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Plant Abiotic Stressfrom signaling to development

2nd meeting of the INPAS*


14-17 May 2009 Tartu Estonia

*International Network of Plant Abiotic Stress (INPAS)


is supported by EU COST action FA0605

Plant Abiotic Stress


from signaling to development
Tartu, Estonia
14-17 May 2009

Scientific Committee:
Antonio F. Tiburcio (Universtat de Barcelona, Spain, chair of INPAS, cost-inpas.org)
Dorothea Bartels (University of Bonn, Germany)
Laszlo Bogre (Royal Holloway University of London, UK)
Pedro Carrasco (Universitat de Valencia, Spain)
Rina Iannacone (Metapontum Agrobios S.S., Italy)
Hannes Kollist (University of Tartu, Estonia)
Csaba Koncz (Max-Planck Institute, Kln, Germany)
lo Niinemets (Estonian University of Life Sciences, Estonia)
Ioanna Stavridou (COST FA0605 Science Officer)
Laszlo Szabados (Biological Research Center, Hungary)
Bernd Wollenweber (Aarhus University)
Aviah Zilberstein (Tel Aviv University, Israel)

Local organizing Committee:


Hannes Kollist
Krisiina Laanemets
Ove Lindgren
Liina Margna
Ebe Merilo
Heino Moldau
Priit Pechter
Irina Puzrjova
Triin Vahisalu
lo Niinemets
Steffen M. Noe
Cover photo by lle Kollist
Design of conference materials by Tanel Vahisalu

Welcome
Plants, unlike animals, cannot move. This requires that adverse changes in their environment are rapidly
recognized, discerned and responded to with adequate reactions. Cold, salinity and drought are among the
major stresses that adversely affect plant growth and productivity. In fact, these abiotic stresses represent
the main cause of crop failure worldwide, dipping average yields for major crops by more than 50%. Since
the world population is increasing at an alarming rate, minimizing these losses is also a major concern for all
nations coping with the increasing food demand.
The overall aim of the conference is to provide a platform for interdisciplinary discussions between scientists
dealing with different aspects of plants abiotic stress from signaling to development. We hope that the
efforts of the speakers will be rewarded by active discussions during the sessions and throughout the whole
conference.
The meeting is organized and supported by the European Cooperation in Science and Technology (COST)
Action FA0605 entitled Signalling control of stress tolerance and production of stress protective compounds
in plants (2007-2011). This is an International Network of Plant Abiotic Stress (INPAS, cost-inpas.org) that
stimulates collaborations between complementary activities of experts working in various fields of stress
biology and is currently composed by 58 partners from 28 participating countries.
We also thank the support from the Estonian Ministry of Education and Research and from our sponsors.
Finally, it is our great pleasure to welcome you to this conference. We hope that these few days in Tartu will
include professional highlights and social events to be remembered for all participants.

Antonio F. Tiburcio (chair of INPAS)


Hannes Kollist (chair of local organizing committee)

Table of Contents
Programme

Abstracts

Oral Presentations

Poster Presentations

39

List of Authors

128

Maps and travel Information

137

Sponsors

139

Programme
DAY 1 (14th of May, Thursday)
17:00-22:00

Registration and poster set up

20:30-22:00

Welcome refreshments

DAY 2 (15th of May, Friday)


8:00-9:00

Registration and poster set up

9:00-9:15

Welcome note by Antonio Tiburcio, chair of INPAS


ABIOTIC STRESS SIGNALLING
Chair: Simon Gilroy

9:15-9:45

lo Niinemets (Estonia): Scaling abiotic stress resistance from molecular mechanisms to


the field

9:45-10:10

Tamas Dalmay (UK): Plant short RNAs and stress

10:10-10:30

Irute Meskiene (Austria): Stress and Plant Developmental control by protein phosphatases

10:30-10:50

Tony Remans (Netherlands): Elucidating the molecular triggers of root developmental


responses to heavy metal stress

10:50-11:10

Teun Munnik (Netherlands): Phospholipid Signaling in Plant Stress & Development

11:10-11:40

Coffee break
Chair: Dorothea Bartels

11:40-12:10

Montserrat Pages (Spain): Drought tolerance in maize, an important crop in agriculture

12:10-12:30

Laszlo Bogre (UK): Signalling pathways regulating the extent and directionality of plant
growth in response to environmental stress factors and during development

12:30-12:50

Dudy Bar-Zvi (Israel): Structure function of tomato ASR1 - a plant specific stress regulated
hydrophilin

12:50-13:10

Natalia Stepanchenko (Russia): Cross-talk between ethylene and abscisic acid signaling
pathways mediates proliferation of Arabidopsis thaliana cultivated cells

13:10-14:30

Lunch
GENETICS AND NATURAL VARIATION
Chair: Laszlo Szabados

14:30-15:00

Matthieu Reymond (Germany): Genetic and molecular basis of plant performance using
natural variation in Arabidopsis thaliana

15:00-15:20

Arnd Heyer (Germany): Mathematical Modelling of Acclimation to low Temperature Reveals Contrasting Strategies in Natural Accessions of Arabidopsis thaliana

15:20-15:40

Ruben Alcazar (Germany): Environmental dependence of genetic epistatic networks


modulating growth, immune responses and speciation processes in Arabidopsis

15:40-16:00

Gad Galili (Israel): Principal transcriptional programs regulating plant metabolism in response to abiotic stresses

16:00-16:30

Coffee break
Chair: lo Niinemets

16:30-17:00

John Doonan (UK): Natural variation in cell growth: roles in adaptation to environmental
stresses

17:00-17:20

Arnould Savoure (France): Opposite stress signalling pathways are present in Arabidopsis
thaliana and in Thellungiella halophila

17:20-17:40

Myriam Olortegui (Netherlands): Natural genetic variation of abiotic multi-stress responses


in Arabidopsis

17:40-18:00

Mary Prathiba (Hungary): A Novel genetic approach for Identifying genes involved in abscisic acid regulation

18:00-19:30

Free time

19:30-22:00

Poster session with beer/wine/buffet

DAY 3 (16th of May, Saturday)


PHYSIOLOGY, BIOCHEMISTRY & METABOLIC PROFILES
Chair: Hannes Kollist
9:00-9:30

Rainer Hedrich (Germany): Guard cells in action

9:30-10:00

Maria Israelsson Nordstrm (Sweden): Keeping up with changing CO2 levels- new
insights from the guard cells

10:00-10:20

Irina Puzrjova (Estonia): Over-expression of ERD15 affects stomatal response to several


atmospheric stimuli

10:20-10:40

Triin Vahisalu (Estonia): Key proteins in governing stomatal response to ozone and induction of reactive oxygen species in plants

10:40-11:00

Kumud Mishra (Czech Republic): Feasibility experiments for developing tools and methodology for non-invasive sensing of drought resistance in tomato transgenics

11:00-11:30

Coffee break
Chair: Antonio Tiburcio

11:30-12:00

Alain Bouchereau (France): A comparative functional analysis of salt and osmotic stress
metabolomes in Thellungiella halophila and Arabidopsis thaliana

12:00-12:20

Teresa Altabella (Spain): Putrescine as signaling molecule involved in the control of stress
responses to cold and drought

12:20-12:40

Claudia Jonak (Austria): High soil salinity: Metabolic adaptation, redox balance and signalling

12:40-13:00

Aviah Zilberstein (Israel): Newly Identified Cytosolic-Mitochondrial Proline-P5C Cycle in


Plants

13:00-14:30

Lunch
ABIOTIC STRESS AND DEVELOPMENT
Chair: Pedro Carrasco

14:30-15:00

Simon Gilroy (USA): Feeling green: mechanotransduction in Arabidopsis growth and


development

15:00-15:20

Seth Davis (Germany): Redox stress is a major component of circadian-clock resetting in


response to dawn

15:20-15:40

Carlos S. Galvan-Ampudia (Netherlands): Osmotic stress-induced signals control root


growth

15:40-16:00

Margarete Mller (Germany): UBP14 is involved in root hair development under phosphate
starvation in Arabidopsis

16:00-16:30

Coffee Break

16:30-18:00

Management Committee Meeting

18:00-20:00

Sightseeing Tour

20:00-03:00

Closing Dinner in Gunpowder Cellar

DAY 4 (17th of May, Sunday)


6:00-12:00

Dismounting posters

6:00-12:00

Checkout from Dorpat hotel

Abstracts

Oral Presentations

SCALING ABIOTIC STRESS RESISTANCE FROM MOLECULAR MECHANISMS TO THE FIELD


lo NIINEMETS
Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1,
51014 Tartu, Estonia
Global climate change implies simultaneous modification of multiple environmental drivers (CO2, water availability and temperature), but the mechanisms of acclimation to interacting environmental stresses are still poorly understood. To understand such responses to interactive environmental variables, structural and physiological controls on photosynthetic and respiratory acclimation and stress-induced volatile organic compound
(volatile plant hormones and volatile compounds produced during oxidative signalling) emissions are studied.
Lab results in various model plants with varying longevity and abiotic stress resistance (wild and mutant genotypes of Arabidopsis, Nicotiana and Populus) are transferred to natural environments by field experiments and
mathematical modelling using scenario analyses to predict plant performance in future climates. The presentation emphasizes the need to study plant abiotic stress resistance in model systems with various levels of
constitutive stress tolerance and potential to acclimate to altered environments.

CENTRAL ROLES OF THE SNRK2 SUBFAMILY OF PROTEIN KINASES IN ABA SIGNALING


Jian-Kang ZHU, Hiroaki FUJII
Department of Botany and Plant Sciences, University of California, Riverside, CA 92521, USA,
jian-kan.zhu@ucr.edu

Abscisic acid (ABA) is an important phytohormone regulating seed dormancy, germination, seedling growth,
and plant transpiration. We report here an Arabidopsis triple mutant that is disrupted in three SnRK2s (SNF1Related Protein Kinase Subfamily 2) and nearly completely insensitive to ABA. These SnRK2s, SnRK2.2,
SnRK2.3 and SnRK2.6 (also known as OST1), are activated by ABA and can phosphorylate the ABF (ABAResponsive Element Binding Factor) family of b-ZIP transcription factors, which are important for the activation
of ABA-responsive genes. Though stomatal regulation of snrk2.6 and seed germination and seedling growth
of the snrk2.2/2.3 double mutant are insensitive to ABA, ABA responses are still present in these mutants, and
the growth and reproduction of these mutants are not very different from those of the wild type. In contrast,
the snrk2.2/2.3/2.6 triple mutant grows poorly and produces few seeds. The triple mutant plants lose water
extremely fast when ambient humidity is not high. Even on 50 M ABA, the triple mutant can germinate and
grow, whereas the most insensitive known mutants cannot develop on 10 M ABA. In-gel kinase assays showed
that all ABA-activated protein kinase activities are eliminated in the triple mutant. Furthermore, the expression
of ABA-induced genes examined is completely blocked in the triple mutant. These results demonstrate that the
protein kinases SnRK2.2, 2.3, and 2.6 have redundant functions, and suggest that ABA signaling is critical for
plant growth and reproduction.

PLANT SHORT RNAs AND STRESS


Tamas DALMAY, Cintia KAWASHIMA
University of East Anglia, Earlham Road, NR4 7TJ, Norwich, United Kingdom, t.dalmay@uea.ac.uk

Plants play an important role in the global sulphur cycle because they assimilate sulphur from the environment
and build it into methionine and cysteine. Several genes of the sulphur assimilation pathway are regulated by
microRNA-395 (miR395) that is itself induced by low-sulphur (-S) environment. Here, we show that the six
Arabidopsis miR395 loci are induced differently. We find that MIR395 loci are expressed in the vascular system
of roots and leaves and root tips. Induction of miR395 by S environment in both roots and leaves suggests
that translocation of miR395 from leaves to roots through the phloem is not necessary for plants growing on
S soil/medium. We also demonstrate that induction of miR395 is controlled by SLIM1, a key transcription
factor in the sulphur assimilation pathway. Unexpectedly, the mRNA level of a miR395 target gene, SULTR2;1,
strongly increases during miR395 induction in root. We show that the spatial expression pattern of MIR395
transcripts in the vascular system does not appear to overlap with the expression pattern previously reported
for SULTR2;1 mRNA. These results illustrate that negative temporal correlation between expression level of a
miRNA and its target gene in a complex tissue cannot be a requirement for target gene validation.

10

STRESS AND PLANT DEVELOPMENTAL CONTROL BY PROTEIN PHOSPHATASES


Irute MESKIENEa, Zahra AYATOLLAHIa, Julija UMBRASAITEa, Tschu-Jie LIUa, Verena
UNTERWURTZACHERa, Alois SCHWEIGHOFERb
a
b

MFPL - Max F.Perutz Laboratories, Dr.Bohrgasse 9/4, A-1030, Vienna, Austria, irute.meskiene@univie.ac.at
Max-Planck-Institute of Molecular Plant Physiology, Vienna, Austria

Abiotic stresses, such as wounding, induce protein phosphorylation cascades, activation of response genes
and mechanisms leading to plant defence.
We found that wound-induced PP2C-type phosphatase, AP2C1 from Arabidopsis controls ET and JA amounts
in plants after wounding (1). PP2C-modified plants demonstrated changes in activation of MAPKs and stress
marker-gene expression by wounding. Consequently with the ET and JA role in plant protection, these plants
demonstrated modulation in stress responses (2). Here, closely related PP2Cs were analyzed in their control
of MAPKs activities or ET production, demonstrating their activities as stress-induced MAPK phosphatases.
MAPKs are also controlled by the dual specificity phosphatases MKPs (3,4,5). Here to follow the cross-talk
between different negative regulators we created and analyzed ap2c1/mkp1 mutant plants.
Our results show that stress-related MAPK activity control by phosphatases is affecting plant development,
where the balance between the kinase activity and the action of the phosphatases is essential to ensure normal
development of plants under environmental conditions. This data contribute in bridging the gap between PP2C
and DSP regulation of stress MAPK activities.
We are supported by Austrian Science Fund (FWF). A.S. is supported by E. Schrdinger and Marie Curie
fellowships. Z.A. and J.U. by fellowships from University of Vienna.
1)
2)
3)
4)
5)

Schweighofer, A. et al. (2007). Plant Cell 19, 2213-2224.


Schweighofer, A. and Meskiene, I. (2008). Molecular BioSystems, 4, 799-803.
Ulm, R., et al. (2002). EMBO J. 21: 6483-6493.
Lee, J.S., and Ellis, B.E. (2007). J Biol Chem 282: 25020-25029.
Lee, J.S., et al. (2008). Plant J.

11

ELUCIDATING THE MOLECULAR TRIGGERS OF ROOT DEVELOPMENTAL RESPONSES TO HEAVY


METAL STRESS
Tony REMANS, Kelly OPDENAKKER, Heidi GIELEN, Els KEUNEN, Marijke JOZEFCZAK, Jaco
VANGRONSVELD, Ann CUYPERS
Hasselt University, Agoralaan D, 3590, Diepenbeek, Belgium, tony.remans@uhasselt.be

Plant root systems show a high developmental plasticity in response to environmental signals. Molecular
parameters for the response to heterogeneous distribution of nutrients in the soil have been identified. For
example, the Arabidopsis thaliana nitrate transporter NRT1.1 in root tips is a component of the nitrate signalling
pathway triggering root colonization of nitrate rich areas [Remans 2006]. Using the same vertical agar plate
system, we aim to identify molecular components of sensing and signalling of heavy metal stress that lead to
interference with the root developmental program. When exposing Arabidopsis thaliana seedlings to Cd, Cu or
Zn in vertical agar plates, we observed a concentration dependent inhibition of primary root growth. In plants
exposed to Zn, also the number and mean length of lateral roots was affected, whereas in plants exposed to
Cd or Cu, the inhibition of primary root growth was accompanied by an increased lateral root length per unit
primary root length. This was due to lateral roots forming closer to the primary root apex, which is similar to
responses to low P (Svistoonoff 2007) and the effect of glutamate on root growth (Walch-Liu 2006). Hence the
response pathway that is activated may be very similar, but the question remains how heavy metal stress is
perceived and how the abiotic stress signal triggers this response pathway. Using a vertical agar plate growth
system and split root experiments, we so far revealed that the inhibition of primary root growth by Cd and Cu is
triggered locally. Furthermore, the metal specific expression patterns that we found for some NADPH oxidase
and lipoxygenase genes may cause metal specific signalling and stress responses. Gene expression will be
correlated with localization of expression of these genes and mutants will be studied for altered responses.
Remans et al 2006 PNAS 50:19206; Walch-Liu et al 2006 Plant&Cell Physiol 47:1045; Svistoonoff et al 2007
Nature Genetics 39:792

12

PHOSPHOLIPID SIGNALING IN PLANT STRESS & DEVELOPMENT


Teun MUNNIK
University of Amsterdam, PI, Science park 904, 1098XH, Amsterdam, The Netherlands, t.munnik@uva.nl

Over the years, we and others have shown that a variety of biotic- and abiotic stresses can activate distinct
lipid signalling pathways, including PLC, PLD and/or certain lipid kinases. Especially, polyphosphoinositides
(PPI) and phosphatidic acid (PA) are emerging as important lipid second messengers. While activations are
fast (sec-min) and lipid responses transient, less is known how these pathways are activated, where in the
cell or plant this takes place, and what the functional significance of the lipid signal is. To start addressing
these questions, new research lines have been initiated, including: i) Arabidopsis KO and OE mutants, ii)
GFP-based lipid biosensors to visualize lipid signalling in vivo, and iii) proteomic approaches to identify and
characterize protein targets for lipid second messengers.
1. Testerink & Munnik (2005) PA - a multifunctional stress-signalling lipid in plants. Trends Plant Sci. 10, 368375.
2. Vermeer et al. (2006) Visualisation of PtdIns3P dynamics in living plant cells. Plant J. 47, 687-700.
3. Bargmann & Munnik (2006) The role of PLD in plant stress responses. Curr. Opin. Plant Biol. 9, 515-522.
4. Van Leeuwen et al. (2007) Visualisation of PtdIns(4,5)P2 in the plasma membrane of tobacco BY-2 cells and
whole Arabidopsis seedlings. Plant J. 52, 1014-1026.
5. Kusano et al. (2008) The Arabidopsis phosphatidylinositol phosphate 5-kinase PIP5K3 is a key regulator for
root hair tip growth. Plant Cell 20, 367-380.
6. Vermeer et al. (2009) Visualisation of PtdIns4P dynamics in living plant cells. Plant J. 57, 356 - 372.
7. Bargmann et al. (2009). Multiple PLDs required for high salinity- and water deficit tolerance in plants. Plant
Cell Physiol. 50, 78-89.
8. Munnik & Testerink (2009). Plant Phospholipid Signalling - in a nutshell. J. Lipid Res. In press

13

DROUGHT TOLERANCE IN MAIZE, AN IMPORTANT CROP IN AGRICULTURE


Montserrat PAGES
Departamento Genetica Molecular de Plantas CRAG (CSIC-IRTA UB) Barcelona, Spain
montse.pages@cid.csic.es

Drought, high salinity or extreme temperatures are responsible for adverse effects on plant growth and seed
production. More precisely, drought and salinity are the major causes of crop loss worldwide. Plants must
adapt to these stress conditions in order to maintain growth and complete their life cycle. This is achieved
by the activation of cascades of molecular networks that lead to physiological, morphological and metabolic
modifications in order to re-establish homeostasis at cellular level.
Tolerance mechanisms allow plants to maintain turgor and volume, to continue metabolism and to maintain
cell membrane stability even at a low water potential. Stress tolerance is thus dependent on long term plant
performance with respect to biomass, yield data and on the degree of recovery from stress.
Cereal seeds embryos can sustain reductions in water content of about 80% at the final stage of seed
maturation, whereas such severe desiccation kills the cells in any other part of the plant. Late embryogenesis
abundant (LEA) proteins are among the most intriguing candidates for biotechnological approaches, since
they do accumulate to elevated extent in the latter stages of seed maturation, and can be induced in vegetative
tissues submitted to water deficit, either associated to drought, salt or cold stresses strongly suggesting that
they are part of the general response of the plant to desiccation.
Because of the complexity of the stress responses several genes will have to be expressed to achieve
biotechnologically useful effects. In this context, we are developing drought tolerant plants by making transgenic
plants with a single regulatory gene (such as a transcription factor) which in turn regulate the expression of
downstream genes involved in the stress response. Understanding of stress adaptive mechanisms in plants
can bring important information in the long term purpose of crop improvement.

14

SIGNALLING PATHWAYS REGULATING THE EXTENT AND DIRECTIONALITY OF PLANT GROWTH IN


RESPONSE TO ENVIRONMENTAL STRESS FACTORS AND DURING DEVELOPMENT
Laszlo BOGREa, Pavla BINAROVAb, Robert DOCZIa, Rossana HENRIQUESc, Alex JONESd, Zoltan
MAGYARa, Christine ZALEJSKIa
Royal Holloway, University of London, Egham Hill, TW20 0EX, Egham, United Kingdom, l.bogre@rhul.ac.uk
Institut of Microbiology, Vdesk, Czech Republic
c
Rockefeller University, New York, United States of America
d
Sainsburys Laboratory, Norwich, United Kingdom
a

Plants continuously and sensitively assess their environment and adapt their physiology, growth and development
accordingly. A large number of signalling mechanisms has been mapped out in recent years, and a large
volume of fragmented data has been attained on the processes these signalling pathways might regulate.
A perhaps surprising emerging picture is that developmental and environmental stress signals might share
common signalling mechanisms and impinge on common cellular mechanisms, such as the regulation of auxin
transport, protein translation and cell growth, cell proliferation. The plasma membrane - cortical microtubule
compartment might provide a platform for initial signalling events for both environmental and stress signals.
The talk will focus on the 3-phosphoinositide-dependent protein kinase (PDK1) and downstream signalling
events to it, including MAP kinase pathways, and will present our recent results how these pathways regulate
auxin transport, protein synthesis and proliferation to modulate the directionality and extent of plant growth in
response to stress and developmental signals.

15

STRUCTURE FUNCTION OF TOMATO ASR1 - A PLANT SPECIFIC STRESS REGULATED


HYDROPHILIN
Dudy BAR-ZVI
Ben-Gurion University, Rager Blvd, 84105, Beer-Sheva, Israel, barzvi@bgu.ac.il

Hydrophilins is a group of proteins defined by high hydrophilicity index and glycine content (> 1.0 and > 6%,
respectively), found mainly in plants, bacteria and yeast. They represent small fraction of the genome and
suggested to be a predictor for responsiveness to hyperosmosis since the steady state levels of the transcripts
of most hydrophilins is elevated in response to water deficit. The largest group of plant hydrophilins is Late
Embryogenesis Abundant (LEA) proteins. Hydrophilins are believed to protect macromolecules and biological
structures against stress-induced damages. The biological activity of most hydrophilins is not understood.
Tomato ASR1 (SlASR1) is a plant specific protein that meets with the hydrophilin definition. SlASR1 levels are
increased by salt stress, water stress and ABA. Overexpressing SlASR1 increases salt tolerance in transgenic
tobacco and Arabidopsis plants. The protein is localized both in the nucleus and in the cytoplasm. SlASR1
possesses a zinc-dependent sequence specific DNA-binding activity. DNA and zinc binding domains were
mapped. SlASR1 was predicted to be mostly unfolded. Structural studies using an array of biophysical methods
showed that the cytosolic form of SlASR1 is mainly unfolded monomeric. The protein readily assumes high
levels of structure and dimerizes upon the binding of zinc ions, or drying the protein solution, suggesting that
nuclear DNA-bound form of SlASR1, as well as ASR1 in pollen and desiccated seeds is an ordered protein.
The unfolded form of SlASR1, possess a chaperone-like activity that is enhanced in the presence of osmolytes.
We thus suggest that SlASR1 is a dual functionality protein.
References: Kalifa et al (2004) Biochem J. 381, 373; Kalifa et al (2004) Plant Cell Environ. 27, 1459; Rom et
al (2006) Biochimie 88, 621; Goldgur et al (2007) Plant Physiol. 143, 617; Konrad and Bar-Zvi (2008) Planta
227, 1213; Shkolnik and Bar-Zvi (2008) Plant Biotechnol. J. 6, 368.

16

CROSS-TALK BETWEEN ETHYLENE AND ABSCISIC ACID SIGNALING PATHWAYS MEDIATES


PROLIFERATION OF ARABIDOPSIS THALIANA CULTIVATED CELLS
Natalia STEPANCHENKO, Galina NOVIKOVA, Alexander NOSOV, Igor MOSHKOV,
Timiryazev Institute of Plant Physiology Russian Academy of Sciences, Botanicheskaya, 35, 127276,
Moscow, Russia, stepashka.nt@gmail.com

In order to study a mutual influence of ethylene and ABA at the cellular level, the cell suspension culture of
wild type of A. thaliana (Col-0) and ethylene insensitive mutants etr1-1 and ctr1-1 have been established. The
preservation of mutated genes in cultivated cell suspensions was confirmed. Having optimized conditions
and media composition for the cultivated cells, we assured stable growth cell suspensions. Cells of all strains
have similar morphology, clusters consist of the typical elongated cell files. Since ethylene biosynthesis is
occurred that can affect the rate of cell proliferation and endoreduplication of nuclear DNA, the heterogeneity
of nuclear DNA contents have been investigated and mixoploidy of cultivated cells was revealed. It is generally
accepted that a regulation of eukaryotic cell cycle is due to activity of Ser/Thr-protein kinases. We have studied
an effect of exogenous ABA on the pattern of phosphorylated proteins in Col-0, etr1-1 and ctr1-1 cells upon
the conditions chosen to avoid changes in the ethylene biosynthesis. Proteomic approach was exploited and
provided intriguing data. According to primary data, in cells in the middle of the exponential phase of growth
when the relative division rate was declined as well as at the stationary phase of growth, characterized by
cessation of cell division, potential candidates involved in ethylene and ABA signal transduction might be
AtMPK3, AtMPK5, AtMPK1, Ca2+-dependent protein kinases and Ser/Thr-phosphatase PP2A. The possible
role of MAPK has been confirmed. It is likely that ABA blocks cells at the G1/S transition, whereas ethylene
stimulates both the endoreduplication and the transition from G1 to S-phase in cell cycle. It is important to
relate changes in the level of phytohormone-regulated protein phosphorylation and the phase of cell cycle.
This issue would be discussed in the presentation.
The work is supported by RFBR, grant 08-04-000643.

17

GENETIC AND MOLECULAR BASIS OF PLANT PERFORMANCE USING NATURAL VARIATION IN


ARABIDOPSIS THALIANA
Matthieu REYMOND, Maarten KOORNNEEF
Max Planck Institute for Plant Breeding Research, Department of Plant Breeding and Genetic, group leader,
Carl-Von-Linn-Weg 10, 50829 Kln, Germany, reymond@mpiz-koeln.mpg.de

Arabidopsis thaliana accessions have been collected from various and contrasting environments in the
northern hemisphere. Genetic diversity present among these accessions is assumed to reflect adaptation to
local environments. This genetic diversity also leads to phenotypic variation in many traits. Among these traits,
growth (from the cell level to the whole plant level) and growth responses to environmental factors (biotic
and abiotic factors) are segregating between accessions of Arabidopsis thaliana. The genetic and molecular
basis of such traits can be revealed by using natural variation and by detecting QTL (for Quantitative Trait
Locus). Understanding the effect of this genetic variation on plant performance under different environmental
scenarios is also relevant for plant breeding because it involves traits determining yield and yield stability
in crops. Examples of QTL involved in the variation of growth and its responses to environment (QTLxE)
using Arabidopsis thaliana natural variation will be presented. In addition, strategies to reveal the genetic and
molecular basis of detected QTL will be proposed.

18

MATHEMATICAL MODELLING OF ACCLIMATION TO LOW TEMPERATURE REVEALS CONTRASTING


STRATEGIES IN NATURAL ACCESSIONS OF ARABIDOPSIS THALIANA
Arnd HEYER, Thomas NAEGELE, Sabine FRANA
University of Stuttgart, Pfaffenwaldring 57, 70569, Stuttgart, Germany, arnd.heyer@bio.uni-stuttgart.de

The term Systems Biology is loosely defined and used by many researchers to describe data mining
approaches dealing with large data sets from so-called omics experiments. However, derived from systems
theory, the concept refers to mathematical modelling of complex sets of relationships composing a system.
We have applied mathematical modelling using sets of differential equations to emulate plant primary
metabolism over diurnal cycles to reveal the account of starch turnover, soluble sugar pools and long distance
transport of assimilates as sinks for CO2 taken up during a light/dark cycle.
We used exposure to low temperature to disturb metabolic homeostasis and followed system responses in
natural accessions of Arabidopsis that show large variation in the capacity for acclimation to low temperatures.
Comparing metabolite dynamics in various accessions, we computed trajectories for metabolic adjustment
in the cold. Parameter sets for the simulations could account for sets of accessions including cold sensitive,
moderately tolerant and tolerant genotypes. However, simulations for the Russian accession, Rschev, and the
Scandinavian, Tenela, did not yield consistent parameter sets, pointing to differences in the trajectories. The
discrepancies result mainly from variation in hexose accumulation during a 14 day exposure to 4 C. Because
hexoses as well as the trisaccharide raffinose are produced from sucrose, and sucrose levels show similar
dynamics, differential changes in enzyme activities must be claimed. This method of analysing metabolic data
demonstrates that metabolite levels cannot be interpreted as isolated parameters but must be included in
dynamic models, which cannot be surveyed intuitively.

19

ENVIRONMENTAL DEPENDENCE OF GENETIC EPISTATIC NETWORKS MODULATING GROWTH,


IMMUNE RESPONSES AND SPECIATION PROCESSES IN ARABIDOPSIS
Rubn ALCAZAR
Max-Planck-Institut fr Zchtungsforschung (MPIZ), Carl-von-Linn-Weg 10, 50829, Kln, Germany,
alcazar@mpiz-koeln.mpg.de

Growth of plants is largely influenced by environmental cues. Although environmental factors have traditionally
been classified as abiotic or biotic, evidence point to extensive crosstalk between these stress signaling
pathways. Here we have used natural variation as source of genetic diversity in Arabidopsis to unravel a novel
epistatic interaction involving 2-3 loci which modulates growth in response to mild changes of temperature
(20 C versus 14 C). Genes underlying one of the interacting loci map to a cluster of RPP1-like TIR-NBLRR (toll/interleukin-1 receptor-nucleotide binding leucine rich repeat), homologs of which are known to
recognize specific pathogen effectors and trigger immune responses. We establish that growth modulation by
temperature is driven by salicylic acid -dependent pathways and this also affects pathogen resistance traits.
The nature of the epistatic interaction and its effects on fitness conform the Dobzhansky-Muller model of genetic
incompatibilities, involved in reproductive isolation and gene-flow barriers between species. As conclusion we
have identified a genetic epistatic interaction modulating growth, immune responses and potential evolutionary
speciation processes in response to mild environmental changes.

20

PRINCIPAL TRANSCRIPTIONAL PROGRAMS REGULATING PLANT METABOLISM IN RESPONSE TO


ABIOTIC STRESSES
Gad GALILI, Hadar LESS, Ruthie ANGELOVICI
The Weizmann Institute of Science, Herzl, 76100, Rehovot, Israel, gad.galili@weizmann.ac.il

We have recently developed a new bioinformatics tool adapted for: (i) analyzing the response of Arabidopsis
thaliana genes controlling plant metabolism to abiotic stresses; and (ii) identifying novel regulatory genes
controlling the operation of the stress-associated metabolism. Using this new approach to analyze publicly
available microarray datasets, we have recently identified novel expression coordination patterns between
gene modules controlling the operation to central amino acid metabolic networks to various abiotic stresses
(1). We have also further developed this bioinformatics tool to elucidate the transcriptional response of genes
encoding the entire set of Arabidopsis metabolic enzymes to the various stress conditions. We will present
the results of this study, which elucidate several novel regulatory principals of plant metabolism in response to
stress conditions.
1. Less, H and Galili, G. (2009) Coordinations between gene modules control the operation of plant amino acid
metabolic networks. BMC System Biology: 3:14: 1-18

21

NATURAL VARIATION IN CELL GROWTH: ROLES IN ADAPTATION TO ENVIRONMENTAL STRESSES?


