Società Italiana di Biologia Vegetale

Transcript

Società Italiana di
Biologia Vegetale
First Congress
Verona 30th June - 02nd July 2009
PROCEEDINGS
Università degli Studi di Verona
Polo Zanotto, V.le dell’Università 34 - Verona
Società Italiana di Biologia Vegetale - I Annual Congress - Verona 30th June 02nd July 2009
TUESDAY JUNE 30th
08:00 - 9:00
PARTICIPANT REGISTRATION, POSTER PUT ON
09:00 - 09:30
OPENING CEREMONY
09:30 - 12:30
Plenary Lecture: DARWIN AND EVOLUTION
AULA MAGNA T3
In collaboration with the Faculty of Mathematical, Physical and Natural
Sciences - University of Verona.
Chairmen: Roberto Bassi and Rodolfo Costa
9:30 - 10:15
ON DARWIN'S FOOTSTEPS: HOT TOPICS IN ANIMAL PHYLOGENY AND
EVOLUTIONARY DEVELOPMENTAL BIOLOGY
A. Minelli. Dipartimento di Biologia, Università di Padova. Padova, Italy
10:15 - 11:00
THE ROLE OF GENOME DUPLICATION IN THE EVOLUTION OF DIVERSITY
11:00 - 11:45
EVOLUTION OF CELL DEFENCE MECHANISMS
11:45 - 12:30
EVOLUTION OF THE HUMAN BRAIN
12:30 - 14:30
LUNCH & POSTER VIEWING
14:30 - 16:30
Plenary Session I: PLANT EVOLUTION
J.H. Postlethwait. University of Oregon, Eugene, OR. USA
G. Macino. Dipartimento Biotecnologie Cellulari ed Ematologia Università di
Roma “La Sapienza”. Roma, Italy
G. Berlucchi. Dipartimento di Scienze Neurologiche e della Visione.
Università di Verona. Verona, Italy
AULA MAGNA T3
Chairman: Rodolfo Costa and Roberto Bassi
14:30 - 15:15
15:15 – 16:00
SALTATIONAL EVOLUTION IN PLANTS: HOPEFUL MONSTERS ARE HERE TO
STAY.
G. Theissen. Department of Genetics, Friedrich Schiller University. Jena,
Germany
PHOTOSYNTHESIS, AN UNINTELLIGENT DESIGN.
16:00 - 16:30
W. Nitschke. Laboratoire de Bioénergétique et Ingénierie des Protéines
(BIP). Institut de Biologie Structurale et Microbiologie (IBSM) CNRS,
Marseille. France
ROUND TABLE: HOTSPOTS ON EVOLUTION OF LIFE ON EARTH
R. Costa, R. Bassi, J. Barber, W. Nitschke, A. Minelli, J.H. Postlethwait
G. Macino G. Berlucchi G. Theissen.
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16:30 - 17:00
COFFEE BREAK & POSTER VIEWING
17:00 - 19:05
Plenary Session II: PLANT GROWTH AND DEVELOPMENT
AULA MAGNA T3
Chairpersons: Rino Cella and Lucia Colombo
17:00 - 17:45
A GENETIC FRAMEWORK FOR THE AUXIN/CYTOKININ CONTROL OF CELL
DIVISION AND DIFFERENTIATION IN THE ROOT MERISTEM.
S. Sabatini Dipartimento di Genetica e Biologia Molecolare, Laboratory of
Functional Genomics and Proteomics of Model Systems, Università di Roma
“La Sapienza”. Roma, Italy
17:45 - 18:30
ORGAN SIZE CONTROL IN ARABIDOPSIS.
18:35 - 19:05
D. Inzé Department of Plant Systems Biology, Flanders Interuniversity
Institute for Biotechnology (VIB), Ghent University, Belgium
Parallel Session I: PLANT GROWTH AND DEVELOPMENT
AULA MAGNA T3
Chairpersons: Lucia Colombo
18:35 - 18:50
PI 14: A VPE GENE IS UP-REGULATED DURING NUCELLUS PROGRAMMED CELL
DEATH IN SECHIUM EDULE SEED.
L. Lombardi. Dipartimento di Biologia. Università di Pisa. Pisa, Italy
18:50 - 19:05
PI 17: ROLE OF GIBBERELLINS IN TWO TOMATO AUXIN SIGNALLING MUTANTS
DURING THE EARLY STAGES OF FRUIT DEVELOPMENT.
F. Mignolli. Dipartimento di Biologia delle piante agrarie. Università di
Pisa. Pisa, Italy
18:35 - 19:05
Parallel Session II: PLANT GROWTH AND DEVELOPMENT
AULA T 1.2
Chairpersons: Rino Cella
18:35 - 18:50
PI 05: DNA REPAIR CAPABILITY AND OXIDATIVE BURST PRODUCTION ARE
IMPAIRED IN TOPO I-DEFICIENT CARROT CELLS.
A. Balestrazzi. Dipartimento di Genetica e Microbiologia.Università di
Pavia. Pavia, Italy
18:50 - 19:05
PIII 09: THE USE OF A NEW GENETICALLY ENCODED PROBE ALLOWS IN VIVO
DETECTION OF H2O2 AT PEROXISOMAL LEVEL IN YOUNG AND SENESCENT
ARABIDOPSIS LEAVES, UNCOVERING A CA2+ -DEPENDENT SCAVENGING SYSTEM.
A. Costa. Dipartimento di Biologia.Università di Padova. Padova, Italy
19:30 -21:00
TOURISTIC TOUR IN VERONA
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WEDNESDAY JULY 1ST
09:00 - 11:00
Plenary Session III: BIOENERGY
AULA T4
Chairmen: Roberto Bassi and Felice Cervone
09:00 –09:50
IF THE LEAF CAN DO IT WE CAN DO IT AND EVEN BETTER.
J. Barber. Imperial College, London. London, UK – Politecnico di Torino,
Torino, Italy
09:50 – 10:40
THE CELL WALL CHALLENGE: DEVELOPING PLANT BIOMASS AS A FEEDSTOCK
FOR BIOFUELS AND BIOREFINERIES.
L. Gomez. Centre for Novel Agricultural Products, Department of Biology,
University of York. York, UK
10:40 – 11:00
PII 01: IMPROVED GROWTH IN PHOTOBIOREACTORS USING CHLAMYDOMONAS
REINHARDTII MUTANTS SELECTION FOR REDUCED ANTENNA SIZE
C. Formighieri. Dip. di Biotecnologie-Università di Verona. Verona, Italy
11:00 - 11:30
11:30 – 13:30
Parallel Session III: SIGNALLING
AULA T4
Chairpersons: Giulia De Lorenzo and Mauro Marra
11:30 – 11:45
PIII 06: HORMONAL CONTROL OF FRUIT DEVELOPMENT: AUCSIA GENES AS NEW
PLAYERS IN AUXIN-MEDIATED FRUIT INITIATION?
B. Molesini. Dipartimento di Biotecnologie - Università di Verona, Verona
11:45 – 12:00
PIII 11: BIOCHEMICAL CHARACTERIZATION OF THE ATYPICAL CHLOROPLASTIC
GLUTAREDOXIN S12.
