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

Strasser2 1Department of Biophysics and Radiobiology, Faculty of Biology, University of Sofia, Sofia, Bulgaria,2Bioenergetics Laboratory, Department of Plant Biology, University of Geneva,

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 Golbeck1, M Antonkine2and D Stehlik2

1Biochemistry and Molecular Biology; Chemistry, The

Pennsylva-nia State University, University Park, PA, USA,2Fachbereich

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 Kouril1, A Zygadlo2, P E Jensen2, N Yeremenko3,

H Matthijs3, H D’Haene4, J Dekker4and E Boekema1

1Department of Biophysical Chemistry, Groningen University, Groningen, The Netherlands,2Agricultural University, Copenhagen, Denmark,3University of Amsterdam, Amsterdam, The Nether-lands,4Vrije Universiteit Amsterdam, Amsterdam, The Nether-lands E-mail: e.j.boekema@rug.nl

Photosystem I (PSI) and Photosystem II (PSII) can form supercom-plexes with various types of antenna proteins One such protein is Light-harvesting complex II (LHCII) In plants it is bound in its tri-meric form to ditri-meric PSII In spinach and Arabidopsis thaliana most dimers bind 2–4 LHCII trimers We have characterized the PSI-LHCII complex from Arabidopsis membranes in state two by single particle electron microscopy at about 16 A˚ PSI binds one trimer at the site of the PsaL and PsaK subunits Cyanobacteria express 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 dissipators Electron microscopy studies on over 130 000 particle projections of these supercomplexes show that photosystem 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 copcop-ies, respectively This shows that IsiA can 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 obligatory compo-nents in the formation of PSI-IsiA supercomplexes

J1-004 Initial charge separation in photosystem II reaction centers identified with femtosecond mid-infrared spectroscopy

M.-L Groot1, N P Pawlowicz1, L J van Wilderen1, J Breton2,

I H van Stokkum1and R van Grondelle1

1Department of Physics and Astronomy, Vrije Universiteit, Amsterdam, The Netherlands,2CEA, 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

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 elecelec-tron 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)

sphaeroideswas 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 Gardiner1, A W Roszak2, J Southall1, C J Law3,

R J Codgell1and N W Isaacs2

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,3L2-009, 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-sulatusand appears to be associated with the LH1 complex facili-tating ubiquinol/ubiquinone exchange between the RC and the cytochrome b/c1complex W is located directly in front of the Qb

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 Andersson1,2, M Heddad1and I Adamska1,2 1

Department of Biochemistry and Biophysics, Stockholm Univer-sity, Stockholm, Sweden,2Department 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

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 Chernev1, I Zaharieva1, V Goltsev1and R J Strasser2

1Department of Biophysics and Radiobiology, Faculty of Biology,

University of Sofia, Sofia, Bulgaria,2Bioenergetics 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 interacinforma-tions 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 Goltsev1, I Zaharieva1, P Chernev1and R J Strasser2

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

fluorescence component is proposed to be a result of radiative

charge recombination in Photosystem II reaction centers in state

Z+PQA-QB-, and its lifetime is determined by the rate of electron

transfer from QA- to QB- The millisecond delayed fluorescence

component is associated with recombination in Z+PQA-QB=

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 QB

-site On the basis of these assumptions and of the

differ-ent share of the three compondiffer-ents 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 monomerization 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 CO2gas 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 CO2 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 Palencar1, F Vacha2,3and M Kuty1

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,2Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic,3Institute

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 partichemic-ally 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

Acknowledgment: This work was supported by MSMT

(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

Res2005; In press

3 Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S

Science2004; 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 Seguin1, G Ajlani1, J.-M Verbavatz1, R Gobin1, A Gall1,2,

M Paternostre1and B Robert1

1Service de Biophysique des Fonctions Membranaires, DBJC/CEA

- URA 2096/CNRS, Gif-sur-Yvette, France,2IBLS/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 reacexcita-tion 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 Toth1, G Schansker1, G Garab2and R J Strasser1

1

Laboratory of Bioenergetics, Department of Plant Biology,

Uni-versity of Geneva, Geneva, Switzerland,2Institute 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´zl1, Z Ja´szay2, L Hulla´n3, T Szarvas4, I Petneha´zy1,

A Csiba5, L Sarkadi6and L To˜ke1

1Department of Organic Chemical Technology, Budapest Univer-sity of Technology and Economics, Budapest, Hungary,2Organic Chemical Technology Research Group, Hungarian Academy of Sci-ences, Budapest, Hungary,3Department of Biochemistry, National Oncological Institute, Budapest, Hungary,4Institute of Isotopes

Co Ltd., Budapest, Hungary,5Veterinary and Food Control Sta-tion, Budapest, Hungary,6Department of Biochemistry and Food Technology, Budapest University of Technology and Economics, Budapest, Hungary E-mail: trezl@oncol.hu

The generally accepted photosynthetic CO2 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 CO2 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 NaH13CO3with certain proteinogenic amino acids (l-lysine, l-arginine, l-glutamine and l-asparagine)

on physiological pH by 13C-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 NaHCO3 solution by GSH and Arg+GSH.To get further evidence for the reduction of CO2 to formic acid via carbamates in the plant leaves we applied photosynthetic14CO2

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

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 Zaharieva1, P C Chernev1, V N Goltsev1and

R J Strasser2

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, millisecsub-millisec-ond 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+PQA-QB-, the millisecond one is associated with recombination in Z+PQA

-QB

=

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

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 funcloss-of-func-tional 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¨t Tu¨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 vescytokinet-icle fusion machinery or proteins required for mcytokinet-icrotubule 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-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´zˇkova´ 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

[Bisˇova´ 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 Bisˇova´ 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 Alunni1, P Mergaert1, N Maunoury1, M Redondo-Nieto1, A.-E Mausset1, T Uchiumi2, A Kondorosi1and E Kondorosi1

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 melilotisoil 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

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 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-bindcGMP-bind-ing 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´zˇkova´1, K Bisˇova´2, J Hendrychova´1, M Vı´tova´1and

V Zachleder1

1Laboratory of Cell Cycles and Biotechnology of Algae, Depart-ment of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences, Tøeboo`, Czech Republic,2The 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-monasis 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 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 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

(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-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