John DOONAN
Department of Cell & Developmental Biology, John Innes Centre, Norwich Research Park,
Colney, Norwich NR4 7UH, UK, john.doonan@bbsrc.ac.uk

Plant growth depends on essentially two cellular processes, cell division and cell expansion. Thus, the ultimate
size of organs such as leaves and petals are the product of the number of cells in the mature organ and the
average size of those cells. As a first step towards understanding how the environment influences cellular
growth processes, we surveyed geographically diverse accessions of the model species, Arabidopsis, for
variation in cell number and variation in cell size, and how these cellular parameters were related to organ
size.
Significant variation in both cellular traits was observed and led us to the conclusion that, unlike the case in
animals where variation in cell number is thought to explain the majority of variation in organ size, variation in
plant organ size depends on both processes. Moreover, we find that the relative contribution to organ size is
related to the geographic origins of the accession. Possible adaptive explanations will be discussed.

22

OPPOSITE STRESS SIGNALLING PATHWAYS ARE PRESENT IN ARABIDOPSIS THALIANA AND IN


THELLUNGIELLA HALOPHILA
Arnould SAVOURa, Kilani BEN REJEBb, Mohamed Ali GHARSb, Luc RICHARDa, Anne-Sophie LEPRINCEa,
Delphine LEFEBVRE-DE VOSa, Marianne BORDENAVEa, Chedly ABDELLYc,
UPMC, 3 rue Galile, 94200, Ivry-sur-Seine, France, arnould.savoure@upmc.fr
CBBC/UPMC, Tunisia/France
c
CBBC, Tunisia
a

Water stress is one of the major environmental constraints that affect plant growth and crop productivity. Plants
respond and adapt to water stress by the synthesis of osmolytes. Among them, proline is one of the most
frequently accumulated compounds in plants. Recently, we have shown that lipid signalling pathways including
phospholipase C (PLC) and D (PLD) are involved in the tight regulation of proline metabolism in Arabidopsis
thaliana (Thiery et al., 2004, J Biol Chem 279: 14812-14818; Parre et al., 2007, Plant Physiol 144: 503-512).
Moreover calcium has been identified as a molecular switch to trigger proline accumulation in response to salt
stress. The Arabidopsis relative Thellungiella halophila, considered as an extremophile, is characterized by
an up regulation of stress genes. In addition, a high proline accumulation is observed in response to abiotic
stress conditions but also in non-stress ones in this plant (Ghars et al., 2008, J Plant Physiol 165: 588-599).
Therefore we investigated the regulation of key signalling regulators upon water stress in T. halophila. We
especially assessed the role of lipid signalling pathways and the reactive oxygen species in the tight control of
proline metabolism. Our results clearly demonstrated the involvement of positive and negative regulators in the
regulation of proline metabolism upon water stresses. The remarkable stress tolerance of T. halophila may be
partially explained by the opposite regulation of stress signalling pathways between the two species.

23

NATURAL GENETIC VARIATION OF ABIOTIC MULTI-STRESS RESPONSES IN ARABIDOPSIS


Myriam OLORTEGUI, Joost KEURENTJES, Dick VREUGDENHIL, Harro BOUWMEESTER
Laboratory of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD, Wageningen, The
Netherlands, myriam.olortegui@wur.nl

Plants, like most living organisms, grow subject to different kinds of biotic and abiotic stresses. The effects
of a combination of stress-factors on crops might be more severe than the effects of the same stress, but
applied separately. Biotic and abiotic stresses affect plants at the genetic and physiological levels. Plants
respond variably to these stresses depending on their genetic background and on the gene-environment
interaction. Elucidating the natural variation in responses and adaptations to the combination of two or more
simultaneous biotic and/or abiotic stresses in the model plant Arabidopsis will help understanding, predicting
and manipulating stress responses in crops. Extensive research on the effects of different single-stress factors
to plants has been carried out. However, the bioinformatics tools for analyzing the complex data generated
by plant systems biology experiments under multiple stressing conditions are being developed only recently.
The aim of this project is to identify and generate information about Arabidopsis natural variation responses to
selected abiotic stresses combinations at the genomic, transcriptomic, metabolomic and proteomic levels, and
to identify the level and functions of stress regulatory networks and crosstalk.
The Arabidopsis accessions Landsberg erecta, Cape verde island and Antwerpen, as well as a triple cross
population (300 lines) derived from those accessions, were subjected to combinations of salt and temperature
stresses. Growth was measured daily (accessions) or every two days (triple cross population) using imaging
software. QTL analysis of the data will be finished in the coming weeks.

24

A NOVEL GENETIC APPROACH FOR IDENTIFYING GENES INVOLVED IN ABSCISIC ACID


REGULATION
Mary PRATHIBA, Laszlo SZABADOS
Hungarian Academy of Sciences, Szeged, Hungary, maryprat@brc.hu

Abscisic acid is the main stress response hormone in higher plants. In the past few decades many stress
regulatory factors were identified which are involved in ABA dependent stress regulation. In order to understand
the complicated regulatory web of ABA signaling the Controlled cDNA Overexpression System have been
developed (COS, Papdi et al., 2008). We have transformed the Arabidopsis Col-0 wild type plants with the
COS library and screened progenies of infiltrated plants for ABA insensitivity in the presence and absence of
estradiol in germination assays. Screening one million seeds (aproximately 25,000 transformed seeds), of T1
generation resulted 156 plants, which were selected based on their germination capacity on high concentration
ABA supplemented media. By testing of T2 generation, estradiol dependent ABA insensitivity was confirmed
in 29 lines. Estradiol dependent ABA insenstitive germination was most notable in A26 and A44 lines, which
were able to germinate in the presence of 5 M ABA, which otherwise completely inhibited the germination
of wild type seeds. Insertions were identified in both lines and corresponded to full-length cDNA encoding
the small heat-shock protein HSP17.6A-cII (A26) and a previously unknown zinc-finger domain containing
transcription factor protein (A44). GFP fusion and HA-tagging experiments showed nuclear localization of the
A44-derived transcription factor. While constitutive overexpression of this transcription factor reduced fertility,
insertion mutants, where transcription of the corresponding gene was abolished, were hypersensitive to ABA.
Our results show, that the COS system is suitable for the identification of novel ABA regulatory factors.

25

GUARD CELLS IN ACTION


Rainer HEDRICH
Julius-von-Sachs Institute of Biosciences, Molecular Plant Physiology and Biophysics, University of
Wrzburg, Germany, hedrich@botanik.uni-wuerzburg.de

Stomata of higher plants close in response to darkness, draught and CO2. This process is induced by the
activation of guard cell anion channels. By mutant screens recently a putative guard cell anion channel or
essential component thereof named SLAC1 was identified. Since SLAC1 has not been functionally expressed
yet, the central question how stomatal closure-related signaling components led to anion channel activation
remains still unanswered. In a split YFP-based protein-protein interaction screen with SLAC1, we identified a
protein kinase and phosphatase within the ABA transduction pathway. Upon coexpression of slac1 with the
protein kinase SLAC1-related anion currents similar to those observed in guard cells appeared. SLAC1 was
characterized by a voltage-independent, anion-selective channel.
Both protein kinase and phosphatase appear are essential for ABA-triggered stomatal closure. Upon
coexpression of the phosphatase with slac1 in oocytes, SLAC1 remained electrically silent, while relative
the kinase/phosphatase dose seem to control the activation state of SLAC1. At the meeting the regulation of
stomatal movement and SLAC1 activation by a calcium-dependent and calcium-independently pathway will
be presented.

26

KEEPING UP WITH CHANGING CO2 LEVELS- NEW INSIGHTS FROM THE GUARD CELLS
Maria ISRAELSSON NORDSTRMa, Honghong HUb, Aurelien BOISSON-DERNIERb, Josef KUHNb, Maik
BOEHMERb, Jan GODOSKIb, Julian I. SCHROEDERb
Stockholm University, Dept of Botany, Stockholm University, SE-10691, Stockholm, Sweden,
israelsson@botan.su.se
b
University of California, San Diego, United States of America

Guard cells form adjustable stomatal pores in the epidermis of plants. Through stomata, carbon dioxide is
taken up for photosynthetic carbon fixation and water is lost through transpiration. The response of stomata to
carbon dioxide is subject to the relative shift of [CO2], where an increase promotes closing and decreased CO2
concentrations induce stomatal opening. The global rise in CO2 reduces stomatal apertures and thereby plants
water use efficiency and the gas exchange between plants and the atmosphere. However, the mechanisms
that mediate CO2 sensing remain unknown. Whether the CO2 signal is perceived and transduced within the
guard cells themselves and/or via the neighbouring mesophyll cells is not entirely known. The involvement of
photosynthesis in CO2-induced stomatal signal transduction is also a matter of present debate. In this study,
we present data describing a gene that function early in CO2 signaling. Our results provide genetic evidence
into the questions whether this CO2 signaling pathway requires photosynthesis and in which cell types CO2
responsiveness occurs.

27

OVER-EXPRESSION OF ERD15 AFFECTS STOMATAL RESPONSE TO SEVERAL ATMOSPHERIC


STIMULI
Irina PUZRJOVA, Heino MOLDAU, Hannes KOLLIST
University of Tartu, Institute of Technology, Nooruse 1, 50411, Tartu, Estonia, irina.puzorjova@ut.ee

EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) is a small acidic protein rapidly induced by various
abiotic stress stimuli in Arabidopsis. It has been shown that over-expression of ERD15 reduced plants ABA
insensitivity and drought and freezing tolerance whereas RNAi silencing had an opposite effect (Kariola et al.
2006). These phenotypes raised the question of ERD15 involvement in plant stomatal regulation.
Here we have studied stomatal responses to CO2, air humidity, ozone and light/dark transitions in plants where
ERD15 level is modulated by over-expression (ERD15ovx) and silencing by RNAi (ERD15RNAi).
Intact A. thaliana plants were used and stomatal conductance patterns were calculated from whole-rosette water
vapour exchange recordings. ERD15ovx had constitutively more open stomata compared to ERD15RNAi and
vector control and stomatal closure induced by raising CO2 concentration from 400 ppm to 800 ppm for 30 min
was delayed in ERD15ovx plants. Reduction of relative air humidity from ~70% to ~30% for 30 min decreased
stomatal conductance in ERD15RNAi, ERD15ovx and vector control plants at similar rates within the first 10
min. Thereafter the conductance in ERD15ovx reached a plateau, whilst conductance in ERD15RNAi and
vector control continued to decrease. Ozone-induced (~350 ppb for 3 min) rapid transient stomatal closure was
also delayed in ERD15ovx, whereas ERD15RNAi had a similar response as vector control. Stomatal response
to 2-hour light deprivation on midday was extremely weak in ERD15ovx. Surprisingly, ERD15ovx had strongly
suppressed stomatal response also after the onset of the normal dark period. Impaired darkness-induced
stomatal closure together with constitutively more open stomata phenotype of ERD15ovx plants might provide
an explanation for the drought sensitivity of ERD15ovx plants.
We conclude that ERD15 is a multi-functional key node in stomatal signalling network that controls responses
to a variety of atmospheric factors vital for plant survival.
Kariola T, Brader G, Helenius E, Li J, Heino P and Palva E.T. (2006) EARLY RESPONSIVE TO DEHYDRATION
15, a Negative Regulator of Abscisic Acid Responses in Arabidopsis. Plant Physiology 142:1559-1573.

28

KEY PROTEINS IN GOVERNING STOMATAL RESPONSE TO OZONE AND INDUCTION OF REACTIVE


OXYGEN SPECIES IN PLANTS
Triin VAHISALUa, Irina PUZRJOVAa, Heino MOLDAUa, Jaakko KANGASJRVIb, Hannes KOLLISTa
a
b

University of Tartu, Nooruse 1, 50411, Tartu, Estonia, triink@ut.ee


University of Helsinki, Helsinki, Finland

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow atmospheric
carbon dioxide influx into leaves in exchange for transpirational water loss to the atmosphere. Stomata also
control the entry of ozone (O3) the major air pollutant. However, information about genetic/biochemical
mechanisms regulating plant stomatal O3-sensing is scarce. Detailed analysis of stomatal responses revealed
that O3 induces a Rapid Transient Stomatal Closure (RTSC) within 6-10 min from the start of exposure and a
subsequent reopening within further 30 min. O3 is needed just for the induction of RTSC, as a single 50 sec O3
pulse induces the process. Repeated O3 pulses during the reopening period did not show any further effect,
indicating that the process is well programmed. In order to study which proteins are involved in the regulation
of plant stomatal responses to O3 we have analyzed RTSC in 40 different Arabidopsis mutants related to
stomatal functioning. Interestingly, RTSC is absent in guard cell signaling mutants abi1, abi2, ost1 and in
slac1. This indicates that these proteins, namely, ABI1, ABI2 protein phosphatases, OST1 kinase and SLAC1,
a protein associated with the guard cell plasma membrane anion channel are all functionally essential for plant
stomatal closure in response to O3.
The involvement of reactive oxygen species (ROS) in plant stomatal signaling has been previously shown. To
address whether the O3-induced RTSC is associated with generation of the intrinsic burst of ROS in guard cells
we have used fluorescence dyes and confocal microscopy to visualize early ROS production. Strong ROS
production was detected 11-12 min after the onset of O3. ROS production patterns were similar in wild-type
plants, slac1, srk2e/ost1, as well as in NADPH oxidase catalytic subunit gene mutant atrbohD/F indicating that
the key proteins blocking RTSC in slac1 and srk2e/ost1 are not necessarily related to O3-induced guard cell
ROS production.

29

FEASIBILITY EXPERIMENTS FOR DEVELOPING TOOLS AND METHODOLOGY FOR NON-INVASIVE


SENSING OF DROUGHT RESISTANCE IN TOMATO TRANSGENICS
Kumud MISHRAa, Rina IANNACONEb, Anamika MISHRAa, Angelo PETROZZAb, Giovanna LA VECCHIAb,
Martin TRTILEKc, Ladislav NEDBALa, Francesco CELLINIb
Institute of Systems Biology & Ecology, ASCR; Institute of Physical Biology, Univ. of South Bohemia, 136
Zamek, 37 333, Nove Hrady, Czech Republic, mishra@greentech.cz
b
Metapontum Agrobios S.S. Jonica 106, 75010 Metaponto, Italy
c
Photon System Instruments, Hogrova 20, 61200 Brno, Czech Republic

Genetically engineered crops that can tolerate drought stress and are expected to improve the quality as well
as yield of the major crops are being developed and studied in various laboratories across the world. However,
current methods to study the characteristics of genetically modified mutants/transgenic plants and their stress
tolerance capacity are based on biochemical methods that are invasive, laborious and time consuming. There
is a need to develop reliable non-invasive methods that can be used as a screening tool and can be able to
determine resistance capacity of genetically engineered crops.
We have conducted two experimental campaigns on greenhouse plants at Agrobios, (Italy) to test the feasibility
of chlorophyll fluorescence imaging for developing tools and methodology for non-invasive sensing of drought
stress in tomato transgenic over-expressing the transcription factor ATHB7. We induced drought to the WT
and transgenic tomato plants for 18 days and daily measured time resolved chlorophyll fluorescence images.
In parallel, we measured leaf and stem water potential, leaf dry weight and the foliar pigments, ABA, proline,
chlorophylls and carotenoids. This experiment confirms earlier results that transgenic tomato over-expressing
transcription factor, ATHB7 is highly resistant against drought stress and shows a high level of recover after rewatering. Initial results reveal that chlorophyll fluorescence imaging is promising and its classical parameters
can be used as a proxy of drought stress. In this meeting, a detailed report of the experimental trials will be
presented.

30

A COMPARATIVE FUNCTIONAL ANALYSIS OF SALT AND OSMOTIC STRESS METABOLOME IN


THELLUNGIELLA HALOPHILA AND ARABIDOPSIS THALIANA
Alain BOUCHEREAUa, Raphal LUGANa, Marie-Franoise NIOGRETa, Laurent LEPORTa, Jean-Paul
GUEGANc, Franois Robert LARHERa, Arnould SAVOUREd, Joachim KOPKAb
INRA-Agrocampus Ouest-Universit de Rennes 1, UMR 118, Amlioration et Biotechnologies Vgtales,
35653 Le Rheu cedex, France, alain.bouchereau@univ-rennes1.fr
c
Ecole Nationale Suprieure de Chimie de Rennes, Campus de Beaulieu, F-35042 Rennes Cedex, France
b
Max Planck Institute of Molecular Plant Physiology, Department Prof. Willmitzer, Am Muelenberg, D-14476
Postdam-Golm, Germany
d
CNRS-Universit Pierre et Marie Curie, UMR 7180, 4, place Jussieu, F-75005 Paris, France
a

Thellungiella halophila (Th.), a Brassicaceae growing naturally in harsh environments, is very tolerant to cold
and high salinity (http://www.thellungiella.org/). Closely related to Arabidopsis thaliana (At.), this species is
arising as the Arabidopsis relative plant model system in the field of abiotic stress tolerance studies. The
present work is devoted to the functional analysis of At. and Th. shoot metabolomes under control, saline
and osmotic stress conditions, assuming that changes in levels of primary metabolites broadly participate in
processes responsible for stress tolerance, such as osmoregulation and osmoprotection.
1
H-NMR fingerprinting and non-targeted GC-MS metabolomics depicted very few qualitative differences
between both species, mainly limited to secondary metabolites. Conversely very important quantitative
differences were found in terms of primary metabolite levels, questioning the involvement of basic metabolite
accumulation in stress tolerance. Subsequently, quantitative profiling of organic and mineral solutes allowed
a nearly comprehensive calculation of osmotic balances in shoots of both species. A relative stability of total
solutes contents was observed since accumulation of salt and organic compounds were compensated by a
roughly equivalent reduction of other mineral solutes. In this respect, Th. shoots displayed constitutively lower
water content than At. and showed a much greater ability to desiccate under stress. Since both organic solutes
accumulation and low water content may challenge optimal cell functioning, the impact of intrinsic properties of
organic compounds was studied. Various physicochemical properties of samples metabolome were calculated
as those of a unique virtual metabolic pool through the weighted average of all the metabolites quantified.
Significant differences were observed between Th. and At. metabolome, related to their global solubility in
water, carbon reduction level, molecular weight or free energy of formation. Osmotic stress was also found
to change those properties in both species, Th. metabolome reinforcing its constitutive properties while At.
metabolome shifted toward a higher compatibility, comparable to that of Th. but in a less efficient way.

31

PUTRESCINE AS SIGNALING MOLECULE INVOLVED IN THE CONTROL OF STRESS RESPONSES TO


COLD AND DROUGHT
Teresa ALTABELLAb, Ruben ALCAZARa, Juan CUEVASb, Xavier ZARZAb, Joan PLANASb, Triambak
SAXENAb, Csaba KONCZa, Antonio F. TIBURCIOb,
a
b

Max Planck Institute for Plant Breeding Research, Kln, Germany


Universitat de Barcelona, Diagonal 643, 08028, BARCELONA, Spain, taltabella@ub.edu

The polyamines (PAs) putrescine (Put), spermidine (Spd) and spermine (Spm) are low molecular organic
cations present in all eukaryotic cells. Put accumulation under abiotic stress conditions has been traditionally
correlated with changes in arginine decarboxylase (ADC) activity in several plant systems.
In Arabidopsis, a model plant missing a functional ornithine decarboxylase pathway, most of the key genes
involved in polyamine biosynthesis are duplicated. This gene redundancy has been related to the involvement
of certain gene isoforms in the response to specific environmental stimuli. We have shown that drought stress
induces ADC2 expression while transcript levels for ADC1 remain constant. In contrast, in response to cold,
although we observed induction of both genes encoding ADC, the transcript levels of ADC1 were higher.
We have also studied the PA profiles of Arabidopsis plants challenged with cold or drought stress. The diamine
Put was found to increase in plants subjected to both abiotic stresses, while no increase was detected in the
levels of Spd and Spm. Despite these and other published data, the functions of Put and other PAs in the
regulation of abiotic stress responses are unknown. To obtain novel insights into these questions, we have
studied the response to drought and cold of Arabidopsis plants with altered Put levels. By using Arabidopsis
mutants defective in Put biosynthesis (adc1, adc2) and transgenic Arabidopsis lines over-expressing the
homologous ADC2 or ADC1 genes, we have shown that the accumulation of Put is essential for proper coldacclimation and survival at freezing temperatures. Over-accumulation of Put also induces drought tolerance in
Arabidopsis, but this effect is only achieved by over-expression of ADC2. The possible mechanisms involved
in these responses, as well as possible crosstalk with other plant growth regulators, will be discussed.

32

HIGH SOIL SALINITY: METABOLIC ADAPTATION, REDOX BALANCE AND SIGNALLING


Claudia JONAK
Gregor Mendel Institute, Dr. Bohr-Gasse 3, 1030, Vienna, Austria, claudia.jonak@gmi.oeaw.ac.at

High soil salinity affects almost all aspects of plant physiology and metabolism. Plant metabolism is highly
flexible and adjusts under conditions of salt stress. ABA is a central hormonal signal that regulates several
aspects of stress response. Regarding metabolic adjustment to high salinity conditions, ABA appears to trigger
stress-induced starch mobilisation while salt-specific signals might be necessary for a complete metabolic
adjustment to high salt concentrations.
Plant responses to environmental constraints are delicately coordinated by integrated signalling pathways that
ultimately result in tolerance or sensitivity. Protein kinases constitute important regulators in these circuits.
For example, MsK4 is a positive regulator of high salt tolerance by adjusting carbohydrate metabolism in
response to environmental stress. In a screen for novel protein kinases important for stress tolerance, we
identified ASK5 as a modulator of redox homeostasis during stress. ask5 knock-out plants are more sensitive
to high soil salinity, whereas plants overexpressing ASK5 display an enhanced tolerance. Consistent with
a positive regulatory role, ASK5 in vivo kinase activity is rapidly induced by high soil salinity. ASK5 activity
mutants have a modified cellular redox state. Various metabolic enzymes have been shown to be regulated
by phosphorylation. Glucose-6-phosphate dehydrogenase (G6PDH) is part of an inducible mechanism of
eukaryotic cells to respond to oxidative stress. It is the rate-limiting enzyme of the oxidative pentose phosphate
pathway delivering reducing equivalents. In detailed molecular and biochemical analyses, we elucidated a
novel mechanism of G6PDH regulation by ASK5-mediated phosphorylation pointing towards an important role
for ASK5 in safeguarding cellular redox balance under environmental stress conditions.

33

NEWLY IDENTIFIED CYTOSOLIC-MITOCHONDRIAL PROLINE-P5C CYCLE IN PLANTS


Aviah ZILBERSTEINa, Gad MILLERb, Arik HONIGa, Hanan STEINa, Ron MITTLERb
a
b

Tel Aviv University, Ramat Aviv, 69978, Tel Aviv, Israel, aviah@post.tau.ac.il
University of Nevada, Nevada, United States of America

Proline (Pro) two-step-oxidation in all eukaryotes is performed in the inner mitochondrial membrane by the
consecutive action of proline dehydrogenase (ProDH) that produces Delta1-pyrroline-5-carboxylate (P5C) and
P5C dehydrogenase (P5CDH) that oxidizes P5C to glutamate (Glu). This catabolic route is silenced in plants
during osmotic stresses, allowing free Pro accumulation. Our results show that overexpression of an ectopic
ProDH in tobacco and Arabidopsis or impairment of P5C oxidation in the Arabidopsis p5cdh mutant did not
change the cellular Pro to P5C ratio under ambient and osmotic stress conditions, indicating that P5C excess
was reduced back to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH and P5C reductase,
exists in animal cells and now unraveled in plants. As a part of the cycle operation Pro oxidation by the ProDHFAD-linked complex delivers electrons to the mitochondrial electron transport chain. Hyper-activity of the cycle,
e.g. when an excess of exogenous L-Pro is provided, generates mitochondrial ROS by delivering electrons
to O2, as was evident by specific MitoSox staining of mitochondrial superoxide ions. The stain has been used
to specifically identify mitochondrial ROS in animal cells and is here examined in plants. In the absence of
P5CDH activity, Pro excess led to higher ROS production under dark and light conditions that also affected the
nuclear membrane integrity, allowing MitoSox penetration into nuclei. Hence normal oxidation of P5C to Glu by
P5CDH is a key step required to negatively regulate P5C/Pro intensive cycling and control ROS production.

34

FEELING GREEN: THE ROLE OF CA2+, REACTIVE OXYGEN SPECIES AND PH DURING
MECHANOSENSING IN ARABIDOPSIS
Simon GILROYa, Gabriele MONSHAUSENa, Tatiana BIBIKOVAb, Sarah SWANSONa, Gregory RICHTERb
Department of Botany, University of Wisconsin - Madison, Birge Hall, 430 Lincoln Drive, 53706, Madison,
WI, United States of America, sgilroy@wisc.edu
b
Department of Biology, PennState University, United States of America

Mechanical stimulation of plants is well characterized as triggering a transient cytoplasmic Ca2+ increase
that is thought to link the touch stimulus to appropriate growth responses. However, the signal transduction
pathways elicited by such a Ca2+-dependent signaling system remain poorly defined. We have found in roots of
Arabidopsis, external and endogenously generated mechanical forces triggered not only a rapid and transient
increase in cytosolic Ca2+, but also a rapid, localized and transient apoplastic alkalinization and cytoplasmic
acidification. Mechanical stimulation likewise elicited apoplastic reactive oxygen species production localized
to the area of touch. These responses showed the same kinetics as mechanically-induced Ca2+ transients
and could be elicited in the absence of a mechanical stimulus by artificially increasing Ca2+ with the ionophore
A23187. Both pH changes and reactive oxygen species production were inhibited by pretreatment with La3+, a
Ca2+ channel blocker, which also inhibited mechanically-induced elevations in cytosolic Ca2+. In the Arabidopsis
rhd2 mutant that lacks a functional NADPH oxidase ATRBOH C, touch stimulation still triggered extracellular
and cytoplasmic pH changes but not the local increase in reactive oxygen species production seen in wild-type
plants. Thus, mechanical stimulation likely elicits Ca2+-dependent activation of ATRBOH C yielding reactive
oxygen species production to the cell wall. This reactive oxygen species production appears to be coordinated
with intra- and extracellular pH changes through the same mechanically-induced cytosolic Ca2+ transient.
These localized changes in pH and ROS may be utilized by plants to rapidly adjust growth and mechanical
stability of the wall in response to mechanical stress, whilst simultaneously triggering cytoplasmic signaling
cascades to affect downstream responses to mechanical stimulation.

35

REDOX STRESS IS A MAJOR COMPONENT OF CIRCADIAN-CLOCK RESETTING IN RESPONSE TO


DAWN
Seth DAVIES
Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, 50829 Kln, Germany,
davis@mpiz-koeln.mpg.de

Plant growth and development requires robust detection of the diurnal environment. This occurs through a
coupling mechanism of light detection to the circadian clock. The result is clock resetting, which is termed
entrainment. Entrainment in Arabidopsis thaliana occurs exclusively at dawn, and previously, investigators
have classified canonical photoreceptors as being the exclusive factors required for entrainment. Here, we
challenge this notion and present the hypothesis that clock-resetting occurs through redox/stress signaling.
Previously, we have characterized TIC as a clock gene that works at dawn. It encodes a nuclear regulator
of morning-clock-gene expression. Under a clock-resetting assay, in a TIC-dependent manner, we profiled
the whole-genome transcriptional changes that occur in response to jetlag, which would lead to a resetting
signal. Stress- and redox-regulated genes were the TIC-dependent responses. We tested if oxidative stress
could directly act on the clock, and found that it does, and does so only at subjective dawn. To understand
biochemically how TIC integrates the dawn-stress signal, we have isolated an interacting kinase. This kinase is
stress and metabolism activated, and can phosphorylate TIC. Furthermore, genetic analysis places the kinase
and TIC in an epistatic complex. Taken together, the resultant conclusion is that the redox/stress changes
that occur at dawn in response to light capture, i.e. photosynthesis, activate a stress-perceiving kinase, which
phosphorylates TIC. This phosphorylation-activation mediates nuclear clock-gene expression by TIC.

36

OSMOTIC STRESS-INDUCED SIGNALS CONTROL ROOT GROWTH


Carlos S. GALVAN-AMPUDIAa, Christine ZALEJSKIc, Laszlo BOGREc, Remko OFFRINGAb, Christa
TESTERINKa
University of Amsterdam, Swammerdam Institute for Life Sciences, Science Park 904, 1098 XH,
Amsterdam, The Netherlands, C.S.GalvanAmpudia@UvA.nl
b
Molecular & Developmental Genetics, Institute of Biology, Leiden University, The Netherlands
c
School of Biological Sciences, Royal Holloway, University of London, London, United Kingdom
a

Plant roots are constantly exposed to a variety of abiotic stresses. Depending on the type and intensity of the
stress, plants have the capability to overcome the stress by initiating signal transduction pathways that lead
to the activation of ion transporters and expression of genes involved in tolerance. An alternative strategy
is to modify root growth in order to avoid the stress source. Although it is known that environmental signals
cause local changes in auxin distribution necessary for the reorientation of growth, the molecular basis of the
avoidance strategy is still poorly understood.
Using physiological assays to measure salt avoidance, and live confocal microscopy of salt-stress Arabidopsis
roots, we set out to find the key players involved. Upon osmotic stress, phospholipid signals are produced, in
particular PA and PIP2. The same lipids have been shown to activate 3-phosphoinositide-dependent kinase
(PDK1), a master regulator of AGC protein kinases (Anthony et al., 2004) and its target PINOID (PID) (Zegzouti
et al., 2006), a key regulator of polar auxin transport. Here, we show that osmotic stress in roots leads to
rearrangements of the microtubule (MT) network and induces changes in the localization of signalling proteins,
including PID and PDK1. Currently, mutants in pid, pdk1 and the phospholipid-metabolizing enzymes that
generate PA, are being tested in a salt avoidance assay. We propose a model on how osmotic stress regulates
root growth through a signalling cascade in which phospholipid signalling and protein phosphorylation play a
central role.

37

UBP14 IS INVOLVED IN ROOT HAIR DEVELOPMENT UNDER PHOSPHATE STARVATION IN


ARABIDOPSIS
Margarete MLLERa, Ott TRJKd, Wolfgang SCHMIDTc, Thomas BUCKHOUTb, Thomas ALTMANNa
Leibniz Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, 06466, Gatersleben, Germany,
muellerma@ipk-gatersleben.de
b
Humboldt University Berlin, Germany
c
Academia Sinica, Taiwan
d
University of Potsdam, Germany

Under phosphate (Pi) starvation, plants increase the absorptive surface area of the root by increasing the
number and length of root hairs. To identify new genes that are involved in this process, we screened an
EMS-mutagenized Arabidopsis population for individuals that have no root hairs under Pi deficiency but
develop normal root hairs under sufficient Pi supply. One of the derived mutant lines was characterized in
more detail. The mutant developed only small bulges instead of root hairs under Pi starvation. Cryo-SEM
images showed that the rhizodermis of the Pi-deficient mutant was deformed and that the root hair bulges had
material accumulations at their tips. In the Pi-sufficient mutant, the shape of the root hairs was regular like in
the wildtype. The number and position of root hairs was not changed in the mutant, neither in the presence
nor in the absence of Pi, indicating that the Pi deficiency-induced root hair elongation is impaired rather than
epidermal cell specification. Also other Pi starvation responses were altered in the mutant. The number of lateral
roots was increased under Pi-sufficient and -deficient conditions. Furthermore, the anthocyanin content in the
leaves of the mutant was increased under Pi deficiency. Backcross experiments showed a co-segregation of
the mutant root hair phenotype with the increased lateral root number and anthocyanin content indicating that
the alterations were caused by the same mutation. Map-based cloning of the mutation revealed a nucleotide
exchange from C to T in the deubiquitinase gene UBP14, which causes a synonymous substitution. By screening
a highly EMS-mutagenized population for further SNPs in the UBP14 gene with TILLING, we identified three
additional mutant alleles causing an impaired root hair elongation under Pi starvation.

