the registration of delayed chlorophyll a fluorescence in native photosynthesizing objects is developed. Using a disc phosphoroscope, we register simulta- neously (i) delayed fluorescence dark relaxation curves (decayed in 0.35–5.5 ms time range) recorded every 11 ms during the transition of the photosynthetic apparatus from dark to light- adapted state and (ii) changes of the intensity of prompt chloro- phyll a fluorescence. A mathematical model that includes the electron carriers between the oxygen-evolving complex and the plastoquinone pool is designed in order to analyze the lumines- cent characteristics of Photosystem II. The registered delayed fluorescence signal is a sum of three components – sub-millisec- ond with life-time about 0.6 ms, millisecond decayed 2–4 ms and slow component with life-time >> 5.5 ms. The sub-millisecond delayed fluorescence component is proposed to be a result of radiative charge recombination in Photosystem II reaction cen- ters in state Z + PQ A - Q B - , the millisecond one is associated with recombination in Z + PQ A - Q B = centers and the slow one – with delayed light emission from closed reaction centers. On the basis of these assumptions and of the mathematical model, an attempt is made to correlate the delayed fluorescence characteristics to particular processes occurring in the Photosystem II complex – proton or electrical gradient accumulation, changes in the redox state of quinone acceptors, changes in the pigment-protein com- plexes caused by different stress factors, for example tempera- ture. J2–Cell Cycle Control in Plants J2–001 CULLIN-based ubiquitin ligases in plants: phytohormones signalling but not much about cell cycle yet M C. Criqui, M. Dieterle, E. Lechner, Y. Parmentier, T. Potuschak, A. Thomann and P. Genschik Laboratoire de Biologie Mole ´ culaire des Plantes, CNRS, Strasbourg France. E-mail: pascal.genschik@ibmp-ulp.u-strasbg.fr The ubiquitin/26S proteasome pathway has dramatically changed our understanding of cellular functions. It is now clear that all eukaryotic cells control a number of central processes by break- ing down key regulatory proteins. In particular, cell cycle pro- gression and many developmental processes are tightly controlled by ubiquitin-dependent protein degradation. Ubiquitylation is achieved through an enzymatic cascade involving the sequential action of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes. Among these enzymes, the E3s play a central role in the selectivity of ubiquitin-mediated protein degradation. CULLIN (CUL)-dependent ubiquitin ligases are structurally related multisubunit E3 enzymes that can be viewed as two functional modules brought together by the CUL- LIN proteins, acting as molecular scaffolds. The first module forms the catalytic centre and is composed by a RING finger domain protein and an E2 enzyme. The second module can be considered as the substrate recognition module, in which a speci- fic protein physically interacts with the target substrate. Members of the CULLIN proteins have been identified in all eukaryotes and based on phylogenetic studies fall into different subfamilies, each forming a different class of E3. Among them, the best-char- acterized complexes are the SCF (SKP1-CUL1-F-box), the ECS (ElonginC-CUL2-SOCS box), the CUL3-BTB complexes, as well as the APC/C (Anaphase Promoting Complex or Cyclosome), which contains a more distant CULLIN member, called APC2. The SCF and the APC/C play critical roles in the control of the cell cycle in fungi and metazoans, by promoting the entry into S-phase and mitotic progression and exit, respectively. The pres- entation will cover our current knowledge on the function of the plant CULLIN-based E3s in cell cycle control and phytohor- mone regulation. Abstracts 454 J2–002 Integration of cell cycle and epigenetic regulation during Arabidopsis development W. Gruissem Institute of Plant Sciences, Swiss Federal Institute of Technology, Zu ¨ rich, Switzerland. E-mail: wilhelm.gruissem@ipw.biol.ethz.ch Animals and plants have evolved complex regulatory mechanisms that direct development and cell differentiation, but the integra- tion of these processes with the cell cycle and chromatin remodel- ling are not well