M. Zaffagnini. Laboratory of Molecular Plant Physiology, Department of
Evolutionary Experimental Biology, University of Bologna. Bologna
12:00 – 12:15
PIII 16: STUDY OF THE OLIGOGALACTURONIDE SIGNALLING PATHWAY USING
PROTEOMIC STRATEGIES AND IN VITRO AND IN VIVO AFFINITY FISHING USING
DERIVATIZED ELICITORS.
B. Mattei. Dipartimento di Biologia Vegetale, Università di Roma “La
Sapienza”. Roma
12:15 – 12:45
ISOPRENE: ONLY A LEAF BY PRODUCT OR A KEY ANTIOXIDANT/SIGNALLING
MOLECULE?
F. Loreto. Consiglio Nazionale delle Ricerche (CNR) – Istituto di Biologia
Agroambientale e Forestale. Monterotondo Scalo (Roma), Italy.
12:45 – 13:15
SPATIO-TEMPORAL FEATURES OF THE ELECTRICAL NETWORK ACTIVITY IN
THE ROOT APEX.
S. Mancuso. LINV, Department of Horticulture, Polo Scientifico, Università
di Firenze, Sesto Fiorentino (Firenze), Italy
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11:30 – 13:30
Parallel session IV: BIODIVERSITY
AULA 1.2
Chairmen: Claudio Varotto and Giovanni Giuseppe Vendramin
11:30 – 12:00
DNA BARCODING AND RECONSTRUCTION OF PAST PLANT COMMUNITIES USING
PERMAFROST SAMPLES
P. Taberlet. Laboratoire d'Ecologie Alpine (LECA), CNRS UMR 5553,
Univ. Joseph Fourier. Grenoble, France.
12:00 – 12:30
12:30 – 12:45
12:45 – 13:00
13:00 – 13:15
CONIFERS: PATTERNS OF GENE DIVERSITY AND RECOMBINATION, AND
MOLECULAR SIGNATURES OF NATURAL SELECTION AND DEMOGRAPHICAL
EVENTS
Santiago C. González-Martínez. Center of Forest Research-INIA. Madrid,
Spain.
PIV 03: EVOLUTION OF FLOWERING TIME AND FRUIT QUALITY TRAITS IN
TOMATO.
G. Falcone, Casaccia Research Center.SM di Galeria( Roma), Italy
PIV 06: MOLECULAR ANALYSIS
ANTHOCYANINS IN THE FRUIT.
OF
TOMATO
MUTANTS
PRODUCING
G. Povero. PlantLab, Scuola Superiore Sant'Anna. Pisa, Italy
PIV 07: DIVERSITY IN THE RESPONSE TO LOW TEMPERATURE IN A SET OF
REPRESENTATIVE BARLEY GENOTYPES CULTIVATED IN EUROPE.
13:30 - 14:30
14:30 - 16:30
16:30 – 17:00
17:00 – 18:15
AULA T4
18:30 – 20:00
AULA 1.2
21:00
C. Lago. CRA-GPG Centro Genomica e Postgenomica Animale e Vegetale.
Fiorenzuola d'Arda (PC), Italy.
LUNCH
POSTER VIEW
GENERAL ASSEMBLY I
1) COMUNICAZIONI;
2) CONGRESSO F.I.S.V 2010;
3) BILANCIO CONSUNTIVO S.I.F.V. 2008
4) SUMMER SCHOOL MARATEA 2009 “MINERAL NUTRITION IN
PHOTOSYNTHETIC ORGANISMS: MOLECULAR, PHYSIOLOGICAL AND
ECOLOGICAL ASPECTS”
5) NOMINA DEL VINCITORE E LECTURE DEL PREMIO FRANCA RASI
CALDOGNO 2009;
6) APPROVAZIONE DELLO STATUTO E DEL REGOLAMENTO DELLA SOCIETÀ
ITALIANA DI BIOLOGIA VEGETALE
GENERAL ASSEMBLY II
1) RINNOVO DELLE CARICHE SOCIALI: ELEZIONE DEL PRESIDENTE SIBV, DEL
SEGRETARIO SIBV, DEL CONSIGLIO SIBV, REVISORI DEI CONTI SIBV
2) AMMISSIONE NUOVI SOCI
3) VARIE ED EVENTUALI
SOCIAL DINNER
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THURSDAY, JULY 2nd
09:00 - 11:00
Plenary Session IV: CROPS FOR THE FUTURE
AULA T4
Chairmen: Mario Enrico Pè and Pierdomenico Perata
09:00 - 09:45
THE IMPORTANCE OF PLANT SCIENCE AND BREEDING IN THE 21ST CENTURY.
09:45 – 10:30
FROM GENOMICS TO PLANT BREEDING: HOW TO MAKE THE BEST USE OF
GENETIC VARIATION.
A. Greenland. The John Bingham Laboratory, NIAB – Cambridge, UK
M. Morgante. Istituto di Genomica Applicata, Parco Scientifico di Udine
and Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine.
Udine, Italy
10:30 - 10:45
10:45 - 10:00
PII 09: TOBACCO CHLOROPLAST TRANSFORMATION: SYSTEM FOR PLANT "BIOFACTORY"
P. Longoni. Dipartimento di Genetica e Microbiologia.Università di Pavia.
Pavia, Italy
PI 31: OVEREXPRESSION OF AQUAPORIN VVPIP2;4 AMELIORATES GROWTH
PERFORMANCES BY MODIFYING WATER METABOLISM OF GRAPEVINES IN
ABSENCE OF WATER OR SALT STRESS, BUT NOT UPON STRESS.
C. Lovisolo. Dipartimento di Colture Arboree. Università di Torino.
Grugliasco (TO), Italy
11:00 - 11:30
11:30 – 13:30
Parallel Session V: PLANT-MICROBE INTERACTIONS
AULA T4
Chairpersons: Paola Bonfante and Matteo Lorito
11:30 – 12:00
PLANT-PATHOGEN INTERACTIONS: DEVELOPMENT OF RESISTANT POTATO
PLANTS THROUGH ADVANCED BREEDING STRATEGIES.
D. Carputo. Department of Soil, Plant, Environmental and Animal
Production Sciences, University of Naples Federico II (ITALY), Portici
12:00 – 12:30
12:30 – 12:45
12:45 – 13:00
THE ROLE OF RNA SILENCING IN PLANT VIRUS-INTERACTION.
J. Burgyan. Istituto di Virologia Vegetale, CNR, Torino, Italy
PVI 16: HOST-DERIVED SIGNALS ACTIVATE PLANT INNATE IMMUNITY .
S. Ferrari. Dipartimento di Biologia Vegetale, Università di Roma “La
Sapienza”. Roma, Italy.
PVI 11: LIPOPEROXIDATIVE EVENTS INFLUENCE OCHRATOXIN A BIOSYNTHESIS
IN ASPERGILLUS OCHRACEUS DURING THE INTERACTION WITH TRITICUM
DURUM SEEDS.