38

Poster Presentations

39

SUMOYLATION IN PLANT ABIOTIC STRESS RESPONSE AND DEVELOPMENT

Isabel ABREUa, Rafael CATALAb, Mafalda RODRIGUESa, Margarida OLIVEIRAa, Nam-Hai CHUAc
ITQB-Instituto de Tecnologia Quimica e Biologica, Av. da Repblica, Estao Agronmica Nacional,
2780-157, Oeiras, Portugal, abreu@itqb.unl.pt
b
CIB-Centro de Investigaciones Biolgicas, Spain
c
Rockefeller University, United States of America
a

Post-translational protein modification allows the fast regulation of the cell proteome. SUMO (Small Ubiquitinrelated MOdifier) is a small protein that can be covalently bound to proteins in a process similar to ubiquitination.
In general, SUMO modification of proteins (SUMOylation) regulates transcription factor activity, alters protein
subcellular localization and induces changes in protein-protein interaction. Plants, in particular, respond with
massive sumoylation when challenged by several types of abiotic stress, but SUMOs function in plant stress
response is still poorly understood. Our results show that plants missing a key ligase in the sumoylation
pathway (AtSIZ-1) are more susceptible to dehydration. This led us to propose that this process mediates
drought response and most probably other types of abiotic stress response (1). On the other hand, both SUMOconjugating-enzyme (AtSCE1a) and AtSIZ1 transcript levels respond to drought treatments, suggesting that
the SUMOylation process itself is somehow controlled by this abiotic stress. We have also observed important
roles for both SUMO-conjugating-enzyme (AtSCE1a) and AtSIZ1 in Arabidopsis development. These results,
together with other reports on SUMOylation in plants, show that this process is important during a plant life
cycle, in particular during embryogenesis and organogenesis, and in protein regulation during abiotic stress
response.

1. Catala R, Ouyang J, Abreu IA, Hu Y, Seo H, Zhang X, Chua N-H. (2007) The Arabidopsis E3 Sumo ligase
Siz1 regulates plant growth and drought responses. Plant Cell 19:2952-2966

40

GENOME-WIDE ANALYSIS OF SHORT RNAS AS MODULATORS IN DEHYDRATION STRESS


TOLERANCE USING THE RESURRECTION PLANT CRATEROSTIGMA PLANTAGINEUM

Ute ACHENBACH, Syed Sarfraz HUSSAIN, Geetha VENKATESH, Tamas DALMAY, Dorothea BARTELS
University of Bonn, Institute of Molecular Physiology and Biotechnology, Ptzlachstr. 103, 51061, Kln,
Germany, achenbach@uni-bonn.de

Plants have developed many different adaptive strategies to withstand dehydration stress. The resurrection
plant Craterostigma plantagineum is able to survive almost complete tissue dehydration, and rehydrate
rapidly on rewatering. The plant miRNAs represent an important class of endogenous small RNAs that guide
cleavage of a mRNA target or repress its translation to control development and adaptation to stresses. An
explosive increase in research reports on plant miRNAs have been witnessed during the past few years. So
far, identification of miRNAs has been limited to a few model plants species such as Arabidopsis, rice and
Populus whose genomes are sequenced.
Our objective is to explore regulatory roles of small RNAs (sRNAs) in dehydration stress tolerance, which is
a critical aspect in understanding this environmental condition. The common responses to different stresses
indicate similar function of gene products for plants under stress conditioning involving water deficit. Thus the
study of genes modulated by dehydration has also potential to improve drought tolerance of crops plants, an
abiotic factor likely to affect crop yields globally.
We used Solexa sequencing to find several millions of sequences which are currently investigated for potential
sRNAs. Next EST data will be available in the course of this project to further confirm true sRNAs and identify
potential targets. The differentially expressed sRNAs and potential targets will be validated by RNA blot and
qRT- PCR.

41

THE POSSIBLE ROLE OF POLYAMINES IN PROTECTION OF SEEDS FROM AGING

Halina ALEKSEICHUK, Anna CHOMYAK, Nicolay LAMAN


V.F.Kuprevich Institute of Experimental Botany of National Academy of Sciences, Academichaskaya str, 27,
220072, Minsk, Belarus, aleks.galina@gmail.com

Aliphatic polyamines (PAs) such as spermidine, spermine and their precursor putrescine are present in most
living cells and involved in different physiological events in plants, including cell division, DNA and protein
synthesis, and protection against different kinds of stress. When seeds deteriorate, they lose vigor and become
more sensitive to stresses at germination. Main biochemical processes which determine this aging are:
membranes become leaky, enzymes lose catalytic activity and chromosomes accumulate mutations (Walters,
1998; McDonald, 1999). A positive correlation between PAs content and stress tolerance in plants creates the
necessary prerequisites for search of correlation between polyamines and rate of seed aging. But studies on
qualitative and quantitative profiles of PAs in response to seed aging are very few. It is known that PAs are
present, but with an unknown function, during embryogenesis and accumulated in relatively high quantities
in mature dry seeds of some species (Matilla, 1996; Puga-Hermida et al., 2006). PAs concentration can be
altered by the stratification and/or osmotic priming. Priming-induced levels of PAs, especially, putrescine and
spermine, were related to the improved seed vigor (Basra et al., 1994). But the biochemical mechanism
binding the PAs to a series of compounds of low and high molecular weight is currently unknown in seeds.
The possibility that control of PAs biosynthesis could be used for the establishment of biochemical methods to
improve seed storage and to control germination of seeds is discussed.

42

POSTTRANSLATIONAL REGULATION OF C4 PEPC IN DROUGHT STRESSED PANICUM


MILIACEUM

Sabrina ALFONSO, Wolfgang BRGGEMANN


Goethe-University Frankfurt, Siesmayerstr. 70, 60323, Frankfurt am Main, Germany,
alfonso@bio.uni-frankfurt.de

Drought stress is one of the main environmental factors that limits photosynthesis and consequently the yield
of plants. Although only 3% of the angiosperm species use C4 photosynthesis, and C4 plants have a great
significant contingent of the global terrestrial primary production. As the first CO2 fixing enzyme in the C4 cycle,
PEPC has a key role in the regulation of C4 photosynthesis.
Besides inhibitors and activators, PEPC is also regulated by means of phosphorylation. The phosphorylation
state influences the sensitivity of the enzyme with regard to activators and inhibitors. This causal correlation
was used to determine the phosphorylation state of PEPC from control and drought stressed P. miliaceum
plants. For both conditions, IC50 values for PEPC induced by the inhibitor aspartate were measured as well in
darkened as in illuminated leaf samples.
In vitro enzyme activities of control and drought stressed plants did not show significant differences in the
absence of aspartate. Light-exposed control plants showed higher IC50 values and thus higher rates of
phosphorylation of PEPC than the darkened ones. In plants treated with drought stress we found similar
IC50 values and consequently the same phosphorylation state as in control plants under light conditions. In
the darkened samples the use of aspartate resulted in a higher IC50 and hence a higher phosphorylation
state of PEPC in the drought stressed plants. Both results show that the phosphorylation state of PEPC in P.
miliaceum is not responsible for non stomatal limitation of photosynthesis under drought stress conditions.
Instead, the measurements indicate that a higher phosphorylation state of PEPC in darkened leaf samples
could be an adaptation of the plants to promote increased assimilation of CO2 by the enzyme under stress
conditions already in the first minutes after illumination.
Additional regulatory mechanisms like aspartate accumulation and pH decrease will be discussed.

43

THE COMPARATIVE ANALYSIS OF PROTECTIVE ACTION OF 24-EPIBRASSINOLIDE AND


CYTOKININ ON WHEAT PLANTS UNDER SALINITY

Azamat AVALBAEV, Ruslan YULDASHEV, Felix URUSOV, Farida SHAKIROVA


Institute of Biochemistry and Genetics, Ufa Scientific Centre, Russian Academy of Sciences, Senior
Researcher, Pr. Oktyabrya 71, 450054, Ufa, Russia, shakirova@anrb.ru

Brassinosteroids (BR) represent a class of plant hormones with high antistress activity. Earlier in our research
it was shown that 24-epibrassinolide (EB), active representative of BR, induced in wheat seedlings 2-fold
increase in the level of cytokinins (CK) which is well-known for their protective action to various stresses.
The important contribution to observed CK accumulation had the EB-induced inhibition of gene expression
and activity of cytokinin oxidase which is responsible for cytokinin degradation. It is possible to suggest that
maintenance of increased level of CK under EB influence might have important role in the EB-induced defence
effect. It was revealed that pretreatment with EB during 24 h prevented the growth inhibiting effect of NaCl on
wheat seedlings. Probably this was due both to the maintenance of increased level of endogenous CK and
reduction of the level of stress-induced ABA accumulation in EB-pretreated seedlings. Pretreatment of seedlings
with cytokinin 6-benzylaminopurine (BAP) had comparable with EB protective effect on growth of seedlings
subjected to salinity. Meanwhile BAP-pretreatment itself led to essential rise in the level of endogenous CK
and this was accompanied by increase in ABA level which however did not prevent growth-stimulating action
of BAP. The influence of salinity on BAP-pretreated seedlings reduces the level of endogenous CK but it
was remained well above control, whereas salinity-induced ABA accumulation fell to the level typical to BAPpretreated seedlings in absence of NaCl. The received data confirm our suggestion about important regulative
role of endogenous CK in EB-induced resistance of wheat seedlings to salinity.
This work is supported by Grant RFFI 08-04-01563 and Grants MK-4081.2008.4 and NSh-915.2008.4.

44

COMBINED EFFECT OF CADMIUM, ANTHRACENE AND CHLORIDAZON ON THE


PLANKTONIC GREEN ALGAE DESMODESMUS SUBSPICATUS

Agnieszka BACIK-REMISIEWICZ, Anna AKSMANN, Wojciech POKORA, Zbigniew TUKAJ


University of Gdansk, Pisudskiego 46, 81-378, Gdynia, Poland, abrem@ocean.ug.gda.pl

Cells of D. subspicatus strain 86.81 (SAG) were used to examine the toxicity of cadmium (Cd), anthracene
(ANT) and chloridazon (CHD) applied individually and in binary combinations. The experiments were performed
according to the standardized test conditions of the ISO protocol 8692 (2004). Measured parameters were:
growth rate (k), cell volume (V), viability of cells, and chlorophyll a fluorescence parameters (PI, performance
index, Po, maximum yield of primary photochemistry). The values of ErC10 and ErC50 (k reduction by 10%
and 50%, respectively) for the chemicals were determined separately. Then, the effect of mixtures of two
substances (ErC10+ErC10, ErC50+ErC50: Cd+ANT, Cd+CHD, ANT+CHD) were characterized. The toxicity
of individual chemicals after 72 h exposition was as follows: ANT (ErC10=0.06; ErC50=0.26); Cd (ErC10=0.12;
ErC50=0.30); CHD (ErC10=2.83; ErC50=9.52 mg L-1). ANT and CHD did not significantly affect the viability
of cells, whereas the viability of algae after exposure to Cd was 72%. All individually applied substances at
ErC50 values reduced the volume of cells within the range from 64 to 76% of the control. The combined effects
of Cd+CHD and ANT+CHD stimulated the volume of cells. The markedly lower values of PI (algae vitality)
were observed in cells exposed to combined chemicals as compared to single substances individually treated
and control cells. The mixtures of Cd+ANT and Cd+CHD slightly diminished the values of Po whereas in the
other experimental variants they were almost the same as in the control. When the substances were applied
at ErC10 values, additive (Cd+ANT, ANT+CHD) and antagonistic (Cd+CHD) effects were observed. However,
all binary mixtures of chemicals used at ErC50 revealed antagonistic interaction.
This work was financially supported by grant (No. N304 092 31/3355) from the Polish Ministry of Science and
Higher Education.

45

INFLUENCE OF CYTOKININ ON CAPACITY AND PROTEIN LEVEL OF MITOCHONDRIAL AOX


PROTEIN IN LUPIN COTYLEDONS

Natalia BELOZEROVA, Alexander G. SHUGAEV, Elena POJIDAEVA, Viktor V. KUSNETSOV


Timiryazev Institute of Plant Physiology RAS, Botanicheskaya str. 35, 127276, Moscow, Russia,
n_belozerova@list.ru

Plant mitochondria may function as a sensor of stresses and initiate cellular responses to specific stresses, for
instance by contributing to altered nuclear gene expression. Changes in transcription of nuclear genes caused
by alternation of mitochondrial state are known as mitochondrial retrograde regulation (MRR). Recent work
in our laboratory has revealed that light and cytokinins regulate nuclear- and plastome-encoded chloroplast
protein gene transcriptional level. The role of cytokinin in MRR is still under the study. Its known that cytokinin
can moderate respiration and activity of the alternative oxidase (AOX) from alternative (cyanide-resistant)
respiratory pathway. We investigated effect of cytokinin with presence (or without) of light on respiration and AOX
capacity in 5-day old cotyledons of Lupinus luteus L. The 22 M of synthetic cytokinin (N6-benzylaminopurine;
BAP) fully inhibited of the alternative pathway in mitochondria isolated from cotyledons incubated with BAP for
12 h in dark. In two separated assay capacity AOX was about 10-times less than in control (water). Western
analysis showed direct correlation between content of AOX in cell and its capacity. We also optimized run-on
assay Lupinus luteus L cotyledons to investigate effect of light and cytokinin in transcription of mitochondria
encoded genes.

46

HEAT SHOCK INDUCED MEMBRANE DAMAGE, VOLATILES EMISSION OF METHANOL


AND LIPOXYGENASE PATHWAY PRODUCTS, AND PROGRAMMED CELL DEATH IN INTACT
LEAVES

Irina BICHELEa, Lucian COPOLOVICIb, lo NIINEMETSb


Department of Biophysics and Plant Physiology, University of Tartu, Riia 23, 51010, Tartu, Estonia,
irina.bichele@ut.ee
b
Department of Plant Physiology, Estonian University of Life Science, Tartu, Estonia

Altered membrane fluidity is direct and rapid response to high-temperature exposure and results in modified
activity of membrane-associated proteins. Polyunsaturated fatty acids cleaved by phospholipases become
substrates for lipoxygenases and are transformed to lipidhydroperoxides and further to a great variety of
secondary products. If enzymatic lipid peroxidation turns to non-enzymatic, alkoxy radicals are converted
to 2,4-dienals, which induce programmed cell death (PCD). The severity of an external impact determines
whether PCD or necrosis is induced.
Fully expanded trifoliate leaves of Phaseolus vulgaris L. plants were exposed to temperatures of 45 to 55 C for
5 min. We examined kinetic trends of the following parameters: - membrane damage, estimated on the basis
of electrolyte leakage;- cell viability, calculated as relative area of Evans blue-stained cells; - lipid peroxidation,
estimated as malondialehyde (MDA) concentrations measured with thiobarbituric acid method; - methanol and
lipoxygenase pathway volatiles emission measured with PTR-MS; - DNA fragmentation as a hallmark of PCD,
separated with agarose gel and visualised with ethidium bromide.
Leaf exposure to high temperatures immediately initiated enzymatic peroxidation of membrane lipids, as was
seen from time-courses of LOx-volatiles, MDA content and dye-stained cells. The reaction was slow at 46-47
C, but above 48 C the fluxes of volatiles rised up to 2 orders of magnitude, MDA concentration increased
by about 70% in the dark, but by 3 times in the light. We suggest that redox homeostasis is disturbed at
temperatures over 48 C in plant cells, inducing a break-point in all measured parameters. Reactive oxygen
species cause the enzyme-controlled lipid peroxidation to turn to a non-enzymatic uncontrolled process,
followed by an apoptosis-like cell death.

47

TOXICITY OF HERBICIDES MCPA, CHLORIDAZON AND THEIR MIXTURES IN LEMNA


GROWTH INHIBITION TEST

Joanna BISEWSKA, Alicja KAWECKA, Zbigniew TUKAJ


University of Gdansk, Pilsudskiego 46, 81-378, Gdynia, Poland, joannag@ocean.ug.gda.pl

The MCPA (auxin-like growth inhibitor) and chloridazon (CHD) (PSII-inhibitor) are commonly used herbicides
to weed control. Extensive use of these chemicals causes potential risk for non-target aquatic organisms
because of their leaching and runoff from fields. Additionally, herbicides are often applied as mixtures; therefore
their harmful interaction to aquatic biota can be expected. The toxic effects of MCPA, CHD and their mixtures
were determined in Lemna minor growth inhibition test according to the procedure of ISO 20079: 2005. The
concentrations of herbicides causing ca. 10-90% growth (b) and growth rate (r) reduction were: 0.8, 4.1, 20.5,
102.4, 512 mg/dm3 for MCPA and 0.7, 2.1, 6.3, 18.9, 56.7 mg/dm3 for CHD (b reduction) and 0.9, 2.4, 6.6, 17.7,
47.8, 129.1, 348.7 mg/dm3 for MCPA and 0.6, 1.3, 2.7, 5.6, 11.7, 24.5, 51.5 mg/dm3 for CHD (r reduction). The
obtained data were used to calculate EC10 and EC50 values. They were: EbC10 - 0.7 mg CHD/dm3 and 0.8
mg MCPA/ dm3, ErC10 2.9 mg CHD/dm3 and 0.8 mg MCPA/dm3, EbC50 10.4 mg CHD/dm3 and 5.7 mg
MCPA/dm3 and ErC50 - 15 mg CHD/dm3 and 52.7 mg MCPA/dm3. Next, the mixtures of herbicides were tested
to assess their joint toxicity. Comparison of EC50 values indicates that MCPA is more toxic to Lemna growth,
but less toxic to the growth rate than CHD. In addition, higher ErC values than EbC ones suggest higher
sensitivity of growth than growth rate in toxicity assessment of both herbicides. The results of combined toxicity
tests clearly show that there is an antagonistic interaction between MCPA and chloridazon.

48

10 OZONE EXPOSURE AND LEAF AGEING, A COMPARISON BETWEEN A STRESS INDUCEDAND A NATURAL PROCESS
Sacha BOHLERa, Isabelle LEFVREa, Sbastien PLANCHONa, Yves JOLIVETb, Lucien HOFFMANNa, JeanFranois HAUSMANa, Pierre DIZENGREMELb, Jenny RENAUTa
Centre de Recherche Public - Gabriel Lippmann, 41, rue du Brill, 4422, Belvaux, Luxembourg,
bohler@lippmann.lu
b
UMR 1137 INRA-UHP Nancy I, France
a

Tropospheric ozone has increased in the last century and peak concentrations on sunny days are frequent. It
is responsible for 10% of yield loss in European forests.
This study deals with the effects of ozone on poplar leaves. Poplar trees were submitted to 120 ppb of ozone for
one month. Young leaves were tagged and followed during their development. Tree growth was barely affected,
yet visual symptoms like necroses and chloroses appeared on mature leaves. Leaf loss and leaf formation
were increased by the treatment. Non photochemical quenching measured by chlorophyll fluorescence was
higher in treated leaves. Quantification of pigments showed a drop in levels of chlorophylls a and b, lutein and
neoxanthin, while zeaxanthin, violaxanthin and beta carotene barely changed.
A proteomic 2D DiGE experiment revealed that, in developing control leaves, most metabolic processes
decline over time. Only photosystem proteins increased, which could be due to a shading effect in the growth
chambers.
In ozone-treated leaves, variation over time mostly followed the same pattern as in controls, but with clear
changes in relative abundance. Proteins involved in photosynthesis and Calvin cycle showed a lesser
abundance compared to controls, while some proteins involved in carbon catabolism increased in abundance.
A general rise in protein folding proteins and an increase in some detoxification and defence proteins was
apparent.
During ozone stress, we observed the same pattern of variation of protein abundance in exposed and in
control leaves, coupled to specific changes in exposed leaves that diverged from the normal ageing process.
This shows that the response to ozone is not merely an acceleration in the leaf-ageing process, but an active
and controlled process with specific changes.

49

11 UNRAVELLING THE INVOLVEMENT OF GENES ENCODING ENZYMES OF AMINO ACID


METABOLIC PATHWAYS IN STRESS RESPONSES IN ARABIDOPSIS THALIANA
Sigrid BRAUC, Geert ANGENON
Vrije Universiteit Brussel (VUB), Pleinlaan 2, 1050, Brussels, Belgium, sigrid.brauc@vub.ac.be

One well known plant mechanism to cope with salt and osmotic stress is the production and accumulation of
osmoprotectants. The role of some amino acids or amino acid derivates herein has already been demonstrated.
Engineering increased contents of proline has been shown to lead to improved osmotolerance. Glycinebetaine,
a glycine derivate, is another well studied compatible solute. Polyamines also have positive effects on salt and
osmotic stress resistance.
All these compatible solutes have amino acids as precursor. Are other amino acids also involved in abiotic
stress response?
From publicly available microarray data we could deduce that several genes involved in amino acid metabolism
are induced after stress exposure in Arabidopsis thaliana. Using quantitative RT-PCR we demonstrated that
those genes are induced early after stress treatment, albeit with different amplitudes. For example, salt conditions
led to nearly 15-fold higher expression of glutamate decarboxylase, catalyzing the conversion of glutamate to
-aminobutyric acid (GABA), while osmotic stress did not strongly influence the expression. GABA has been
shown to be an osmoprotectant and could also be a signalling molecule. Lysine-ketoglutarate reductase/
saccharopine dehydrogenase is induced up to 10-fold after salt and osmotic stress treatment. This enzyme
catalyzes the first steps in the lysine catabolic pathway, leading to the production of glutamate, which is in
turn a precursor of other amino acids, e.g. proline. Also the expression of arginase, the enzyme catalyzing the
first step in polyamine biosynthesis, showed a slight increase after salt and osmotic stress treatment. Several
amino acid transferases are likewise affected by abiotic stress treatment, though with a small amplitude.
All these expression profiles showed an early induction of genes involved in amino acid metabolism. Further
analyses will unravel the involvement of these genes in stress defence mechanisms.

50

12 FLOODING INDUCED EMISSIONS OF VOLATILE SIGNALING MOLECULES IN SPECIES WITH


DIFFERING WATERLOGGING TOLERANCE
Lucian COPOLOVICI, lo NIINEMETS
Estonian University of Life Sciences, Fr.R. Kreutzwaldi 1, EE-51014, Tartu, Estonia,
lucian.copolovici@emu.ee

To gain insight into immediate response and acclimation to spoil waterlogging, volatile compounds synthesized
in tissues under anoxia (ethanol, acetaldehyde) and under stress (methanol, lipoxygenase (LOX) pathway
products), and NO emission patterns were studied in temperate trees species Alnus glutinosa, Populus tremula
and Quercus rubra (ranked from highest to lowest flooding tolerance). Root zone flooding resulted in emissions
of ethanol and acetaldehyde by all species, due to ethanol produced in the roots by alcoholic fermentation.
In Alnus glutinosa, the most flooding-tolerant species, methanol, was emitted at constant low level for three
days, after which the emissions constantly increased over the flooding period. In contrast, in less-tolerant
Quercus rubra and Populus tremula, methanol emissions increased sharply within the first three days of the
flooding period, and then declined to a significantly lower level. Emission of LOX pathway products exhibited
first an emission burst 1-2 h after the start of flooding follow by an exponential decreasing for all species. The
pattern of NO emission was similar to LOX emission, but large differences in the timing and time-dependent
reduction in LOX and NO emissions were observed across the species reflecting species differences in stress
tolerance. Foliage photosynthesis rates initially declined in all species, and recovered to pre-stress value at
day 5 in Alnus, while stabilizing at intermediate value in Populus and lowest value in Quercus. These results
suggest that Alnus glutinosa is well-adapted to flooded environments, fully acclimating in five days. Populus
tremula and Quercus rubra responded to stress more sensitively and only partly recovered. These results
demonstrate that volatile organics and NO can be used as quantitative measures of stress tolerance and
acclimation kinetics in temperate trees.

51

13 ECOTILLING ANALYSIS OF DROUGHT RELATED CANDIDATE GENES IN BARLEY


Andras CSERIa, Andras PALAGYIc, Matyas CSERHATIb, Janos PAUKc, Dnes DUDITSb, Ott TRJKb
Biological Research Center of Hungarian Academy of Sciences, Temesvri krt. 62, H-6726, Szeged,
Hungary, cseriandras84@freemail.hu
b
Institute of Pant Biology, BRC HAS, Szeged, Hungary
c
Cereal Research Non-Profit Company, Szeged, Hungary
a

In Hungary terminal drought is a main factor limiting the yield in barley. In this investigation the Ecotilling
technology was used as a polymorphism discovery tool to examine DNA variation in barley candidate genes
for drought tolerance. Gene targets were selected based on gene expression and mapping studies. SNPs
discovery and haplotype analysis was performed in a set of 96 barley cultivars and wild germplasm containing
drought tolerant and sensitive genotypes. EcoTILLING reactions were performed in one-well format using
fluorescently labeled nucleotides and Cel-1 treated products were visualized on ABI PRISM 377 sequencer.
Until now 17 EcoTILLING screens were performed for 9 genes (e.g.: HvARHGN, DREB1, HvDRF1, HVA1,
SRG6 etc.) analyzing more than 1,500,000 base pairs. Unique haplotypes (2-9 haplotypes / amplicon) were
sequenced and the obtained SNPs and INDELs were collected in a web-based database. A set of informative
polymorphisms were converted into easily detectable genetic markers allowing the detection and separation
of the main haplotypes in three candidate genes.

52

14 A COMPARATIVE STUDY OF PROTEIN EXTRACTION PROTOCOLS OF POPULUS DELTOIDES


X (TRICHOCARPA X DELTOIDES) FOR TWO DIMENSIONAL GEL ELECTROPHORESIS
ANALYSIS
Joke DUPAE, Ann CUYPERS, Jana BOULET, Nele WEYENS, Jaco VANGRONSVELD
University of Hasselt, agoralaan - gebouw D, 3590, Diepenbeek, Belgium, joke.dupae@uhasselt.be

Through its low economic and environmental costs, phytoextraction is believed to be one of the most
promising techniques to clean up metal contaminated soils. Recent studies indicate that the combination of
poplar trees with plant-associated bacteria can improve the phytoextraction efficiency. However, before the
in situ application, more fundamental knowledge of mechanisms behind the effects of toxic metals and plantassociated bacteria on poplar is required.
To reflect the functional state of an organism, 2-DE (2 dimension electrophoresis) can be used and is therefore
an efficient technique to monitor the impact of external influences at the proteome level. Proteomic analysis
of plant tissues appears to be especially challenging due to low protein content and high protease activities.
On top, other compounds such as phenols, terpenes, organic acids, oxidative and proteolytic enzymes, etc.
can interfere with 2-DE and result in vertical or horizontal streaking, smearing or reduction in the number of
distinctly resolved spots. This concludes that the extraction step is very critical in proteome research and
therefore it is important to invest sufficient time to find an optimal extraction procedure.
TCA/acetone extraction, phenol extraction and a combination of the former are often used for protein extraction
of recalcitrant plant tissues. Several studies comparing these extraction methods have been published and
they conclude that phenol extraction or a combination of phenol extraction and TCA/acetone extraction is most
suited for recalcitrant plant tissues. However, every extraction method has its limitations, advantages and
disadvantages; therefore these methods were compared before selecting a proper protocol. Proteome studies
on poplar have been published, but up to date there is no comparative study on protein extraction of poplar
released. Therefore, the objective of this study was to optimize a protein extraction method for poplar leaves.

53

15 GENE EXPRESSION PROFILING IDENTIFIES ROS-RELATED DETERMINANTS OF CHILLING


TOLERANCE
John EINSET
Norwegian University of Life Sciences, Drbaksveien, 1432, Aas, Norway, john.einset@umb.no

It has now been demonstrated that treatment of Arabidopsis thaliana plants with glycine betaine (GB) improves
tolerance to chilling stress by regulating gene expression. This finding provides the opportunity to identify new
stress determinants using gene expression profiling with microarrays to identify candidate genes followed by
functional confirmation of the involvment of candidate genes via mutant studies. The first gene identified by
this approach was the gene for RabA4c GTPase (At5g47960), which is expressed in roots and is involved in
vesicle trafficking from the golgi to the plasma membrane. We have also identified the FRO2 ferric reductase
(At1g01580), which is localized on the plasma membrane of root epidermal cells, as another component of the
GB-regulated system and suggested that enhanced production of reductant in the cell wall also plays a role in
chilling tolerance.
J. Einset and E. L. Connolly 2009 Glycine betaine enhances extracellular processes blocking ROS signaling
during stress. Plant Signaling & Behaviour 4: 197-199;
J. Einset, Winge, P., Bones, A. and E. L. Connolly 2008 The FRO2 Ferric Reductase is Required for Glycine
Betaines Effect on Chilling Tolerance in Arabidopsis Roots. Physiol. Plant. 134: 334-341;
J. Einset, E. Nielsen, E. L. Connolly, A. Bones, T. Sparstad, P. Winge and J.-K. Zhu 2007 Membrane Trafficking
RabA4c Involved in the Effect of Glycine Betaine on Recovery from Chilling Stress in Arabidopsis. Physiol.
Plant. 130: 511-518;
J. Einset, P. Winge and A. Bones 2007 ROS Signaling Pathways in Chilling Stress. Plant Signaling & Behaviour
2: 5, 365-367.

54

16 THE EFFECT OF HIGH CONCENTRATION CARBON DIOXIDE AND HYPOXIA ON REACTIVE


OXYGEN SPECIES FORMATION AND LIPOXIGENASE ACTIVITY IN PLANTS
Antonina ERSHOVA, Olga BERDNIKOVA
Voronezh State Pedagogical University, Lenin Street 86, 394043, Voronezh, Russia, aershova@vspu.ac.ru

Plants often fall in oxygen deficit conditions (hypoxia) which is accompanied by carbon dioxide accumulation.
It is shown (Crawford et al., 1996) that under hypoxia in plant the lipid peroxidation processes get activated
which might be induced by reactive oxygen species (ROS) accumulation. An impact of high concentration
carbon dioxide (100%) and 3-24h hypoxia on ROS formation and lipoxigenase (LOX) activity in plants with
different tolerance was investigated.
Constant increase discovered for superoxide anion in pea seedlings (intolerant) under hypoxia and CO2media was up to 150% of aerated plants. In soybean seedlings (midtolerant) it was slightly increasing in CO2media only. The hydroperoxides content in pea seedlings under 3h hypoxia was close to normal, but to essay
end it increased on 60% and under CO2-media on 70% to control. In soybean seedlings the hydroperoxides
content almost did not change under all hypoxia periods and CO2-media. The hydrogen peroxide content,
the most long lived ROS form, in pea increased 2-fold already in first hours of hypoxia. Under CO2-media the
concentration of hydrogen peroxide in cells was 200% and to essay end increased 2-fold. In soybean seedlings
the hydrogen peroxide content increased on 10-20% under hypoxia and CO2-media. The LOX activity involved
in lipoperoxides formation was analyzed in plant homogenates. It was discovered that LOX activity increased
under 3-9h hypoxia and CO2-media in pea on 50-70% and sharply fall to 15-25% of control. In soybean after
small increase LOX activity returned to control level.
The results showed that under hypoxia in plants the processes of all types ROS formation get activated which
is dependent on their tolerance. It was discovered that LOX enzyme strengthens these processes through
accumulation of lipoperoxides in cells, but only in first hours of hypoxia and CO2-media. It was noted that high
concentrations of CO2 strengthen all effects of hypoxia on ROS formation processes in plants.

