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:
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:

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:
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:
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:
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:
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:
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:
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:
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:
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: fia.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:
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:
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:
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:
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:
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:
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:
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: fia.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:
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:
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:
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:
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:
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:
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:
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:
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: ;
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:
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:
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:
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:
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
J3–Circadian Rhythm Control in Plants
J3–001
Detecting the boundaries of one day
S. J. Davis
Davis Laboratory, Department of Plant Developmental Biology,
Max Planck Institute for Plant Breeding Research, Cologne,
Germany. E-mail:
Proper plant growth and development requires a robust detec-
tion of the diurnal environment. This occurs through a coupling
mechanism of light detection and the circadian clock. We have
been characterizing in the model plant Arabidopsis thaliana fac-
tors that mediate this coupling. We have genetically isolated the
TIC gene required for proper detection of the dark to light
transition of dawn. Our recent cloning of this gene provides
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 frame-
work of the clock. We have also uncovered an allelic series of
elf3 and elf4 mutations. Characterization of these mutations at
physiological and biochemical levels is assisting our understand-
ing of the protein function of these pioneer proteins. The collec-
tive use of genetics and protein analyses will enhance our
understanding of the biochemical mechanisms of detecting the
boundaries of one day.
J3-002
Post-transcriptional control in the Arabidopsis
circadian system
D. Staiger
Biology Department, University of Bielefeld, Bielefeld, Germany.
E-mail:
The ATGRP7 (Arabidopsis thaliana glycine-rich RNA-binding
protein) transcript encoding a protein with an N-terminal
RNA-binding domain and a glycine-rich C-terminus undergoes cir-
cadian oscillations, peaking at the end of the day. Constitutive
overexpression of ATGRP7 in transgenic plants depresses oscilla-
tions of the endogenous transcript, indicating that the transcript
and the protein are pat of a negative feedback loop. The molecular
mechanism of the negative autoregulation at the post-transcrip-
tional level will be discussed as well as how ATGRP7 is involved in
modulating the expression of a heterologous transcript.
J3–003
Circadian regulation of signalling in plant cells
A. Webb
Department of Plant Sciences, University of Cambridge,
Cambridge, UK. E-mail:
The circadian clock is the internal timekeeper of plants. This

clock regulates most aspects of plant physiology. This 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 becom-
ing refined with detail, especially at the level of molecular proces-
ses that underlie timekeeping (the circadian oscillator), and the
synchronization of the oscillator with the external day-night cycle
(entrainment). However, the signal transduction pathways that
communicate endogenous estimates of external time from the
molecular oscillator(s) to clock-controlled components of bio-
chemistry and physiology remain poorly understood. We are
investigating the hypothesis that the circadian clock regulates cel-
lular physiology via a Ca
2+
-based signalling cascade. We have
evidence that demonstrate how the circadian clock encodes infor-
mation in oscillations of cytosolic free Ca
2+
. We have identified
new components of the circadian signalling network using auto-
mated measurements of physiology and microarray analysis.
These data have allowed is to demonstrate why plants have circa-
dian clocks and to quantify and characterize the benefits of pro-
cessing a circadian clock.
J3–004
The circadian clock gates shade avoidance
responses in Arabidopsis
K. A. Franklin, V. S. Larner, L. Doyle, M. G. Salter and
G. C. Whitelam

Department of Biology, University of Leicester, Leicester, UK.
E-mail:
The phytochromes are a family reversibly photochromic plant
photoreceptors that perceive spatial and temporal informational
signals and initiate appropriate adaptive developmental strategies.
One such strategy involves the phytochrome-mediated perception
of the far-red radiation (700–800 nm) reflected/scattered from the
leaves of nearby vegetation plants, providing an early warning of
potential shading, and leading to a series of developmental
responses, the ‘‘shade-avoidance’’ syndrome, that can result in
the overgrowth of those neighbours. Thus, on sensing a reflected
far-red light signal, a shade-avoiding plant very rapidly exhibits
enhanced elongation growth. If the rapid elongation strategy is
unsuccessful and the far-red light signal persists, then other
aspects of the shade-avoidance syndrome cause accelerated
flowering and early production of seeds, enhancing the probabil-
ity of survival. Despite its adaptive significance, little is known
about the molecular events that connect light perception with
increased elongation growth in shade avoidance. Using microar-
rays, we have shown that an early event following the perception
of the far-red light signal is the altered expression of numerous
genes. For several of these genes, altered expression in response
to far-red light signals is gated by the circadian clock. Further-
more, the rapid shade avoidance elongation growth response is
gated by the circadian clock, being most apparent around dusk.
One of the rapidly far-red-responsive genes encodes a basic helix-
loop-helix protein, PIL1 that is essential for the rapid elongation
growth response.
Abstracts
459