A. Ricelli. Istituto di Chimica Biomolecolare, CNR, Roma
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13:00 – 13:15
13:15 – 13:30
PVI 06: INTRACELLULAR CALCIUM CHANGES IN THE SYMBIOTIC SIGNALING
PATHWAY ACTIVATED BY FLAVONOIDS IN RHIZOBIA.
L. Navazio. Dipartimento di Biologia, Università degli Studi di Padova.
Padova, Italy
PVI 08: IDENTIFICATION OF THE EFFECTS OF FUSARIUM VERTICILLOIDES ON
MAIZE TRANSCRIPTOME IN RELATION WITH HOST RESISTANCE.
A. Marocco. Institute of Agronomy, Genetics and Crop Sciences, Università
Cattolica del Sacro Cuore. Piacenza, Italy
11:30 – 13:30
Parallel session VI: MEMBRANE DYNAMICS AND FUNCTIONS
Aula 1.2
Chairpersons: Anna Moroni and Alessandro Vitale
11:30 – 12:00
ON THE ROLE OF THE OUTER CHLOROPLAST ENVELOPE IN REGULATION OF
METABOLITE EXCHANGE BETWEEN THE CHLOROPLAST AND CYTOPLASM.
R. Wagner. Biophysics, Department of Biology/Chemistry, University
Osnabrueck, Osnabrueck, Germany.
12:00 – 12:30
RETICULONS AND PLANT ENDOPLASMIC RETICULUM MORPHOLOGY.
12:30 – 12:45
PVII 04: BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION
CHLOROPLAST-LOCATED PLANT GLUTAMATE RECEPTOR.
L. Frigerio. Department of Biological Sciences, University of Warwick,
Coventry, UK
OF
A
Ildikò Szabò. Dipartimento di Biologia, Università degli Studi di Padova.
Padova, Italy
12:45 – 13:00
PVII 05: CALCIUM PERMEATION IN PLANT CATION CHANNELS DETERMINED BY
A NOVEL FLUORESCENCE/PATCH-CLAMP APPROACH
A. Carpaneto. Istituto di Biofisica, Consiglio Nazionale delle Ricerche
(CNR). Genova, Italy
13:00 – 13:15
PVII 14: PLANT CELLULAR DYNAMICS ASSOCIATED TO ROOT COLONIZATION BY
ARBUSCULAR MYCORRHIZAL FUNGI.
A. Genre. Dipartimento di Biologia Vegetale, Università di Torino/IPPCNR, Torino, Italy.
13:15 – 13:30
PVII 10: MODELLING OF PROTEIN-PROTEIN INTERACTIONS WITHIN THE
PHOTOSYSTEM II CORE COMPLEX AND ANTENNA SYSTEM IN GRANA
MEMBRANES OF PLANT CHLOROPLASTS
S. Zorzan. Dipartimento di Biotecnologia, Università degli Studi di Verona.
Verona, Italy
13:30
CLOSING REMARKS
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PLENARY LECTURES – Darwin and Evolution
On Darwin’s footsteps: hot topics in animal phylogeny and
evolutionary developmental biology
A. Minelli
Dipartimento di Biologia, Università di Padova. Padova, Italy
It took a century since the Origin for systematics to adopt sound methods of phylogenetic
reconstruction. More recently, the comparison of protein and nucleic acid sequences has
been vigorously shaking the phylogenetic tree. Major changes of perspectives have been
broadly accepted, e.g. the notion of Ecdysozoa (Arthropoda with Nematoda) replacing the
previously fashionable Articulata (Arthropoda with Annelida), but we still do not know
whether the sponges, the crustaceans and the hexapods are monophyletic groups, how the
first bilaterians did look like, or whether the Chaetognatha belong in the system. Despite the
many big problems still remaining with the reconstruction of phylogeny, we have started at
last reconstructing, against the revised phylogenetic scenarios, the evolution of animal
organization (e.g. body axes and their polarity, segmentation, body cavities, brain etc.) and
animal life cycles. This has been largely possible because of the resurrection of the long
abandoned dialogue between evolutionary biology and developmental biology, with the
advent of an integrated approach (evo-devo) which is contributing a revised understanding
of evolvability, and thus of the gross patterns of evolution, including exaptation and
convergence.
The role of genome duplication in the evolution of diversity
J. H. Postlethwait
University of Oregon, Eugene, OR. USA
Genome duplication has been a recurring theme in the evolution of plants and animals.
Gene and genome evolution have been imagined to act as engines of lineage diversification
and increasing complexity. What are the similarities and differences in the origins and
consequences of genome duplication in plants and animals? Like many plant species,
humans are octaploids. Compared to humans, teleost fish, the largest group of vertebrates,
experienced an additional genome duplication. We will explore the principles that guided the
evolution of gene functions after three rounds of whole genome duplication in animals and
contrast the results with similar trajectories of genome duplication in plants.
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PLENARY LECTURES – Darwin and Evolution
Evolution of cell defence mechanisms.
G. Macino
Dipartimento Biotecnologie Cellulari ed Ematologia Università di Roma “La Sapienza”
In early ninety there were several report in Plants and fungi about an unknown mechanism
of gene silencing affecting gene expression in transgenic organisms. Since then much is now
known. RNA silencing is a natural mechanism (RNAi) of gene regulation in eukaryotic cells
and is used as defence against transposons and viruses. Small double-stranded RNA
molecules 20-28 nucleotides long (siRNA) trigger the degradation of target RNA or DNA,
thereby reducing specific gene expression. Furthermore in recent years very abundant small
RNA molecules called MicroRNA (miRNA)were found in mostly of the eukaryotic cells
encoded by endogenous genes although miRNA in animals and plants seems to have
evolved independently. Conservation of the key proteins involved in RNAi suggests that the
last common ancestor of modern eukaryotes possessed siRNA-based mechanisms. MicroRNA
are now considered the most promising regulators of gene expression in normal and
pathological tissues. Since then an enormous number of groups joined the field of RNA
silencing producing an impressive acceleration to the studies and their possible applications.
Evolution of the human brain.
G. Berlucchi
Dipartimento di Scienze Neurologiche e della Visione. Università di Verona
The evolutionary line leading to Homo sapiens split from that leading to the living great apes
around 5-6 million years ago in Africa. The brain of Homo sapiens is three times as large as
expected in a hypothetical primate of the same body size as living humans. The striking
increase in brain size is supposed to have occurred about 2.5 million years ago with the
emergence of the genus Homo, possibly in association with the development of tool
manufacture and migration out of Africa. It is generally thought that the adaptations of our
hominin forebears to a hunter-gatherer existence on the African savannah are reflected in a
brain which eventually enabled the existence of the unique characteristics of the mind of
modern humans: language, episodic and prospective memory, mental time travel and
theory of mind. The general plan of brain organization specific to our species has remained
unchanged since the appearance of Homo sapiens at least one hundred thousand years ago.