55

17 ALTERED ASCORBATE REDOX STATE IN TOBACCO PLANTS RESULTS IN DELAYED DARKINDUCED SENESCENCE IN DETACHED LEAVES
Vasileios FOTOPOULOSa, Angelos KANELLISb

a
b

Cyprus University of Technology, PO Box 50329, 3603, Limassol, Cyprus, vassilis.fotopoulos@cut.ac.cy


Aristotle University of Thessaloniki, Limassol, Cyprus

Ascorbate oxidase (AO) is an apoplastic enzyme that uses oxygen to catalyze the oxidation of ascorbate
(AA) to dehydroascorbate (DHA) via the unstable radical monodehydroascorbate (MDHA). Here, we report
that transgenic tobacco plants (Nicotiana tabacum L. cv. Xanthi) with an in vivo lowered apoplast AA redox
state through increased AO expression demonstrate signs of delayed dark-induced senescence compared
with wild-type plants, as shown by chlorophyll loss and lipid peroxidation assays. In situ localization of
H2O2 suggests that although transgenic plants have higher constitutive levels of H2O2 under normal growth
conditions, imposed dark-induced senescence results in smaller induction levels of H2O2, an observation which
correlates with increased antioxidant enzyme activities and an induction in the expression of AA recycling
genes compared with that in wild-type plants. Our current findings, combined with previous studies which
showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in AA redox state
in the leaf apoplast of these transgenic plants results in an increase in the endogenous levels of H2O2, which
provides a form of acquired tolerance to oxidative stress imposed by dark-induced senescence.

56

18 ANTIOXIDANT RESPONSES TO SALT STRESS IN MEDICAGO TRUNCATULA LINES


Vasileios FOTOPOULOSa, Haythem MHADHBIb, Photini MYLONAc, Mohamed AOUANIb, Alexios
POLIDOROSd
Cyprus University of Technology, Athinon & Anexartisias 57, 3036, Limassol, Cyprus,
vassilis.fotopoulos@cut.ac.cy
b
Centre de Biotechnologie, Tunisia
c
Agricultural Recearch Center of Northern Greece, Greece
d
Institute of Agrobiotechnology, Greece
a

Legumes are important in sustainable agriculture providing high value protein, supporting meat and dairy
production and are unique in their ability to improve soil fertility. However, legumes are sensitive to abiotic
stresses most significant of which are water deficit and soil salinity. Modern agriculture encounters salinity
as a major factor limiting crop productivity worldwide. Soil salinity induces water deficit that leads to nutrient
deficiencies. Aside of water and ionic stress, salinity is accompanied by generation and accumulation of high
levels of reactive oxygen species (ROS), known as oxidative stress. Plants deploy antioxidant mechanisms to
alleviate the deleterious effects of elevated ROS. The aim of this study was to explore, elucidate and decipher
the role of antioxidant genes/enzymes and mechanisms under salt stress in the model legume Medicago
truncatula. Three lines of M. truncatula with differential tolerance to salinity have been used to study the
antioxidant responses. The M. truncatula lines used were: Jemalong A17; tolerant to salinity, TN1.11; very
tolerant to salinity, and TN6.18; sensitive to salinity. Enzyme activities of catalase, superoxide dismutase,
ascorbate peroxidase and guaiacol-peroxidase were determined along with their gene expression profiles
by qRT-PCR method, in roots and leaves after 24 and 48 hours of salt stress. Enzyme activities increased
in response to salt stress in roots while in leaves a differential pattern was exhibited for each line examined.
Following, gene expression profile in roots and leaves followed a differential pattern in each line. Our data show
that antioxidant responses to salt stress are concentration, tissue, time and genotype specific. In conclusion,
highly regulated and finely tuned antioxidant mechanisms operate in roots and leaves of M. truncatula in order
to effectively protect the plant from increased levels of ROS generated by the imposed salt stress.

57

19 IN VITRO SCREENING TOOLS USING CELLULAR AND ENZYMATIC SYSTEMS LINKED


TO INFLAMMATION TO STUDY THE ANTIOXIDANT ACTIVITIES OF PLANT EXTRACTS:
APPLICATION TO PRUNUS AVIUM SHOOTS CULTURED IN VITRO
Thierry FRANCKa, Claire KEVERSb, Ginette DEBY-DUPONTa, Didier SERTEYNa
Center for Oxygen, Research and Development, University of Lige, Sart-Tilman, B6a, 4000, Lige,
Belgium, t.franck@ulg.ac.be
b
Plant Molecular Biology and Biotechnology, University of Lige, Lige, Belgium

Stress in plants involves disequilibrium between oxidant and antioxidant systems and leads to a disruption
of the metabolic homeostasis. We describe methods able to measure the antioxidant and anti-inflammatory
capacity of plant extracts by studying their effects on two key elements involved in inflammatory response: the
neutrophil, a white-blood cell involved in reactive oxygen species (ROS) production, and myeloperoxidase, an
oxidant enzyme found in neutrophil (2). The antioxidant effect of the extracts on ROS production by neutrophils
was quantified by the measurement of the lucigenin-dependent chemiluminescence (CL), and the catalytic
effect of the extracts on the activity of purified MPO was assessed by SIEFED an original method allowing to
study the interaction of a drug with the enzyme without interference due to the reaction medium (2).
These techniques were applied on extracts obtained from Prunus avium shoots cultured in vitro on agar
(normal shoots, NS) and gelrite (hyperhydric shoots, HS) during a 21 days period. Hyperhydricity of in vitro
cultured plant was considered as an adaptive stress response to abnormal in vitro culture conditions (3).
Results showed that the antioxidant activities of the extracts from NS and HS were stable during the first week
of culture. Thereafter, a slight higher antioxidant activity was measured at day 14 and a higher inhibition of
MPO activity was observed at day 21 for the extract of HS.
Our results show that the adaptive response of stress plants could be measured by techniques using
inflammatory cells and enzymes and suggest that extracts from stressed plants could be a potential source
of molecules having an interest in animal and human health, especially in the modulation of inflammatory
response.
1) Kevers C. et al. (2004) Plant Cell Tiss Organ Cult 77, 181-191.
2) Franck T. et al. (2008) Physiol Res 57, 577-587.
3) Franck T. et al. (2004) Plant Physiol Biochem 42, 519-527.

58

20 BIOLOGICAL BASIS OF MODE OF ACTION OF THE PROTECTIVE ROLE OF ASAHI SL


AGAINST PLANT STRESSORS
Helena GAWROSKAa, Arkadiusz PRZYBYSZa, Elbieta SZALACHAa, Mariola WROCHNAa, Adam
SOWISKIb, Boguslaw ZRALYc
Laboratory of Basic Research in Horticulture, Warsaw University of Life Sciences, Nowoursynowska 159,
02 - 776, Warsaw, Poland, helena_gawronska@sggw.pl
b
Arysta LifeScience Co., Ltd, Poland
c
Asahi Chemical Mfg., Co., Ltd, Japan
a

Major task in modern agriculture today is to ensure near optimal growth conditions and/or to improve/repair
the status of plants that experienced stresses. Biostimulators, including Asahi SL, have a protective effect on
plants grown under stresses via an increase of the plants ability to cope with stress. The protective role of
Asahi SL has been shown both by researchers and farmers but no much is known on the mode of action of
this biostimulator. An attempt was made to evaluate the effect of Asahi SL on Arabidopsis thaliana L., Brassica
napus L. var. oleifera and Amaranthus sp. plants grown under drought, salinity, cadmium or frost stress.
Depending on experiment plants, grown in growth chambers or greenhouse were subjected to drought,
cadmium or salinity stress. Field grown oilseed rape plants experienced spring frost. Asahi SL was applied
either as supplement to nutrients solution or as foliar spray. Data on plant growth and development, biomass
accumulation, efficiency of photosynthetic apparatus, water status, ion accumulation were collected. Also
profile gene expression in A. thaliana grown under optimal conditions as affected by Asahi SL using microarray was monitored.
Asahi SL plays a protective role against imposed stresses that was manifested by: (i) more advanced
development especially generative, (ii) greater biomass accumulation, (ii) improved efficiency of photosynthetic
apparatus (iv) stable plant water status despite of higher transpiration, (v) increased activity of anti-oxidizing
system enzymes to greater degree than of and O2-level. Depending on Amaranthus cultivar, Asahi SL caused
both decrease or increase of toxic Na+ and Cl- ions accumulation. Results of micro-array showed that Asahi
SL changed gene expression in A. thaliana plants even under optimal conditions and part for drought is in
progress.
This study was financially supported by Arysta LifeScience Co., Ltd.

59

21 DROUGHT TOLERANCE OF AN EUCALYPTUS GLOBULUS L. CLONE BY PHYSIOLOGICAL


AND GENETIC APPROACHES.
Victor GRANDAa, Milln CORTIZOa, Candela CUESTAa, Rubn ALVAREZa, Ana RODRIGUEZa, Mara Luz
CENTENOb, Ricardo J. ORDASa, Isabel FEITOc, Beln FERNANDEZa
Oviedo University, C/Rodrigo Uria, s/n, E-33071, Oviedo, Spain, biovgg@gmail.com
Len University, Spain
c
SERIDA, Spain

Water stress is the most important factor affecting the crops survival and production, and the physiological
and genetic responses of plants to this sort of stress are well reviewed for most of the economically important
crops. In fact, obtaining tolerant genotypes to water stress is a main objective in the plant breeding programs.
However, these efforts came up against the long life cycle of forest crops. Therefore, in order to achieve an
early detection of water stress tolerant genotypes in woody plants, a prior understanding of the physiological
and the genetic responses is required.
The aim of the present study is the establishment of the relationship between the physiological responses and
genetic profiles of a drought tolerant clone of Eucalyptus globulus L. (C14 clone) under water stress, focusing
on the expression changes in early adaptation to water stress. For that purpose, a new experimental design
was conducted by accommodating C14 clone to hydroponic culture. The fast induction of water stress was
achieved by adding polyethilenglycol 8000 (PEG) to the culture medium. The plant growth regulator abscisic
acid (ABA) was selected as physiological marker of water stress, studying its dynamics under the water stress
conditions.
Considering the relevance of early-response genes of C14 clone on water stress, a SSH library was built.
Apical stem samples were collected following the ABA dynamics. The genetic profile of this clone in a severe
drought condition was obtained, and the genes implicated in signal transduction and hormonal responses were
selected for further genetic expression studies (e.g. RT-PCR).
This work was supported by the projects of Ministerio de Educacin (AGL2006-13912-C02-01 and GEN200627791-C2-1E/VEG). V. Granda is funded by a predoctoral grant by Ministerio de Educacin y Ciencia (FPI
BES-2007-15663). The C14 clone was provided by ENCE group.

60

22 ROLE OF SUGAR METABOLISM IN VASCULAR DEVELOPMENT AND FUNCTION


David GRANOTa, Shimon RACHAMILEVITCHd, Roni ALONIb, Marcelo GERMANa, Shabtai COHENa, Maciej
ZWIENIECKIc, Michele HOLBROOKc, Hila DAMARI-WEISSLERa
Agricultural Research Organization, The Volcani Center, P.O. Box6, 50250, Bet-Dagan, Israel,
granot@agri.gov.il
b
Tel-Aviv University, Israel
c
Harvard University, United States of America
d
Ben Gurion University, Israel

Vascular development requires sugar metabolism, but how sugar metabolism regulates vascular development
and function is not known. We study the role of the initial sugar metabolism genes in vascular development of
tomato plants. Sucrose, the photoassimilate transported in the phloem of many plants including tomato must
be cleaved by either invertase into glucose and fructose, or by sucrose synthase (SuSy) into fructose and
UDP-glucose. While UDP-glucose may be used for cellulose synthesis, the hexoses glucose and fructose
must first be phosphorylated by hexose phosphorylating enzymes prior to their utilization. There are only
two groups of hexose phosphorylating enzymes in plants, hexokinases (HXKs) and fructokinases (FRKs),
which have different intracellular locations and distinct biochemical characteristics. We found that LeFRK2,
the major FRK expressed in the vascular system of tomato plants plays a major role in vascular development
and function. Suppression of LeFRK2 results in a significant reduction in the size of vascular cells and slows
fiber maturation. The vessels in stems of LeFRK2-antisense plants are very narrow and have thin secondary
cell walls. Although the cambium produces rounded secondary vessels, these vessels become deformed
during the early stages of xylem maturation. Water conductance is then reduced in stems, roots and leaves,
suggesting that LeFRK2 influences xylem development throughout the entire vascular system. Interestingly,
the build-up of positive xylem pressure under static (no-flow) conditions was also decreased. Suppression of
LeFRK2 also reduced the length and width of the sieve elements, and reduced phloem transport. However,
triple graft experiments demonstrated that restricted upward water transport through the xylem and not the
interruption of downward sugar transport in the phloem is the underlying cause of the inhibited growth and
wilting observed in LeFRK2-antisense plants.

61

23 PHYSIOLOGICAL AND SPECTRAL CHARACTERISTICS OF GREEN ALGAE


CHLAMYDOMONAS ACTINOCHLORIS DEASON AT BOLD UNDER THE INFLUENCE OF
MICROWAVE IRRADIATION
Olesia GRYGORIEVA, Maryana BEREZOVSKAJA, Olexandr DACENKO
National Kyiv Taras Shevchenko University, 32 Glushkova str., apt.9, 03187, Kyiv, Ukraine, allegro@ukr.net

According to the critical level of environmental pollution by various abiotic factors using of the spectral and
fluorescent methods is very important. Those methods provide an express-diagnostics of the cells state. The
aim of this work was to study the influence of various doses of microwave radiation in the decimetre range
(generation frequency 2450 MHz) on Ch. actinochloris samples, staying in different initial physiological states
motile and palmelloid. The current functional state of the samples was controlled by the efficiency of chlorophyll
photoluminescence (the two bands with maxima at 685 and 710 720 nm). Luminescence was initiated by
argon laser radiation with wavelength 488 nm and with 19 mW power.
Analysis of data on the seventh day of the experiment showed that the 20-second exposure of algae (dose 85
J g-1) in motile state causes significant stimulation of luminescent processes; however 30-second exposure
(dose 130 J g-1) almost completely reduces them. The luminescence intensity of the samples in the palmelloid
state irradiated during 20 s was slightly higher than control and significantly surpassed the other options. In
both cases, irradiation at a dose 130 J g-1 caused the luminescence degradation compared to the control and,
on the contrary, a dose 85 J g-1 caused increase of luminescence.
Thus, microwave irradiation affects various intracellular processes, but the nature of this influence depends on
the dose of absorbed radiation, and on the physiological state of the plant.
Radiation dose approximately 85 J g-1 clearly stimulates intracellular processes, as evidenced by the increasing
biomass of Ch. actinochloris and increase the intensity of luminescence; radiation dose approximately 130 J
g-1, on the contrary, suppresses them.

62

24 RAPID SCREENING OF LEAF METAL CONTENT WITH INNOV-X HANDHELD XRF ANALYZER
Pauliina HALIMAA, Viivi HASSINEN, Sirpa PERNIEMI, Sirpa KRENLAMPI, Arja TERVAHAUTA
University of Kuopio, Yliopistonranta 1E, 70211, Kuopio, Finland, pauliina.halimaa@uku.fi

Metal accumulating plants have received extensive interest since their potential in remediating metal
contaminated soils. However, many metal accumulators are small in size and thus not well-suited for costefficient cleaning. Scientists are trying to develop, by molecular biotechnology means, plants that are better
suited for this purpose. However, screening of plant individuals from a large set of candidate plants can be both
labor- and time-consuming, as well as high-priced.
Innov-X Systems offers a set of X-ray fluorescence measuring devices, combined with a pocket PC, to
be used for detecting element content from various sources. It has been used for example in mining and
environmental monitoring applications, in detecting lead from paints and toxic metals from toys, and in quality
control in pharmaceutical and biomedical industry. We used Innov-X handheld XRF for rapid screening of metal
accumulating ecotypes of Finnish Thlaspi caerulescens populations. It allows measurement of metal content
without detaching leaves and measurement can be done in natural habitats of plants. In the experiment, the
populations were exposed to zinc and after 9 days, two leaves were collected and the leaf metal content was
analyzed with XRF. The same leaves were analyzed also with AAS verify XRF results. The XRF results are,
as expected, relative, but comparative with AAS and it was possible to distinguish better metal accumulating
populations from other populations.

63

25 ERD15 MODULATES ABIOTIC AND BIOTIC STRESS TOLERANCE IN ARABIDOPSIS


Elina HELENIUS, Tarja KARIOLA, Kukka AHO, Anne KUJANP, Jing LI, Ville PENNANEN, Mantas
SURVILA, Pekka HEINO, Gnter BRADER, Tapio PALVA
University of Helsinki, Dept of Biological and Environmental Sciences, Genetics, Viikinkaari 5 D, 00014,
Helsinki, Finland, elina.helenius@helsinki.fi

ERD15 (EARLY RESPONSIVE TO DEHYDRATION 15) is rapidly induced in response to various abiotic and
biotic stress stimuli in Arabidopsis thaliana. We have previously demonstrated that ERD15 is one of the key
negative regulators of responses to abscisic acid (ABA). Plants overexpressing ERD15 are insensitive to
ABA and impaired in their abiotic stress tolerance, while silencing of ERD15 results in hypersensitivity to
ABA and increased tolerance to drought and freezing. The ABA insensitivity of the ERD15 overexpressors
is also manifested in their deficiency to close stomata in response to external cues (see Puzrjova et al.,
this meeting). Interestingly, the plants overexpressing ERD15 show enhanced induction of SAR and have
improved resistance to the plant pathogens Erwinia carotovora and Pseudomonas syringae DC3100. This
pathogen resistance is suppressed by exogenous ABA. The function of the ERD15-protein is still unknown.
The presence of a PAM2-motif in the ERD15 sequence indicates interaction with poly-A-binding proteins
(PABP), suggesting a function in RNA metabolism. By split-ubiquitin based yeast 2-hybrid method we have
demonstrated that ERD15 is interacting with PABPs with differential affinities towards different members of the
PABP family in Arabidopsis.

64

26 STOMATAL DEVELOPMENT RESPOND TO THE ENVIRONMENTAL CONDITIONS. THE ROLE


OF AIR DIFFUSIVITY.
Marie HRONKOVAa, Martina VASKOVAb, Marie SIMKOVAa, Jiri SANTRUCEKb
Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Plant Molecular Biology,
Branisovska 31, 370 05, Ceske Budejovice, Czech Republic, hronkova@umbr.cas.cz
b
Faculty of Science, University of South Bohemia, Czech Republic
a

Stomata, as specialised epidermal structures, control the exchange of water and carbon dioxide between
plants and surrounding atmosphere. Their number and function (aperture) are the key factors allowing
maximal photosynthetic CO2 fixation at minimal water losses. Changed diffusivity of the atmosphere when
nitrogen is substitute by helium (Helox atmosphere) changed both the number of stomata on developing
leaves and their patterning. Low CO2 concentration at high humidity increased number of stomata and number
of stomatal clusters. The mechanism of this response is not known, but it seems to be dependent on both CO2
concentration (in and out of the leaf) and transpiration, which regulate leaf temperature. Germinating plants of
Lepidium sativum L. and Arabidopsis thaliana L. were used in our experiments. Analysis of naturally occurring
stable isotopes 13C in plant organic matter (long-term effect) and 2H and 18O in plant water (short-term effect)
and interpretation of isotopic data together with accumulation of dry mass accumulated by plants and water
content in plants can help us to understand stomatal sensing of environmental signals. Stomatal aperture of
mature leaves and stomatal density and patterning on developing leaves are influenced by environmental
conditions.
This work was funded by Grant Agency of Czech Republic 206/08/0787, Czech Academy of Sciences AVOZ
50510513 and Ministry of Education, Youth and Sports of the Czech Republic.

65

27 HIPPs, A NEW CLASS OF PROTEINS INVOLVED IN ABIOTIC STRESS RESPONSE OF PLANTS


Klaus HUMBECK, Olaf BARTH, Stefan EHNERT, Sebastian FRSTER, Wiebke ZSCHIESCHE
Martin-Luther-University Halle, Weinbergweg 10, 06120, Halle, Germany,
klaus.humbeck@pflanzenphys.uni-halle.de

HIPP proteins are characterized by a heavy metal associated domain (HMA-domain) and an isoprenylation
motif at the C-terminus. Members of this functionally not yet further characterized protein family could be
identified in Arabidopsis thaliana and Hordeum vulgare. Expression studies show that members of the HIPP
family are induced during different abiotic stress conditions including drought, cold and heavy metal stress.
The nuclear localization of some HIPPs could be confirmed in onion epidermal cells overexpressing GFP-HIPP
constructs. Experiments with modified HIPPs indicate that the isoprenylation motif is important for the spatial
distribution in the nucleus. Furthermore, using promoter-GUS constructs a high expression could be detected
in vascular tissues. By yeast-two hybrid approaches interaction partners of HIPP proteins could be identified.
A strong interaction could be detected with the zinc finger homeodomain transcription factor ATHB29, which is
known to play a role in dehydration stress response. This was confirmed by GST pull-down assays.
In order to functionally characterize several HIPP proteins Arabidopsis thaliana mutant lines were established
and analyzed under drought stress conditions showing a decrease in drought tolerance as indicated by
physiological parameters. Additionally, it could be shown that knock-out of a HIPP protein affects expression
of stress related genes which are regulated by ATHB29, indicating again the functional relationship between
HIPP proteins and this central drought stress related transcription factor.

66

28 VARIABLE TEMPERATURE DEPENDENCIES OF DARK RESPIRATION IN FOLIAGE OF


POPULUS TREMULA. HOW DO SUGARS PROTECT MITOCHONDRIAL PROCESSES?
Katja HVEa, Irina BICHELEb, lo NIINEMETSa, Mari TOBIASa, Bahtijor RASULOVb
Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1,
51014, Tartu, Estonia, shueve@gmx.de
b
Department of Plant Physiology, University of Tartu, Tartu, Estonia

Alteration of leaf sugar concentration is a possible mechanism of short-term adaptation of leaf photosynthesis
and respiration to temperature changes in the field, but the influence of sugars on heat sensitivity of respiration
is not known. Temperature responses of leaf dark respiration in relation to natural variation in leaf sugar
concentration and to enhanced sugar levels achieved by sugar-feeding were studied in the temperate tree
Populus tremula L. Rapid temperature response curves were obtained by increasing the leaf temperature with
a rate of 1 C min-1. We demonstrate that respiration, similarly to chlorophyll fluorescence, has a break-point at
high temperatures, where respiration starts to increase with a faster rate than below the critical temperature.
This break-point is possibly correlated with start of breakdown of starch as respiration substrate. Furthermore,
the onset of oxidation of membrane fatty acids is accompanied by an increase in respiration. The observed
break-points of respiration (TRD) versus temperature curves occur at a similar temperature as the well-known
fluorescence break-points (TF0). At a given temperature, feeding with sucrose resulted in a major increase in
respiration rates, but respiration was much less affected by feeding with a non-metabolisable sugar, sorbitol.
However, both sucrose and sorbitol affected TF0 and TRD, shifting break-points to higher temperatures. Further,
TF0 and TRD correlated with leaf osmotic potential to some degree. At high sugar concentrations, the break
point was abolished. Shift of the respiration break-point to higher temperatures by enhanced concentrations
of soluble sugars may have different reasons, like non-specific osmotic protection of membranes, provision
of respiration substrate, or specific sugar-activated or deactivating metabolic pathways. These reasons are
discussed.

67

29 RESPIRATION AND STARCH DEGRADATION IN LEAVES OF ARABIDOPSIS UNDER HIGH


TEMPERTURE STRESS
Hiie IVANOVA, Olav KEERBERG, Tiit PRNIK
Eesti Maalikool, Instituudi tee 11, 76902, Harku, Estonia, hiie.ivanova@emu.ee

Raising the temperature of Arabidopsis leaves above 40 C an abrupt increase in the rate of respiration was
detected, both in the dark and in the light. To elucidate the mechanisms of this phenomenon the consumption
of primary and stored photosynthates in respiratory processes was studied. For this purpose the rates of
respiration and degradation of pulse-labeled starch were measured in parallel in the dark and in the light
under different irradiances and temperatures. Starch degradation and respiratory decarboxylation of stored
photosynthates showed similar responses to short-term changes in irradiance and temperature. Both processes
were suppressed at low irradiances (below 60 mol m-2 s-1). At irradiance higher than 100 mol m-2 s-1 degradation
of starch was blocked and reversed to its additional synthesis from cytosolic soluble photosynthates, the
substrates of respiration were derived exclusively from primary and soluble stored photosynthates, mainly from
sucrose. In the light breakdown of starch took place only in conditions where photosynthetic CO2 assimilation
was suppressed (in CO2-free air and/or under supraoptimal temperatures). In the dark the rates of respiration
and degradation of starch increased with temperature, most abruptly at temperatures higher than 40 C. In the
light at moderately high temperatures (20-40 C) the rate of decarboxylation of soluble stored photosynthates
did not depend on temperature, the rise in total respiration was due to the temperature-mediated increase
in respiratory decarboxylation of primary photosynthates. Under these conditions degradation of starch is
blocked. The block was removed at temperatures higher than 40 C where an extensive degradation of starch
and respiratory decarboxylation of stored photosynthates were followed also in the light. Thus, the abrupt rise
in respiration under high temperature stress could be explained by the increased availability of substrates
derived from degradation of starch.

68

30 EFFECT OF DROUGHT STRESS ON DIFFERENT DEVELOPMENTAL CHARACTERISTICS AND


ON THE TRANSCRIPT PROFILING OF HUNGARIAN RICE (ORYZA SATIVA L.) VARIETIES
Mihaly JANCSa, Zoltan ZOMBORIb, Ibolya SIMON-KISSa, Janos GYRGYEIb, Janos PAUKc
Research Institute for Fisheries, Aquaculture and Irrigation, Anna liget 8., H 5540, Szarvas, Hungary,
jancsom@haki.hu
b
Biological Research Center of the Hungarian Academy of Sciences, Hungary
c
Cereal Research Non-Profit Ltd., Hungary
a

Breeding for drought tolerance of rice (Oryza sativa L.) is carried out since 1984 in Hungary. Depending on
the research and technological development, a new aerobic rice cropping system (SANORYZA) was patented
and several aerobic rice varieties (Karmina, Ringola (HSC55), Sandora, Janka, Abel) were released. Using
these achievements the water consumption compared to conventional paddy fields can be decreased by
45-55 percent without loss of quality and quantity. A small-scale experiment was started in 2006 parallel on a
controlled drought-prone and a paddy field. Varieties tolerant to drought stress produced significantly higher
yield (bel 5.44 t*ha-1, Janka 4.75 t* ha-1, Bioryza H 5.02 t* ha-1 and Sandora 5.51 t* ha-1) than the sensitive
variety (Marilla 0.81 t* ha-1).
Several developmental characteristics of these varieties (e.g. root- and shoot growth, yield) were also studied
in a drought test system installed in a glasshouse. Sandora was found the most drought-tolerant variety and it
was superior to the other varieties by root dry-mass and root-length.
Using the experimental material generated in glasshouse experiments, transcript profiling of the roots of
Sandora cultivar has been performed on oligonucleotide DNA chip. Several gene categories were found which
exhibit drought induced, diurnal changes or both in roots of rice.

69

31 REDUCING POWER DEPENDENT ON METABOLIC CHANGES COULD IMPROVE THE


DETERMINATION OF OZONE RISK THRESHOLD FOR HIGHER PLANTS
Yves JOLIVET, Didier LE THIEC, Matthieu BAGARD, Emilien DELACOTE, Marie Paule HASENFRATZ
SAUDER, Marie Noelle VAULTIER, Joelle GERARD, Jacques BANVOY, Pierre DIZENGREMEL
UMR 1137 Forest Ecology and Ecophysiology, INRA/ Nancy-Universit, Bd des Aiguillettes, 54506,
Vandoeuvre les Nancy cedex, France, jolivet@scbiol.uhp-nancy.fr

Tropospheric ozone is considered as one of the most important air pollutants affecting crop and forest vegetation.
Necrosis may be the result of a long term exposure of plants to low ozone concentrations, but biochemical
processes are affected prior to any detectable symptoms of injury. Initially, the critical level of ozone exposure
associated to biomass losses was based on the seasonal sum of the external concentrations of the pollutant
above 40 nL L-1 (AOT 40). Recently, a novel concept of effective ozone flux has been proposed. This concept
takes into account the ozone flux in the leaf through stomata and the internal defense capacity of the foliar
tissues. Among defense processes, the detoxification reactions play a prominent role and we suggested to also
consider some parameters linked to the generation/state of the reducing power which is essential to sustain the
functioning of the detoxifying systems. In this report, we will present preliminary results on poplar and wheat
species exposed to chronic ozone fumigations. In the leaves of both species, the pool of NAD(P)H appeared
quite well maintained. This result could be the consequence of a stimulatory effect on the activity of enzymes
implied in the biosynthesis of the reduced pyridine nucleotides. The stimulation of the phosphoenolpyruvate
carboxylase (PEPc) activity in these tissues validates this hypothesis: PEPc is involved in the biosynthesis of
malate, a mobile form of reducing power in the cell. In addition, we have shown that the activity of enzymes
involved in NADPH biosynthesis (malic enzymes, glucose-6-phosphate dehydrogenase, etc.) was stimulated
in different plant species exposed to a chronic ozone fumigation. We are running additional experiments to
precise their level of regulation (transcriptional, translational or post-translational) and to use some of these
key enzymes as indicators to improve the risk assessment for plants exposed to ozone.

70

32 DIFFERENTIAL RESPONSE OF PHLEUM PRATENSE, P. BERTOLONII AND P. PHLEOIDES TO


DROUGHT STRESS
Kristina JONAVIIEN, Vanda PAPLAUSKIEN, Sigitas LAZAUSKAS, Gintaras BRAZAUSKAS
Lithuanian Institute of Agriculture, Instituto al. 1, 58344, Akademija, Kdainiai dstr., Lithuania, kristinaja@lzi.lt

Due to climate change droughts and plant water stress will increase so it is important to identify water-efficient
and drought-tolerant grass species. In order to establish the response of grasses to drought stress, three
Phleum species: P. pratense L., P. bertolonii DC. and P. phleoides (L.) H. Karst. were compared in a humidity
and temperature test chamber study. Relative water content (RWC) in leaves and maximum potential quantum
efficiency (Fv/Fm ratio) was chosen as drought stress indicators. The RWC was measured by weighing method
and Fv/Fm with chlorophyll fluorometer OS-30p (Opti-Science, USA). A total of 6 days was considered to be
a whole drought stress period where the RWC and Fv/Fm rates in all Phleum species were unaffected for the
first 3 days of the experiment, however, later values of these indicators declined and drought stress symptoms
became apparent. RWC of P. pratense decreased by 34% and P. bertolonii by 32%, while that of P. phleoides
declined by only 17% to reach final RWC values of 53, 58 and 73% respectively. Fv/Fm ratio decreased from
0.712 to 0.490 for P. pratense and from 0.711 to 0.457 for P. bertolonii, with no significant differences between
those two species (p>0.05), but large differences were observed between unstressed and stressed plants
(p<0.001). ANOVA results indicated no significant differences between P. phleoides unstressed and stressed
plants (p>0.05), however P. phleoides differed significantly (p<0.01) from both P. pratense and P. bertolonii. The
correlation between Fv/Fm and RWC was found to be high (r=0.95). The results of our study indicate differential
response to drought stress among three Phleum species under investigation with higher drought sensitivity of
P. pratense and P. bertolonii than that of P. phleoides. Further analysis of differential gene expression will be
performed to identify drought response genes in these species.