J4–Nutriton/Metabolite Sensing in Plants
J4–001
Multilevel genomics analysis of carbon-
nutrient interactions in arabidopsis
M. Stitt
Max Planck Institute, Potsdam, Germany.
E-mail:
A multilevel phenotyping platform including growth systems or
‘‘gauntlets’’ that display phenotypes that are related to changes
in carbon and nitrogen supply, near whole-genome 22K ATH1
arrays for routine expression profiling, an in-house platform
that allows sensitive and quantitative profiling over >1200 tran-
scription factors by multiplexed real time RT-PCR, a robotized
platform for the measurement of enzyme activities, and robo-
tized enzymic assays and metabolite profiling has been used to
provide a detailed molecular characterization of the response of
Arabidopsis thaliana to changes in the nitrogen, phosphate and
carbon supply. Changes of the carbon and nutrient supply lead
to rapid and coordinated changes of gene expression in many
sectors of primary metabolism, to reprogramming of secondary
metabolism and to coordinated regulation of processes that are
involved in growth like protein synthesis and cell wall extensi-
bility. These play a major role in the regulation of gene expres-
sion not only in adjustment to chronic deficiency, but also
during the diurnal cycle in non-stressed plants. The impact of
changes in gene expression depends upon whether these lead to
changes in the levels of the encoded protein. It will be discussed
how this can be investigated using robotized enzyme assays and
quantitative proteomics. Genes that could regulate responses of
cabon and nutrients include genes involved in the synthesis of

trehalose-6-phosphate, in hormone synthesis and sensing, in pro-
tein turnover, and in the regulation of transcription. First exam-
ples will be presented of the functional analysis of selected
candidate genes, using ectopic overexpression of heterologous
genes to alter the levels of potential signaling molecules, or
inducible over-expression of regulatory genes, followed by
detailed molecular and biochemical characterization of the
transformants.
J4–002
Metabolite sensing in plants: a role for
trehalose metabolism in seed development
and embryo development
L. D. Gomez, S. Baud and I. A. Graham
CNAP, Graham laboratory, Biology, University of York, York,
North Yorkshire UK. E-mail:
We previously showed that Trehalose-6-phosphate synthase 1
(TPS1), which catalyses the first step in trehalose synthesis, is
essential for embryo maturation in Arabidopsis (Eastmond et al.,
Plant J. 2002; 29: 225–235). The tps1 mutant embryos develop
more slowly than wild type. Patterning in the tps1 embryos
appears normal but they do not progress past the torpedo stage
to cotyledon stage, which is when storage reserves start to accu-
mulate in the expanding cotyledons. Our initial data led to the
hypothesis that trehalose metabolism plays a key role in regula-
ting storage reserve accumulation by allowing the embryo to
respond to the dramatic increase in sucrose levels which occurs
at the torpedo stage of embryo development. We have carried
out a comprehensive analysis of the transcriptome of torpedo
stage and cotyledon stage wild type embryos and compared these
with the transcriptome of tps1torpedo stage embryos. We have

also performed extensive electron microscopic, enzyme and meta-
bolite analysis of the same three stages. Our data demonstrates
that at the transcriptome, biochemical and EM levels, tps1 tor-
pedo stage embryos more closely resemble cotyledon stage than
torpedo stage wild type embryos. This data demonstrates that
separate programmes operate during embryo development. In the
tps1 mutant developmental progression of embryos from torpedo
to cotyledon stage is blocked but the metabolic programme gov-
erning the accumulation of storage reserves proceeds in a similar
fashion to wild type.
J4–003
A conserved uORF mediates sucrose-induced
repression of translation.
A. Wiese, N. Elzinga, B. Wobbes, J. Hanson and S. Smeekens
Molecular Plant Physiology, Utrecht University, Utrecht,
The Netherlands. E-mail:
Sugars as signalling molecules regulate the expression of numer-
ous plant genes. Most genes are controlled via transcriptional
regulation but other mechanisms have been uncovered as well.
These include the control of mRNA stability and translation, and
protein stability. A sucrose specific translation control mechanism
was recently discovered, which controls translation of the group S
bZIP-type transcription factor ATB2/AtbZIP1.1 This control
requires the very long 5’UTR of the gene. The elements involved
in translational control were identified by studying point muta-
tions and deletions in the 5’UTR. A highly conserved uORF that
encodes 42 amino acids is required for sucrose repression. This
uORF is conserved in 5’UTRs of bZIP-transcription factors from
four other Arabidopsis genes and many other plant species. The
AtbZIP11 gene shows a restricted expression patterns and seems

mainly associated with vascular tissues. However, the sucrose
repression system is operating throughout the plant as shown by
ectopic expression of a 5’UTR construct using the ubiquitin 10
promoter. The other Arabidopsis conserved uORF encoding tran-
scription factors (AtbZIP1, 2, 44, 53) show similar sucrose-
induced repression of translation. A general sucrose specific trans-
lational mechanism is suggested, dependent on the presence of the
conserved uORF. The target genes of these transcription factors
are most likely regulated in a sucrose-dependent way. Such target
genes are currently being identified using the novel DamID sys-
tem, which uses directed methylation of promoter regions.
J4–004
14–3–3s as signalling integrators in plant and
human cells
C. MacKintosh, M. Pozuelo Rubio, K. Geraghty, B. Wong,
N. Wood, J. Murphy, F. Dubois and J. Harthill
MRC Protein Phosphorylation Unit, School of Life Sciences,
University of Dundee, Dundee, Tayside UK.
E-mail:
14-3-3 proteins exert an widespread influence on cellular proces-
ses in all eukaryotes. They bind to specific phosphorylated sites
on diverse target proteins, thereby forcing conformational chan-
ges or influencing the interactions of their targets with other mol-
ecules [1,2]. Hundreds of phosphoproteins can bind to 14-3-3s
[3], so 14-3-3s must connect signaling pathways to the control of
many cellular processes, including metabolism, actin dynamics,
trafficking and proliferation, which signalling pathways promote
Abstracts
460
the interactions of which targets with 14-3-3s? In plants, we are