While some characteristics of the human mind may have evolved because of the increase in
brain size, complex manual skill and language appear to have required a brain
reorganization involving the allocation of different functions to the right and left cerebral
hemispheres. In addition, human evolution may have required the division of the brain into
modules specialized for different cognitive functions, such as language production, language
comprehension, grammatical competence, face recognition and so forth. The merits and
weaknesses of this hypothesis will be reviewed in the light of modern neurophysiological
evidence on brain-mind relations.
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PLENARY LECTURES – Plant Evolution
Saltational evolution in plants: hopeful monsters are here to stay
G. Theissen
Department of Genetics, Friedrich Schiller University Jena, Philosophenweg 12, 07743 Jena
Charles Darwin argued that evolution proceeds always in a countless number of very small
steps, a view termed „gradualism‟. Darwin was afraid that, „if it could be demonstrated that
any complex organ existed, which could not possibly have been formed by numerous,
successive, slight modifications‟, his „theory would absolutely break down‟. Almost all
contemporary biologists will agree that gradual changes represent the most frequent mode
of evolution, but whether it is the only one has been hotly debated since Darwin‟s time.
Several lines of evidence, ranging from paleontology to molecular biology and genomics,
suggest that profound („saltational‟) changes also may have been crucial evolutionary
events, especially for the establishment of novelties. In my talk I will report about homeotic
changes in the flower of the angiosperms, which make it appear likely that saltational
events indeed happened during evolution. Comparing tulips, orchids and Arabidopsis as
examples I will show that changes in organ identity that occurred many millions of years
ago can be reconstructed and their molecular developmental genetic basis be understood.
But how could organisms with a profound mutant phenotype initially survive and establish
new evolutionary lineages? To better understand the performance of such „hopeful
monsters‟ in natural populations in the wild we study molecular, morphological and
ecological details of a floral homeotic variant of Capsella bursa-pastoris (Shepherd‟s purse)
that has all petals replaced by stamens. Such studies of saltational evolution „in statu
nascendi‟ may help us to clarify exactly how homeotic transitions contribute to
macroevolution. Since saltational changes have the potential to establish profound novelties
initiating adaptive radiations, they could be very important for the origin of biodiversity,
even though they are possibly very rare events. From that perspective, however, saltational
changes are not more bizarre scenarios of evolutionary change than whole genome
duplications, endosymbiosis or impacts of meteorites. In conclusion I argue that the
complete dismissal of saltational evolution is a historical error of evolutionary biology tracing
back to Darwin that needs to be rectified.
Photosynthesis, an un-intelligent design
Nitschke W.A.
Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP). Institut de Biologie
Structurale et Microbiologie (IBSM) CNRS, Marseille, France.
The observation that (almost) all life on our present planet ultimately depends on
photoautotrophic organism has in the past led to the firm and persistent conviction that the
photosynthetic mechanism must have been closely linked to the origin of life. Results from
palaeogeochemistry, microbial diversity and molecular phylogeny have during the last two
decades completely overthrown this concept. The new vision of the early evolution of life on
Earth now acknowledges a “relatively” late appearance of photosynthesis and its integration
into full-fledged chemoautotrophic and heterotrophic chemiosmotic membranes. In parallel,
the depth and details of the evolutionary pathway from simple (anoxygenic?) roots to the
oxygen-evolving Z-scheme started to unfold in front of our eyes. Although many questions
remain, a large part of the previous inventory of evolutionary scenarios must now be
discarded based on empirical evidence and many hitherto enigmatic and counterintuitive
properties of photosynthesis are quite naturally rationalised via its Darwinian evolution
within a genuinely non-photosynthetic background.
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PLENARY LECTURES – Plant growth and development
A genetic framework for the auxin/cytokinin control of cell division
and differentiation in the root meristem.
R. Dello Ioio1, K. Nakamura2, L. Moubayidin1,4, S. Perilli1,4, M. Taniguchi2, M.
T. Morita3, T. Aoyama2, P. Costantino1, S. Sabatini1*
1
Dipartimento di Genetica e Biologia Molecolare, Laboratory of Functional Genomics and Proteomics of
Model Systems, Università La Sapienza, Rome. Italy. 2 Institute for Chemical Research, Kyoto
University, Uji, Kyoto 611-0011, Japan. 3 Graduate School of Biological Sciences, Nara Institute of
Science and Technology, Ikoma, 630-0101, Japan.
Plant post-embryonic development takes place in the meristems. In the root meristem a stem cell
niche generate transit-amplifying cells, which undergo additional division in the proximal meristem,
and differentiate in the distal meristem transition zone that encompasses the boundary between
dividing and expanding (differentiating) cells in the different cell files. For meristem maintenance, and
therefore continuous root growth, the rate of cell differentiation must equal the rate of generation of
new cells: how this balance is achieved is a central question in plant development. While the molecular
mechanisms involved in stem cell positioning and activity are partially comprehended, the regulatory
networks controlling the shift from transit-amplifying identity to differentiation are still poorly
understood. We have previously shown that in the Arabidopsis root meristem the hormone cytokinin
controls the differentiation rate of transit-amplifying cells by antagonizing the activities of a diffusible
input in the vascular tissue of the transition zone. Here we demonstrate that this diffusible input is
auxin, and that the balance between cell differentiation and cell division, necessary for controlling root
meristem size and root growth is the result of the interaction between cytokinin and auxin through a
simple regulatory circuit converging on the SHY2 gene. In particular, in the vascular tissue of the
transition zone, a primary cytokinin-response transcription factor, ARR1, activates the gene SHY2, a
repressor of auxin signaling that negatively regulates the PIN genes that encode auxin transport
facilitators. Thus, cytokinin causes redistribution of auxin, prompting cell differentiation. Conversely,
auxin mediates degradation of the SHY2 protein, sustaining the activity of the PIN genes and
prompting cell division.
Organ size control in Arabidopsis
Dirk Inzé* and Nathalie Gonzalez
Department Plant Systems Biology, VIB, and Department Plant Biotechnology and Genetics,
Ghent University, 9052 Gent, Belgium
Understanding the mechanisms that govern tissue, organ and organism size are amongst the
most mysterious and fascinating open questions in biology. However, despite its general
importance little is known on the mechanisms controlling organ size. Our long term goal is to
unravel the molecular pathways that govern leaf size in Arabidopsis. One of our approaches is
based on studying the action mechanisms of genes which enlarge leaf size (hereafter called
“intrinsic yield genes” (IYG)) (Gonzalez et al., 2009, Curr. Opin. Plant Biol., in press). Such
analysis is likely to shed light on the various instructor systems governing leaf size.
Currently, we have confirmed the positive effect of 13 IYGs on Arabidopsis leaf size. These
genes operate in seemingly unrelated pathways such as transcriptional regulation; hormone
signaling; tonoplast proton transport; ubiquination and proteolysis (for references see
Gonzalez et al., 2009; our unpublished results). Some of those genes might affect the overall
production of nutrients and/or hormones and thereby indirectly enhance organ size while
others might directly influence the growth process itself e.g. by promoting cell proliferation. In
all cases examined so far enlarged leaf size results from an increased cell number without any
significant effect on cell size. At least in the case of plants with enhanced expression of GRF5
and BRI we could demonstrate that during leaf development cell division continues for a
longer time as compared to wild-type. Our results indicate that, by a yet unknown
mechanism, the instructor network must affect the developmental timing of cell division. Cell
cycle control genes (Inzé and De Veylder, 2006 Annu. Rev. Genet. 40, 77-105) are likely
targets for the instructor genes. Various approaches are now being used to decipher how leaf
size is determined. The long-term goal is to develop computational models describing the
molecular basis of organ size and to use these models to improve crop productivity.