71

33 ROLE OF SALICYLIC ACID AS A SIGNAL MOLECULE IN RETARDING LEAF ROLLING IN


PLANTS UNDER DROUGHT STRESS
Asim KADIOGLUa, Tuba ACETa, Aykut SAGLAMa, Neslihan SARUHANb, Rabiye TERZa
a
b

Karadeniz Technical University, Kalkinma, 61080, Trabzon, Turkey, kadioglu@ktu.edu.tr


Rize University, Turkey

Effects of salicylic acid (SA) on leaf rolling and antioxidant system were investigated under drought stress.
For this reason, some antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), ascorbate
peroxidase (APOX), guaiacol peroxidase (GPOX), dehydroascorbate reductase (DHAR), monodehydro
ascorbate reductase (MDHAR), glutathione reductase (GR)), reactive oxygen species (hydrogen peroxide and
superoxide) and lipid peroxidation were determined during the rolling in the leaves of Ctenanthe setosa (Rosc.)
Eichler (Marantaceae). The plants were subjected to 30 days drought period by withholding water and a group
of plants was treated with 10-6 M SA. Samples were taken from all plants having rolled leaves on 0th (unrolled)
4th, 8th and 10th days after the treatment.
SA application retarded leaf rolling in comparison with the application without SA. In addition. SA more induced
all antioxidant enzyme activities than untreated plants during leaf rolling period. In all plants, SOD, CAT, APX
and GR increased up to 8th day after the treatment but they decreased a little at the 10th day. There was a
similar trend in MDHAR activity in untreated plants but its activity continuously increased in SA treated plants
during rolling period. DHAR activities also continuously increased in all plants. SA application resulted in a
higher increase in reactive oxygen species than untreated plants at the beginning of the treatment. Then a
sharp decrease occurred in SA treated plants but not untreated plants. However, SA treatment prevented the
peroxidation while lipid peroxidation increased in untreated plants.
The results showed that SA retarded leaf rolling and this delay may be due to the effect of SA on antioxidant
system in plants. In conclusion, SA may be one of the important signal molecules in controlling leaf rolling in
plant under drought stress.

72

34 PHOTOSYNTHESTIC CARBON METABOLISM IN LEAVES OF COLD STRESSED AND COLD


ACCLIMATED WILD TYPE ARABIDOPSIS AND OF ITS TRANSFORMANT OVEREXPRESSING
SUCROSE PHOSPHATE SYNTHASE
Olav KEERBERG, Hiie IVANOVA, Tiit PRNIK
Eesti Maalikool, Instituudi tee 11, 76902, Harku, Estonia, olav.keerberg@emu.ee

To elucidate the consequences of accelerated sucrose synthesis on the cold acclimation of photosynthetic
apparatus the rates of carbon fluxes and pool sizes of metabolites in the reaction system of photosynthetic
carbon metabolism were determined in leaves of cold-shocked (grown at 25 C, transferred to 5 C and measured
at 5 C), cold-developed (grown and measured at 5 C) and control (grown and measured at 25 C) plants of
wild type (WT) Arabidopsis and of its transformant overexpressing sucrose phosphate synthase (SPS+ plants,
constructed by Ch. Foyer, UK)). The freezing tolerance and the rate of sucrose synthesis of SPS+ plants were
significantly higher than of WT plants. Responses of photosynthetic carbon metabolism to low temperature
were in SPS+ plants similar to those in WT. The rate of photosynthesis, rates of photorespiration and of carbon
flux through the glycolate cycle, the activity of glycine decarboxylase complex, and the enzymatic capacity of
the regenerative phase of the reductive pentose phosphate cycle were suppressed by cold shock but partially
or fully recovered during acclimation at low temperatures. The pools of sugar phosphates increased at low
temperatures having the highest values in cold-developed plants. Low temperatures stimulated synthesis of
sucrose and suppressed synthesis of starch in WT but not in SPS+ plants of Arabidopsis. The rate of sucrose
synthesis in non-acclimated SPS+ plants was the same as in acclimated WT. The results obtained enable to
ascertain the reactions of photosynthetic carbon metabolism affected by cold shock and/or modified during cold
acclimation of plants. It is concluded that genetic modification of sucrose metabolism represents a possible
approach for the development of freezing tolerance and the over-wintering capacity of plants.

73

35 LONG TERM IMPACT OF OZONE ON PROTEIN EXPRESSION PROFILES OF EUROPEAN


BEECH SAPLINGS (FAGUS SYLVATICA L).
Ren KERNER, Gerhard MLLER-STARCK
Department of Forest Genetics, Am Hochanger 13, D-85354, Freising, Germany, kerner@wzw.tum.de

Ozone, one of the most hazardous phytotoxic air pollutants, is creating stress environments for plants including
crops as well as forest trees. A lysimeter study was performed to monitor long term effects of ozone on
European beech saplings. An additionally infection with the pathogen Phytophthora citricola was performed
under ambient and elevated ozone conditions. A proteomic study was conducted using highly sensitive twodimensional differential gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) in order to
identify indicative proteins for ozone impact in European beech plants. Furthermore, the present work allows
studying plant-parasite interactions under ambient and elevated ozone conditions. Approximately 750 spots
were detected by two dimensional gel electrophoresis. We identified several leaf proteins whose abundance
was regulated during elevated ozone and after the inoculation with the pathogen P. citricola. The most evident
changes were observed in the presence of the pathogen under twice ambient ozone.

74

36 PROFILING OF AN ARABIDOPSIS MUTANT WITH ALTERED PROLINE/CARBOHYDRATE


RATIO
Sarita KESKI-SAARI, Ella NUKARINEN, Ilkka PORALI, Nina SIPARI, Markku KEINNEN
University of Joensuu, Box 111, FI-80101, Joensuu, Finland, sarita.keski-saari@joensuu.fi

The Arabidopsis mutant rcd1 (radical-induced cell death1) was originally isolated on the basis of its sensitivity
to ozone. An rcd1 suppressor mutant line 311 was later isolated on the basis on its tolerance to ozone. Both
rcd1 mutant and the suppressor mutant 311 have higher proline content than the wild type. The proline contents
in rcd1 mutant and the suppressor mutant 311 are about 80% higher and about 40-fold higher than in the Col-0
wild type, respectively.
In our research project, we will gain insight to the molecular basis of regulation of proline metabolism in
Arabidopsis, and elucidate the role of proline in abiotic stresses, especially regarding the signaling components
involved. The main objectives are to perform positional cloning of the gene causing proline accumulation in
the rcd1 suppressor mutant line 311, and characterize the mutant, specifically in relation to oxidative stress
responses, including salt and cold tolerance. So far, we have backcrossed the line 311 once to the Col-0 wild
type, and analyzed F2 plants for the presence of rcd1 mutation and for proline and total carbohydrate contents.
The plants without the rcd1 mutation were screened and only a few plants with high proline content were
found. Surprisingly, we found instead plants with lower proline content than the wild type, but with much higher
total carbohydrate content than the wild type. The low proline and high carbohydrate accumulating mutants are
also characterized by higher anthocyanin content. Fine structural analysis and more detailed chemical profiling
will be described.

75

37 VARIOUS TECHNIQUES TO MEASURE THE ANTIOXIDANT CAPACITY OF STRESSED


PLANTS: CHANGES DURING INDUCTION OF HYPERHYDRICITY IN PRUNUS AVIUM SHOOTS
CULTURED IN VITRO.
Claire KEVERS, Thierry FRANCK, Jacques DOMMES
University of Liege, Boulevard du rectorat, 27, 4000, Liege, Belgium, c.kevers@ulg.ac.be

In vitro culture is a source of stress conditions (1) that can specially lead to the phenomenon of hyperhydricity
(abnormal shoots with a translucent aspect generally due to a chlorophyll deficiency and higher water content)
during micropropagation. Hyperhydricity of P. avium was induced and expressed in one 4 weeks in vitro
multiplication cycle simply by replacing agar by gelrite (2). Previous works had shown that the enzymatic
defence systems have lower activities in hyperhydric shoots than in normal ones. Nevertheless markers of lipid
peroxidation were measured at a lower level in hyperhydric shoots with an unchanged free fatty composition
suggesting that hyperhydric shoots had an alternate capacity of defence (3, 4).
The variations of the antioxidant capacity were measured by several tests during the culture of P. avium shoots
in normal and hyperhydric conditions. Five methods allowing to measure the antioxidant capacity were selected
so as to span a diversity of technical approaches: TEAC (radical ABTS), DPPH (radical used to measure
reducing capacity), ORAC (Oxygen Radical Scavenging Capacity), red blood cell hemolysis (protection of
biological sample), and ESR (for direct free radical evaluation). First, the antioxidant capacities of standard
compounds were measured by the various assays, showing significant differences in free radical scavenging
activity according to the method used. To standardize reporting on antioxidant capacity, it was proposed to use
a weighted mean of the values obtained using four assays.
The antioxidant capacity was generally stable during the 2 first weeks in normal and hyperhydric conditions; at
day 21, the decrease of this capacity was more important in normal conditions. The more important antioxidant
capacity in hyperhydric tissues, observed after 21 days of culture in gelrite, can belong to a process of defence.
In further study, we will study the evolution of the antioxidant capacity after a longer time of culture.
1. Kevers C et al (2004) Plant Cell Tiss Organ Cult 77, 181-191.
2. Franck T et al (1995) Plant Growth Regul 16, 253-256.
3. Franck T et al (1998) J Plant Physiol 153, 339-346.
4. Franck T et al (2004) Plant Physiol Biochem 42, 519-527.

76

38 GENE EXPRESSION RESPONSES OF PAPER BIRCH (BETULA PAPYRIFERA) TO ELEVATED


CO2 AND O3 DURING LEAF MATURATION AND SENESCENCE
Sari KONTUNEN-SOPPELAa, Juha PARVIAINENd, Hanna RUHANENe, Mikael BROSCHb, Markku
KEINNENa, Ramesh C. THAKURc, Mikko KOLEHMAINENd, Jaakko KANGASJRVIb, Elina OKSANENa,
David F. KARNOSKYc, Elina VAPAAVUORIe
University of Joensuu, Faculty of Biosciences, PL 111, 80101, Joensuu, Finland,
sari.kontunen-soppela@joensuu.fi
b
University of Helsinki, Faculty of Biosciences, Department of Biological and Environmental Sciences,
Helsinki, Finland
c
Michigan Technological University, School of Forest Resources and Environmental Science, United States
of America
d
University of Kuopio, Department of Environmental Sciences, Kuopio, Finland
e
Finnish Forest Research Institute, Suonenjoki Unit, Finland
a

Gene expression responses of paper birch (Betula papyrifera) leaves to elevated concentrations of CO2 and
O3 were studied with microarray analyses from three time points during the summer of 2004 at Aspen FACE
experiment, in Rhinelander, WI, USA (http://aspenface.mtu.edu/). Normalized microarray data were analyzed
with clustering techniques, Self-organizing Maps, K-means clustering and Sammons mappings, to detect similar
gene expression patterns within sampling times and treatments. Most of the alterations in gene expression
were caused by elevated [O3], alone or in combination with elevated [CO2]. Ozone induced defensive reactions
to oxidative stress and earlier leaf senescence, seen as decreased expression of photosynthesis- and carbon
fixation-related genes, and increased expression of senescence-associated genes. Transport and proteolysis
genes were activated, indicating that at least some remobilization of nutrients from senescing leaves for
storage was completed. The effects of elevated [CO2] reflected surplus of carbon that was directed to synthesis
of secondary compounds. In the combined elevated [CO2]+[O3] treatment gene expression changes were
treatment-specific, or similar to O3 treatment, indicating that CO2 cannot totally alleviate the harmful effects
of O3. Some GE changes were specific to the combined elevated [O3]+[CO2] treatment, showing differential
expression of steroid biosynthesis genes, for example. This result emphasizes the fact that experiments with
ozone or CO2-exposure alone are not sufficient to predict plant responses to these gases together, and that
field experiments with multiple variables are pivotal in order to understand responses to future environmental
conditions.

77

39 PHOSPHATIDIC ACID CONTENT CHANGES IN MAIZE ROOT MEMBRANES IN RESPONSE TO


SALT TREATMENT
Olga KONTURSKA, Tatiana PALLADINA
M.G. Kholodny Institute of Botany, National Academy of Science of Ukraine, 2 Tereschenkivska str, 01601,
Kyiv, Ukraine, konturska@ukr.net

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second
messenger phosphatidic acid. It can be generated by two independent signaling pathways via phospholipase
D (PLD) and via phospholipase C (PLC). The synthetic compound treatment is used in order to increase
the plant salt tolerance. To this effect we used preparation methyure (uracil derivative) and ivine (pyridine
derivative). The aim of the present study was to evaluate the effects of low and high concentration of NaCl and
synthetic preparation on phosphatidic acid content and PLD activity.
Seeds of maize were treated by 10-7 M methyure and ivin. 7-old-days seedlings were exposed at 0.05M and
0.1M NaCl presence. PLD activity in tissue of root was assayed spectrophotometrically. Lipid fraction was
resolved via thin layer chromatography.
The salt exposition to 0.05M NaCl increased the phosphatidic acid content in plasma membrane and the PLD
activity. The effect was observed by 1-day salt exposition and 10-day salt exposition. The salt exposition to
0.1M NaCl also increased the phosphatidic acid content in plasma membrane respectively non-salt control.
However, the PLD activity decreased under such conditions. The synthetic compounds treatment increased
the PLD activity under the salt condition. It was shown that methyure treatment decreased the phosphatidic
acid content in plasma membrane. The ivin treatment had contrast effect.

78

40 STRUCTURAL CHARACTERIZATION OF CYTOKININ OXIDASE/DEHYDROGENASE MUTANTS


David KOPENa, Hana POPELKOVAe, Catherine MADZAKd, Pierre BRIOZZOb, Marek EBELAa, Ivo
FRBORTa, Amel MAJIRAc, Michel LALOUEc, Nicole HOUBA-HRINc
Palack University, Faculty of Science, lechtitel 11, 783 71, Olomouc, Czech Republic,
kopecny_david@yahoo.co.uk
b
UMR INRA-INAPG 206 de Chimie Biologique, Institut National Agronomique Paris-Grignon, 78850
Thiverval-Grignon, France
c
Laboratoire de Biologie Cellulaire, INRA, Route de Saint-Cyr, F-78026 Versailles Cedex, France
d
Laboratoire de Microbiologie et Gntique Molculaire, INRA-CNRS-INAPG, F-78850 Thiverval-Grignon,
France
e
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor,
MI 48109-1048, United States of America

FAD-containing cytokinin oxidases/dehydrogenases (CKO) catalyze the oxidative breakdown of isoprenoid


cytokinins to adenine/adenosine and the corresponding unsaturated aldehydes. Oxygen as well as quinones
can re-oxidize FAD reduced during catalytic reaction. While cytokinin substrate binds at the active site, the
binding of electron acceptor is not fully understood. Site-directed mutagenesis with subsequent X-ray analysis
provides a powerful tool to study amino acid residues of interest and to clarify their function and importance.
A CKO from Zea mays (ZmCKO1) was cloned and expressed in the yeast Yarrowia lipolytica using pINA1267
vector. After site-directed mutagenesis, ZmCKO1 mutants were obtained in the same way and their kinetics
was analyzed. Three mutants were prepared based on the sequence similarity to quinone-binding motif of
fumarate reductase C. However, none of the mutants exhibited the expected lower ratio dehydrogenase versus
oxidase activity compared to the wild-type ZmCKO1. Active-site mutants pointed out the importance of D169
as a crucial catalytic residue. Mutation of residues located at the entrance or inside the active site like E381,
P427 or V378, strongly affected the substrate specificity, sensitivity to inhibition by N-phenyl-N-pyridylurea
derivatives as well as reaction rates with various electron acceptors. Mutant H105A containing noncovalent
FAD was active and showed both reduced reaction rates and affinity to natural substrates. Several mutants
were crystallized, crystals infiltrated with substrates or inhibitors and then X-ray data were collected up to 1.8
resolution. Refinement of the structures is underway.
Supported by grants No. MSM 6198959215 from the Ministry of Education, Youth and Sports of the Czech
Republic and No. 522/08/P113 from the Czech Science Foundation.

79

41 REMOVAL OF ANTIBIOTIC RESISTANCE GENE FROM TRANSGENIC PLANTS USING TISSUE


SPECIFIC PROMOTERS
Jana LIBANTOVA, Jana MORAVKOV, Eva BOSZORDOV, Martin JOPK, Ildik MATUKOV
Institute of Plant Genetics and Biotechnology, Department of Molecular Biology and Biotechnology,
Akademicka 2, 950 07, Nitra, Slovakia, jana.libantova@savba.sk

Transgenic plants are considered to be powerful tool to combat various forms of the environmental stress. They
usually contain the desired gene representing by the transgene(s) reducing the impact of certain type of stress
and selectable marker gene. The latter is introduced into the plants with the aim to prefer the regeneration
of the plants only from transgenic cells on media containing selectable agent. Despite the large number of
marker genes that exist for plants, the antibiotic resistance genes are the most efficiently used markers for
selection of the transformants. After completing their role in the process of transgenic regeneration they become
superfluous. In addition their permanent presence in plants that are intended to be released into environment is
undesired due to bio-safety concerns. Although there are several methods how to prepare selectable markerfree transgenic plants, most of the current excision methods are based on site-specific recombination systems.
Self-excision variant of Cre/lox recombination system is based on the excision of the selectable marker gene
and the cre recombinase at once. It requires the application of tissue specific promoter that controls the
expression of cre recombinase and at the same time excision event. For this purpose we tested excision event
in transgenic plants of tobacco that was controlled by seed specific cruciferin promoter. At present we focused
on isolation and characterization of new tissue specific promoters applicable in this strategy.

80

42 EXPRESSION OF A BACTERIAL BIFUNCTIONAL -GLUTAMYLCYSTEINE LIGASE


- GLUTATHIONE SYNTHETASE IN TOBACCO RESULTS IN MASSIVE GLUTATHIONE
ACCUMULATION AND IN TOLERANCE TO OXIDATIVE AND HEAVY METAL STRESS
Verena LIEDSCHULTEa, Andreas WACHTERb, Thomas RAUSCHa
University of Heidelberg, Heidelberg Institute for Plant Science, Department of Molecular Physiology, Im
Neuenheimer Feld 360, 69120, Heidelberg, Germany, vliedschulte@hip.uni-heidelberg.de
b
University of Tbingen, Center for Molecular Biology of Plants, Department of General Genetics, Germany
a

The tripeptide glutathione (GSH) has various well-described functions in plant stress defence. Besides acting as
cellular redox buffer and in detoxifying reactive oxygen species, it sequesters heavy metal ions and xenobiotics
from the plant cell. Consequently, in order to improve plant stress tolerance, several attempts were made to
elevate plant GSH content by introducing -glutamylcysteine ligase (GCL), alone or together with glutathione
synthetase (GS), into different plant species. A moderate increase in GSH content often resulted in elevated
heavy metal tolerance, however, resistance to oxidative stress was unaltered or even impaired in comparison
to wild type plants. Possible limitations of previous approaches may have been the strong GSH feedback
inhibition of the ectopically expressed GCL enzymes and/or an accumulation of the intermediate -EC caused by
unbalanced expression of the two GSH biosynthetic enzymes. In the present study, we explored the possibility
to introduce both enzymatic functions simultaneously, as provided by bifunctional enzymes displaying both
GCL and GS activity. The GCL-GS enzyme from Streptococcus thermophilus seemed a promising candidate
for transformation, based on its kinetic parameters and a very weak feedback inhibition by GSH. Expression of
StGCL-GS in the cytosol or in the chloroplasts of Nicotiana tabacum increased GSH content up to twenty-fold
in the F0 generation and up to thirty-fold in the F1 generation compared to untransformed plants. To test for
performance under oxidative stress, transgenic tobacco plants were exposed to paraquat. Compared to wild
type, transformed seedlings and leaf discs from mature transformants showed improved growth and reduced
bleaching, respectively, in response to paraquat treatment. Furthermore, under Cd2+ stress an increased
production of phytochelatins was observed in the transformants, resulting in higher fresh weight and in higher
pigment content compared to wild type.

81

43 A METHOD FOR FINDING NOVEL STOMATA MUTANTS IN ARABIDOPSIS THALIANA


Ove LINDGRENa, Aurore COINCEa,b, Kristiina LAANEMETSa, Liina MARGNAa, Heino MOLDAUa, Hannes
KOLLISTa
Plant Signal Research Group, Institute of Technology, University of Tartu, Nooruse 1, Tartu 50411, Estonia,
Leif.Ove.Lindgren@ut.ee
b
UMR Ecologie et Ecophysiologie Forestieres 1137 INRA - UHP Nancy 1, France
a

Our research team is primarily focusing on molecular mechanisms of stomata function. We have recently
been able to establish a novel method for finding open stomata mutants in Arabidopsis thaliana. The method
consists of three different ways of measuring water-loss of cut (rosette) leaves. Each step is narrowing down
the number of mutant candidates until only the most convincing individuals remain. A mutant of SLAC1 (SLOW
ANION CHANNEL-ASSOCIATED 1) is used as a positive control in the screen. The slac1-3 mutant has no
obvious visible phenotype under normal or dry growth conditions (i.e. no wilting). Cut-off leaves from the mutant
are rapidly loosing water though, with a clear weight-loss difference to wildtype (50-80% as compared to 1530% within 90 minutes). We have found two putative mutant individuals with a strong water-loss phenotype.
One of these, cb1 (cool breath1) displays an even stronger water-loss phenotype than the slac1 mutants.
Grown at normal conditions cb5 is also displaying fused leaves, a character found in many cuticle-affected
mutants. Toluidine blue treatment revealed a strong staining in mutant rosette leaves further suggesting an
impaired cuticle. Interestingly, both toluidine blue staining and waterloss phenotypes are only observed in
older (2-3 weeks) rosette leaves, but not in younger true leaves, cotyledons or cauline leaves, suggesting
that the responsible gene is only active or necessary after a switch in leaf cuticle development. Further
characteristics of cb5 are currently being analyzed by using our gas-exchange equipment. cb1 also displayed
a strong water-loss phenotype in excised leaves but also had an extremely delayed bolting phenotype. Since
we were screening a 35S insertion line library, the affected genes are now being identified by TAIL-PCR.
Furthermore, we also have 40 EMS-mutagenized lines that were found in an earlier ozone-sensitivity screen
by Jaakko Kangasjrvi at the University of Helsinki. These already established mutants are currently being
analyzed using our gas-exchange equipment which is very sensitive to the state of stomata induction and
closure. We have established that at least one line does not display water loss in excised leaves, but is still
showing impaired stomata response in gas-exchange analysis.
This work is supported by grants from The Estonian Science Foundation (ETF).

82

44 ISOLATION AND CHARACTERIZATION OF KEY PLAYERS IN RICE COLD STRESS RESPONSE


Tiago LOURENCO, Nelson SAIBO, Cndido PINTO RICARDO, Margarida OLIVEIRA
ITQB, ITQB/IBET, Lab. Eng. Gentica de Plantas, Quinta do Marqus., 2784-505, Oeiras, Portugal,
tsantos@itqb.unl.pt

Abiotic stress is one of the major causes of productivity loss in rice worldwide. It is a complex trait poorly
understood and difficult to engineer. Transcription factors have been shown to play key regulatory roles in
a range of abiotic stresses, including cold, drought and salinity. Rice (Oryza sativa L.) production is mainly
limited to warmer temperatures, which may indicate that rice does not cold acclimate very efficiently. However,
the mechanisms underlying cold-stress tolerance in rice are still not fully understood. In this work, we isolated
the rice orthologue of the HOS1 gene which is involved in the cold signal transduction in Arabidopsis. The
isolated rice gene was named OsHOS1 and its function studied through a RNA interference (RNAi) transgenic
approach. The RNAi::OsHOS1 plants showed, under cold stress, a higher expression of the OsDREB1A
gene and of other stress-responsive genes. Furthermore, we analyzed the transcriptional regulation of this
gene through Yeast-One hybrid screenings and identified two transcription factors with an ERF-AP2 domain.
Although, we could not prove a specific interaction between the OsHOS1 and OsICE1, the rice HOS1 encodes
a protein with a function apparently similar to the one observed in Arabidopsis. Further analysis must be
performed to fully understand if/how the OsHOS1 regulates the cold transduction pathway in rice.

83

45 ATRBOH D, F AND JASMONIC ACID ARE KEY PLAYERS IN REGULATING LIGNIN


BIOSYNTHESIS
Priyadharshini MADHOU, Thorsten HAMANN, Mark BENNETT
Imperial College London, Sir Alexander Fleming Building, South Kensington, SW7 2AZ, London, United
Kingdom, pmadhou@gmail.com

The plant cell wall determines cell shape and is the main barrier against abiotic and biotic stresses. Our
laboratory is investigating, using Arabidopsis thaliana as a model system, the mechanisms and signalling
pathways regulating cell wall composition/structure. Previous research in our lab has shown that CBI resulted
in lignin accumulation after 4-6h of cellulose biosynthesis inhibition (CBI) in the primary root tip and was
preceded by activation of lignin biosynthesis genes (Hamann et al. 2008). Lignin deposition was significantly
reduced by a mutation in a NADPH oxidase and enhanced in the JASMONIC ACID RESISTANT 1 (JAR1)
background. Phytohormone measurements revealed increases in JA after 12h of CBI. The CBI-induced
transcriptional /phenotypic effects and increases in JA did not occur in the absence of hexoses (metaboliseable
and non-metaboliseable) or in the presence of osmotic support. These results implicate hexoses in CWI
signaling processes and suggest a key role for ROS and JA in the cell wall stress (CWS) response. We want
to understand the functions of reactive oxygen species (ROS) and jasmonic acid (JA) in regulating lignin
biosynthesis in response to CBI.
Results from this ongoing research will be presented at the conference.

84

46 EFFECTS OF OXIDATIVE SIGNALING AND GLOBAL WARMING ON CARBON RETENTION


PROCESSES IN BIRCH BETULA PENDULA AND ASPEN POPULUS TREMULA
Maarit MENPa, Sari KONTUNEN-SOPPELAa, Matti ROUSIc, Timo OKSANENb, Kaisa HARTIKAINENb,
Johanna RIIKONENb, Elina OKSANENa
University of Joensuu, Yliopistokatu 7, 80101, Joensuu, Finland, maarit.maenpaa@joensuu.fi
University of Kuopio, Kuopio, Finland
c
Finnish Forest Research Institute, Rovaniemi, Finland
a

The levels of tropospheric ozone (O3), an environmental pollutant, are predicted to rise simultaneously with
global warming at an annual rate of 0.5 - 2.5%. Both, the rising temperature and tropospheric O3 concentration
are likely to affect the carbon allocation processes. We studied in open field conditions the combined impacts
of this oxidative stress and warming on two fast-growing tree species, birch (B. pendula) and aspen (P.
tremula).
Experiment was arranged at University of Kuopio experimental area in 2007 with four 10m elevated ozone
exposure fields and four control study fields. Each field was split into two parts, with either elevated temperature
or not. Ozone exposure was realized with vertical perforated pipes around the field, releasing generated ozone
online from upwind direction according to ambient exposure. Infra-red heaters were used to create open-field
elevated temperature treatment. Plants were measured for growth, net photosynthesis, stomatal conductance
and gas exchange profiles along the plant axis. In addition, senescence was observed.
The results showed that the net photosynthesis was decreased in both species by elevated ozone. Elevated
temperature increased photosynthesis only slightly but total leaf area had remarkable influence on whole
seedling net C assimilation level. Temperature treatment increased the length of the growing season. The
experiment was continued with birches on season 2008 and gene expression and metabolisms were studied.

85

47 PHYSIOLOGICAL AND BIOCHEMICAL DIFFERENCES IN PARENTAL WHEAT FORMS CHINESE


SPRING AND SQ1 AS A BASIS OF STUDIES OF DOUBLED HAPLOID MAPPING POPULATION
LINES
Izabela MARCINSKAa, Ilona CZYCZYLO-MYSZAa, Edyta SKRZYPEKa, Agata STAWICKAa, Steve
QUARRIEb
Institute of Plant Physiology, Polish Academy of Sciences (IPP PAS), Niezapominajek 21, 30-239, Krakow,
Poland, i.marcinska@ifr-pan.krakow.pl
b
Consultative Bureau for International Projects, Serbia
a

The F1 hybrids of hexaploid wheat parental lines: Chinese Spring (CS) and SQ1 (a high abscisic acid-expressing
breeding line) were used to prepare the mapping population of DH lines (CSDH) consisted of 94 lines and
mapped with more then 400 molecular markers. This genetic map can be used to identify quantitative trait loci
(QTLs) for various response genes, also involved in drought resistance. In order to localize such genes on a
map, it is necessary to find a phenotype features (physiological, developmental or biochemical parameters)
which clearly differentiate between parental lines under water deficit conditions. The aim of these studies was
to show differences between Chinese Spring and SQ1 in the response to two water stress regimes. The yield
components, content of chlorophyll and carotenoids, kinetics chlorophyll fluorescence in this experiment were
carried out. The data have been completed under the tillering stage. Statistically significant changes, between
genotypes experiencing the drought stress, were noticed. On the basis of the results, the SQ1 genotype can be
considered as a drought resistant, due to higher activity of its photosynthetic apparatus, the higher decrease
of the content of chlorophyll and carotenoids, after drought treatment, as compared to CS genotype. These
parameters have been correlated with selected phenotypic traits and yield components of both stressed/non
stressed CS and SQ1 forms. All determined parameters can represent the potential traits, to identify the
drought resistance genes by using CSDH genetic map, which is currently analyzed.

86

48 EFFECT OF THE LATITUDE ON THE BIOCHEMICAL COMPOSITION OF TWO NATURAL


PRODUCTS: JUNIPER (J. COMMUNIS) NEEDLES AND BILBERRY (V. MYRTILLUS) LEAVES
Francoise MARTZa, Riitta JULKUNEN-TIITTOb, Sari STARKa
a
b

Finnish Forest Research Institute, Etelranta 55, 96300, Rovaniemi, Finland, Francoise.martz@metla.fi
University of Joensuu, Joensuu, Finland

Juniper (Juniperus communis) shoots and bilberry (Vaccinium myrtillus) leaves are natural products important
in food, pharmaceutical and cosmetic industries. Their demand has rapidly increased these last years and is
still expected to increase. Both species are widely collected in Finland but their biochemical composition is
expected to vary geographically. To study the effect of the growing conditions (latitude ranging form 59N to
69N) on the quality of juniper needles and bilberry leaves, 129 samples were collected all over Finland and
analyzed for their biochemical composition.
Juniper is known as a plant rich in terpenoids and phenolics and the content of both type of compounds
significantly increased with the latitude. Compared to southern samples, northern samples were richer in
monoterpenoids, proanthocyanidins and flavonols but poorer in apigenin derivatives.
Bilberry leaves are particularly rich in hydroxycinnamic acids (mainly chlorogenic acid) and flavonol glycosides.
Similarly to what was observed in juniper needles, the latitude has a significant effect on the composition of
bilberry leaves, the major effect detected in this study being an increase in the proportion of flavonol glycosides
and proanthocyanidins in higher latitudes. A higher radical scavenging activity was also detected in higher
latitudes. An increase in flavonol glycosides and a decrease in p-coumaric acids were also observed during
leaf maturation. The increase in flavonol glycosides was however detected only in plants growing under high
light conditions.
As a conclusion, important differences in the composition of juniper needles or bilberry leaves were measured
depending on the latitude. It is likely that the harsh growing conditions in high latitudes but as well N-poor soils
and high light in summer time are all abiotic factors affecting the secondary metabolisms of plants, and in this
case, improving the quality of the natural products.