dissecting roles for 14-3-3s in a dark-induced signalling pathway
that prevents dangerous metabolic imbalances when photosyn-
thesis is suddenly inhibited [1]. In human cells, we have identified
several target proteins (in translation, vesicle trafficking and
other processes) that bind to 14-3-3s in response to IGF-1 in vivo
and PKB phosphorylation in vitro [4]. Finding such an array of
14-3-3 targets in the PI 3-kinase/PKB pathway makes us wonder
whether the relative strength of signalling through to different
downstream targets of PKB is influenced by the spectrum of
14-3-3 isoforms in the cell. Do cells give kinetically different
responses to PKB signalling depending on their complement of
14-3-3 isoforms? In some cases, 14-3-3s only bind after targets
have been phosphorylated by two different kinases, suggesting
that 14-3-3s can act as ‘‘signalling integrators’’ or ‘‘coincidence
monitors’’ that engage and trigger a response when dual signals
are received.
References
1. MacKintosh, C Biochem J 2004; 381: 329–342
2. Kulma A, Villadsen D, Campbell DG et al. Plant J 2004; 37:
654–667
3. Pozuelo Rubio M, Geraghty KM, Wong BHC et al. Biochem
J 2004; 379: 395–408
4. Pozuelo Rubio M, Peggie M, Wong BHC et al. Embo J 2003;
22: 3514–3523
J4–005
Crystal structure of potato tuber ADP-glucose
pyrophosphorylase catalytic subunit and
conversion of the regulatory subunit into a
catalytic subunit
M. A. Ballicora

1
, X. Jin
1,2
, C. H. Devillers
1
, J. R. Dubay
1
,
J. H. Geiger
2
and J. Preiss
1
1
Department of Biochemistry & Molecular Biology, Michigan
State University, East Lansing, Michigan USA,
2
Department of
Chemistry, Michigan State University, East Lansing, Michigan
USA. E-mail:
ADP-glucose pyrophosphorylase catalyzes a major regulatory step
in starch biosynthesis. It is allosterically activated by 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-ter-
minal 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 mech-
anism and allosteric regulation of the enzyme. The native enzyme
is composed of two subunits as a heterotetramer. S, the small sub-
unit, is the catalytic subunit. L, the large subunit is the regulatory

subunit. The enzyme is made inactive (0.2%) by substituting Asn
for Asp residue 145 of the S subunit. This inactive enzyme can be
made catalytically active, by mutagenizing Lys 44 to Arg and Thr
54 to Lys of the L subunit. The purified mutagenized enzyme S,
D145N, L, K44R/T54K, had 18% of the catalytic activity of the
wild-type enzyme. The mutant enzyme was still activated by
3PGA and inhibited by Pi as the wt enzyme and the apparent
affinities of the substrates are the same as the wt enzyme. Muta-
tion of the large subunit residue D160, which is homologous to
the D145 of the catalytic (small) subunit to Asn in the mutagen-
ized S, D145N, L K44R/T54K enzyme abolished its activity indi-
cating that catalysis was now occurring in the large subunit. These
results confirm the evolutionary relationship between the S and L
subunits and suggest the ancestral catalytic activity of the L sub-
unit. The conversion of the large subunit to function in a strictly
regulatory capacity may be due to the necessity of different plants
for acquiring multiple regulatory properties.
J4–006
Plant responses to cadmium stress: a
proteomic approach
P. Deckers
1
, A. R. Craciun
2
, A. Azmi
1
, E. Witters
1
, K. Laukens
1

,
N. Verbruggen
2
and H. Van Onckelen
1
1
Laboratory of Plant Biochemistry and Physiology, Department of
Biology, University of Antwerp, Antwerp, Belgium,
2
Laboratoire
de Physiologie et Ge
´
ne
´
tique Mole
´
culaire des Plantes, Universite
´
Libre de Bruxelles, Brussels, Belgium.
E-mail:
Using a proteomic approach, we investigated cadmium (Cd) allo-
cation in roots and leaves. After selection, we screened 11 Cd
sensitive (S) and 18 Cd hypertolerant (T) plants. Proteomic ana-
lysis was performed on both pools S and T, with (+Cd) and
without (-Cd) Cd treatment (10 lM). Two-dimensional gel elec-
trophoresis 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 1000 spots were detected of which 6% seemed to have
an altered protein expression. Relevant spots were picked and
analyzed using Matrix-assisted Laser Desorption/Ionization
Time-of-flight (MALDI Tof-Tof) mass spectrometry. After sub-
mission to several databases using Mascot, 17 differently
expressed proteins were identified between T and T+Cd. For
example, a putative glycoprotein (EP1) (a possible membrane
protein involved in Cd transport mechanism) is up-regulated and
a glutathione S-transferase PM24 is down-regulated after Cd
treatment. In order to corroborate our findings, these results were
compared to microarray data. With 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. Budagovskaya
1
and V. I. Guliaev
2
1
Institute of Plant Physiology, Russian Academy of Sciences,
Moscow, Russian Federation,
2
All-Russian Institute of Genetics
and Breeding of Fruit Plants, Russian Academy of Agricultural
Sciences, Michurinsk, Russian Federation.
E-mail:
Unfavourable nutritional conditions such as deficiency of mineral
elements, salinity cause disturbances in metabolism, decreasing in