understood. Current models suggest that mammalian Rb, and perhaps the plant homolog RBR1, has a dual role in regulating cell cycle progression and cell differenti- ation. Rb family proteins are co-repressors of the E2F/DP family of transcription factors, and together they control various aspects of cell cycle progression. Rb also functions to maintain the differ- entiation status of several cell types and to protect cells from apoptosis, perhaps via chromatin remodelling complexes. For example, mammalian Rb and plant RBR interact with RbAp48/ MSI1, a WD40 protein found in several complexes that function in chromatin-related processes. Complete loss of Arabidopsis RBR1 or MSI1 function is gametophytically lethal. Nuclei in the region of the female egg apparatus continue to proliferate after megagametogenesis, indicating that RBR1 is required to arrest the nuclei of the egg apparatus prior to fertilization. The endo- sperm nucleus also proliferates in RBR1 or MSI1 loss-of-func- tion mutants, thus establishing a functional link between these two proteins. MSI1 interacts with RBR1 and FIE, which is part of a complex with MEA, a protein similar to the Drosophila Polycomb group (PcG) protein E(Z). But in contrast to RBR1, the egg apparatus does not overproliferate in MSI1 or other fer- tilization independent seed (fis) mutants, suggesting that nuclear proliferation is controlled by different mechanisms in the egg apparatus and endosperm. MSI1 is also a subunit of the CAF-1 chromatin assembly factor complex. Although CAF-1 is well defined in vitro, the precise function of the complex in vivo is still poorly understood. Loss of Arabidopsis CAF-1 function results in developmental alterations. Partial loss of MSI1 function results in ectopic expression of genes for histone H3.3 variants (among others), suggesting that MSI1 may have an important develop- mental function in controlling the deposition of H3.3 variants and gene activation. J2–003 The control of endoreduplication in Arabidopsis L. De Veylder and D. Inze Plant Systems Biology, VIB/UGent, Ghent, Belgium. E-mail: dirk.inze@psb.ugent.be Although our knowledge on how the different cell cycle transi- tions are regulated has increased dramatically during the last years [De Veylder et al., 2003; Inze ´ , 2005), it is still unclear how a dividing cell exits its division programme and enters the differ- entiation pathway. A major cause explaining the lack of informa- tion on this important aspect of development is the unavailability of good differentiation markers. Using the Arabidopsis leaf as a model system we found that the exit of the mitotic cell cycle of leaf cells coincides with the onset of endoreduplication, being a modified cell cycle during which DNA is duplicated in the absence of mitosis [Boudolf et al., 2004; Vlieghe et al., 2005]. As such, understanding how the mitosis-to-endocycle transition is regulated might help to unravel how cell differentiation is initiated. Analysis of leaf development in transgenic plants mis-expressing cell cycle genes illustrated that the onset of endo- reduplication involves nothing more than loss of M-phase cyclin- dependent kinase (CDK) activity. This decrease in activity was found to be controlled by the interplay of both positive and neg- ative regulators of CDKs. Control mechanisms include the E2F/ DP pathway, activated protein destruction, and post-transcrip- tional activation of CDK inhibitory proteins. A mathematical simulation of this complex regulatory circuit will be presented. References: 1. Boudolf et al., 2004; Plant Cell 16:2683–2692 2. De Veylder et al., 2003; Curr Opin Plant Biol 6:536–543 3. Inze ´ , 2005; EMBO J 24: 657–662 4. Vlieghe et al., 2005; Curr Biol 15: 59–63 J2–004 Cytokinesis in Arabidopsis: rush-hour traffic during cell division G. Ju ¨ rgens ZMBP Entwicklungsgenetik, Universita ¨ tTu ¨ bingen, Tu ¨ bingen, Germany. E-mail: gerd.juergens@zmbp.uni-tuebingen.de Cytokinesis partitions the cytoplasm of a dividing cell between the forming daughter nuclei. In higher plant cytokinesis, a new stretch of plasma membrane is laid down from the centre to the periphery of the cell. Initially, a plant-specific cytoskeletal array, the phragmoplast, forms in the centre