87

49 THE MAIZE CHLOROPLASTIC CU/ZN SUPEROXIDE DISMUTASE IS ASSOCIATED WITH THE


CYTOSOLIC INTERFACE OF THE CHLOROPLAST ENVELOPE
Sergio MAUROa, Mathieu BOXUSb, Jean-Marie VANDERWINDENd, Yordan MUHOVSKIa, Alain MICHELc,
Bernard WATILLONa
Centre Wallon de Recherches Agronomiques, Chausse de Charleroi,234, 5030, Gembloux, Belgium,
mauro@cra.wallonie.be
b
Facult des Sciences Agronomiques de Gembloux, Gembloux, Belgium
c
Biological Chemistry Dept, Mons University, Belgium
d
Laboratoire de Neurophysiologie, Universit Libre de Bruxelles, Belgium
a

Maize chloroplastic Cu/Zn superoxide dismutase SOD1 was previously identified as the most abundant SOD
isozyme found in green leaves and the only SOD isozyme located in the chloroplasts (1). However not all
experimental approaches concur to associate SOD1 with either or both types of chloroplasts found in mature
maize leaves (2,3).
To further extend these localization studies intact bundle sheath and mesophyll chloroplasts were subjected
to fluorescence-activated cell sorting (FACS) using a polyclonal monospecific peptide antibody raised against
a 15-amino acid portion of SOD1 and a fluorescent secondary antibody. FACS analysis indicated that SOD 1
was present in both chloroplast types. Furthermore confocal laser scanning microscopy analysis revealed that
SOD1 is associated with the cytosolic interface of the chloroplast envelop.
1. Kernodle S., Scandalios J. (2001) Arch. Biochem and Biophysics, 391:137-147.
2. Pastori G., Foyer C., Mullineaux (2000) J. Exp. Bot. 51: 107-113.
3. Majerana W., Caia Y., Qi Sunb, van Wijka K. J. (2005) 3111-3140

88

50 THE PHYSIOLOGY OF BLACKBERRIES MICROPLANTS STORED IN VITRO AT LOW


TEMPERATURE
Sergey MEDVEDEVa, I.S. SAMATOVAa, T.A. GAVRILENKOb, E.I. SHAROVAa, O.Y. ANTONOVAb, S.E.
DUNAYEVAb, S.M. SCHIPAREVa
St. Petersburg State University, Department of Plant Physiology and Biochemistry, Universitetskaya str.,
7 / 9, 199034, St. Petersburg, Russia, ssmedvedev@mail.ru
b
N.I. Vavilov Research Institute of Plant Industry, Russia
a

It was established that the viability of blackberries microplants stored at +5 C under in vitro conditions is
inversely proportional to the level of hydrogen peroxide and is directly proportional to the peroxidase activity.
During the first 4 months of storage, the content of free proline in plants increased from 0.9 to 1.7 mg/(g of wet
weight) and then gradually decline to 0.6 mg/g by 12 months of storage. At that the content of ascorbic acid
has been reduced by 10 times in 4 month of storage. The degree of free proline accumulation, the reduce of
ascorbic acid content, as well as the dynamics of hydrogen peroxide may serve as a test for determining the
resistance of microplants to low temperature storage conditions. None of the used 24 RAPD-primers did not
reveal the differences between the spectra of amplicons of in vitro plants stored at low temperature, their field
analogs, as well as the microplants before and after 12 months of storage when significantly elevated levels of
hydrogen peroxide has been observed.
The study was supported by grant of Biovariety International / Vavilov Genebank / LoA 07/053 and grants of
Russian Foundation of Fundamental Research ( 05-04-49619; 08-04-00566).

89

51 INFLUENCE OF PHENANTHRENE ON GROWTHA AND ROOT EXUDATION OF SORGHUM


BICOLOR (L.) MOENCH
Wolfgang MERBACHa, Anna MURATOVAb, Neeru NARULAc
University of Halle-Wittenberg, Julius-Khn-Strae 25, D - 06112, Halle (Saale), Germany,
merbach@landw.uni-halle.de
b
Institute of Biochemistry and Physiology of Plants and Microorganisms Saratov, Russia
c
Haryana Agricultural University Hisar, Dept. Microbiology, India

The objective of this paper was to study the effect of phenanthrene (Ph) on growth and root exudation of
Sorghum bicolor (L.) Moench in pot experiments. Following results were obtained:
- Ph concentration up to 10 ppm did not cause significant effects on plant survival and growth. 100 ppm of Ph
had substantial toxic effects of Sorghum: plant survival was decreased by almost 50%, shoot and root biomass
production was strongly inhibited.
- Root exudation was strongly affected by a Ph induced damage of root system. The total amount of exuded
compounds was reduced by 78%. This was mainly a result of reduced root biomass. Only of lower exudation
per unit root surface could be observed.
- High Ph concentration resulted in a reduced exudation of organic acids (malate, transaconitate, citrate). In
average, the acid exudation was reduced by 77%.
- The neutral exudates fraction (fructose, glucose, maltose, galactose) was reduced under the influence of
high Ph concentration by 84%, first of all a result of root biomass inhibition. Low Ph concentration seems to
stimulate the carbohydrate exudation.
- High Ph concentration resulted in a substantial decrease of amino acid exudation (alanine, leucine, serine,
glycine), whereas low Ph concentration did not affect their amount significantly.
- The protein content in the rhizosphere of Sorghum plants was increased by Ph application.
- Related to mg protein in root exudates, enzymatic turnover of peroxidase(s) increased strongly as a result of
Ph treatment. Oxidase and tyrosinase activities were under the influence of Ph treatment increased also.

90

52 RELATIONSHIPS BETWEEN RHIZOSPHERE MICROFLORA AND BITUMEN IN SOIL


Wolfgang MERBACHa, Anna MURATOVAc, Neeru NARULAd, Peter KUSCHKb
University of Halle-Wittenberg, Julius-Khn-Str. 25, D - 06112, Halle (Saale), Germany,
merbach@landw.uni-halle.de
b
UFZ Centre of Environmental Research Leipzig-Halle, Germany
c
Institute of Biochmistry and Physiology of Plants and Microorganisms Saratov, Russia
d
Haryana Agric. University, Department of Microbiology, Hisar, India
a

The microbial communities and their degradative potential in rhizosphere of alfalfa (Medicago sativa) and
reed (Phragmites australis) and in unplanted soil in response to bitumen contamination of soil were studied
in pot experiments. According to the results of fluorescence microscopy, over a period of 27 months, bitumen
contamination of soil reduced the total number of microorganisms more significantly (by 75%) in unplanted
than in rhizosphere soil (by 42% and 7% for reed and alfalfa, respectively) and had various effects on some
important physiological groups of microorganisms such as actinomycetes as well as nitrogen fixing, nitrifying,
denitrifying, ammonifying, phosphate solubilizing, sulphuroxidizing, celluloytic and hydrocarbon degrading
microorganisms. The changes in the physiological structure of the microbial community under bitumen
contamination were found to hinge on not merely the presence of plants but also their type. It was noted that
the rhizosphere microflora of alfalfa was less inhibited by hydrocarbon pollution and had a higher degradative
potential than the rhizosphere microflora of reed.

91

53 FACTORS BEHIND NATURAL VARIATION IN OZONE SENSITIVITY AMONG ARABIDOPSIS


THALIANA ACCESSIONS
Ebe MERILOa, Mikael BROSCHEb, Florian MAYERc, Priit PECHTERa, Irina PUZRJOVAa, Gnter
BRADERb, Jaakko KANGASJRVIb, Hannes KOLLISTa
University of Tartu, Institute of Technology, Nooruse 1, 50411, Tartu, Estonia, ebe.merilo@ut.ee
University of Helsinki, Helsinki, Finland
c
Institute of Agronomy and Plant Breeding, Gregor Mendel-Strae 33, A-1180 Wien, Austria

Ozone sensitivity of 93 Arabidopsis accessions collected throughout the world was analysed together with
five so far identified ozone sensitive Arabidopsis mutants. Dramatic differences in O3 sensitivity, expressed as
visual leaf damage after 7hr O3 exposure with 300 ppb, were evident. The variation in leaf injury was much
higher among different naturally occurring accessions than among so far identified O3 sensitive mutants and
corresponding wild types, illustrating the value of genetic information provided by naturally occurring variation.
No significant correlation was detected between O3-induced leaf damage and climatic conditions (temperature
and precipitation) of the geographical origin for accessions.
We selected nine accessions with varying O3 sensitivity and tested their whole-plant stomatal conductance and
concentrations of antioxidants and stress hormones in order to detect factors behind different O3 sensitivity.
No sole factor was able to explain the variation in O3 sensitivity among accessions. In Cvi-0, by far the most
O3 sensitive accession, high stomatal conductance was responsible for significant visible injury. However, in
8 accessions (all but Cvi-0), higher initial levels of abscisic acid and salicylic acid were correlated with higher
O3 sensitivity.
We also tested the correlation between stomatal status and O3-induced damage of all 93 accessions. Significant
correlations between O3-induced leaf damage and either total leaf (abaxial+adaxial) stomatal conductance or
fresh weight loss of detached leaves were found, indicating the role of stomatal openness. In conclusion,
stomatal conductance was an important factor in controlling natural variation in ozone sensitivity among
Arabidopsis accessions, whereas the initial high levels of some stress hormones also tended to be involved.

92

54 LOW CO2 PREVENTS THE OZONE-TRIGGERED RAPID TRANSIENT STOMATAL CLOSURE


Heino MOLDAU, Irina PUZRJOVA, Hannes KOLLIST
University of Tartu, Nooruse 1, 50411, Tartu, Estonia, heino.moldau@ut.ee

Increased permeability of voltage-gated ion channels in guard cell plasma membrane under acute exposure to
a stress stimulus is a common event in electrophysiological experiments in vitro. The result is intense efflux of
osmotic ions, membrane depolarization and rapid stomatal closure. An intriguing example observed in vivo is
the ozone-triggered Rapid Transient Stomatal Closure (RTSC) in intact Arabidopsis leaves where the recovery
occurs even under O3 exposure continuation (Nature, 452: 487-491, 2008).
To test the necessity of sufficient plasma membrane depolarization for triggering the RTSC, we put forward the
following prediction. If guard cell plasma membrane is hyper-polarized before the onset of an ozone pulse, the
O3-triggered depolarization may be insufficient to reach the membrane potential where the ion efflux channels
are activated. In this case the RTSC may be absent.
To hyper-polarize guard cells differentially, we exposed intact A. thaliana rosettes to various CO2 concentrations
in the range from 400 to 10 ppm for up to 60 min. Then, the given CO2 still on, the rosette was allowed to
absorb about 4.5 mol O3 per m2 leaf area (exposure to 250 - 350 ppb O3 for 3 4 min, depending on stomatal
conductance).
The O3-triggered RTSC was clearly less pronounced under 200 ppm CO2 than under 400 ppm CO2. Under CO2
100 ppm the RTSC was entirely absent. Even a 2-min exposure to 100 ppm CO2 was sufficient to prevent the
RTSC. If the absorbed O3 dose was doubled, the RTSC appeared also in rosettes exposed to 100 ppm CO2,
but it was still absent when CO2 was 50 ppm.
These data support the conception that a low potential on guard cell plasma membrane is a prerequisite for
the O3-triggered RTSC to occur in intact plants. To quantify the roles of different voltage-gated ion channels
in shaping the RTSC, their activity windows should be determined during free-running membrane potential
recordings under combined exposures to CO2 and O3.

93

55 ANALYSIS OF THE NATURAL VARIABILITY OF RICE (ORYZA SATIVA L.) THROUGH


ECOTILLING IN SALT TOLERANCE GENES
Sonia NEGRAOa, Cecilia ALMADANIMa, Kenneth Mcnallyb, M.Margarida OLIVEIRAa
ITQB, Av. Republica- Estacao Agronmica Nacional, 2780- 157 Oeiras, Oeiras, Portugal,
snegrao@itqb.unl.pt
b
IRRI, The Philippines

Rice (Oryza sativa L.) is the most important food crop, feeding almost half of the worlds population. Salinity
is one of the major obstacles to increasing production in rice growing areas worldwide, being rice rated as a
salt-sensitive crop. Recent advances in molecular marker techniques, together with functional genomics and
bioinformatics show that salt tolerance previously described as a complex trait controlled by quantitative trait
loci (QTL), can now be associated with major loci that contribute to quantitative traits.
In order to clarify gene function and identification, and apply this information to breeding and conservation of
natural diversity, it is necessary to rely on the genome sequence of one or a few reference lines. However,
sequence data on few lines may not be informative enough to refine gene function. To identify allele variants
associated to salinity tolerance, we are using the EcoTILLING technique. With this strategy it is possible
to characterize natural alleles at a specific locus, across numerous germplasm accessions, enabling SNP
discovery and haplotyping of candidate loci.
We are targeting 5 genes already described and characterized as being related to the increase of salinity
tolerance in rice and we will present the preliminary results obtained so far.

94

56 METABOLISM OF VARIOUS NITROGEN FORMS IN HIGHER PLANTS


Marek NEUBERGa, Milan PAVLIKb, Daniela PAVLIKOVAa, Ji BALKa, Regina KALISZOVAa
Czech University of Life Sciences, Kamycka 129, 16521, Prague, Czech Republic,
euronymos666@seznam.cz
b
Academy of Science of the Czech Republic, Czech Republic

The effects of NH4+, NO3- and mixture of both on growth, and nitrogen (N) uptake kinetics of maize (Zea
mays L.) and spinach (Spinacia oleracea L.) were studied in the plot experiment. Also the effect of different
fertilization systems, especially CULTAN (Controlled Uptake Long Term Ammonium Nutrition) was investigated
in this study. Uptake of various form of nitrogen was measured as levels of certain physiological (free) amino
acids compared with protein hydrolysates of plants cultivated on the same concentration of nitrogen and
determined by GS-MS. Derivatization and cleaning steps in amino acid isolation procedure were made using
Phenomenex sets for analyzing of physiological (free) amino acids protein hydrolysates.
As modeling plant were used maize (Zea mays L.) and spinach (Spinacia oleracea L.) because of its very
well known levels of both (free amino acids and amino acids in proteins). Significant differences in the levels
of free amino acids were found. Levels of alanine and glutamine of maize aboveground biomass growing on
treatment with urea ammonium nitrate solution (DAM 390) were higher compared with individuals treated by
ammonium nitrate. Levels of alanine and glutamine (in spinach) were lower compared with those in maize
and depending on form of utilized nitrogen. By comparison conventional system of fertilization with system
CULTAN were detect differences in level of proline suggesting that CULTAN system can affect osmotically
balance of treated individuals.
A financial support through the grant NAZV 1G58027 (National Agency for Agricultural Research) and CIGA
21140/1313/3107 (Czech University of Life Sciences Prague) is gratefully acknowledged.

95

57 PARTIALLY REDUNDANT FUNCTIONS OF CLASS XI MYOSINS IN DIFFERENTIATING


ARABIDOPSIS TRICHOMES AND ROOT HAIRS
Eve-Ly OJANGU, Kristel JRVE, Erkki TRUVE, Heiti PAVES
Tallinn University of Technology, Akadeemia tee 15, 12618, Tallinn, Estonia, eve-ly.ojangu@ttu.ee

Myosins form a large superfamily of molecular motors that move along actin filaments. Class XI myosins are
characterized by the large number of IQ domains and long C-terminal tail. In 2007, we described the first
abnormal phenotype for loss-of-function mutant of class XI myosin - XIK. Homozygous XIk mutants were
having short root hairs and stem and leaf trichomes with twisted shape and irregular size. Others have later
reported that also Arabidopsis plants missing mya2 gene have shorter root hairs and that mya2/XIb double
knockouts have root hairs with the length of only 15% of the wild type value. In addition, it was demonstrated
that the motility of Golgi stacks, peroxisomes and mitochondria is reduced in XIk as well as in mya1 and mya2
homozygous knockouts. Here we report that the root hair phenotype of mya2 knockout plants is dependent on
growing conditions. The root hairs of double knockout line XIk/mya2 has stably short root hairs. Moreover, the
stem and leaf trichomes of XIk/mya2 as well as XIk/mya1 plants are severely malformed whereas mya1 single
knockout lines do not reveal any detectable phenotypic changes. These data indicate that class XI myosins
have partially redundant functions in the epidermal differentiation of Arabidiopsis.

96

58 INCREASING NIGHT-TIME TEMPERATURES INCREASE VOLATILE COMPOUND EMISSIONS


FROM BETULA PENDULA AND POPULUS TREMULA THROUGH COMPLEX CHANGES IN
GENE ACTIONS
Elina OKSANENa, Mohamed IBRAHIMc, Maarit MENPa, Viivi HASSINENb, Sari KONTUNEN-SOPPELAa,
Lukas MALECd, Matti ROUSIe, Liisa PIETIKINENb, Jarmo HOLOPAINENc
University of Joensuu, Faculty of Biosciences, Yliopistokatu 7, 80101, Joensuu, Finland,
Elina.oksanen@joensuu.fi
b
University of Kuopio, Department of Biosciences, Kuopio, Finland
c
University of Kuopio, Department of Environmental Science, Kuopio, Finland
d
Institute of Chemical Technology, Technicka, Prague, Czech Republic
e
Finnish Forest Research Institute, Vantaa Research Station, Finland
a

Plants emit several volatile organic compounds (VOC) that have crucial role in adaptation to high temperatures.
In this experiment impacts of increasing night-time temperature on terpenoid emissions and related gene
expressions in silver birch (Betula pendula) and European aspen (Populus tremula) clones were studied. The
plants were growing under five different night-time temperatures (6 C, 10 C, 14 C, 18 C and 22 C) while
day-time temperature was kept constant (22 C). VOC emissions were collected in the morning at 22 C and
analysed by GC-MS. In birch, emissions of DMNT, several sesquiterpenes, non-terpenes (Z)-3-hexenylacetate
and methyl salicylate increased with temperature, while in aspen increases were recorded in emissions of
DMNT and several monoterpenes and non-terpenes. Profiles of volatile emissions were specific for each
genotype in both species. We also demonstrated temperature-depended roles of genes encoding DXR, DXS,
IPP and IspS in regulation of monoterpene and isoprene biosynthesis. Our results indicate that increasing
night temperature may strongly affect the quantity and quality day-time VOC emissions of northern deciduous
trees through changes in gene expression pattern. Genetic regulation seems to be complex, involving several
negatively controlled components.

97

59 MOLECULAR MECHANISMS OF HEAVY METAL STRESS IN ARABIDOPSIS THALIANA


Kelly OPDENAKKER, Karen SMEETS, Tony REMANS, Jaco VANGRONSVELD,
Ann CUYPERS
Hasselt University, Agoralaan Building D, 3590, Diepenbeek, Belgium, kelly.opdenakker@uhasselt.be

During the last decades, heavy metal pollution is a problem of increasing concern as a result of anthropogenic
activities. Metals such as cadmium (Cd) and copper (Cu) are highly toxic and interact with physiological
processes in all living organisms, even at low concentrations. In plants, processes such as growth,
photosynthesis and transpiration are disturbed which results in visible symptoms such as chlorosis, necrosis
and growth impairment. At the cellular level, exposure to either metal result in a production of ROS, a condition
called oxidative stress. The importance of the MAPK (mitogen-activated protein kinase) pathway in the cellular
defence against heavy metal stress was hypothesised several times during the last few years. Nevertheless,
detailed information regarding the specific role of these MAPK components during different metal treatments
remains rather scarce. To unravel the complexity of these signals during stress responses induced by different
metals, the role of OXI1 in heavy metal induced oxidative signalling was investigated on the gene expression
level after Cd and Cu exposure in both wild type and oxi1 mutants. Our results indicate a highly important role
for OXI1 in the cellular redox-signalling during Cu-induced toxicity in the roots of Arabidopsis thaliana that
was not the case for Cd stress. Cu-induced responses in the roots of these mutants influenced interorgan
regulatory mechanisms and root-to-shoot signalling. Hydrogen peroxide and lipoxygenases were revealed as
potential downstream targets of OXI1 under both metal conditions.

98

60 DECIPHERING HOST/ENDOPHYTE INTERACTIONS IN MYCORRHIZA FORMATION. FROM


STRESS TO SYMBIOSIS. A TRANSCRIPTOME AND METABOLOME ANALYSIS
Maria Salom PAIS, Monica SEBASTIANA
BioFIG, Campo Grande, 1749-016, Lisbon, Portugal, mpais@ymail.com

Mycorrhizae are symbiotic associations resulting from the interaction of soil fungi and the roots of vascular
plants. Ectomycorrhiza establishment encompasses a series of global changes in both organisms. In the
early hyphaeroot contact stage, host plants release critical metabolites to the rhizosphere that trigger spore
germination, hyphae growth towards the root, and early steps of mycorrhiza formation. The question is how
these stages are regulated and what factors trigger their development. They involve the expression of genes
encoding developmental proteins, enzymes and transporters that regulate symbiosis development. Dramatic
alterations occurring in gene expression are required for symbiosis development. Plant and fungal genetic
switches necessary for ECM development remain unidentified to date and little is known on the initial contact
phase. The model system used is the mycorrhizal association between Castanea sativa and Pisolithus tinctorius.
Transcriptome and metabolome profilings were performed for early steps of mycorrhiza establishment. The
metabolome profiling results and the integrated maps generated, reveal that the main metabolites crucial for
mycorrhizae formation are, at the plant level, stress and defence related amino acids (proline, alanine and
glycine); cell wall synthesis and degradation related metabolites; saccharides (glucose, fructose and sucrose);
galacturonic acid (an indicator of cell wall destruction); aromatic amino acids and signalling related metabolites
(myo-inositol, polyamines and galactinol); at the fungi level: trehalose-fungi (marker of fungi presence);
gluconic acid (promoter of ectomycorrhizae formation); glutamine (marker of mantle development and Hartig
net formation). Transcriptome profiling revealed among up regulated transcripts, genes encoding for stress
and defence response proteins (a cystatin, a cystatin-like protein, a defensin and a universal stress protein).

99

61 INVESTIGATION OF CANDIDATE WHEAT TRANSCRIPTION FACTORS RESPONSIBLE FOR


FROST TOLERANCE
Istvan PAPPa, Fruzsina SZIRAc, Csilla DEAKa, Gergely BALAZSa, Barnabas JENESb, Andrea BALOGHb,
Gabor GALIBAc
Department of Plant Physiology and Plant Biochemistry, Corvinus University of Budapest, Mnesi u, 1118,
Budapest, Hungary, istvan.papp2@uni-corvinus.hu
b
Agricultural Biotechnology Center, 2100 Gdll, Hungary
c
Agricultural Research Institute of the Hungarian Academy of Sciences, 2462 Martonvasar, Hungary

C repeat binding factor (CBF) genes are master regulators of cold stress response and frost tolerance in a
number of species. A cluster of (CBF) genes was recently mapped to a frost resistance locus (Fr-Am2) in
einkorn wheat (Triticum monococcum). Three of these genes were chosen for further analysis. The cDNAs
coding for these genes were cloned into appropriate expression vectors under a constitutive promoter and
transformed into Arabidopsis. Transgenic plants expressing the transgenes were evaluated for morphological
as well as for stress tolerance traits. One of the candidate CBF genes had an effect on gross morphology
of Arabidopsis inducing dwarf phenotype. According to preliminary results the same transgenic Arabidopsis
lines also exhibited increased frost tolerance. The other tested CBF genes had no appreciable effect on the
plants survival in frost, nor did they induce any morphological alteration. Further experiments are underway to
address possible changes in responses to other stress factors in the transgenic Arabidopsis lines.

100

62 FATTY ACID AMIDE HYDROLASE-MEDIATED INTERACTIONS ON N-ACYLETHANOLAMINE


AND ABSCISIC ACID SIGNALING IN PLANTS
Priit PECHTER, Joosep SEDMAN, Yuh-Shuh WANG
Institute of Technology, University of Tartu, Nooruse 1, 50411, Tartu, Estonia, priit.pechter@ut.ee

N-acylethanolamines (NAEs) are a group of bioactive lipid mediators that modulate both animal and plant
development. Their signaling activity is terminated as NAEs are hydrolyzed by the fatty acid amide hydrolase
(FAAH). We have demonstrated that the Arabidopsis FAAH (AtFAAH) was capable of modulating endogenous
NAE levels as well as plant sensitivities to exogenous NAE. We also reported interaction between NAE and
abscisic acid (ABA) signaling in wild-type Arabidopsis. When NAE and ABA were applied together, a synergistic
growth inhibitory effect was observed. However, this interaction appeared to be altered in transgenic plants
overexpressing the AtFAAH. Instead of showing normal or reduced sensitivity to ABA, as would be predicted
based on their lower endogenous NAE content, plants overexpressing the AtFAAH displayed significant ABA
hypersensitivity and tendency to wilt faster. Our preliminary results suggested that the C-terminal domain of
the AtFAAH, rather than its amidase activity was crucial for the NAE/ABA interaction as plants expressing an
AtFAAH-GFP fusion exhibited slight ABA resistance and tolerance to exogenous NAE. In this study, plants
expressing a GFP fused to either N- or C-terminus of the AtFAAH are examined for their responses to NAE/
ABA in order to verify whether a free AtFAAH C-terminus is required for interaction with ABA signaling. A splitubiquitin membrane yeast two-hybrid approach will be applied on identification of potential protein partners.
In addition, suppressor screens on mutants with the AtFAAH overexpressor background will be conducted to
assist on discovery of additional players on NAE/ABA interaction. Using pharmacological and physiological
assays, as well as molecular genetics approaches, we anticipate results from this project to provide new
insight into the mode of action on both NAE and ABA signaling.

101

63 COPPER UPTAKE BY ELSHOLTZIA SPLENDENS AND ALLIUM SATIVUM


Sarka PETROVAa, Petr SOUDEKa, Lukas SEDLACEKb, Tom VANKa
Institute of Experimental Botany AS CR, Rozvojova 263, 16502, Prague, Czech Republic,
petrova@ueb.cas.cz
b
Charles University, Czech Republic
a

Copper as one of essential elements is being associated with various proteins with important roles in redox
reactions. However, at high concentrations copper can become toxic to plants and affect a wide range of
biochemical and physiological processes. It is known to damage cell membranes by binding to the sulfhydryl
groups of membrane proteins and inducing lipid peroxidation. Furthermore, the toxicity of copper can be
considered an oxidative stress mediated by reactive oxygen species. Some plant species actively take up
and accumulate metals to high levels in the above ground tissues, far exceeding the levels detected in the
soil. Plants that accumulate high level of metals have mechanisms to protect themselves from metal toxicity.
Metal binding peptides such as phytochelatins and metallothioneins serve as one of the many mechanisms for
coping with elevated metal stress in some plants. Copper strongly interacts with ligands containing N and S
and can also displace and substitute other metal-cofactors. Allium sativum is well known for its characteristic
resistance to biotic and abiotic environmental stresses, such as viral, bacterial and oxidative stress. On the
other hand, Elsholtzia splendens is a Chinese native Cu-tolerant plant species which can tolerate total levels
of 1500 mg/kg Cu in soil. In this study, we investigated the temporal sequence of physiological reactions,
including changes in chlorophylls contents and antioxidative enzymes activities and also copper contents in
roots and shoots of A. sativum and E. splendens seedlings after copper exposure.
Supported by project AVOZ 50380511 and COST FA 0605 (COST.FA 0605 OC 9039).

102

64 ARE STRESS-RESPONSIVE MIRNAS SYSTEMICALLY MOBILE?


Janin PIERITZa, Julia KEHRb
Max Planck Institute of Molecular Plant Physiology, AM Mhlenberg 1, D-14476, Potsdam-Golm, Germany,
pieritz@mpimp-golm.mpg.de
b
Centro de Biotecnologia y Genmica de Plantas (UPM-INIA), Campus de Montegancedo, M40 (km38),
28223 Pozuelo de Alarcn/ Madrid, Spain

The recently discovered microRNAs (miRNAs) have emerged as important players in plant stress responses.
miRNAs are endogenous, around 22 nucleotide long, small non-coding regulatory RNAs. Plant miRNAs usually
down-regulate the amount of their highly complementary target mRNAs post-transcriptionally. miR395, for
example, is accumulating during sulfate starvation and down-regulates its target genes: a low-affinity sulfate
transporter and a plastid localized ATP sulfurylase. During phosphate deficiency miR399 is up-regulated and
down-regulates the mRNA of its targets: an ubiquitin-conjugating E2 enzyme, which is involved in targeting
proteins to the degradation pathway, and a phosphate transporter. There is increasing evidence that systemic
signaling from shoot to root is an important component of the network regulating nutrient allocation. However,
if miRNAs could directly be involved in the information transfer is unknown and whether miRNAs are mobile
between cells and even long distance is still a matter of debate. To demonstrate that nutrient-stress responsive
miRNAs are phloem-mobile in vivo, we performed reciprocal micrografting experiments in Arabidopsis thaliana
between wildtype and hen1-1 mutants that are defective in miRNA methylation and thus produce only reduced
amounts of miRNAs. The successful grafts were transferred to hydroponic cultures deficient in various nutrients.
RNA was extracted and analyzed by RNA gel blots. We could detect a transport of several stress-related
miRNAs from wildtype shoot to hen1-1 roots, but not in the opposite direction, indicating that the miRNAs
follow the phloem assimilate transport from source to sink. In parallel samples, we could not detect an increase
of any miRNA precursors. We also analyzed whether the increase in miRNAs in the hen1-1 rootstocks led to a
down-regulation of the target-mRNAs, and could observe a decrease of several targets. These results indicate
that different stress-responsive miRNAs can indeed move from shoots to roots in grafting experiments, and
can affect the levels of their target mRNAs at the destination.

103

65 THE ROLE OF F-BOX-PROTEINS IN ABIOTIC STRESS AND STRESS-RELATED SIGNALING IN


ARABIDOPSIS THALIANA
Maria PIISIL, Gnter BRADER, M. Ali KEELI, Tarja KARIOLA, E. Tapio PALVA
University of Helsinki, Haagan urheilutie 18 B 18, 00320, Helsinki, Finland, maria.piisila@helsinki.fi

Arabidopsis genome encodes roughly 700 F-box proteins that function in protein degradation pathway as part
of the ubiquitin proteasome system (UPS). They are involved in various processes including plant metabolism,
hormonal responses as well as responses to environmental stresses. One example of the role of F-box
proteins in abiotic stress response is TUBBY-like protein 9 (TLP9) involved in abscisic acid (ABA) signaling
pathway. TLP9 interacts with XERICO that confers drought tolerance through increased ABA biosynthesis (Ko
et al., 2006). Moreover, F-box protein DOR was found to act as a negative regulator of the guard cell ABA
response and is also likely involved in regulation of ABA biosynthesis under drought stress in Arabidopsis
(Zhang et al. 2008). The objective of this study is to identify and characterize F-box proteins involved in abiotic
stress signaling in Arabidopsis. Using reverse genetic screen approach, we are screening mutants of F-box
proteins belonging to C1-C4 subfamilies based on the classification of Gagne et al. (2002). These subfamilies
contain many proteins associated with stress signaling (e.g. ORE9/MAX2, TLP9). The screening program
includes testing freezing and ozone tolerance as well as ABA responses. Freezing tolerance was studied
by exposing plants to -8 C. For testing ozone sensitivity, plants were subjected to 300 ppb of ozone. Seed
germination was studied in the presence of ABA. Preliminary results indicated several mutant lines showing
ozone sensitivity and altered sensitivity to ABA. The function of F-box genes potentially involved in altered
stress tolerance phenotypes will be further characterized by independent mutants, genetic complementation
and by transcriptome analysis using microarrays.

104

66 GENETIC BASIS OF ALUMINIUM RESISTANCE IN AVENA STRIGOSA SCHREB


Kornelia POLOK, Roman ZIELINSKI
Department of Genetics UWM, Plac Lodzki 3, 10-967, Olsztyn, Poland, kpolok@moskit.uwm.edu.pl

Aluminium, constituting 7.5% of the earth crust, belongs to the most abundant minerals in soil. Because of its
pH-dependent solubility, aluminium toxicity occurs only at soil pH values below 5.5. It is estimated that 40%
of the arable soils of the world are acidic, mainly as a result of acid rains and acidity caused by fertilisers.
The attempts to find Al-tolerance sources have been undertaken in all major cereals but most of them rarely
tolerate Al and if, only modest doses (an average 50 M in laboratory tests). The only tolerant cereal is rye
(up to 300 M Al), however it is of low interest for animal feed due to anti-nutritional factors. We demonstrated
that A. strigosa is the most tolerant species, being able to grow at Al concentrations from 200 to 600 M in
laboratory conditions without significant root damages. This feature makes the species interesting both to
sustainable agriculture and as a source of useful genes for other crops. Tolerance to aluminium in A. strigosa
seems to be of quantitative inheritance controlled by major QTLs. Comparative genomic approaches can
identify A. strigosa orthologs to the main Al tolerance gene found in Triticeae using recently mapped and
cloned genes including the ALTM1 gene in wheat, Alp in barley and ScALTM1 in rye. However, genetic studies
in A. strigosa are limited due to the lack of Al sensitive genotypes as exemplified by 56 accessions from world
collection that tolerated 400 M of Al in our studies. Thus, the aim of present research was to induce variation
in aluminium tolerance through chemical mutagenesis (MNH and NaN3) and use mutants to identify Al tolerant
genes in A. strigosa. Seven lines sensitive to 200 M of Al and two lines sensitive to 400 M but tolerant to
200 M were selected from 109 mutants studied. All sensitive lines demonstrated significant root and seedling
length reduction. Moreover, DNA markers (ISJ) linked with Al tolerance were identified, which will be useful in
genes identification.