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PLENARY LECTURES – Bioenergy
If the leaf can do it we can do it and even better.
James Barber FRS
Imperial College London and Politecnico di Torino
Global energy consumption is projected to increase, even in the face of substantial declines in
energy intensity, at least two-fold by midcentury relative to the present because of population
and economic growth. This demand could be met, in principle, from fossil energy resources,
particularly coal. However, the cumulative nature of CO2 emissions in the atmosphere demands
that holding atmospheric CO2 levels to even twice their preanthropogenic values by midcentury
will require invention, development, and deployment of schemes for carbon-neutral energy
production on a scale commensurate with, or larger than, the entire present-day energy supply
from all sources combined. Among renewable energy resources, nuclear fusion energy or solar
energy are by far the largest exploitable resource. However in both cases technological
breakthroughs are required with nuclear fusion being very difficult. On the other hand, one hour
of sunlight falling on our planet is equivalent to all of the energy consumed by humans in an
entire year. If solar energy is to be a major primary energy source, it must be stored and
dispatched on demand to the end user. An especially attractive approach is to store solarconverted energy in the form of chemical bonds as occurs in natural photosynthesis. However a
technology is needed which has a year-round average efficiency significantly higher than current
plants or algae, to reduce land-area requirements and to be independent of food production.
Therefore the scientific challenge is to construct an “artificial leaf” able to efficiently capture and
convert solar energy and then store the energy in the form of chemical bonds, producing
oxygen from water and a reduced fuel such as hydrogen, methane, methanol, or other
hydrocarbon species. The “artificial leaf” must be robust and constructed of common materials
and with effort there is no reason why such technology can not be created for future prosperity,
sustainability and harmony of the human race.
The Cell Wall Challenge: Developing Plant Biomass as a Feedstock
for Biofuels and Biorefineries
Leonardo D. Gomez.*, Simon McQueen-Mason
Centre for Novel Agricultural Products, University of York, United Kingdom, YO10 4HH.
With oil reserves diminishing and the effects of industrial emissions on the global climate, there
is an immediate need for renewable carbon-neutral industrial feedstocks. First generation
biorefineries, producing biofuels and bioplastics by the fermentation of sugar or starch, are
seeing a rapid expansion and are adding stress to food supplies. A more sustainable option is to
use plant biomass from agricultural by-products, or dedicated biomass crops. Conversion of
these polysaccharides to sugars will provide cheap and abundant raw materials for industrial
biotechnology. The use of plant biomass in this way is hampered by the high cost of
saccharification due to the recalcitrance of cell walls to enzymatic hydrolysis. At the Centre for
Novel Agricultural Products we are working in several areas to improve the conversion of cell
walls into industrial products. These include an enzyme discovery program as part of the
Sustainable Bioenergy Center, the use of waste for the production of biofuels, and a large EUF7
Consortium (RENEWALL) aiming to identify and modify the structural features of plant cell walls
that make them difficult to process. This European partnership brings together outstanding
biologists, chemists, and enzymologists, as well as industrialists from the plant breeding and
biotechnology sectors, who can take an integrated multidisciplinary approach to solving this
fundamental problem. The Consortium is composed of 17 partners including Academic
Institutions from six European countries, American partners and industry. We will identify the
molecular barriers to saccharification, and the genes that can be manipulated to lower these
barriers. These may be plant genes involved in cell wall biosynthesis or other (often microbial)
genes that can modify wall properties or degrade wall polymers when expressed in plants.
These genes can then be used directly in GM approaches to breed improved plant feedstocks for
biorefining.
- 11 -
PARALLEL SESSIONS – Signalling
"Isoprene: only a leaf byproduct or a key antioxidant/signaling
molecule?"
F. Loreto
Consiglio Nazionale delle Ricerche (CNR) – Istituto di Biologia Agroambientale e Forestale.
Via Salaria Km 29,300 – 00015 Monterotondo Scalo (Roma), Italy.
Plants have evolved an extraordinarily diverse suite of protective mechanisms against biotic
and abiotic stresses. Non-volatile isoprenoids are known to be powerful antioxidants but the
role of volatile isoprenoids (isoprene, monoterpenes and sesquiterpenes) in abiotic stress
responses remains controversial. Here I note that abiotic stress responses generically
involve production of reactive oxygen species in plant cells, that volatile isoprenoids are
reactive molecules whose biosynthesis is elicited by stress conditions, and that volatile
compounds generally exert signaling functions. I will review evidence that isoprene, the
primary and most abundant volatile isoprenoid, a) primes plant response to stress by
activating H2O2 signaling; b) quenches reactive oxygen species in vitro and in vivo; c)
reacts with NO, indirectly modulating the signaling of programmed cellular death upon
stress occurrence; and d) intercalates into cell membranes and strengthens them when
attacked by reactive oxygen species. Finally, I propose that isoprene mitigates the effects of
oxidative stress by mediating, directly or indirectly, the oxidative status of the leaf.
Spatio-temporal features of the electrical network activity in the root
apex
S. Mancuso
LINV, Department of Horticulture, Polo Scientifico, Università di Firenze, Viale delle idee 30,
50019 Sesto Fiorentino (FI) - Italy
Electrically excitable cells are present in many multicellular organisms, especially in brains
of animals, but they are present also in lower animals lacking central nervous system as
sponges or in animals having excitable epithelia, which can conduct signals in addition to
neurons. Conducted electrical events serve for translation of environmental parameters and
cues, obtained via sensory systems, into biological information and processes. In plants,
most cells are electrically excitable and active, releasing and propagating action potentials
(APs), which are believed to play a central role in intercellular and intracellular
communication at all levels of evolution from algae, to bryophytes and higher plants. By
using multi-electrode arrays (MEAs) the spatio-temporal characteristics of the electrical
network activity of the root apex characterised by intense spontaneous electrical activities
as well as stimulation-elicited bursts of locally propagating action potentials, have been
observed. Propagation of APs indicates the existence of excitable travelling waves in plants,
similar to those observed in animal electrogenic non-nervous tissues. Moreover, data
recorded with MEAs reveal synchronous electric activities of root cells, which emerge within
specific root apex region. The dynamic electrochemical activity of root apex cells is proposed
to continuously integrate internal and external signalling for developmental adaptations in a
changing environment.