of the division plane and mediates trafficking of Golgi-derived vesicles which fuse with one another to form a transient membrane compartment, the cell plate. Subsequently, the microtubules of the phragmoplast are depolymerised underneath the cell plate and new microtubules polymerise along the remaining ones at the margin, thus trans- forming the compact phragmoplast into a hollow cylinder. As a result, new membrane vesicles are trafficked to, and fuse with, the margin of the cell plate. Expansion of the cell plate proceeds in concert with lateral translocation of phragmoplast microtu- bules until the expanding cell plate fuses with the lateral plasma membrane of the dividing cell. We are using the Arabidopsis embryo as an assay system for identifying genes involved in cell division. Two classes of mutants have been obtained. Cytokinesis mutants are defective in cell-plate formation whereas cell-division mutants stop dividing altogether at a very early stage of embryo- genesis. The genes identified encode components of the cytokinet- ic vesicle fusion machinery or proteins required for microtubule formation or reorganization. Our current studies address membrane dynamics and specificity of vesicle fusion during cyto- kinesis. J2–005 The role of CDC20 isoforms in A. thaliana cell cycle regulation and development A. Kroll, Z. Kevei, Z. Kelemen, E. Kondorosi and A. Kondorosi Laboratory of Adam Kondorosi, Institut des Sciences du Ve ´ ge ´ tal (ISV), Centre National de la Recherche Scientifique CNRS UPR2355, Gif-sur-Yvette, France. E-mail: a_kroll@web.de In animal systems, CDC20 has been shown to be of crucial importance for the cell cycle during mitosis by activating the Anaphase Promoting Complex (APC) E3 ubiquitin ligase. The APC by interacting with the activator subunits, Cdc20 and Cdh1 controls ordered destruction of various cell cycle proteins – including mitotic cyclins – through the 26S proteasome. The APC substrates contain characteristic destruction motives such the D-, KEN, A- and GxEN boxes. Their recognition is medi- ated by the CDC20 and CDH1 proteins. CDC20 expression as well as protein activity are subject to cell cycle dependent regu- Abstracts 455 lation mediated by transcription factors (Mcm1, SFF), phos- phorylation (Bub1, MAPK), and protein–protein interaction (Mad2). Unlike animals and yeast, CDC20 is represented by five isoforms in Arabidopsis thaliana whose function has not been explored yet. Although the protein structure is highly con- served among the plant and animal CDC20 homologs, only two of the five plant isoforms display a significantly higher similarity to the animal CDC20 proteins and contain the conserved APC binding motifs and the KEN box required for destruction of CDC20 by the APC-CDH1 complex. Whether all of the five plant proteins are functional is not known. To elucidate the function of the multiple A. thaliana CDC20 isoforms, we have been investigating the expression pattern the csd20 genes in transgenic A. thaliana carrying the Atcdc20 promoter-reporter gene fusions as well as the interaction of the AtCDC20 proteins with the A. thaliana APC components and potential targets such as different types of mitotic cyclins. These data as well as the knowledge on the plant Cdh1-type APC activators Ccs52 proteins are expected to give an insight in the plant APC functions. J2–006 Kinase activity of cyclin-dependent kinase complexes in the cell cycle of chlorococcal algae J. Hendrychova ´ ,M.Vı ´ tova ´ ,M.E ` ı ´ zˇ kova ´ and V. Zachleder Laboratory of Cell Cycles and Biotechnology of Algae, Depart- ment of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences, Tøeboo ` , Czech Republic. E-mail: jahen@alga.cz Cyclin-dependent kinases (CDK) are enzymes, which require binding to a cyclin subunit for their phosphorylation activity. CDK function is involved in proper timing of cell cycle processes, e.g. mitosis. In contrast to the single CDK (Cdc2) in yeast, six classes of CDKs referred to as CDKA-F have been reported for higher plants. Genes coding for CDKA-E were found in the gen- ome of unicellular alga Chlamydomonas reinhardtii whose CDKA was the first proven plant homologue of the key cell cycle regulator Cdc2. We demonstrated