105

67 THE ROLE OF SPPA PEPTIDASES IN THYLAKOIDS ACCLIMATION UPON LIGHT STRESS


Elena POZHIDAEVAa, Irina STERRb, Anna SOKOLENKO-NIEBLINGb
Timiryazev Institute of Plant Physiology RAS, Botanicheskaya str. 35, 127276, Moscow, Russia,
alenapoj@hotmail.com
b
Department fur Biologie I, Bereich Botanik, LMU, Germany
a

Intracellular proteolysis is important for regulation of metabolic and signaling pathways as well as protein
homeostasis and viability of cells and organelles, including chloroplasts. Previously, we identified a lightregulated protease SppA that is located in chloroplast thylakoids of Arabidopsis thaliana. To gain in function of
SppA we screened the T-DNA insertion collection established by Syngenta Biotechnology. BLAST searching
of the genomic sequence for SppA against this T-DNA insertion collection identified one potentially tagged
line. PCR-based genotyping and Western analysis confirmed that the plants were homozygous for a T-DNA
insertion.
We also used cyanobacterium Synechocystis sp. PCC 6803 as a model organism for study chloroplastregulated processes.Two genes, sppA1 and sppA2, are found in Synechocystis genome that encodes
homologues of the chloroplast SppA. Inactivation of the corresponding genes showed that both proteins,
SppA1 and SppA2, are required for light adaptation. We found that upon acclimation to higher light intensity
in the sppA1 mutant cleavage of the LR33 and LCM99 linker proteins does not occur. The SppA1-mediated
phycobilisome degradation is contributed in the mechanism responsible for a light stress adaptation.
The function of SppA2 which has half-size of SppA A. thaliana still unknown. Similar to sppA gene from plants
the expression of sppA2 gene is enhanced at higher light. However, SppA2 protein is rapidly degraded with a
half-life of 3 h under 350 E/m2 s (HL) and recovered after 4 h of exposure to 50 E/m2 s (LL). We found that
the SppA2 half-life is regulated by SppA1 or SppA1-controlled protease. The degradation of SppA2 is specific
to light and not to other stress conditions. These results suggest that SppA2 plays a role during cell recovery
from HL to LL and may be involved in the degradation of proteins required for photoprotection.

106

68 ROOT ARCHITECTURE IN POTATO: POTENTIAL FOR IMPROVING RESOURCE CAPTURE


AND QTL MAPPING
Ankush PRASHAR, Jane WISHART, Timothy GEORGE, Lawrie BROWN, Jacqueline THOMPSON, Gavin
RAMSAY, John BRADSHAW, Philip WHITE, Glenn BRYAN, Peter GREGORY
SCRI, Errol Road, Invergowrie, DD2 5DA, Dundee, United Kingdom, Ankush.Prashar@scri.ac.uk

Potato is a relatively sensitive crop in terms of yield, quality and pathogen resistance under limited water
supply. Potato is also considered to be inefficient in resource acquisition, using a disproportionate amount of
water and fertilizer, which conflicts with environmental protection measures and agricultural sustainability.
This study involves screening of a broad range of potato genotypes including the European tetraploid potato
(S. tuberosum Gp Tuberosum), primitive cultivated types (S. tuberosum Gp Phureja, Neotuberosum clones
selected from S. tuberosum Gp Andigena) and diverse germplasm from Commonwealth Potato Collection
(CPC) under field conditions to assess variation in rooting traits associated with resource capture and as the
initial stage in understanding their genetic framework.
Significant differences in root number, length and surface area were found. Total root length per plant varied
from 38.63m (22.76m) for the Tuberosum var. Pentland Dell to 123.02m (19.70m) for the Phureja var.
Mayan Twilight. Tuber yield, for the Tuberosum Group, was related to root length, while for the Phureja Group
yield was related to root number. Multivariate clustering based on rooting characters enabled separation of
S. tuberosum Groups Andigena, Tuberosum and Phureja suggesting potential genetic differences in resource
partitioning.
Absorption of water and nutrients is also determined by the proximity of the nutrients to the root surfaces together
with their availability and mobility. Thus, significant variability in root characteristics amongst potato genotypes
will help in determining efficiency of resource acquisition. Extreme genotypes will now be tested under drought
conditions to establish whether longer roots make whole plants more efficient. Molecular characterisation will
further enable resource capture improvement through genotypic and/or phenotypic selection of appropriate
root traits and their expression in relevant commercial germplasm.

107

69 EFFECTS OF SALINITY AND OSMOTIC STRESS ON GERMINATION, GROWTH,


PHOTOSYNTHESIS AND POTASSIUM FLUXES OF SELECTED GRASS SPECIES
Martin REICH
University Frankfurt/Main, Emil-von-Behring Str. 27, 60439, Frankfurt am Main, Germany,
reich.frankfurt@web.de

The onward salinisation of precious soils in arid and semiarid densely populated areas through irrigation
calls for new and sustainable techniques in agriculture. One of the big challenges to modern ecophysiology
is to contribute to the interception of those effects by identification and characterization of alternative crops
which grow and yield on oversalted soils. To define and measure the respective tolerance of a plant proper
parameters are needed.
Against this background the ambition of this work is to analyze a selection of Poaceae species in terms of
their potential to grow under different salt concentrations and to describe observed differences considering
physiological mechanisms.
Comparable experiments are arranged with one Panicum species of the C3 type (P. bisulcatum), two C4
species (P. bulbosum (NADP-ME), P.miliaceum (NAD-ME)) and the halophytic C4 grass Spartina anglica.
Further on Zea mays as a common agricultural crop and C4 model plant in botanical research is included.
C4 grasses are known to have a higher water use efficiency than C3 grasses which leads to a tolerance
to osmotic stress. Whether they are also tolerant to increasing salt concentrations is an important question
because of the reasons mentioned above.
Experiments are performed with different isosmotic concentrations of NaCl and PEG6000. This polymer is not
taken up by plant roots. Thereby the effects of the osmotic component of salt stress can be distinguished from
the physiological effects of NaCl accumulation in the plant tissue.
As indicators for tolerance to salt and osmotic stress the rate of germination, growth and photosynthetic
parameters are measured.
Furthermore the difference in the changes of potassium fluxes as a parameter for salt tolerance in the examined
species is proved by measurements with ion selective microelectrodes.

108

70 FUNCTIONNAL ANALYSIS OF GABA TRANSAMINASE (POP2) REVEALS THAT GABA IS


INVOLVED IN SALT STRESS RESPONSES AND PLANT DEVELOPMENT IN ARABIDOPSIS
THALIANA
Hugues RENAULTa, Abdelhak EL AMRANIc, Valrie ROUSSELb, Matthieu ARZELb, Alain BOUCHEREAUb,
Carole DELEUb
Plant Genetics and Biotechnologies, UMR 118 INRA-Universit, Campus de Beaulieu, Bat 14A, case
14.01, 35042, Rennes cedex, France, hugues.renault@univ-rennes1.fr
b
INRA, Agrocampus Ouest, Universit de Rennes 1, France
c
CNRS, Universit de Rennes 1, France
a

Among stress-responsive metabolites, -aminobutyric acid (GABA) occupies a singular place in plants. Indeed,
since decades it is mentioned that GABA accumulates in response to a wide range of environmental stimuli;
however its functions in plants remain unclear. Most of studies have pointed to the metabolic function of GABA,
especially in stressful conditions. Nevertheless, several lines of evidence also attest of a signaling role of
GABA as shown for pollinic tube growth and guidance, nitrogen absorption or 14-3-3 genes regulation.
In the present work, we investigated both metabolic and signaling functions of GABA in Arabidopsis thaliana by
performing a functional analysis of POP2 (Pollen-Pistil Incompatibility 2), encoding the first enzyme of GABA
catabolism. Combining genomics tools (pop2 mutants, POP2-overexpressing plants, pPOP2::GUS lines),
GABA feeding of plants and NaCl treatments, we designed plant systems with wide range of GABA content.
Among relevant results we obtained, we showed that both NaCl and GABA treatments affect pop2 mutants
but with different symptoms. In these mutants, GABA is significantly overaccumulated upon GABA and NaCl
treatments. Transcriptomic analysis of pop2 mutant treated with either GABA or NaCl revealed that GABA
clearly impacts plant development and particularly cell elongation but in two different ways, depending on
treatment. This property of GABA to repress cell elongation was linked to POP2 expression pattern which
is reconfigured during NaCl treatment. Possible involvement of GABA in organ expansion during stressful
conditions in Arabidopsis thaliana will be discussed.
Key words: Abiotic stress, GABA-T, pop2 mutant, POP2 overexpression, cell elongation

109

71 BIOACCUMULATION OF CD, PB, ZN, MO AND S FROM POLLUTED AND UNPOLLUTED SOIL
IN BRASSICA NAPUS L. VAR. NAPUS
Nadja ROMIH, Cvetka RIBARI-LASNIK, Botjan GRABNER
Institute of the Environment and Spatial Planning Research, Ipaveva 18, 3000, Celje, Slovenia,
nadja.romih@iop.si

The study was conducted at three locations in the Savinjska region of Slovenia (Medlog, kofja vas, Ponikva),
where soil is contaminated with heavy metals due to the zinc industry (Cinkarna Celje). In Medlog the soil to
a depth of 30 cm contains 0.9 mg kg-1 Cd, 23 mg kg-1 Pb and 86 mg Zn kg-1, in kofja vas 6.7 mg kg-1 Cd, 215
mg kg-1 Pb and 1400 mg kg-1 Zn and in Ponikva 0.5 mg kg-1 Cd, 25 mg kg-1 Pb and 70 mg kg-1 Zn. pH at the
selected sites was between 7.3 and 7.6. In the beginning of September 2006 two hybrids of Brassica napus
L. var. napus, PR 45D01 (a semi-dwarf hybrid with a distinctive reduced crop height and tolerance to different
ecological conditions, suitable for biodiesel) and PR 46W31 (the first hybrid sort in Slovenia, with a high yield
of seeds and oil, a high tolerance to drought and stable in different ecological conditions, suitable for biodiesel)
from Pioneer Hi-Bred International, Inc., were sown. After 300 days in Ponikva, 277 days in Medlog and kofja
vas plants were collected. Parts of plants (root, shoot and seed) were divided and determined with ultra trace
ICP mass spectroscopy of 37 elements. We compared two different hybrids, their uptake of Cd, Pb, Zn, Mo and
S in divided parts of plants and S/R (shoot/root) ratio. Our results favour hybrid PR45 D01, which had lowest
S/R ratio on all three location. The ratio between seed and root was also determined, for Zn 0.28 1.07, Cd
0.08 0.25, Pb 0.01 0.27, Mo 0.22 1.13 and S 1.65 3.26.

110

72 CRITICAL PROMOTER ELEMENTS IN UV-B INDUCED EXPRESSION OF ANAC13


Judit SAFRANY, Veronika HAASZ, Gza DALLMANN
Agricultural Biotechnological Centre, Gdll, Vaskapu u. 10-14., 1097, Budapest, Hungary,
jcrocus@freemail.hu

As an intrinsic part of the sunlight UV-B radiation reaches the Earths surface increasing level in the last
decades. Plants use at least three photoreceptor systems: red/far red sensing phytochromes, blue/UV-A
sensing cryptochromes and phototropins, but the UV-B specific photoreceptor and the signalling pathway are
not yet known. The aim of our work is to analyse new components of UV-B response in Arabidopsis thaliana.
ANAC13 transcription factor is a component of UV-B signal transduction. We have identified EMS mutants
defective in UV-B responsiveness in ANAC13::Luc transgenic Arabidopsis thaliana and we have determined a
new element taking part in the UV-B signal transduction.

111

73 DROUGHT STRESS AND POLYAMINES OF SCOTS PINE SEEDLINGS


Tytti SARJALAa, Riina MKELa, Jaana VUOSKUb, Hely HGGMANb, Markku SAARINENa, Juha
HEISKANENa
a
b

Finnish Forest Research Institute, Kaironiementie 54, FI 39700, Parkano, Finland, tytti.sarjala@metla.fi
University of Oulu, Oulu, Finland

Frequency of diverging temperature and drought stress in boreal forests has been proposed to increase due
to climate change. We are interested in the adaptation of Scots pine to the increasing frequency of drought
episodes during the most vulnerable stages of development.
Our aim is to link the changes in polyamine metabolism on metabolite and gene expression levels to the
changes in Scots pine seedling growth under drought stress.
Scots pine seedlings were grown on peat with three different moisture levels. Growth of the seedlings was
measured and samples for endogenous polyamine analysis and realtime RT-PCR were collected to analyze
expression of two polyamine synthesis genes (arginine decarboxylase, spermidine synthase). The drought
stress changed root/shoot ratio, decreased the biomass of the seedlings and number of the root tips,
especially mycorrhizae. Realtime PCR analyses are in progress. Our first results show that drought stress
induced changes mainly in soluble conjugated fraction of polyamines in the needles. The results will be used
to estimate changes in below- and above ground carbon allocation due to drought stress and to elucidate the
possible role of polyamines in the process.
Key words: polyamines, drought, Pinus sylvestris

112

74 DISTRIBUTION OF PIP-AQAPORINS IN STEROL-ENRICHED PLASMA MEMBRANE DOMAINS


IMMOBILE UNDER SHORT-TERM OSMOTIC STRESS
Taisiya SHEVYREVA, Marina S. TROFIMOVA
Institute of Plant Physiology of Russian Academy of Science, Botanicheskaya 35, 127276, Moscow, Russia,
shevyreva@mail.ru

Aquaporin mediated water membrane permeability is considered to regulate by aquaporin gating, expression
of their genes and traffic of these proteins. However, little is known about the conditions determining what
mechanism of the regulation of the aquaporin activity is realized. Earlier, we found that membrane subjected
to short-term osmotic induced endo- and exocytosis is poor by PIP-aquaporins in suspension-cultured sugar
beet mesophyl protoplasts. According to this evidence we suggested that PIP-aquaporins are localized
predominantly in membrane structure with low mobility, for example lipid rafts. Lipid rafts can be characterized
as sterol-enriched domains being resistance to solubilization of non-ionic detergent Triton X-100. To check
this hypothesis two experimental approaches were used. First, we analyzed PIP-aquaporin distribution in the
plasmalemma fractions after solubilization by Triton X-100 and their subsequent partition by flotation in the
OptiPrep discontinuous gradient. Second approach was to observe traffic of sterol-enriched membranes marked
by filipin in protoplasts under osmotic stress. It was found that the Triton-resistant fraction of plasmalemma is
characterized by a high sterol/protein ratio and has considerably higher content of aquaporins. In addition it
was shown that filipin stained plasma membrane structures were becoming close under hyperosmotic stress
and were spaced out under hypoosmotic stress. Taken together, these results allow us to conclude that the
observed low mobility of the PIP-aquaporins under osmotic stress is due to their localization in sterol-enriched
domains of plasmalemma, with the incorporation of these integral proteins in any membrane domains may be
considered as a possible mechanism of the regulation of their vesicular transport including one induced by the
stress action.

113

75 CARRY-OVER OF ABIOTIC STRESS SIGNALS IN THE FOLIAGE OF DECIDUOUS TREE


CANOPY THROUGH BUD FORMATION.
Anu SBER, Tairi TAPPO, Eve EENSALU
a

University of Tartu, Institute of Ecology and Earth Sciences, Lai 40, 51005, Tartu, Estonia, asober@ut.ee

Bud size and leaf area formation from buds in 4-7 year old deciduous forest (regenerating vegetatively in
clear-cut area) was studied during canopy closure. Measured species were Populus tremula and Tilia cordata.
Stand contained also other species (e.g. Betula pendula and, Quercus robur). Buds on upper and lower lateral
branches were examined.
Usually buds of upper branches were bigger than buds of lower branches. After summer with water stress, the
normal distribution in bud size (both in volume and mass) was shifted towards smaller buds: the probability to
occur was highest for smallest buds. This effect was maximal in upper branches of Populus tremula (after dry
summer buds on upper branches were even smaller than buds on lower branches in this case). The similar
shift in bud size towards prevalence of small buds occurred in lower branches of both species during canopy
closure.
Significant part of small buds died before bud-burst (especially on lower branches).
The length of surviving buds (measured in early spring) correlated positively with their volume and mass, but
also with leaf area which formed from the same buds (r2 > 0.6 in both species and branch positions). Buds
with the same size gave more leaf area in lower branches than in upper branches in both species. The rate
of chlorophyll formation, (but not final chlorophyll content of leaves) correlated with N-concentration of buds.
Stomatal density was nearly constant during expansion of leaves, being slightly different in different canopy
positions.
The effects of previous year environmental conditions on development of leaf area, structure and function are
discussed.

114

76 URANIUM AS AN ABIOTIC STRESS FACTOR FOR SELECTED HIGHER PLANTS


Petr SOUDEKa, Zuzana LHOTAKOVAb, Sarka PETROVAa, Martin BUZEKa, Ondej LHOTSKa, Jana
ALBRECHTOVAb, Tom VANKa
Institute of Experimental Botany AS CR, v.v.i., Drnovska 507, 16105, Prague, Czech Republic,
soudek@ueb.cas.cz
b
Department of Plant Physiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic

The area of land contaminated with heavy metals has increased during the last century due to mining, smelting,
manufacturing, urban and other industrial activities. These elements can be leached into the surface water or
groundwater, taken up by plants, released as gases into the atmosphere, or bound semi-permanently by soil
components such as clay or organic matter, and later affect human health. Heavy metals represent high stress
factor for environment and for human. Their toxicities are expressed in many different effects and they can
cause indigestions, different dermatitis, changes in the blood count, damage of fundamental organs (brain,
liver and kidney), cancerous processes etc. Therefore the decrease of toxic metals to the natural level is one
of the important subject to deal with.
This project is focused on an accumulation and translocation mechanisms of uranium in plants under laboratory
conditions. We concentrate to uranium accumulation in plant body and we are looking for stress responses in
plants on different levels.
Acknowledgements: Supported by project AVOZ 50380511 and COST FA0605 (COST.FA0605OC9039).

115

77 DROUGHT TOLERANCE OF BARLEY DH LINES DERIVED FROM MUTANT CROSSES


Roman ZIELINSKI, Kornelia POLOK
Department of Genetics UWM, Plac Lodzki 3, 10-967, Olsztyn, Poland, rzielin@moskit.uwm.edu.pl

In Europe, severe water shortage at specific location is expected once every 50 years. However, scarcely 5
years go by without serious droughts across at least some part of Europe and some of these events can last
for several years, thus being the major limitation to plant productivity. During evolution plants have acquired
different adaptive strategies to cope with abiotic stresses. Exposure of plants to a water-limiting environment
during various developmental stages activates physiological and developmental changes, understanding of
which is still a major challenge in biology. Among cereals, barley is a good genetic model to study tolerance
to abiotic stresses owing to huge genetic resources, autogamy, diploidy and a wide range of adaptability.
Quantitative and molecular genetics approaches enable the dissection of this complex trait, identification of
QTLs connected with drought tolerance however suitable genetic stocks with different alleles at QTLs must be
available. In our studies we developed and characterized at DNA level drought tolerant DH lines that can be
used in studies of underlying molecular mechanisms. In total 47 DH lines developed from three heterotic F1 of
mutant crosses were selected for two year studies differed in rainfall conditions. A complete block design with
three blocks and ten replications was applied. Four genotypes (A63, A78, D161, M43) were found to be tolerant
to drought at the meiosis and anthesis as well as they were superior in grain yield from 15 to 170%, especially
compared with the parent cultivar. These lines did not suffer from chlorosis, neither. Drought resistant lines
have also revealed a number of mutations at DNA level including loss and gain of new BARE1 insertion sites.
Our results emphasize the possibility to develop drought tolerant and high yielding lines by selection in cross
combinations derived from otherwise low yielding and poorly perform mutants.

116

78 INFLUENCE OF PPD GENES ON FROST HARDINESS OF WINTER WHEAT (TRITICUM


AESTIVUM L.) UNDER DIFFERENT PHOTOPERIOD CONDITIONS.
Alexander ZUBRYCH, Olga AVKSENTYEVA, Vasily ZHMURKO
V. N. Kharazin National University of Kharkov, Department of Plant Physiology and Biochemistry, Svoboda
sq., 4, 61077, Kharkov, Ukraine, vasily.v.zhmurko@univer.kharkov.ua

Photoperiodic sensitivity in wheat (Triticum aestivum L.) is controlled by Ppd genes. It is known about
their pleiotropic effects on frost hardiness (FH), though physiological mechanisms of this influence are still
studied insufficiently. According to the data of our previous studies Ppd genes may influence metabolism of
carbohydrates, which play important role in resistance of cold-hardy plants to frosts. According to above-stated
we hypothesized that effects of Ppd genes on FH may be connected with their influence on carbohydrate
content in crown tissues and/or on number of tillers.
Mono-dominant near-isogenic lines (NILs) on Ppd genes of winter wheat (cultivar Mironovskaya 808) were
grown in field conditions. During the first stage of cold acclimation ( October - November ) one half of plants
was exposed to shortened 8 h days (SD) within 30 days while another one was grown at natural long 11-10
h days (LD). The sampling for analysis of carbohydrates content in crown tissues and for tillers count was
executed after the end of vegetation period. In February monolithic blocks of soil with the plants were kept
exposed to 17 within 24 h in a climatic room. The FH was expressed as percentage of survived plants
after re-growing.
The results have shown that FH of all NILs from LD conditions were equal. Under SD conditions NILs with
dominant Ppd 22, Ppd 33 genes and NIL with all recessive Ppd 112233 genes except NIL with dominant Ppd
11 genes lowered their FH significantly. Also all NILs excluding NIL with dominant Ppd 11 genes had reduced
intensity of tillering under SD conditions, whereas soluble carbohydrate content was lower after SD in all NILs
including the one with dominant Ppd 11 genes. Thus relative FH of NIL with dominant Ppd 11 genes under SD
conditions should be attributed to the insensitivity of its tillering intensity to short photoperiod.

117

79 PHOSPHATIDIC ACID DIRECTS THE PROTEIN KINASES IN THE PLANTS RESPONSE TO


SALT
Christa TESTERINK
University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands, c.s.testerink@uva.nl

Phosphatidic acid (PA) has been identified as a lipid second messenger in both plants and animals. In plants,
PA accumulates in response to several biotic and abiotic stress stimuli, such as drought, salinity, cold, wounding
and pathogen infection. Although PAs mode of action is not clear yet, an important discovery is that several
protein kinases appear to have PA-binding affinity. These include the phosphoinositide-dependent protein
kinase 1 (PDK1), the negative regulator of ethylene signalling CTR1 and two individual SNF1-related protein
kinases (SnRK2.4 and 2.10).
The SnRK2 subfamily is plant specific and most family members were shown to be activated by salt in
protoplasts. Using hydroponically grown Arabidopsis plants, we were able to instantly monitor SnRK2 activity
and study their localization in root tissue in response to salt treatment. Interestingly, not all SnrK2 isoforms
have affinity for PA. Knock-out mutants of both PA-binding isoforms, as well as a double mutant, are currently
being characterized with respect to salt tolerance.
Another line of research focuses on the role of phosholipid signaling in directing osmotic stress-induced loss
of gravitropism. We observed that osmotic stress in roots leads to rearrangements of the microtubule (MT)
network and induces changes in the subcellular localization of signalling proteins, including the protein kinases
PINOID and PDK1. We hypothesize that a signalling cascade involving phospholipid signalling and protein
phosphorylation directs root growth away from high salt concentrations. Currently, mutants pid, pdk1 and the
phospholipid-metabolizing enzymes that generate PA, are being tested in a salt avoidance assay.

118

80 THE WLIM1 IS AN ACTIN REGULATORY PROTEIN POTENTIALLY INVOLVED IN THE


MECHANICAL STRESS-INDUCED RESPONSES IN PLANT CELLS
Stephane THOLL, Andr STEINMETZ, Clment THOMAS
CRP-Sant Luxembourg, 84, Val Fleuri, L-1526, Luxembourg, Luxembourg, stephane.tholl@crp-sante.lu

Plants are continuously exposed to a myriad of environmental mechanical cues, such as gravity, wind and
wounding. Several observations suggest that the actin cytoskeleton participates in the perception and
transmission of mechanical signals, although the underlying mechanism remains unclear. The data presented
here suggest that a LIM domain-containing protein could be part of this mechanism. The tobacco WLIM1
protein is an actin-binding protein that stabilizes and bundles actin filaments into cables. We submitted tobacco
BY2 cells that ectopically express a WLIM1-GFP fusion protein to different types of mechanical treatments
and evaluated the effects on the actin cytoskeleton organization using the confocal microscope. Transgenic
cells placed in an open cell growth chamber (control conditions) exhibited an only weak actin cytoskeleton
labeling and rather fine actin bundles. In contrast, when these cells were transferred between slide and
cover slip (stress type I), WLIM1 massively localized to the actin filaments and obviously increased the actin
bundle thickness. Such cytoskeletal modifications were clearly observable after 3 minutes, indicating that the
process is relatively rapid. Similar observations were made by submitting BY2 cells to centrifugal forces (stress
type II), indicating that mechanical stress is the inductive signal. Interestingly, removing the cell wall of BY2
cells provoked both the accumulation of WLIM1 on actin filaments/bundles and actin bundle reinforcement,
independently of mechanical treatment. This points out a crucial role for the cell wall in mechanical stress
perception and/or transduction.
Together our observations suggest that WLIM1 is a mechanical stress responsive protein involved in the
reinforcement of actin cytoskeleton. We are currently conducting functional studies to test this hypothesis in
the context of whole plant tissues and organisms.

119

81 INVESTIGATION ON THE INTERACTIONS AMONG ABSCISIC ACID (ABA), REACTIVE OXYGEN


SPECIES AND ANTIOXIDANT DEFENSE SYSTEM IN TRANSMISSION OF DROUGHT STRESS
SIGNALING IN ABA-DEFICIENT ARABIDOPSIS THALIANA MUTANT (ABA1) AND WILD-TYPE
ARABIDOPSIS THALIANA ECOTYPE
Ismail TURKAN, Ceyda OZFIDAN, Askm Hediye SEKMEN, Burcu SECKIN
Department of Biology, Science Faculty, Ege University, Bornova, 35100, Izmir, Turkey,
ismail.turkan@ege.edu.tr

Increasing evidence suggests that ABA might have a role in oxidative stress in plant cells. However, the
interaction of the ABA, ROS (reactive oxygen species) and antioxidant defence system still needs further
explanation. In our study, we investigated the interaction among abscisic acid (ABA), ROS and antioxidative
defence system in the transduction of drought stress signaling by using aba1 mutant of A. thaliana. For this,
wild type (Columbia) and aba1 mutant were applied exogenously with 50M ABA and plants were exposed
to drought stress induced by 40% polyethylene glycol (PEG 8000; -0.7 MPa) for 6, 12 and 24 h (hours) and
then time course analysis of the activities and isozymes of several antioxidant enzymes such as superoxide
dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), peroxidase (POX; EC 1.11.1.7), ascorbate
peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) were determined. Treatment with
ABA under optimal conditions did not affect activities of antioxidant enzymes in aba1 mutant and wild type. The
activities of POX, APX, and GR in aba1 at 6 h of drought stress showed an increase with ABA application in
comparison to only PEG treated group. However, ABA treatment increased the only GR activity of wild type at
6 h drought stress. Moreover, at 24 h of drought treatment, levels of CAT, APX and GR activities in the ABA+
PEG group of aba1 were remarkably lower than in PEG group and were almost at the same level of control
group. On the contrary, activities of CAT, APX, GR in ABA treated wild type were increased at 24 h of drought
stress in comparison to control group. In addition, lipid peroxidation level of ABA-deficient mutant (aba1) under
drought treatment showed a decrease with ABA treatment. Therefore, we conclude that ABA protects aba1
mutant from drought-induced oxidative stress.

120

82 CLONING AND IDENTIFICATION OF A NOVEL FUNCTION OF PEA LECTIN RECEPTOR-LIKE


KINASE IN SALINITY STRESS TOLERANCE
Narendra TUTEJA, Amita JOSHI, Neha VAID, Hung QUANG-DANG
International Centre for Genetic Engineering & Biotechnology (ICGEB), Aruna Asaf Ali Marg, 110067, New
Delhi, India, narendra@icgeb.res.in

The plant lectin receptor-like kinases are involved in various signaling pathways but their role in abiotic stress
tolerance in plant has not heretofore been described. Among abiotic stress, salinity stress is one of the major
factors which negatively effects plant growth/productivity and threatens food security worldwide. Based on
functional gene-mining assay, we have isolated 34 salinity tolerant genes out of one million Escherichia coli
(SOLR) transformants containing pea cDNAs. Sequence analysis of one of these revealed homology to lectin
receptor-like kinase (LecRLK), which is a N-linked glycoconjugate. Structurally the plant RLKs are similar
to animal receptor kinases because both consist of: 1) N-terminal, extracellular, ligand-binding domain, 2)
a hydrophobic transmembrane domain and 3) a C-terminal intracellular, kinase catalytic domain (Ser/Thr).
The salinity tolerance of LecRLK in bacteria was further confirmed by using another strain of E. coli (DH5)
transformants. In planta, the expression of LecRLK cDNA was also upregulated in response to salinity stress.
This suggests that the cellular response to high salinity stress is conserved across prokaryotes and plant
kingdom. Overall, this study urges to develop novel concepts about the role of plant LecRLK in high salinity
stress tolerance and this study shall provide a highly significant new contribution for our better understanding
of stress tolerance in plants. It remains to be tested whether the expression of these genes will confer durable
resistance to high salinity tolerance in crops, but the successful identification of the salinity stress induced
gene reveals a clear new pathway for the direction for further experimentation.

121

83 CHARACTERIZATION OF AMINOALDEHYDE DEHYDROGENASES FROM PEA AND TOMATO


Martina TYLICHOVAa, David KOPENa, Pierre BRIOZZOb, Solange MORRAc, Jacques SNGAROFFb,
Marek EBELAa
Palack University, Faculty of Science, lechtitel 11, 783 71, Olomouc, Czech Republic,
martina.tylichova@seznam.cz
b
UMR206 Laboratoire de Chimie Biologique, INRA-AgroParisTech, F-78850 Thiverval-Grignon, France
c
Laboratoire dEnzymologie et de Biochimie Structurales, CNRS, F-91198 Gif-sur-Yvette Cedex, France
a

Aminoaldehyde dehydrogenases (AMADH, EC 1.2.1.19) oxidize omega-aminoaldehydes arising from


polyamine degradation. Based on their amino acid sequences, the enzymes belong to the same group as
betaine aldehyde dehydrogenases (BADHs, EC 1.2.1.8), which participate in plant response to osmotic stress.
Two isoenzymes from Pisum sativum (PsAMADH1 and PsAMADH2) and two isoenzymes from Lycopersicon
esculentum (LeAMADH1 and LeAMADH2) were expressed in E. coli and subsequently purified to homogeneity
by immobilized metal ion affinity chromatography and ion-exchange chromatography. Enzyme kinetics was
performed with all recombinant enzymes. The obtained results show that PsAMADH2 has much better affinity
to the best substrate 3-aminopropanal compared with PsAMADH1 and that the compound is also oxidized more
efficiently. Interestingly, all enzymes, but especially LeAMADH1, are able to oxidize pyridine carboxaldehydes
and some other heterocyclic aldehyde compounds in addition to omega-aminoaldehydes and aliphatic C3
to C7 aldehydes. Also various NAD+ analogs functioned as effective coenzymes. To get insight into AMADH
catalysis and specificity, site-directed mutagenesis of PsAMADH2 has been performed together with kinetic
characterization of the mutants. Both pea AMADHs were successfully crystallized with and without NAD+. Xray data were collected up to 2.4 resolution for PsAMADH1. Molecular replacement using human aldehyde
dehydrogenase as a template confirmed that recombinant PsAMADH1 is a dimer. The crystal structure is
currently under refinement.
Supported by grants No. 522/08/0555 and 522/08/H003 from the Czech Science Foundation.