- 12 -
PARALLEL SESSIONS – Biodiversity
DNA barcoding and reconstruction of past plant communities using
permafrost samples
P. Taberlet
Laboratoire d'Ecologie Alpine (LECA), CNRS UMR 5553, Univ. Joseph Fourier, BP 53
38041 Grenoble Cedex 9, France.
DNA barcoding corresponds to the identification of species using a short DNA fragment. If
the DNA fragment is sufficiently short (i.e. 100-150 bp), it can be used to identify plant
species from ancient DNA remains. For this purpose, we set up a plant identification sytem
based on the P6 loop of the chloroplast trnL (UAA) intron. The protocol used consist to
extract DNA from a permafrost sample, to amplify the P6 loop using universal primers, and
to sequence the PCR product using the 454 sequencer. Using this system, in the Arctic, 33%
of the sequences can be identified to the species level. Salicaceae, Poaceae, Cyperaceae,
and Asteraceae are problematic families, showing a relatively low resolution power of the P6
loop. The trnL approach can be complemented for Poaceae, Cyperaceae, and Asteraceae
with an ITS1 fragment, leading to a much better resolution. However, the system cannot be
improved for Salicaceae. I will present a few preliminary results for permafrost samples
dating from approximately 25,000 years ago. The same approach has many other potential
applications.
Conifers: patterns of gene diversity and recombination, and
molecular signatures of natural selection and demographical events
Santiago C. González-Martínez
Center of Forest Research-INIA, Madrid, Spain
In this talk, I will review recent research on conifers candidate genes for adaptive traits, in
particular about approaches based on sampling of natural populations aiming at the
identification of signatures of natural selection on DNA sequence patterns of polymorphism.
First, a brief description of the particular characteristics of conifers at the gene level
(nucleotide diversity, LD and recombination) will be provided. Second, demographical
models and patterns of natural selection are described for some Mediterranean pines and
future research in these species is outlined. In particular, I will summarize research on local
adaptation while colonization of the western European range of Aleppo pine (Pinus
halepensis) and on persistence in western glacial refugia and signatures of selection for
drought-response genes of maritime or cluster pine (Pinus pinaster). Finally, current
approaches to detect adaptive variation based on natural or breeding populations are
described, in particular those related to the identification of outliers for genetic
differentiation, the correlation between SNP-allele frequencies and environmental variation
and genetic association studies. Examples in tree model species, such as loblolly pine (Pinus
taeda) and Douglas-fir (Pseudotsuga menziesii), would be used to illustrate this part.
Finally, insights on the use of adaptive markers for the use and conservation of forest
genetic resources will be discussed.
- 13 -
PLENARY LECTURES – Crops for the future
Challenges for Plant Science and Breeding in the 21st Century
Andy Greenland
The John Bingham Laboratory, The National Institute of Agricultural Botany, Huntingdon
Road, Cambridge, CB3 0LE, UK.
Most analysts agree that global food production has to double by 2050; land is limited and
future needs cannot be met by simply increasing the area used for agriculture. The big
challenge will be in the delivery of new genetic effects. For the foreseeable future plant
breeding will be the principal delivery mechanism in crop improvement. Since 1982, over
90% of the yield increases in UK winter wheat have resulted from the release of new
varieties. For this to continue the opportunities provided by genomics-led research have to
be fully exploited. New genes that increase crop yield and improve food quality are needed;
investment now in the development of genetic tools and resources such as MAGIC
populations, high-throughput marker platforms and techniques in genomic selection should
not be overlooked. Most genetic gains in crops will be incremental although some, such as
conversion from C3 to C4 photosynthesis and resistance to soil-borne diseases could lead to
dimension changing effects on yield. GM technology will also play a part in securing future
food supplies and the barriers that prevent appropriate exploitation of transgenic crops in
Europe need to come down. This will clear the way for investment in this critical area of crop
research to be reinstated so that technologies that make introduction of genes facile and
completely predictable are developed.
From genomics to plant breeding: how to make the best use of
genetic variation
Michele Morgante
Istituto di Genomica Applicata, Parco Scientifico di Udine, Via J. Linussio 51, 33100 Udine,
Italy - Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, Via delle Scienze
208, 33100 Udine, Italy
The genomics revolution of the last 15 years has improved our understanding of the genetic
make up of living organisms. Together with the achievements represented by complete
genomic sequences for an increasing number of species, high throughput and parallel
approaches are available for the analysis of DNA sequence variation, transcripts, proteins.
The use of genomic tools has allowed us to start to unravel the genetic make up of traits
that are relevant to plant breeding. At the same time a deeper understanding of what
natural variation is at the sequence level has also been achieved, allowing us to realize that
nature can sometime have much greater fantasy and inventiveness than any laboratory
scientist and that genetic variation is continuously created in crop species. The pace at
which we can analyze natural sequence variation has recently been greatly accelerated
thanks to the advent of new DNA sequencing technologies and today the bottleneck is still
represented by our ability to genetically dissect complex traits and identify the genes
underlying them. Finally, after more than seventy years of separation coincided with the
development of quantitative genetics, plant breeding is being reunited to genes thanks to
the opportunities offered by genomics for the identification of genes responsible for
quantitative trait variation. A new phase in crop evolution of targeted modifications is on the
horizon thanks to the progresses in genomics: we will describe the perspectives in this area
using examples from different crop species.
- 14 -
PARALLEL SESSIONS – Plant-Microbe Interactions
Plant-pathogen interactions: Development of resistant potato plants
through advanced breeding strategies.
D. Carputo*1, M. Iorizzo1, A. Barone1, B.P. Millett2, D.S. Mollov2, L.
Frusciante1, J. M. Bradeen2
1
Department of Soil, Plant, Environmental and Animal Production Sciences, University of
Naples Federico II (ITALY), Via Università 100, 80055 Portici. 2 Department of Plant
Pathology, University of Minnesota. St. Paul, Minnesota, 55108 (USA).
The numerous (about 200) wild potato species have evolved several genetically controlled
mechanisms to endure invading pathogens. By contrast, the cultivated potato S. tuberosum has a
comparatively narrow genetic basis. Therefore, tuber-bearing Solanums provide an excellent and
unique genetic diversity for potato breeding purposes. Although the potato is a genetically difficult
organism to work with, genomic approaches have now impacted several components of breeding
programs, allowing breeders to better deploy genes and species. This presentation will report two
examples on the exploitation of incongruent Solanum species to confer pathogen resistance to S.
tuberosum: (1) Genetic engineering was employed to transfer gene RB, conferring resistance to
Phytophthora infestans, from S. bulbocastanum into the cultivated potato. Several resistant
transgenic lines were identified following artificial inoculations. Molecular assays demonstrated a
general trend of enhanced resistance with increasing copy numbers and increasing transcript
levels of the transgene. Research also suggested that RB is transcribed under a wide range of
environmental conditions, and that disease resistance declines with potato plant age. (2) Bridge
ploidies and genomic engineering were used to transfer Ralstonia solanacearum resistance from
S. commersonii to S. tuberosum. Analysis of hybrids showed that latent bacterial colonizations
occurred in roots of symptomless hybrids, whereas no bacterial populations were detected within
stems. A molecular study with AFLP markers provided evidence that resistant hybrids were more
similar to cultivated S. tuberosum than to the wild parent. Transcriptomic data are being analyzed
to identify differentially expressed transcripts between S. commersonii and S. tuberosum, before
and after infection. DArT markers have been developed and used to construct a genetic map for
both species. Their use and usefulness in research on potato-pathogen interaction will be
discussed.