CDK-like kinase activities in protein extracts of the green alga Scenedesmus quadricauda [Bis ˇ ova ´ et al., 2000]. Recently we have focused attention on sep- arated CDK complexes. We found out that the CDKA occurs at least in three different complexes in cells of Scenedesmus quadric- auda, but only one of them contains a cyclin subunit and has a kinase activity. The protein related to Rb was detected in this complex as its putative substrate. The amount of this CDKA complex increases during growth phase and its maximum corre- lates with mitotic kinase activity. In an attempt to analyse kinase activity of CDK complexes, we modified a method of in-gel kin- ase assay. Besides the active CDKA complex we have detected two more complexes with the CDK-like kinase activity. However none of them show the PSTAIR epitope characteristic for CDKA. We deduce that these non-PSTAIR complexes could be responsible for CKS (suc1)-bound kinase activity. Function of different types of CDKs and their complexes in cell cycle regula- tion of algae is discussed. Acknowledgment: This work was supported by grants from the GAASCR (KJB5020305) and the GACR (204/02/1438). Reference: 1. Bis ˇ ova ´ K, Vı ´ tova ´ M, Zachleder V 2000 The activity of total histone H1 kinases is related to growth and commitment points while the p13(suc1)-bound kinase activity relates to mitoses in the alga Scenedesmus quadricauda. Plant Physiology and Biochemistry 38: 755–764 J2–007P Big family for successful living: NCR oligopeptides for coordinated cell differentiation in legume-Rhizobium symbiosis B. Alunni 1 , P. Mergaert 1 , N. Maunoury 1 , M. Redondo-Nieto 1 , A E. Mausset 1 , T. Uchiumi 2 , A. Kondorosi 1 and E. Kondorosi 1 1 Groupe Interactions Rhizobium-Le ´ gumineuses, Institut des Sciences du Ve ´ ge ´ tal, CNRS UPR2355, Gif-sur-Yvette, France, 2 Laboratory of Molecular Biology of Plant-Microbe Interactions, Department of Chemistry and BioScience, Faculty of Science, Kagoshima University, Kagoshima, Japan. E-mail: Benoit.Alunni@isv.cnrs-gif.fr The symbiotic interaction between Medicago truncatula and Sino- rhizobium meliloti soil bacteria leads to the formation of nitrogen fixing root nodules. In the infected nodule cells, rhizobia are con- verted into nitrogen fixing bacteroids. Differentiation of the pro and eukaryotic symbiotic cells is strikingly similar, manifested by cell division arrest, cell enlargement and genome amplification. This suggests coordinated development and signalling events between the plant cells and rhizobia. These signals are expected to be nodule specific and present at distinct stages of develop- ment. A recently identified gene family composed of more than 300 members corresponds to these criteria. They encode Nodule- specific Cystein-Rich (NCR) polypeptides [Mergaert et al., 2003] with resemblance to antimicrobial defensins and scorpion toxins. The NCRs are composed of a highly conserved signal peptide (SP) and a variable mature oligopeptide of 25–55 amino-acids containing four or six conserved cystein residues. Nodule tran- scriptomics groups NCRs in five large clusters. All tested NCRs from the distinct groups expressed exclusively in the infected cells and in distinct cell layers or nodule zones. The NCRs are likely targeted to the bacteria. Thus, expression pattern, localization and high diversity of NCRs support their role in communication between the eukaryotic and prokaryotic cells at distinct stages of development. In addition, co-expression of a nodule specific sig- nal peptide peptidase (SPP) with a certain group of NCRs sug- gests that further cleavage of NCR SPs by SPP may release peptide fragments with potential signalling role, as in animals [Weihofen et al., 2002]. References: 1. Weihofen et al. 2002; Science 296: 2215–8. 2. Mergaert et al. 2003; Plant Physiol. 