protein synthesis, slowing down in plant growth. In the course of
adaptation of plants to the mentioned stress factors synthesis of
stress 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 of NaCl in
increased concentration to the root zone of plants caused a two
phase response reaction of leaves: decrease and the following
increase in their growth rate in each phase. Duration of the first
phase was threefold shorter than of the second. Growth rate of
leaves was restored in the course of adaptation by the end of the
second phase (few hours after addition of NaCl). The first phase
may be related to rapid adaptive reactions and changes in leaf
turgor, the second – to slower adaptive processes – de novo syn-
thesis of protectors. The presence of calcium channels blocker
verapamil at the root zone caused a decrease in growth rate of
Abstracts
461
leaves and stems of plants. Introduction of verapamil in low con-
centrations resulted in partial restoration of growth rate of
shoots in 2–3 h due, apparently, to synthesis of protectors. Addi-
tion of antioxidant ambiol to verapamil treated plants led to
rapid restoration of growth rate of shoots. Addition of ambiol to
plants grown under mineral deficiency 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.
J4–008P
The nitrogen fixation of some lichens
O. V. Blinkova
1
and A. Davydov
2
1
Laboratory of Classification and Identification of Microorganisms,
The Centre of Bioengineering RAS, Moscow, Russian Federation,
2
Laboratory of Microbiology, Department of Fora and Vegetation,
Polar-Alpine Botanical Garden-Institute, Kirovsk, Russian
Federation. E-mail:
The biological fixation is the largest source of nitrogen in many
pristine ecosystems. Certain prokaryote microorganisms use the
nitrogenase enzyme to break the triple bonds of atmospheric N2
and fix it into more soluble nitrogen species. Some lichens (about
15 genus) influence biological Ns fixation by forming facultative
symbioses with cyanobionts. We investigated nitrogen fixation
and Ns content in the several plant communities. We used the
acetylene reduction method for detection of nitrogen fixation of
cyanobacteria and the Kjehldahl’s method for determination of
N2 content. We estimate the linear connection between nitrogen
fixation and Ns content of Nd fixing lichens: 0.04 nitrogen is for
1 mkg Ns/twenty-four hours. The nitrogen fixation for Ns accu-
mulation is more important for lichens from north territories
then south.

J4–009P
Synthesis of plant cell proteins from
endogenous and exogenous substrates
V. I. Chikov
1
, G. G. Bakirova
2
and S. N. Batasheva
3
1
Laboratory of the Apoplast Biochemistry, Institute of Biochemis-
try and Biophysics, Kazan Scientific Centre of The Russian
Academy of Sciences, Kazan, Tatarstan Russian Federation,
2
Laboratory of the Apoplast Biochemistry, Institute of Biochemis-
try and Biophysics, Kazan Scientific Centre of The Russian
Academy of Sciences, Kazan, Tatarstan Russian Federation,
3
Laboratory of the Apoplast Biochemistry, Institute of Biochemis-
try and Biophysics, Kazan Scientific Centre of The Russian
Academy of Sciences, Kazan, Tatarstan Russian Federation.
E-mail:
In tobacco callus, soybean cell suspension culture and different
tissues of whole flax and wheat plants,
14
C incorporation from
14
CO
2
and

14
C-amino acids into proteins of different fractions
and localizations – cytoplasmic or cell wall – has been studied.
40 min incubation of callus cells with labelled substrates
(NaH
14
CO
3
or
14
C-amino acid mixture) revealed that relatively
more
14
C incorporated from hydrocarbonate into water-salt-sol-
uble and cytoplasmic proteins compared to the variant with
labelled amino acids as substrates. The latter compounds were
largely used for synthesis of cell wall proteins. Cytoplasmic to
cell wall protein specific radioactivity ratio in the case of exo-
genous substrates (
14
C-amino acids) was about 1.0; in the case
of endogenous substrates (synthesized from
14
CO
2
) the radioac-
tivity of cytoplasmic proteins was twice that of cell wall pro-
teins. In wheat plants, the stem, the spike or the upper leaf
sheath was exposed to
14

CO2 and in 2, 6 or 22 h
14
C incor-
poration into different protein fractions of the leaf and its
sheath was investigated. It was shown that from own sub-
strates (after
14
CO
2
assimilation) considerably more label was
incorporated into water-salt-soluble proteins, and from exogen-
ous substrates (those transported from other assimilating
organs) more label was incorporated into sparingly soluble
proteins. In flax plants, whole plants were exposed to
14
CO
2
when they were 12 cm in height (
14
C-donor part of the shoot),
and in 12 and 21 days
14
C incorporation into leaf, cortex and
wood proteins in
14
C-donor and
14
C-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.
J4–010P
A new role for tyrosinase in the biosynthesis
of the plant pigments betalains
F. Gandı
´
a-Herrero, M. Jime
´
nez-Atie
´
nzar, J. Cabanes,
F. Garcı
´
a-Carmona and J. Escribano
Department of Biochemistry and Molecular Biology A, University
of Murcia, Murcia, Spain. E-mail:
Betalains are an important class of water-soluble pigments, which
exhibit radical scavenging capacity and are characteristic of
plants belonging to the order Caryophyllales. In contrast to other
families of plant pigments, the betalain metabolic pathway is still
to be fully clarified. Tyrosinase or polyphenol oxidase (EC
1.14.18.1) is the key enzyme in melanin biosynthesis and in the
enzymatic browning of vegetables. The role of tyrosinase in the
secondary metabolism of plants remains unclear but its implica-
tion in betalains biosynthesis has been proposed. In this work,
the biosynthetic scheme of betalains is reviewed and a new
branch is proposed based on the description of new substrates
for tyrosinase. The yellow pigments tyrosine-betaxanthin (portu-
lacaxanthin II) and dopaxanthin are characterized as physiologi-
cal substrates of the enzyme. Evidences of interconversion