122

84 COMPARISON OF THE DYNAMICS OF HEAT AND DROUGHT STRESS RESPONSES IN


TOBACCO PLANTS
Radomira VANKOVA, Milena CVIKROVA
Institute of Experimental Botany AS CR, Rozvojova 263, 165 02, Prague 6, Czech Republic,
vankova@ueb.cas.cz

Heat stress represents very fast, acute stress associated with the decrease of leaf water potential. Initial phase
of heat stress (2 h at 40 C) coincided with transient elevation of bioactive cytokinin isopentenyladenosine
as well as transient decrease of abscisic acid (ABA), associated with elevation of ABA catabolite - phaseic
acid. Heat stress was associated with an increase in free polyamines (PAs), especially of putrescine (Put),
spermidine (Spd), norspermidine and cadaverine, which correlated with a stimulation of the activity of arginine
decarboxylase, ornithine decarboxylase and S-adenosylmethionine decarboxylase (ADC, ODC, SAMDC).
After 6 h of heat stress, decrease in free polyamines, especially of Put, coincided with down-regulation of the
activity of PA biosynthetic enzymes and stimulation of diamine oxidase (DAO). During the acclimation phase,
decrease in cytokinin levels was associated with stimulation of the activity of cytokinin oxidase/dehydrogenase
(CKX). No effect on malondialdehyde (MDA), product of lipid peroxidation, was observed within 6 h of heat
stress.
Prolonged drought coincided with considerable drop in plant water potential and vast increase of ABA content.
Strict regulation of ABA content was reflected by accumulation of its metabolites (derivatives of phaseic acid
and glucosylester). Gradual decrease of bioactive cytokinins was accompanied by elevation of their glucosides
and later on with stimulation of the activity of CKX. Drought stress coincided with a significant increase in free
PAs, especially of Spd and Spm. Activity of ADC and ODC correlated with levels of Put; activity of SAMDC with
Spd and Spm. High stimulation of DAO was observed. Levels of MDA increased substantially during drought
in lower leaves and roots.
Re-watering coincided with a fast elevation of cytokinins, especially of trans-zeatin type, down-regulation of
ABA, accompanied by decrease in Spd and Spm contents and, on the contrary, increase in the level of Put.

123

85 LONG-TERM EFFECTS OF LOW CO2 CONCENTRATION, LIGHT INTENSITY AND HUMIDITY ON


STOMATAL DEVELOPMENT AND PATTERNING IN THE LEAVES OF ARABIDOPSIS THALIANA
Martina VAKOVAa, Marie HRONKOVAb, Marie SIMKOVAb, Ji ANTREKa
University of South Bohemia, Braniovsk 31, 37005, esk Budjovice, Czech Republic,
mastavas@centrum.cz
b
Biology Centre of the ASCR, Czech Republic
a

Stomata are pores on the leaf surface which facilitate gas exchange between the plant and the atmosphere. The
number and size of stomata vary among different species of plants and depend strongly on the environmental
conditions. We investigated stomatal development and patterning in the leaves of Arabidopsis thaliana (wild
ecotypes Col and C24 and stomatal mutants sdd1 and tmm1) grown in the artificial atmosphere of Helox
(79% He, 21% O2, 200 ppm CO2) under different light conditions (80 and 200 mol m-2 s-1) and varying relative
humidity (70 and 100 %). Within the experiment we found out differences in the number and spacing of
stomatal pores, leaf temperature, tissue 13C signal, mass increment and water content of seedlings among
treatments and individual ecotypes and mutants as well. 4 weeks after germination we altered the atmosphere
from Helox to Nitox (79% N2, 21% O2 and 400 ppm CO2). Afterwards we observed stomatal number and
patterning modification especially in C24 and Col plants, when more stomatal clusters appeared. Therefore
it is evident that environmental conditions alter not only number of stomata but have significant effect also on
stomatal distribution.

124

86 ACTIVITY OF GLUTATHIONE PEROXIDASE IN WILD AND SELENIUM-RESISTANT STRAINS


OF THE ALGA SCENEDESMUS QUADRICAUDA GROWN IN THE PRESENCE OF INORGANIC
SELENIUM COMPOUNDS
Milada VITOVAa, Dasa UMYSOVAa, Ji MACHTb, Vilm ZACHLEDERa
a
b

Institute of Microbiology ASCR, Opatovicky mlyn, 37981, Trebon, Czech Republic, vitova@alga.cz
Masaryk University, Czech Republic

Selenium is a trace element performing important biological functions in many organisms including humans.
It usually affects organisms in a strictly dosage-dependent manner being essential at low and toxic at higher
concentrations. The impact of selenium on mammalian and land plant cells has been quite extensively studied.
Information about algal cells is rare despite of the fact that they could produce selenium enriched biomass for
biotechnology purposes.
We studied the impact of selenium compounds on the green chlorococcal alga Scenedesmus quadricauda.
Both the dose and chemical forms of Se were critical factors in the cellular response. We selected three
strains of Scenedesmus quadricauda specifically resistant to high concentrations of inorganic selenium
added as selenite - strain SeIV, selenate - strain SeVI or both - strain SeIV+VI. The total amount of Se and
selenomethionine in biomass increased with increasing concentration of Se in the culturing media. In both
the wild type and Se-resistant strains, the activity of glutathione peroxidase (GPx) an antioxidative selenoenzyme was studied. The GPx activity increased rapidly in the presence of the form of selenium for which the
given algal strain was not resistant.
The selenium effect on the green alga Scenedesmus quadricauda was not only dose dependent, but the
chemical form of the element was also crucial. The amount of selenium and SeMet in algal biomass was
dependent on both the type of compound and its dose. The activity of glutathione peroxidase was affected by
selenium treatment in dose-dependent and toxic-dependent manner. The findings implied that the increase in
GPx activity in algal cells was a stress response to selenium cytotoxicity.
This work was supported by the GA ASCR and GACR (A600200701, 525/09/0102), projects EUREKA MEYS
CR (OE221, OE09025) and by Inst Res Concept AV0Z50200510.

125

87 COMPENSATORY RESPONSES IN CUTICULAR MUTANTS OF ARABIDOPSIS THALIANA


Derry VOISINa, Christiane NAWRATHd, Andrea FAUSTb, Benni FRANKEb, Sergey KURDYUKOVc, Nadia
EFREMOVAa, Isa WILLa, Lukas SCHREIBERb, Alexander YEPHREMOVa
Max-Planck Institute for Breeding Research, carl-von-Linne weg 10, 50829, Kln, Germany,
voisin@mpiz-koeln.mpg.de
b
University of Bonn, Germany
c
ARC Centre of Excellence for Integrative Legume Research, Australia
d
University of Lausanne, Switzerland
a

In plants, a lipidic layer, the cuticle, is deposited on the surface of all aerial organs and plays an insulating role,
protecting plants against abiotic and biotic stress factors present in their natural environment. The cuticle also
defines boundaries of organs and is essential for normal development. The major structural components of the
cuticle, a polyester matrix, cutin, and wax, are mainly composed of fatty-acid derivatives. Cuticular mutants
provide an efficient means for identifying genes involved in the biosynthesis, transport and assembly of the
cuticle layer and for studying the response to cuticular damage. We investigated three cuticular mutants,
in Arabidopsis thaliana, bodyguard (bdg), fiddlehead (fdh) and lacerata (lcr) that display secondary mutant
phenotypes, such as leaf deformation and ectopic organ fusion. Strikingly, it appeared that the mutants
overaccumulate typical cutin monomers and wax; however, the putative enzymic functions of the corresponding
proteins could not account for these changes, suggesting a compensatory response to cuticular deficiency. To
study this further, we analysed their gene expression profiles using high-density Affymetrix ATH1 GeneChip
microarrays. This analysis supports the notion that bdg, lcr and fdh are able to respond to the loss of cuticle
integrity by specifically altering the expression of genes involved in cuticle homeostasis, cell wall remodelling
and stress response mechanisms. By comparing the lists of differentially expressed genes (DEG) from these
mutants to DEG lists from more than 600 microarray experiments using MASTA (MicroArray overlap Search
Tool and Analysis), we identified a suppressor of the bdg and lcr mutations.

126

88 THE EFFECT OF WOUND STRESS ON SOLUBLE PHENOLS AND ANTIOXIDANTS IN


POTATOES
Christina B. WEGENER
Julius Khn-Institute, Institute for Resistance Research and Stress Tolerance, Rudolf-Schick-Platz 3,
D-18190 Sanitz, OT Gro Lsewitz, Germany, christina.wegener@jki.bund.de

In potato breeding a high level of tolerance to varying types of environmental stress is a major challenge in
future. Above all, wound stress is a continuous threat for potatoes in agricultural practice. In this study, 32
potato genotypes comprising three cultivars (cvs), four lines of cv Dsire expressing a pectate lyase (PL3)
gene and several progenies derived from crosses between such PL3-potato lines and the cvs Agave and
Adretta were examined for total soluble phenols in tuber tissue and their antioxidant activity generated in
response to wounding the tubers. Pectate lyases are enzymes that release oligogalacturonides (OG) from
plant cell wall pectin, compounds eliciting various physiological responses in plants.
For assaying total phenols and antioxidant activity a first extract sample was taken from fresh tuber tissue,
and a second was prepared 24 h after wounding the tubers. After wounding, all potato genotypes exhibited
significantly increased amounts of soluble phenols in tuber tissue associated with an elevated antioxidant
capacity (P<0.001, n=32, both) measured as ascorbic acid equivalent (ACE). PL3-active lines of cv Dsire
exceeded the non-transformed controls, as well as the cvs Agave and Adretta in their phenol and ACE values
detected after wounding. Also PL3-expressing progenies revealed on average a stronger increase of their
phenol and ACE levels in response to wounding than the PL-inactive progeny group. This implies that the
heterologous PL3 protein strengthened the wound response of potatoes, obviously through a release of OG
elicitors from tissue cell walls. Finally, it may improve the formation of wound periderm.

127

89 SIGNAL REGULATION BY PHA OF CELL DIVISION IN THE BEAN SEEDLINGS UNDER


SALINITY
Marina BEZRUKOVA, Alsu LUBYANOVA, Rimma FATKHUTDINOVA, Farida SHAKIROVA
Institute of Biochemistry and Genetics, Ufa Scientific Centre, Russian Academy of Sciences, pr. Oktyabrya 71,
450054 Ufa, Russia, shakirova@anrb.ru

Earlier we have revealed that growth-stimulating and defense effect to salinity of 1 mg/l wheat germ agglutinin
(WGA) on wheat, barley and rice plants are due to its influence on hormonal system. Based on high degree
homology of WGA with barley and rice lectins it is possible to suggest that realization of growth-stimulating
and protective effects of these cereal lectins along with acceleration of reparation of growth processes by
them is also connected with their influence on hormonal status of barley and rice plants. Is manifestation
of these properties characteristic for other phytolectins 4-d-old bean seedlings were incubated on 1 mg/l
phytohemagglutinin (PHA). The contents ABA, IAA and cytokinins were measured in excised roots of bean
seedlings by ELISA. Mitotic index (MI) was determined using 3000 cells per treatment. We have revealed that
treatment with PHA has resulted in the increase of MI of bean seedling roots which was due to the essential
rise in IAA and cytokinin levels. Similarly to WGA action on cereals, treatment of bean seedlings with PHA also
promoted the acceleration of the reparation of MI of roots after the action of 1.5% NaCl. Thus, the ability to
regulate cell division is typical property to phytolectins and it is realized in specific or related for them plants.
This property is due to the active influence of phytolectins on state of hormonal system.
Key words: WGA, PHA, bean

128

90 TRANSCRIPTIONAL AND METABOLIC PROFILING DURING RIPENING OF TRNCADEIRA


GRAPES
Ana M. FORTESa, Marta S. SILVAb, Ali KASHIFFc, Federica MALTESEc, Young H. CHOIc, Lisete SOUSAd,
Jos M. MARTINEZ-ZAPATERe, Rob VERPOORTEc, and Maria S. PAISa
ICAT, BioFIG, FCUL, University of Lisbon, Campo Grande, 1749-016 Lisboa, Portugal,
margafortes@yahoo.com
b
Dep. Chemistry and Biochemistry, FCUL, Lisboa
c
Division of Pharmacognosy, Section Metabolomics, Institute Biology Leiden, Leiden, The Netherlands
d
Dep. Statistics and Operational Research, CEAUL, FCUL, Lisboa
e
Centro Nacional de Biotecnologia, CSIC, Madrid, Spain
a

Grapes (Vitis vinifera cultivars) are economically the most important fruit crop worldwide. Trincadeira is a
late ripening cultivar widely used in Portugal to make appreciated spicy wine. However, this cultivar which in
certain years gives rise to unique wines presents a very irregular ripening in between seasons and is extremely
susceptible to fungi infections.
We are interested in gathering knowledge on ripening of Trincadeira grapes in particular on the pathways
leading to synthesis of flavour-related compounds and growth regulators as a way to control both processes
with important biotechnological applications. For that purpose we have analyzed samples at pre-veraison (EL
32, EL34), veraison (EL35) and ripe (EL36, EL38) stages during two seasons (2007 and 2008).
Transcript profiling using Affymetrix GrapeGen genome array containing 23096 probesets revealed differential
regulation of genes grouped into functional categories ranging from carbohydrate, amino acid, hormones and
terpenoid metabolism to stress response, signal transduction, metabolite transport facilitation, among others.
Metabolic profiling using 1H NMR spectroscopy enabled a thorough characterization of the developmental
stage by giving information on the content of amino acids, sugars and organic acids.
The results confirmed data already obtained for grape ripening of other cultivars but also provided new
information that may be cultivar specific and/ or related to climate conditions in Southern Portugal. Carbohydrate
and secondary metabolism, stress response and hormone control of ripening will be discussed in detail.

129

91 SP1, A NOVEL STRESS-ASSOCIATED PROTEIN, IS SYMOYLATED UPON SALT STRESS IN


POPULUS EUPHRATICA, AND IN CONSEQUENTLY RELOCATED FROM THE CYTOSOL TO
THE PLASMA MEMBRANE AND TO THE NUCLEUS
Arie ALTMANa, Basia J VINOCURb, Oded SHOSEYOVa
a
b

The Hebrew University of Jerusalem, Herzel, 76100, Rehovot, Israel, altman@agri.huji.ac.il


Evogene Inc, Israel

Populus. tremula plants are salt sensitive whereas Populus euphratica are tolerant, as revealed by biomass,
growth rate, osmotic potential and chlorophyll data. A 12.4 kDa novel protein (PeSP1), ortholog of Populus
tremula SP1 (Wang et al. 2002, Dgany et al. 2005), was identified and characterized in Populus euphratica,
showing 70.5% similarity and 61% identity to SP1. PeSP1 is also a dodecamer homo-oligomeric protein of
around 148 kDa, highly expressed in the plant, and shared the same biophysical characteristics with SP1.
PeSP1 (and SP1) were up-regulated upon NaCl stress in both the salt tolerant P. euphratica and the salt
sensitive P. tremula plants; however the expression of PeSP1 in P. euphratica was higher than in P. tremula
under non-stressed conditions. In situ sub-cellular immuno-gold localization of the corresponding orthologs in
both P. tremula and P. euphratica leaves shows almost homogenous distribution throughout the cytosol in the
control. However, under salt stress, PeSP1 is clearly restricted to and localized in the plasmalemma and the
nucleus of the tolerant P. euphratica, but no changes were observed in its cellular distribution in P. tremula.
Additionally, we show that PeSP1 undergoes a post-translational modification upon stress, due to sumoylation
at specific sites, as confirmed by xxx. Based on reported studies that SUMO conjugation is involved in altering
protein function through changes in activity or cellular localization, we suggest that the present results may
indicate a salt-induced sumoylation and redistribution of the protein in a salt tolerant Populus species but not
in its related salt sensitive one.

130

92 CALLOSE DEPOSITION AND GLUCANASE ACTIVITY IN HEAVY METAL-STRESSED MAIZE


AND SOYBEAN
Beta PIRELOVa, Silvia FLUCHb, Jana LIBANTOVc, Ildik MATUKOVc, Jana MORAVKOVc
Faculty of Natural Sciences, Dept. Botany and Genetics, Constantine the Philosopher University, researcher,
Nbreie mldee 91, 949 74, Nitra, Slovakia, bpirselova@ukf.sk
b
Austrian Research Centres GmbH, Department of Biogenetics/Natural resources, Seibersdorf
c
Institute of Plant Genetics and Biotechnology, Department of Molecular Biology and Biotechnology, SAS,
Nitra

Plant responses to heavy metals from the soil and atmosphere depends on an interrelated network of
physiological and molecular mechanisms. We studied the effect of cadmium (300 mg l-1), lead (500 mg l-1), and
arsenic (100 mg l-1) on the roots of maize (Zea mays L. cv. Quintal) and soybean (Glycine max L. cv. Korada).
All three metals caused after 48h exposure inhibition of root growths as well as loss of cell viability within the
stressed tissue. Soybean was most sensitive to arsenic, least to lead. Maize showed similar levels of sensitivity
to As and Cd, while was relatively tolerant to lead. We also observed significant callose deposition in As, Cd
and Pb-treated maize, while in case of soybean only a decrease was observed in As-treated tissue. Activities
of total glucanases, that form the group 2 of so-called pathogenesis-related proteins and are able to degrade
callose, was increased in soybean stressed with lead and cadmium. In contrast, it was repressed by arsenic in
both species. Accumulation of least 2 glucanase isoforms was detected in response of maize to heavy metal
stress. Our data suggest that defence mechanisms activated in the stressed plants by tested concentrations
of arsenic are different from those triggered by lead and cadmium.
Keywords: metal detoxification, PR proteins, plant defense

131

List of Authors
Name
ABDELLY Chedly
ABREU Isabel
ACET Tuba
ACHENBACH Ute
AHO Kukka
AKSMANN Anna
ALBRECHTOV Jana
ALCZAR Rubn
ALEKSEICHUK Halina
ALFONSO Sabrina
ALMADANIM Cecilia
ALONI Roni
ALTABELLA Teresa
ALTMAN Arie
ALTMANN Thomas
LVAREZ Rubn
ANGELOVICI Ruthie
ANGENON Geert
ANTONOVA O.Y.
AQUANI Mohamed
ARZEL Matthieu
AVALBAEV Azamat
AVKSENTYEVA Olga
AYATOLLAHI Zahra
BAGARD Matthieu
BALAZS Gergely
BALIK Ji
BALOGH Andrea
BANVOY Jacques
BAR-ZVI Dudy
BARTELS Dorothea
BARTH Olaf
BACIK-REMISIEWICZ Agnieszka
BELOZEROVA Natalia
BEN REJEB Kilani
BENNETT Mark
BERDNIKOVA Olga
BEREZOVSKAJA Maryana
BEZRUKOVA Marina
BIBIKOVA Tatiana
BICHELE Irina
BINAROVA Pavla
BISEWSKA Joanna
BOEHMER Maik
BOGRE Laszlo

132

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BOHLER Sacha
BOISSON-DERNIER Aurelien
BORDENAVE Marianne
BOSZORDOV Eva
BOUCHEREAU Alain
BOULET Jana
BOUWMEESTER Harro
BOXUS Mathieu
BRADER Gnter
BRADSHAW John
BRAZAUSKAS Gintaras
BRAUC Sigrid
BRIOZZO Pierre
BROSCH Mikael
BROWN Lawrie
BRGGEMANN Wolfgang
BRYAN Glenn
BUCKHOUT Thomas
BUZEK Martin
CATALA Rafael
CELLINI Francesco
CENTENO Mara Luz
CHOI Young H.
CHOMYAK Anna
CHUA Nam-Hai
COHEN Shabtai
COINCE Aurore
COPOLOVICI Lucian
CORTIZO Milln
CSERHTI Mtys
CSERI Andrs
CZYCZYLO-MYSZA Ilona
CUESTA Candela
CUEVAS Juan
CUYPERS Ann
CVIKROVA Milena
DACENKO Olexandr
DALLMANN Gza
DALMAY Tamas
DAMARI-WEISSLER Hila
DAVIES Seth
DEAK Csilla
DEBY-DUPONT Ginette
DELACOTE Emilien
DELEU Carole
DIZENGREMEL Pierre
DOCZI Robert
DOMMES Jacques
DOONAN John
DUDITS Dnes
DUNAYEVA S.E.
DUPAE Joke
EENSALU Eve
EFREMOVA Nadia

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EHNERT Stefan
EINSET John
EL AMRANI Abdelhak
ERSHOVA Antonina
FATKHUTDINOVA Rimma
FAUST Andrea
FEITO Isabel
FERNNDEZ Beln
FLUCH Silvia
FORTES Ana M.
FOTOPOULOS Vasileios
FRANA Sabine
FRANCK Thierry
FRANKE Benni
FRBORT Ivo
FUJII Hiroaki
FRSTER Sebastian
GALIBA Gabor
GALILI Gad
GALVN-AMPUDIA Carlos S.
GAVRILENKO T.A.
GAWROSKA Helena
GEORGE Timothy
GERARD Joelle
GERMAN Marcelo
GHARS Mohamed Ali
GIELEN Heidi
GILROY Simon
GODOSKI Jan
GRABNER Botjan
GRANDA Vctor
GRANOT David
GREGORY Peter
GRYGORIEVA Olesia
GUEGAN Jean-Paul
GYRGYEI Jnos
HASZ Veronika
HALIMAA Pauliina
HAMANN Thorsten
HARTIKAINEN Kaisa
HASENFRAZ SAUDER Marie Paule
HASSINEN Viivi
HAUSMAN Jean-Franois
HEDRICH Rainer
HEINO Pekka
HEISKANEN Juha
HELENIUS Elina
HENRIQUES Rossana
HEYER Arnd
HOFFMANN Lucien
HOLBROOK Michele
HOLOPAINEN Jarmo
HONIG Arik
HOUBA-HRIN Nicole

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HRONKOVA Marie
HU Honghong
HUMBECK Klaus
HUSSAIN Syed Sarfraz
HGGMAN Hely
HVE Katja
IANNACONE Rina
IBRAHIM Mohamed
ISRAELSSON NORDSTRM Maria
IVANOVA Hiie
JANCS Mihly
JENES Barnabas
JOLIVET Yves
JONAK Claudia
JONAVIIEN Kristina
JONES Alex
JOPK Martin
JOSHI Amita
JOZEFCZAK Marijke
JULKUNEN-TIITTO Riitta
JRVE Kristel
KADIOGLU Asim
KALISZOVA Regina
KANELLIS Angelos
KANGASJRVI Jaakko
KARIOLA Tarja
KARNOSKY David F.
KASHIFF Ali
KAWASHIMA Cintia
KAWECKA Alicja
KEELI Ali M.
KEERBERG Olav
KEHR Julia
KEINNEN Markku
KERNER Ren
KESKI-SAARI Sarita
KEUNEN Els
KEURENTJES Joost
KEVERS Claire
KOLEHMAINEN Mikko
KOLLIST Hannes
KONCZ Csaba
KONTUNEN-SOPPELA Sari
KONTURSKA Olga
KOORNNEEF Maarten
KOPEN David
KOPKA Joachim
KUHN Josef
KUJANP Anne
KURDYUKOV Sergey
KUSCHK Peter
KUSNATSOV Viktor V.
KRENLAMPI Sirpa
LA VECCHIA Giovanna

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LAANEMETS Kristiina
LALOUE Michel
LAMAN Nicolay
LARHER Robert Franois
LAZAUSKAS Sigitas
LE THIEC Didier
LEFEBVRE-DE VOS Delphine
LEFVRE Isabelle
LEPORT Laurent
LEPRINCE Anne-Sophie
LESS Hadar
LHOTKOV Zuzana
LHOTSK Ondej
LI Jing
LIBANTOV Jana
LIEDSCHULTE Verena
LINDGREN Ove
LIU Tschu-Jie
LOURENO Tiago
LUBYANOVA Alsu
LUGAN Raphal
MACHT Ji
MADHOU Priyadharshini
MADZAK Catherine
MAGYAR Zoltan
MAJIRA Amel
MALEC Lukas
MALTESE Federica
MARCINSKA Izabela
MARGNA Liina
MARTINEZ-ZAPATER Jos M.
MARTZ Francoise
MATUKOV Ildik
MAURO Sergio
MAYER Florian
MCNALLY Kenneth
MEDVEDEV Sergey
MERBACH Wolfgang
MERILO Ebe
MESKIENE Irute
MHADHBI Haythem
MICHEL Alain
MILLER Gad
MISHRA Anamika
MISHRA Kurnud
MITTLER Ron
MOLDAU Heino
MONSHAUSEN Gabriele
MORAVKOV Jana
MORRA Solange
MOSHKOV Igor
MUHOVSKI Yordan
MUNNIK Teun
MURATOVA Anna

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MENP Maarit
MKEL Riina
MLLER Margarete
MLLER-STARCK Gerhard
MYLONA Photini
NAEGELE Thomas
NARULA Neeru
NAWRATH Christiane
NEDBAL Ladislav
NEGRAO Sonia
NEUBERG Marek
NIINEMETS lo
NIOGRET Marie-Franoise
NOSOV Alexander
NOVIKOVA Galina
NUKARINEN Ella
OFFRINGA Remko
OJANGU Eve-Ly
OKSANEN Elina
OKSANEN Timo
OLIVEIRA Margarida
OLORTEGUI Myriam
OPDENAKKER Kelly
ORDS Ricardo J.
OZFIDAN Ceyda
PAGES Montserrat
PAIS Maria Salom
PALGYI Andrs
PALLADINA Tatiana
PALVA Tapio E.
PAPLAUSKIEN Vanda
PAPP Istvan
PARVIAINEN Juha
PAUK Jnos
PAVES Heiti
PAVLIK Milan
PAVLIKOVA Daniela
PECHTER Priit
PENNANEN Ville
PERNIEMI Sirpa
PETROZZA Angelo
PETROV rka
PIERITZ Janin
PIETIKINEN Liisa
PIISIL Maria
PINTO RICARDO Cndido
PIRELOV Beta
PLANAS Joan
PLANCON Sbastien
POJIDAEVA Elena
POKORA Wojciech
POLIDOROS Alexios
POLOK Kornelia
POPELKOVA Hana

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PORALI Ilkka
POZHIDAEVA Elena
PRASHAR Ankush
PRATHIBA Mary
PRZYBYSZ Arkadiusz
PUZRJOVA Irina
PRNIK Tiit
QUANG-DANG Hung
QUARRIE Steve
RACHAMILEVITCH Shimon
RAMSAY Gavin
RASULOV Bahtijor
RAUSCH Thomas
REICH Martin
REMANS Tony
RENAULT Hugues
RENAUT Jenny
REYMOND Matthieu
RIBARI-LASNIK Cvetka
RICHARD Luc
RICHTER Gregory
RIIKONEN Johanna
RODRIGUES Mafalda
RODRGUEZ Ana
ROMIH Nadja
ROUSI Matti
ROUSSEL Valrie
RUHANEN Hanna
SAARINEN Markku
SFRNY Judit
SAGLAM Aykut
SAIBO Nelson
SAMATOVA I.S.
SANTRUCEK Jiri
SARJALA Tytti
SARUHAN Neslihan
SAVOUR Arnould
SAXENA Triambak
SCHIPAREV S.M.
SCHMIDT Wolfgang
SCHREIBER Lukas
SCHROEDER Julian I.
SCWEIGHOFER Alois
SEBASTIANA Monica
SECKIN Burcu
SEDLACEK Lukas
SEDMAN Joosep
SEKMEN Askim Hediye
SERTEYN Didier
SHAKIROVA Farida
SHAROVA E.I.
SHEVYREVA Taisiya
SHOSEYOV Oded
SHUGAEV Alexander G.

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SILVA Marta S.
SIMKOVA Marie
SIMON-KISS Ibolya
SIPARI Nina
SKRZYPEK Edyta
SOWISKI Adam
SMEETS Karen
SNGAROFF Jacques
SOKOLENKO-NIEBLING Anna
SOUDEK Petr
SOUSA Lisete
SZABADOS Laszlo
SZALACHA Elbieta
SZIRA Fruzsina
STARK Sari
STAWICKA Agata
STEIN Hanan
STEINMETZ Andr
STEPANCHENKO Natalia
STERR Irina
SURVILA Mantas
SWANSON Sarah
SBER Anu
ANTREK Ji
EBELA Marek
IMKOV Marie
ZACHLEDER Vilm
ZALEJSKI Christine
ZARZA Xavier
ZHMURKO Vasily
ZHU Jian-Kang
ZIELINSKI Roman
ZILBERSTEIN Aviah
ZOMBORI Zoltn
ZRALY Boguslaw
ZSCHIESCHE Wiebke
ZUBRYCH Alexander
ZWIENIECKI Maciej
TAPPO Tairi
TERZI Rabiye
TERVAHAUTA Arja
TESTERINK Christa
THAKUR Ramesh C.
THOLL Stephane
THOMAS Clment
THOMPSON Jacqueline
TIBURCIO Antonio F.
TOBIAS Mari
TROFIMOVA Marina S.
TRTILEK Martin
TRUVE Erkki
TUKAJ Zbigniew
TURKAN Ismail
TUTEJA Narendra

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TRJK Ott
TYLICHOV Martina
UMBRASAITE Julija
UMYSOVA Da
UNTERWURTZACHER Verena
URUSOV Felix
WACHTER Andreas
VAHISALU Triin
VAID Neha
VANDERWINDEN Jean-Marie
VANK Tom
WANG Yuh-Shuh
VANGRONVELD Jaco
VANKOVA Radomira
VAPAAVUORI Elina
VAKOV Martina
VERPOORTE Rob
VINOCUR Basia J.
WATILLON Bernard
VAULTIER Marie Noelle
WEGENER Christiane B.
VENKATESH Geetha
WEYENS Nele
WHITE Philip
WILL Isa
WISHART Jane
VITOVA Milada
VOISIN Derry
VREUGDENHIL Dick
WROCHNA Mariola
VUOSKU Jaana
YEPHREMOV Alexander
YULDASHEV Ruslan

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Maps and travel information


Arrival
1. Arrive in Tallinn.
2. Take a taxi to Tallinn Bus Station. Costs about 80-100 EEK from the airport.
3. Take express bus from Tallinn to Tartu. Ticket costs 150 EEK, bus ride takes 2 hours and 25 minutes.

Walk to Dorpat hotel (Soola str 6, Tartu)


Walk to Pallas hotel (Riia str 4, Tartu)
Walk 1.2 km or take a taxi to Hansa hotel (Aleksandri str 46, Tartu)

Departure
1. Take express bus from Tartu to Tallinn airport.
NB! Majority of Tartu-Tallinn buses stop in Tallinn airport. To be sure ask check this out from the bus driver.
For more travel information see www.ut.ee/inpas/estonia

Map of Tallinn
141

Conference Centre
Dorpat Conference Centre is located at Turu str 2, on the 4th floor. There is a large sign Dorpat Konverentsikeskus above the main entrance.

All talks take place in Struve auditorium (Struve I and Struve II).
Lunches, poster session buffet and coffee are served in the conference centre hall.
Closing Dinner (Saturday, 16th of May) is served in
Gunpowder Cellar.

Map of Tartu
142

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International Network of Plant Abiotic Stress 2009