132: 161–173 J2–008P Purification of cAMP-binding proteins from tobacco BY-2; cloning, expression and characterization of a cAMP-binding nucleoside diphosphate kinase 1 T. De Vijlder, L. Roef, H. Van Onckelen and K. Laukens Laboratory of Plant Biochemistry and Physiology, Department of Biology, University of Antwerp, Antwerp, Belgium. E-mail: thomas.devijlder@ua.ac.be Cyclic AMP plays an important role in the regulation of the euk- aryotic cell cycle. In tobacco ‘‘Bright Yellow 2’’ (BY-2), cAMP levels show stage-dependent oscillations. Inhibition of cAMP-syn- thesis results in a block in cell cycle progression [Ehsan et al. 1998]. Previous work in our laboratory using affinity chromatog- raphy led to the purification of three cAMP-binding proteins from tobacco BY-2, which were identified as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and two nucleoside diphosphate kinase isoforms (NDPK1 and NDPK3) [Laukens et al. 2001]. Besides their noted enzymatic activities, these Abstracts 456 proteins are known to perform a number of alternative actions in a variety of organisms. NDPK isoforms in particular, play roles in phytochrome and oxidative stress signal transduction. Some NDPK isoforms are also capable of phosphorylating protein sub- strates (including themselves) and interact with a number of regu- latory proteins. To enable detailed characterization of the, presumably cytosolic, tobacco NDPK1, the cDNA was cloned using a PCR strategy. The coding sequence was introduced into the Gateway Ò system (Invitrogen) and expressed NDPK1 was purified from E. coli lysate using a cleavable His-tag. The charac- terization of the BY-2 NDPK1 gene product will focus on the characterization of its potential functions, its interactions with other proteins and the effect of cAMP. The status of the ongoing NDPK1 characterization will be presented. Besides GAPDH and the NDPK isoforms, a number of other proteins were shown to interact with immobilized cAMP, albeit in much lower abun- dance. Their identities and potential functions will also be dis- cussed. References: 1. Ehsan et al. 1998; FEBS Lett. 422: 165–169 2. Laukens et al. 2001; FEBS Lett. 508: 75–79 J2–009P Affinity chromatography isolation and characterization of soluble cGMP-binding proteins fromavenaL satival. seedings L. V. Dubovskaya and I. D. Volotovski Laboratory of Molecular Biology of Cell, Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk, Belarus. E-mail: lpcp@biobel.bas-net.by; dubovsk@mail.ru Guanosine 3¢,5¢-cyclic monophosphate (cGMP) was shown to play a crucial role in light, phytohormone and nitric oxide sig- nal transduction in plants. Rhythmic oscillation of its concen- tration and stimulation of floral induction by cGMP was detected in higher plants. The specificity of cellular responses to cGMP is based on cGMP-binding activities of target proteins. Until now the elucidation of cGMP-binding activity and identi- fication of cGMP targets in a plant cell are at the initial stage of investigation. We report the attempt to reveal the targets for cGMP action in Avena sativa L. seedlings using the affinity purification and electrophoresis identification of cGMP-binding proteins that were not previously demonstrated in higher plants. To elucidate the early molecular events associated with biologi- cal action of cGMP its binding to the structural components of Avena sativa L. plant cell has been studied. cGMP was shown to be bound specifically to proteins located predominantly in soluble cytosolic fraction. The Scatchard plot analysis indicated the presence in Avena sativa L . cells of two classes of cyclic GMP-specific binding sites with high and low affinity for cGMP. The heating and the treatment of samples with trypsin and pronase suppressed the ability of the sites to bind to cGMP. Together with the dependence of the binding activity on pH these results suggest the protein nature of cGMP-bind- ing sites. About ten specific cGMP-binding proteins were detec- ted in cytosol with help of cGMP-agarose affinity purification procedure followed by SDS-PAGE. They showed an apparent molecular weight of 15 and 18 kDa, about 30–40 kDa and 53, 58 and 72 kDa. The possible nature of purified proteins was discussed. J2–010P Cyclin-dependent kinases of the green alga Chlamydomonas reinhardtii. M. E ` ı ´ z ˇ kova ´ 1 , K. Bis ˇ ova ´ 2 , J. Hendrychova ´ 1 ,M.Vı ´ tova ´ 1 and V. Zachleder 1 1 Laboratory of Cell Cycles and Biotechnology of Algae, Depart- ment of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences, Tøeboo ` , Czech Republic, 2 The Salk Institute of Biological Sciences, La Jolla, CA 92037 USA. E-mail: majka.p@pobox.sk The biflagellate unicellular alga Chlamydomonas reinhardtii is used as a model system for cell cycle studies. Its uniqueness is grounded mainly in cell division, which occurs by non-canonical mechanism termed multiple fission. In spite of this, Chlamydo- monas is simply handling model thanks to its autotrophic unicell lifestyle and to possibility of culture synchronization. Moreover, its genome has recently been sequenced. Eukaryotic cell cycle is controlled by a set of conserved proteins. This set includes the cyclin-dependent kinases (CDK), which have a key role in coordinating the cell division and in integrating diverse growth- regulatory signals. In spite of the fact that kinases have been studied for many years, investigation of CDKs has not been completed yet, especially in photo-autotrophic organisms. We decided to study the capability of CDKs from C. reinhardtii to complement yeast cdc28 mutant and the intracellular localization of these algal kinases in the course of the cell cycle. We have inserted genes coding algal CDKA and CDKB into pENTR vec- tor. The resulting entry clone (pENTR-CDK) was ready for recombination with any destination vector to create an expres- sion clone. We employed the destination vector specialized in expression in yeast allowing us to perform a complementation test of a S. cerevisiae cdc28 mutant. Algal CDK was shown to complement cdc28 mutation in S. cerevisiae. We have constructed a vector with the cgfp gene fused in frame to a Gateway Ò cas- sette, which is under control of the rbcS2 promoter and termina- tor. The gene encoding green fluorescent protein adapted to the codon usage of C. reinhardtii (cgfp), was used from pMF124cGFP plasmid. The Gateway Ò cassette contains attR recombination sites flanking a ccdB gene and a Cmr gene. We assume that this vector will serve as a useful tool to visualize syn- thesis of different cell cycle proteins and their localization in vivo in the alga C. reinhardtii. Acknowledgment: Supported by grants from GAASCR (KJB5020305) and from GACR (204/02/1438). J2–011P Protein complexes through the plant cell cycle: their composition and dynamics N. Remmerie, L. Roef, K. Laukens, H. Van Onckelen and E. Witters Plant physiology and biochemistry, CEPROMA, Biology Univer- sity Antwerp, Antwerp, Belgium. E-mail: noor.remmerie@ua.ac.be By means of a gel based proteome strategy using blue native gel electrophoresis, we investigated the dynamics of protein com- Abstracts 457 plexes during the Nicotiana tabacum cv. Bright Yellow-2 (BY-2) cell cycle by studying changes in their concentration and subunit build up, and by looking at their post-translational modifications. Samples of synchronized cell suspensions were taken at different phases throughout the cell cycle and subjected to non-denaturing blue native polyacrylamide gelelectrophoresis (BN-PAGE) to sep- arate protein complexes based on their size while preserving the quaternary structure. Samples were subsequently subjected to a denaturing second dimension, SDS-PAGE, in which the complexes break up into their constituents, or to a native pH gradient separating protein complexes according to their pI. Visualization of 2DGE-separated proteins was done by ruthen- ium-based fluorescent staining. In order to follow the dynamics of complexes during their progression through the plant cell cycle, we investigated the possibilities of combining the BN PAGE technique with 2D difference in gel electrophoresis (2D- DIGE)(Amersham Biosciences). This gel based proteome display can easily be further combined with specific staining methods like Pro-Q diamond and Pro-Q Emerald (Invitrogen) to reveal post- translational modifications such as phosphorylation and glycosy- lation that are needed for complex formation or activity. Proteins revealing significant quantitative differences or post-translational modifications were submitted to mass spectrometric identifi- cation. The identity, composition and differential regulation of the complexes will be discussed in relation with cell cycle physiology. J2–012P Buchnania lanzan extract administration increases the life span of rats with hepatocellular carcinoma D. G. Reddy, R. Kartik, V. C. Rao, K. M. Unnikrihsnan and