The role of RNA silencing in plant virus-interaction
Pantaleo V1., Csorba T.2, Lakatos L.2, Burgyan J.1,2*
1
Istituto di Virologia Vegetale, CNR, Torino, Italy. –
Godollo, Hungary.
2
Agricultural Biotechnology Center,
Viruses are inducers, as well as targets, of RNA silencing-based antiviral defence.
Replication intermediates or folded viral RNAs activate RNA silencing, generating small
interfering RNAs (siRNAs), which are the key players in the antiviral response. We have
analysed tombusvirus derived siRNAs, which are primary siRNAs, generated by plant dicers
and guide the RNA-induced silencing complex (RISC) to target viral genome expression.
However, viruses are able to counteract RNA silencing by expressing silencing-suppressor
proteins. We have demonstrated that many of the identified silencing-suppressor proteins
bind long double-stranded RNA or siRNAs and thereby prevent assembly of RISC, which
targets the corresponding viral RNA for degradation. Other viral suppressor proteins such as
Beet western yellows virus P0 and the P1 protein of Sweet potato mild mottle virus have
been shown to interact with the protein components of silencing machinery preventing the
target viral RNA degradation. We have also shown that both P0 and P1 target Argonaute1
(AGO1) protein. While P0 targets AGO1 the slicer component of RISC for degradation and
prevents RISC assembly P1 interacts with si- and miRNA loaded AGO1 proteins and inhibits
its gene inactivation activity. The molecular bases of the silencing suppressor strategies and
their effects on endogenous silencing pathways will be also discussed.
- 15 -
PARALLEL SESSIONS – Membrane dynamics and functions
On the role of the outer chloroplast envelope in regulation of
metabolite exchange between the chloroplast and cytoplasm
R. Wagner
Biophysics, Department of Biology/Chemistry, University Osnabrueck, Barbarastr. 13, 49076
Osnabrueck, Germany.
The plastid organelle family conducts vital biosynthetic functions in every plant cell.
Chloroplasts carry out photosynthesis, which converts atmospheric carbon dioxide to carbohydrates like triosephosphate, starch, sucrose and others. These and other biosynthetic
pathway products and intermediates are steadily exchanged with the surrounding cell by the
assistance of specific carrier proteins, localized in the plastidic inner envelope and solute
channels in the outer envelope. While the inner envelope carrier proteins, e. g. the
triosephosphate-phosphate translocator, the dicarboxylic acid translocator, or the hexose
phosphate carrier show a distinct substrate selectivity and specificity, it remains up to now
elusive to which extent the transport through the outer membrane channels is regulated.
The outer envelope membrane has been assumed for a long time to be freely permeable for
most small molecular weight solutes up to 10 kD. Correspondingly, it is believed that the
osmotic barrier against the cytosol is formed exclusively by the inner envelope membrane.
However, we discovered and characterized four specific pore-forming proteins (OEP16,
OEP21 and OEP24, OEP37) in the outer envelope, each exhibiting differences in substrate
specificity and channel characteristics. Their distinct substrate specificities indicate separate
roles in different metabolic processes, challenging the notion that they are general diffusion
pores. In summary our results indicate that the inter-membrane space of chloroplasts is not
freely accessible to low molecular weight solutes.
Reticulons and plant endoplasmic reticulum morphology
N. Tolley1, I. Sparkes2, C. Hawes2 and L. Frigerio1*
1
2
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. –
School of Cell Biology, Oxford Brookes University, Oxford, UK
Reticulons are a family of integral membrane proteins mostly located in the membrane of
the tubular endoplasmic reticulum (ER). Reticulons are hypothesised to help shape ER
tubules by virtue of their unusual transmembrane topology, which imposes curvature onto
the membrane. The Arabidopsis genome encodes 21 reticulon isoforms. This abundance of
reticulons may correlate with the existence of plant-specific ER domains, such as the
desmotubule in plasmodesmata and ER-derived oil bodies in seeds. We have recently found
that a small, seed-specific reticulon isoform (RTN13) can drastically alter ER tubule
morphology in vivo when overexpressed. Downregulation of RTN13 results in increased oil
storage capacity of Arabidopsis seeds. We will present data showing that expression of
RTN13 is sufficient to convert ER membrane sheets into tubules in vivo, and that the length
of the transmembrane regions of RTN13 correlates directly with its tubule-forming capacity.
We will also show initial results on the expression mapping of other Arabidopsis reticulon
isoforms by YFP tagging of their genomic sequences.
- 16 -
ABSTRACTS - Session I: Plant Growth and Development
PI01
A LEAFMINER HELPS US UNDERSTANDING LEAF HYDRAULIC
DESIGN.
ANDREA NARDINI1, FABIO RAIMONDO2, MARIA A. LO GULLO2, SEBASTIANO SALLEO1.
1
Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste. – 2
Dipartimento di Scienze della Vita, Università di Messina, Salita Sperone 31, 98166 Messina S. Agata,
Italia.
Keywords: leaf, hydraulic architecture, bundle sheath, suberin, transpiration stream.
Leaf hydraulics of Aesculus hippocastanum L. was measured over the growing season and during
extensive leaf mining by the larvae of Cameraria ohridella Deschka et Dimic that specifically destroy
the palisade tissue. After leaf expansion was complete, the hydraulic resistance of leaves and the
partitioning of resistances between vascular and non-vascular compartments, remained unchanged
despite extensive disruption of the palisade, suggesting that water flow from the petiole to the
evaporation sites did not directly involve this tissue. The temperature-dependence of Rlamina revealed
that at least one transmembrane step was involved in water transport outside the leaf vasculature.
Anatomical analysis suggested that this symplastic step may be located at the bundle sheath where the
apoplast is interrupted by hydrophobic thickenings of cell walls. Our findings support the view of a
compartmentalization of leaves into well-organized water pools. The transpiration stream would
involve veins, bundle sheath and spongy parenchyma, while the palisade tissue would be largely bypassed thus protecting cells from short-term fluctuations in water status.
PI02
COPPER TOLERANCE IN SILENE PARADOXA L.: AN EPR
INVESTIGATION.
MILUSCIA ARNETOLI1, FRANCESCO DI BENEDETTO2, GIORDANO MONTEGROSSI3,
ANTONELLA BUCCIANTI3, MAURO ROMANELLI2, CRISTINA GONNELLI1, ROBERTO
GABBRIELLI1.
1
Department of Plant Biology, Università di Firenze, via Micheli 1, 50121 Firenze, Italy. – 2
Department of Chemistry, Università di Firenze, via della lastruccia, 3, 50019 Sesto Fiorentino (FI),
Italy. – 3 Department of Earth Science, Università di Firenze, via La Pira 4, 50121 Firenze, Italy.
Keywords: heavy metal tolerance, copper, EPR, Silene paradoxa.