between betaxanthins catalyzed by an enzyme are 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 parame-
ters were determined and showed a high affinity of tyrosinase for
these physiological substrates.
Acknowledgments: This work was financially supported by
MCYT (Spain) and FEDER (AGL2003-05647); and Fundacio
´
n
Se
´
neca, Consejerı
´
a de Agricultura, Agua y Medio Ambiente
(Spain) and FEDER (AGR/11/FS/02). F. Gandı
´
a-Herrero holds
a fellowship from Fundacio
´
nSe
´
neca (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, University of Tehran,
Tehran, Tehran Iran. E-mail:
Salicylic acid (SA) is an inducer of disease resistance in differ-
ent species and accumulates in the infected and uninfected tis-
Abstracts
462
sues, inducing Systemic Acquire Resistance (SAR). In this
study, we have investigated the time course response of potato
cell suspension to different concentrations of salicylic acid,
superoxide anion production and peroxidase activity to eluci-
date salicylic acid mechanisms. The results show that different
concentrations of salicylic acid increased superoxide anion pro-
duction and peroxidase activity after 1 and 3 h of culture.
2.5 mm of salicylic acid showed the highest superoxide anion
production after 2 h of treatment whereas 1 mM and 5 mM of
salicylic acid reached to their high values after 4 h. Analysis of
extracted peroxidase from the treated and controls showed nine
isozymes with different activities on the native gel. Also, per-
oxidase activity determination demonstrated that the effect of
salicylic acid was dose dependent as after 3 h of treatment
while after 6 h peroxidase activity at 2.5 mM was the highest
one. The results suggest that the formation of superoxide
anion and peroxidase activity is salicylic acid dose – dependent
and 2.5 mM of salicylic acid is the critical concentration. For-
mation of superoxide anion was followed by increase in per-
oxidase activity indicating that superoxide anion is converted
to H
2
O

2
in potato cell suspension enzymetically or non
enzymatically. It seems that production of reactive O
2
and
H
2
O
2
are the intermediate signals through a peroxidase-cata-
lyzed reaction in potato cell suspension.
J4–012P
Characterisation of a highly hydrophobic
polyphenoloxidase from artichoke bracts
R. Zamora, L. Sidrach, J. N. Rodriguez-Lopez,
F. Garcia-Canovas and J. Tudela
( Dept. Bioquimica-A, Facultad de
Quimica, Universidad de Murcia, Murcia, Murcia Spain.
E-mail:
The highly hydrophobic isoform of tyrosinase or polyphenoloxi-
dase (hhPPO, EC 1.14.18.1) from bracts of artichoke (Cynara
scolymus L.) has been characterised. The agroindustrial wastes of
artichoke were supplied by ARTBIOCHEM S.L., a spin-off busi-
ness of the University of Murcia. The isolation has involved the
plant tissue fragmentation with phosphate buffer, and the frac-
tional precipitation with ammonium sulphate, by using a Beck-
man Avanti J20-XP preparative centrifuge. The fractions were
concentrated by tangential flow ultrafiltration with a Millipore
TFF system. The purification has consisted of several chromatog-
raphies with a Pharmacia A

¨
kta Explorer-100 FPLC system. The
IEXC were on carboxymethylcellulose and diethylaminoethylcel-
lulose, with increasing sodium chloride gradients. The HIC was
on phenylsepharose with decreasing sodium chloride gradient.
The advance of the process has been checked with assays of
PAGE and IEF, by using a Pharmacia Phast System. The
enzyme hhPPO is multimeric, it has Mr = 140 kDa by SDS-
PAGE and GFC, pI = 4.0 by IEF, and neutral optimum pH.
The diphenolase and monophenolase activities of hhPPO
have been measured with 3,4-dihydroxyphenylpropionic and
p-hydroxyphenylpropionic acids, respectively, in the presence of
the chromophoric nucleophile MBTH. Other biotechnological
properties are being studied. This communication has been parti-
ally supported by grants RTA AGL2002-01255 ALI CICYT
(F.G.C.), AGR/10/FS/02 Fundacio
´
nSe
´
neca (Murcia) – Conse-
jerı
´
a de Agricultura, Agua y Medio Ambiente (F.G.C.), and
PI-79/00810/FS/01 Fundacio
´
nSe
´
neca – Consejerı
´
a de Educacio

´
n
y Cultura (J.T.). L.S. has a FPU-MEC predoctoral fellowship.
R.Z. has a Fundacio
´
nSe
´
neca – Consejerı
´
a de Trabajo y Polı
´
tica
Social predoctoral fellowship.
J4-013P
Tomato introgression lines as tool to identify
regulatory steps of methionine synthesis
S. Krueger
1
, H. Hesse
1
, N. Schauer
1
, D. Zamir
2
and R. Hoefgen
1
1
Hoefgen, Department of Willmitzer, Max Planck Institute of
Molecular Plant Physiology, Potsdam, Brandenburg Germany,
2