P. Pusphangadan Laboratory of Ethnopharmacology, Department of Pharmacology, NBRI, Lucknow, UttarPradesh India. E-mail: dayanandr@yahoo.com The effect of Buchnania lanzan bark extract administration after induction of hepatocellular carcinoma (H.C.C) by N-nitrasodieth- ylamine (NDEA) was studied in wistar rats. Administration of ethanolic extract of B. lanzan was found to significantly increase the survival of H.C.C. harbouring animals. All untreated rats died of tumor burden by 37.4 ± 1.9 weeks. Administration of B. lan- zan extract (200 mg/kg b.w) after tumor development increased the survival of animals to an average of 52 ± 2.5 weeks. Serum gama glutamyl transpeptidase activity which was elevated to 185 ± 20 u/l by NDEA administration was lowered to 110 ± 19 u/l by the administration of B. lanzan extract. Similarly elevated glutathione S-transferase activity (1445 ± 113 nmol/ min/mg protein) and glutathione (24.3 ± 2.0 nmol/mg protein) levels in the NDEA administered group were found to be lowered to 1001 ± 80 nmol/min/protein and 12.5 ± 2.5 nmol/mgprotein respectively. B. lanzan J2–013P Effect of Diphenylmethyl selenocynate on N-nitrosodiethylamine-induced hepatocarcinogenesis in rats. C. V. Rao, S. K. Ojha, A. K. S. Rawat, D. G. Reddy, R. Kartik and P. Pushpangadan Laboratory of Ethnopharmacology, Department of Pharmacognosy and Ethnopharmacology, N B R I, Lucknow, Uttar Pradesh, India. E-mail: chvrao72@yahoo.com Aim: Effect of Diphenylmethyl selenocynate on N-nitrosodiethyl- amine-induced hepatocarcinogenesis in rats. Methods: Rats were given a single intraperitoneal injection of N-nitrosodiethlyamine (200 mg/kg body wt) followed by subcuta- neous injection of carbon tetrachloride (3ml /kgbody wt/week) for 6 weeks.The animals were randomized and grouped into experimental and control rats (n = 6 in each group). Group I (control) rats were treated with 0.9% normal saline through out the study. Group II rats received single injection of N-nitro- sodiethlyamine (200 mg/kg body wt) followed by subcutaneous injection of carbon tetrachloride (3ml /kgbody wt/week) for 6 weeks. Group III rats were injected with N-nitrosodiethlyamine as in group II and treated with Se compound (2 mg/kg body wt in 5.5% propylene glycol p.o) daily. Group IV rats were treated with Se compound (2 mg/kg body wt in 5.5%propylene glycol p.o). The experiment was terminated after 20 weeks and all ani- mals were killed by cervical dislocation after an overnight fast. Blood was collected and serum was separated. Liver tissues were taken in ice-cold container for biochemical determinations. Results: The serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) which is the liver injury marker enzyme was significantly (P < 0.01)elevated in NDEA treated group and was significantly (P < 0,01) reduced after treatment with Se compound in group III (47% decrease in SGOT and 53% decrease in SGPT level when compared to NDEA treated group). The Alkaline phospha- tase (ALP) which was reduced in Group III compared with NDEA group (49% reduction in the level of ALP). The tumour marker enzyme, gamma glutamyl transpeptidase(GGT) was sig- nificantly higher in NDEA group both in serum and liver was drastically (P < 0.001) reduced upon treatment with Se com- pound in group III (36% decrease in GGT level both in serum and liver). The Gluthathione S-transferase(GST) is an detoxifying enzyme in conjugation with reduced gluthathione (GSH) was ele- vated in wide variety of tumour, the decrease in the level (81% decrease in GST and 51% reduction in GSH) which showed its affinity to detoxifying and protect the liver against NDEA induced hepatocarcinogenesis. 5¢-nucleotidase activity was observed higher in liver cancer, the Se compound potentiate the decrease in the level of 5¢-nucleotidase (44% reduction in the level when compared to NDEA treated group) Conclusion: In the present study, the hepatocarcinogenic inhibi- tory effect of diphenylmethyl selenocynate as evident from the result against various aspects of NDEA-induced hepatocarcino- genesis is due to the upregulation of phase II detoxifying enzymes and inhibition of lipid peroxidation. Abstracts 458 [...]