Zamir, Field Crops,Vegetables and Genetics, Faculty of Agricul-
ture, The Hebrew University of Jerusalem, Jerusalem, Israel.
E-mail:
Methionine, an important essential amino acid, is underrepre-
sented in most crop species for human and animal needs. Methi-
onine receives its carbon skeleton from aspartate while its
sulphur moiety derives from cysteine. Both pathways for cysteine
and methionine synthesis underlay a complex regulation. In
plants the branch point of methionine and threonine synthesis is
O-phosphohomoserine (OPHS), which represent the common
substrate for both threonine synthase (TS) and cystathionine
gamma-synthase (CgS), respectively. With respect to the CgS/TS
branch point, overexpression of a feedback insensitive TS in
tobacco plants (Muhitch, 1997) resulted in a fivefold increase in
threonine, however no effect in methione level was observed. On
the other hand, reducing of the endogenous TS activity in Ara-
bidopsis thaliana (Bartlem et al., 2000) and potato leads to in
decrease of threonine while methionine levels were dramatically
increased by 60- to 200-fold, respectively (Zeh et al. 2001). Sur-
prisingly, the accumulation of methionine was highly variable
and developmentally regulated, as methionine contents dropped
upon flowering in Arabidopsis. CgS also plays a significant regu-
latory role in methionine biosynthesis in plants. A mutation in
the N-terminal region of the Arabidopsis CgS led to a significant
increase in the methionine biosynthesis which led to a novel
hypothesis on post-transcriptional and post-translational regula-
tion of methionine synthesis in Arabidopsis (Amir and Galili,
2002; Chiba et al., 1999; Inaba et al., 1994). The biochemical
control of cysteine and methionine biosynthesis is partially under-
stood, a major lack of knowledge occurs concerning signal per-

ception and signalling cascades, which control cysteine and
methionine homeostasis. The introgression lines (Zamir) provide
a unique resource to obtain novel insights into amino acid bio-
synthesis in plants, especially in tomato fruits. By analysis of
these introgression lines it was possible to identify plants with
altered methionine contents in fruits. Results will be discussed.
J4–014P
Induction of heme oxygenase-1 by a natural
sweetener, Momordica grosvenori Swingle,
attenuates the imbalance of cellular immune
functions in alloxan-induced diabetes mellitus
F. Song
1
, W. Chen
2
, P. Yao
1
, X. Zhang
1
, W. Jia
1
, X. Sun
1
and
L. Liu
1
1
Department of Nutrition and Food Hygiene, School of Public
Health, Tongji Medical College, Huazhong University of Science
and Technology, Wuhan, PR China,

2
Department of Food Science
and Technology, Huazhong University of Agriculture, Wuhan,
PR China. E-mail:
Heme oxygenase-1 (HO-1) is among the acute phase proteins,
which play an important role in the inflammatory process and
have anti-inflammatory activities with their antioxidant proper-
ties. Induction of HO-1 results in protection from cytokine-
induced apoptosis and oxidative stress in various cell types and
animal models, exhibiting potential to treat immune-related dis-
ease, including insulin dependent diabetes mellitus (IDDM). The
hypothesis Momordica grosvenori Swingle (MG), a traditional
Abstracts
463
medicinal herb in China, used as substitute sugar for obese and
diabetic patients, could modulate the imbalance of cellular
immune system and prevent the progression of IDDM, via induc-
tion of HO-1 protein expression was investigated. We demonstra-
ted that alloxan-induced male BALB/C IDDM mice presented
marked differences in lymphocyte subpopulations compared to
non-diabetic controls, as can be seen from a prominently high
level of CD8+ T cells and the lower CD4/CD8 ratio. Among
Th1 cytokines, percentage of IFN-c- and TNF-a-expressing sple-
nic cells significantly ascended after alloxan challenge, suggesting
an increased Th1 cytokine production in IDDM. Concomitantly,
alloxan herein resulted in a prominent lower level of HO-1 pro-
tein expression, indirectly confirming the protective effect of HO-
1. Nevertheless, MG administration effectively attenuated the
early immunity imbalance in IDDM mice, principally by up-regu-
lating the CD4+ T lymphocyte subsets and CD4/CD8 ratio, and

remodeling the intracellular cytokine profiles (reducing the
expression of pro-inflammatory Th1 cytokines towards a benefi-
cial Th2 pattern), ascribed to its induction of HO-1 protein
expression. Thus, MG exhibited anti-diabetic effects and may be
used as a new natural drug for the prevention and treatment for
IDDM.
Abbreviations: TNF-a, tumor necrosis factor-a; IFN-c, inter-
feron-c; IL, interleukines; TPA, 12-O-tetradecanoylphorbol-13-
acetate; EBV-EA, Epstein-Barr virus early antigen; LDL, low
density lipoprotein; STZ, streptozotocin.
J4–015P
Influence of viral infection on transgenic plants
containing genes for nonstructural potyviral
proteins HC-Pro and P3
H. Ryslava
1
, V. Doubnerova
1
, M. Janoskova
1
, Z. Subr
2
,
S. Novakova
2
and N. Cerovska
3
1
Deparment of Biochemistry, Faculty of Natural Sciences, Charles
University, Prague, Czech Republic,

2
Institute of Virology, Slovak
Academy of Sciences, Bratislava, Slovakia,
3
Laboratory of
Virology, Institute of Experimental Botany, Czech Academy of
Sciences, Prague, Czech Republic. E-mail:
The sensitivity to viral infection and its influence on enzymes of
anaplerotic pathways in transgenic plants containing genes for
nonstructural potyviral proteins HC-Pro and P3 was studied.
Gene for P3 was inserted into tobacco plants (Nicotiana tabacum
L. cv. Petit Havana SR1; gene for HC-Pro in plants of Nicotiana
benthamiana (kindly provided by dr. Savenkov, SLU Uppsala,
Sweden). Plants were inoculated by isolate of Potato virus Y
(PVY), NTN strain. In studied plants, the activities of phosphoe-
nolpyruvate carboxylase (PEPC; EC 4.1.1.31.), NADP-malic
enzyme (NADP-ME; EC 1.1.1.40.), and pyruvate phosphate dik-
inase (PPDK; EC 2.7.9.1) were measured. Previously we proved
that the activities of PEPC, NADP-ME, and PPDK are in non-
transgenic infectious plants higher. In experimental plants the P3
gene did not influence the sensitivity to potyviral infection, the
activities of PEPC, NADP-ME, and PPDK are also higher, the
increasing of the activities is comparable with that one in non-
transgenic tobacco plants. Transgenic plants of Nicotiana bentha-
miana containing gene for HC-Pro protein are more sensitive to
infection caused with PVYNTN than non-transgenic controls. In
course of viral infection we did not determined the increasing of
activities of PEPC, NADP-ME, and PPDK. Native electrophor-
esis with NADP-ME activity detection of extracts of studied
plants showed that in Nicotiana benthamiana plants occurred this

enzyme in two isoforms. In infectious plants the activity of
NADP-ME isoform with the higher mobility is increased (trans-
genic and non-transgenic), in transgenic non-infectious plants the
higher activity of isoform with lower mobility is proved.
Acknowledgments: This work was supported by grants : GA
UK 428/2004, GA E
`
R 206/03/0310, APVT-51-011802
J4–016P
Thiamine phosphate pyrophosphorylase – the
key enzyme in thiamine biosynthesis in plants
A. Golda, A. Kozik and M. Rapala-Kozik
Department of Analytical Biochemistry, Faculty of Biotechnology,
Jagiellonian University, Cracow, Poland.
E-mail:
Thiamine pyrophosphate (TDP) is an essential cofactor of carbo-
hydrate and branched-chain amino acid metabolism. Although its
mechanistic role is well understood, the biosynthesis of thiamine
has only recently been described in bacteria and yeasts. Plant thi-
amine synthesis pathways have not been well characterized and
none of the enzymes involved have been isolated. Here we report
the purification and kinetic characterization of thiamine phosphate
pyrophosphorylase (TMPS) from maize seedlings. This enzyme is
responsible for the condensation of 2-methyl-4-amino-5-hydrox-
ymethylpyrimidine pyrophosphate and 4-methyl-5-(2-hydroxyeth-
yl)thiazole phosphate to produce thiamine monophosphate (TMP).
TMPS is a monomeric protein (20 kDa), with optimal activity at
pH 9, which requires a bivalent cation (Mg>Zn>Co>Cu). The
K
m

values for pyrimidine diphosphate and thiazole phosphate are
0.88 and 0.45 lM, respectively. ATP, TDP and PCMB show an
inhibitory effect on TMPS activity. A possibility of additional func-
tion of TMPS as pyrimidine- or thiazole kinase has been tested.
The TMPS activity appears at the third day of germination and
rises up to the tenth day. TMP, produced by TMPS, is the best sub-
strate for a phosphatase (65 kDa) that is induced with a similar
time profile and is active both at pH 5 and 9. This phosphatase is
inhibited by fluoride, vanadate and molybdate. Resulting thiamine
is further pyrophosphorylated to its cofactor form, TDP, by the
pyrophosphokinase (TPK) – a monomeric enzyme (30 kDa) with
pH optimum at 9. This enzyme requires the presence of a bivalent
cation (Mg) and phosphate donors (GTP>ATP>UTP>CTP).
The K
m
value for thiamine is 0.28 lM.
Acknowledgment: This work was supported by grant No. 2
PO4C 017 27 from the State Committee for Scientific Research
(Poland).
J4–017P
Purification of hydrophobic
polyphenoloxidases from artichoke bracts
L. Sidrach, R. Zamora, J. N. Rodriguez-Lopez,
F. Garcia-Canovas and J. Tudela
( Dept. Bioquimica-A, Facultad de
Quimica, Universidad de Murcia, Murcia, Murcia Spain.
E-mail:
The isolation and purification of tyrosinases or polyphenoloxid-
ases (PPO, EC 1.14.18.1) from bracts of artichoke (Cynara scoly-
mus L.) have been carried out. The agroindustrial wastes of

artichoke were supplied by ARTBIOCHEM S.L., a spin-off busi-
ness of the University of Murcia. The isolation has involved the
plant tissue fragmentation with phosphate buffer, and the frac-
tional precipitation with ammonium sulphate, by using a Beck-
man Avanti J20-XP preparative centrifuge. The fractions were
concentrated by tangential flow ultrafiltration with a Millipore
TFF system. The purification has consisted of several chromatog-
raphies with a Pharmacia A
¨
kta Explorer-100 FPLC system. The
IEXC were on carboxymethylcellulose and diethylaminoethylcel-
lulose, with increasing sodium chloride gradients. The HIC was
on phenylsepharose with decreasing sodium chloride gradient.
The advance of the process has been checked with assays of
Abstracts
464
PAGE and isoelectrofocusing, by using a Pharmacia Phast Sys-
tem. They have been obtained three enzymatic isoforms with
low, medium and high hydrophobicity. The diphenolase and
monophenolase activities of PPOs have been measured with 3,4-
dihydroxyphenylpropionic and p-hydroxyphenylpropionic acids,
respectively, in the presence of the chromophoric nucleophile
MBTH. Other biotechnological properties are being studied.
Acknowledgments: This communication has been partially sup-
ported by grants RTA AGL2002-01255 ALI CICYT (F.G.C.),
AGR/10/FS/02 Fundacio
´
nSe
´
neca (Murcia) – Consejerı