... height (14C-donor part of the shoot), and in 12 and 21 days 14C incorporation into leaf, cortex and wood proteins in 14C-donor and 14C-acceptor (above the donor) parts of the shoot was estimated The relationships observed were the same as in wheat and the differences were more pronounced in high light O V Blinkova1 and A Davydov2 1 Laboratory of Classification and Identification of Microorganisms, The... reported and the transformation of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are characterized The identity of the reaction products was studied by HPLC and ESI-MS and data indicated that dopaxanthin-quinone was obtained and evolved to more stable species, related to the violet pigment betanidin, by intramolecular cyclization Kinetic parameters were determined and. .. MCYT (Spain) and FEDER (AGL2003-05647); and Fundacion ´ Seneca, Consejerı´ a de Agricultura, Agua y Medio Ambiente (Spain) and FEDER (AGR/11/FS/02) F Gandı´ a-Herrero holds ´ ´ a fellowship from Fundacion Seneca (Spain) J4–011P Response of potato cell suspension to salicylic acid: superoxide anion production and peroxidase activity L Faravardeh and A Rabbani Institute of Biochemistry and Biophysics,... proteins, glycinebetaine, proline and other protectors takes place Use of a highly sensitive method (accuracy 0.07 lm) – laser interference auxanometry – made it possible to study rapid (min) and slower (h) response reactions of leaves and stems of wheat, oat, barley, rice and buckwheat plants on increase in NaCl concentration in nutrient medium, blockage of calcium channels and effect of antioxidants Addition... 3-P-glycerate and inhibited by Pi We report the first crystal structure of the ADPGlc PPase catalytic subunit The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain adopts a left-handed parallel ß-helix that is involved in a unique oligomerization and in cooperative allosteric regulation The structure provides insights into the catalytic mechanism and allosteric... performed on both pools S and T, with (+Cd) and without (-Cd) Cd treatment (10 lM) Two-dimensional gel electrophoresis was performed and the gels were stained either with standard colloidal coomassie or modified ruthenium II tris (batho-phenanthroline disulfonate) (RuBPS) protocol Using Imagemaster Platinum software, we compared four situations: S with T, S with S+Cd, T with T+Cd and S+Cd with T+Cd Around... this proteomic approach we want to find the link between sink and storage of Cd transport J4–007P Rapid and slow response reactions of plants on changes in composition of mineral nutrition N V Budagovskaya1 and V I Guliaev2 1 Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russian Federation, 2All-Russian Institute of Genetics and Breeding of Fruit Plants, Russian Academy of Agricultural... molecular tools towards understanding the protein activity of the encoded locus As well, we are working on the ELF3 and ELF4 loci required for detection of the light to dark transition of dusk Various molecular-physiological analyses have been use to place the ELF3 and ELF4 proteins within a model framework of the clock We have also uncovered an allelic series of elf3 and elf4 mutations Characterization... includes those cellular and ion transport processes controlling responses as diverse as stomatal, leaf and floral movements, hypocotyl elongation, nutrient uptake and inter- and intracellular transport The consensus model for plant circadian clock function is becoming refined with detail, especially at the level of molecular processes that underlie timekeeping (the circadian oscillator), and the synchronization... increased growth rate of shoots during two phases of response reaction: rapid (min) and slow (2–3 h after addition of antioxidant) The data obtained provide information on dynamics of response reactions of shoots, related and non-related to synthesis of protectors at increase of NaCl concentration at the root zone, effect of calcium channel blocker and antioxidant The biological fixation is the largest source . J1–Photosynthetic Light Harvesting and Reaction Center Complexes J1-001 Cocrystals of photosystem I with. efficiency and live at lower light- regimes and greater depths than most other algae. The pathways for energy transfer between the light harvesting systems and