´
a de Agri-
cultura, Agua y Medio Ambiente (F.G.C.), and PI-79/00810/FS/
01 Fundacio
´
nSe
´
neca – Consejerı
´
a de Educacio
´
n y Cultura (J.T.).
L.S. has a FPU-MEC predoctoral fellowship. R.Z. has a Funda-
cio
´
nSe
´
neca – Consejerı
´
a de Trabajo y Polı
´
tica Social predoctoral
fellowship.
J4–018P
Regulation by magnesium of potato tuber
mitochondrial respiratory activities
J. A. F. Vicente
1
, V. M. C. Madeira
2

and A. E. Vercesi
3
1
Laboratory of Biochemistry, Department of Botany, University of
Coimbra, Coimbra, Portugal,
2
Laboratory of Bioenergetics,
Department of Biochemistry, University of Coimbra, Coimbra,
Portugal,
3
Laboratory of Bioenergetics, Patologia Clı
´
nica,
UNICAMP, Campinas, S. Paulo Brazil. E-mail:
Dehydrogenase activities of potato tuber mitochondria and cor-
responding phosphorylation rates were measured for the depend-
ence on external and mitochondrial matrix Mg
2+
. Magnesium
stimulated state 3 and state 4 respirations, with significantly dif-
ferent concentrations of matrix Mg
2+
required for optimal activ-
ities of the several substrates. Maximal stimulation of respiration
with all substrates was obtained at 2 mM external Mg
2+
. How-
ever, respiration of malate, citrate, and a-ketoglutarate requires
at least 4 mM Mg
2+

inside mitochondria for maximization of
dehydrogenase activities. The phosphorylation system, requires a
low level of internal Mg
2+
(0.25 mM) to reach high activivity, as
judged by succinate-dependent respiration. However, mitochon-
dria respiring citrate or alpha-ketoglutarate only sustain high lev-
els of phosphorylation with at least 4 mM matrix Mg
2+
.
Respiration of succinate is active without external and matrix
Mg
2+
, although stimulated by the cation. Respiration of a-keto-
glutarate was strictly dependent on external Mg
2+
required for
substrate transport into mitochondria, and internal Mg
2+
is
required for dehydrogenase activity. Respiration of citrate and
malate also dependent on internal Mg
2+
but, unlike a-ketogluta-
rate, some activity still remains without external Mg
2+
. All the
substrates revealed insensitive to external and internal mitoch-
ondrial Ca
2+

, except the exogenous NADH dehydrogenase,
which requires either external Ca
2+
or Mg
2+
for detectable activ-
ity. Calcium is more efficient than Mg
2+
, both having cumulative
stimulation. Unlike Ca
2+
,Mn
2+
could substitute for Mg
2+
,
before and after addition of A23, showing its ability to regulate
phosphorylation and succinate dehydrogenase activities, with
almost the same efficiency as Mg
2+
.
J4–019P
Proteomics of cold acclimation and
photoperiod in woody plants: differential
in-gel electrophoresis (2-D DiGE) of soluble
proteins extracted from peach bark tissue.
M. Wisniewski
1
, J. Renaut
2

, J F. Hausman
2
, E. Witters
3
,
H. Van Onckelen
3
, C. Bassett
1
and T. Artlip
1
1
USDA-ARS, Kearneysville, West Virginia USA,
2
CREBS,
Centre de Recherche Public – Gabriel Lippmann, Luxembourg,
Luxembourg,
3
Laboratory for Plant Biochemistry, Department of
Biology, University of Antwerp, Antwerp, Belgium.
E-mail:
Regarding the physiology of environmental stress, proteomics
allows one to develop an understanding of the relationship
between gene and protein expression, the role of post-transla-
tional regulation of proteins in plant adaptation, and the impact
of ectopic gene expression on plant metabolism. In the present
study we discuss the use of DiGE technology to study the pro-
teomics of cold acclimation and photoperiod in peach trees. One-
year old, seedlings of peach were subjected to either a short day
(SD) or (LD) photoperiod at either warm (25 °C) or low (5 °C),

cold-acclimating temperatures for up to 5 weeks. Total soluble
proteins were extracted from bark tissues with a TCA/acetone
precipitation. Extracted proteins were then subjected to differen-
tial in-gel electrophoresis (2-D DiGE), which allows one to mix
and run three samples in the same bidimensional electrophoresis
gel. In the present experiment, five batches of triplicated gels
were used to carry out the total analysis of nine labeled samples
in addition to the internal standard. The extracted soluble pro-
teins were quantified and labeled with fluorescent dyes (Cy2, Cy3
and Cy5) and run on pH 4–7 isoelectrofocusing gels, followed by
a 12% PAGE. Images were analyzed with DeCyder Differential
Analysis Software. Comparisons were made in protein expression
between SD, LD, 5 °C, and 25 °C. Additional comparisons were
made within a treatment at different time points. Computerized
analysis of 2-D gels revealed changes in abundance of more than
13% of the spots (109/824). Among these proteins, some are rela-
ted to PR proteins (e.g. Major allergen Pru av 1), chaperonins
(e.g. HSP70), glycolysis (e.g. 2,3-bisphosphoglycerate-independent
phosphoglycerate mutase), photosynthetic pathway (e.g. PS II
oxygen evolving complex protein 1), amino acid metabolism (e.g.
cysteine synthase), energy metabolism (e.g. ATP synthase beta
unit), cell structure (e.g. beta tubulin), protein metabolism (e.g.
20S proteasome alpha subunit B) or plant hormone response
(e.g. auxin-binding protein 20), etc. A more comprehensive analy-
sis of the results is in progress.
Abstracts
465