EPR spectroscopy has been applied to investigate a potential role of the cell wall in Cu tolerance in
Silene paradoxa. Plants from Fenice Capanne (FC, Cu tolerant population) mine waste and Colle Val
D'Elsa (CVD, sensitive population) uncontaminated soil were grown in hydroponics and exposed to 10
µM CuSO4 for 3 days. When collected, half of the roots was rinsed with PbNO3 to desorb metals
adhering to the cell wall. In roots and shoots Cu concentration was measured and X-band spectra (~9,5
GHz) recorded. Cu concentrations in roots and shoots increased with Cu exposure. Only in CVD roots,
PbNO3 not treated samples showed a significantly higher metal concentration in respect to the PbNO 3
treated ones. EPR spectra were constituted of a main signal centred to ~3400 G, attributable to monoor poli-nuclear Cu2+ ion complexes. Also ~2000 G signals were present and attributable to Fe3+ ions or
to Cu2+ clusters. Spectra of roots with and without PbNO 3 treatment remained unchanged only in FC.
Results suggested that a low cell wall ability to bind Cu can probably concur to generate the
tolerant/excluder phenotype of the Silene paradoxa Cu tolerant population.
- 17 -
PI03
GENETIC ORGANIZATION AND EXPRESSION PATTERN OF GENES
CODING FOR LEAF ISOFORMS OF THE PLANT TOXIN SAPORIN.
ANDREA TARTARINI1, DONATO GIANNINO2, LAURA SPANÒ1.
1
Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio 67010 Coppito
(L'Aquila), Italy. – 2 Institute of Agricultural Biology and Biotechnology Rome Unit, at the CNR
Research Area, Via Salaria km 29,300, Monterotondo Scalo (Roma), Italy.
Keywords: Saponaria officinalis; saporins; gene expression.
Ribosome-inactivating proteins (RIPs) are potent inhibitors of protein synthesis that accumulate in
different tissues of many plant species. The soapwort plant (Saponaria officinalis L.) produces different
type 1 RIPs, that are collectively named saporins and that are encoded by a small gene family. In a
previous work we have purified from the extra (EC) and intracellular (IC) leaf fractions two saporin
gene products that were biochemically characterized. Corresponding full length clones were isolated,
allowing the first determination of the complete sequence of IC saporin, which diverged from EC at the
identity and phylogenetic level. Both IC and EC genes belonged to gene families, each consisting of at
least 4 members, and did not harbour any intron. The messages of both genes were abundant in leaves
and seeds, while scarce in stems and roots. IC transcription increased of ca 1.7 fold from apical to basal
leaves, whilst that of EC did not significantly vary with growth. The differential regulation of the two
genes suggest that they may have distinct roles.
PI04
RESPONSES OF ANTIOXIDANT SYSTEMS TO EXPOSITION TO RARE
EARTH ELEMENTS AND THEIR ROLE IN ABIOTIC STRESSES IN
COMMON DUCKWEED (LEMNA MINOR L.) AND IN TOMATO
(LYCOPERSICON ESCULENTUM L. CV. MARMANDE).
M. P. IPPOLITO1, C. FASCIANO1, L. D'AQUINO2, F. TOMMASI1.
1
Department of Plant Biology and Pathology, University of Bari, Via Orabona 4, 70125 Bari, Italy. – 2
ENEA Portici Research Center, Via Vecchio Macello, 80055 Portici (Napoli), Italy.
Keywords: Rare earth, abiotic stress, antioxidant systems, environmental impact, common duckweed,
tomato.
Rare earth elements (REE) include 15 elements in the Periodic Table, also known as lanthanides, that
are naturally present in the environment and whose environmental entry is increasing because of their
utilization in industry, in agriculture and zootechnics, thus inducing an increasing concern about the
possible impact on both terrestrial and aquatic ecosystems. Over the last years, interest has been
growing about the effects of REE in increasing plant resistance to environmental stresses, since some
works suggest that La3+, at suitable concentrations, can promote plant resistance to such stresses by the
stimulating the antioxidant systems involved in the control of the reactive oxygen species levels in
plants. The aim of this work was to evaluate the effect of treatments with REE on some antioxidant
systems in two model species, tomato and common duckweed, subjected to drought stress and chilling,
respectively. Following treatments with REE, a stimulation of the antioxidant systems was evidenced,
but it did not induce any improvement in the stress responses of plants and it seemed to be only a
consequence of the unbalance of the cell metabolism due to REE.
- 18 -
PI05
DNA REPAIR CAPABILITY AND OXIDATIVE BURST PRODUCTION ARE
IMPAIRED IN TOPO I-DEFICIENT CARROT CELLS.
BALESTRAZZI A.1, LOCATO V.2,3, BOTTONE M.G.4, DE GARA L.2,3, BIGGIOGERA M.4,
PELLICCIARI C. 4 AND CARBONERA D.1.
1
Department of Genetics and Microbiology, University of Pavia, Via Ferrata 1, I-27100, Pavia, Italy; Department of Plant Biology and Pathology, University of Bari, Via Orabona 4, I-70125, Bari, Italy; 3
Interdisciplinary Center for Biomedical Research (CIR) Università Campus Biomedico Via Longoni
83, I-00155 Roma, Italy; - 4 Department of Animal Biology, University of Pavia, Piazza Botta 10, I27100 Pavia, Italy.
2
Keywords: Antisense, Ascorbate metabolism, Daucus carota, 8-oxo-dG, Necrosis, Programmed Cell
Death, ROS, Topo I, UV-C radiation.
At present, little is known about the sensing of DNA damage and the antioxidant response in the
nucleus in plants. In animal cells, recent studies have emphasized the role played by DNA
topoisomerase I (topo I) both as a cofactor of DNA repair complexes and/or damage sensor. In the
present work, the response to oxidative stress using topo I-defective carrot (Daucus carota L.) cell
suspension cultures (line AT1-b/22) exposed to UV-C radiation was investigated. The AT1-b/22
cultures were more sensitive to UV-C than control cells, as evidenced by Evans blue staining and
quantitative evaluation of 8-oxo-dG. Interestingly, the oxidative burst was impaired in the topo Idepleted cells. The UV-C treatment did not alter the ascorbate metabolism, suggesting that necrosis was
the predominant cell death pathway. This was also confirmed by cytofluorimetric analyses. Only a
reduced population (< 4%) of AT1-b/22 cells was entering PCD, as demonstrated by the annexin V
assay. Finally, the strong sensitivity to mitomycin C observed in AT1-b/22 cells suggests for an active
role of topo I in DNA repair processes induced by UV-C-mediated damage
PI06
PROLIFERATION AND MATURATION PHASES OF ABIES CEPHALONICA
LOUD. EMBRYOGENIC CELLS: EFFECTS OF HUMIC AND FULVIC
ACIDS.
MARCO ZANCANI1, ELISA PETRUSSA1, ALBERTO BERTOLINI1, JANA KRAJŇÁKOVÁ2
ALESSANDRO PICCOLO3, FRANCESCO MACRÌ1, ANGELO VIANELLO1
1
Sezione di Biologia Vegetale, Dipartimento di Biologia e Protezione delle Piante, Università di
Udine, via delle Scienze 91, I-33100 UDINE, Italy. – 2 National Forestry Centre, Forest research
Institute, T.G. Masaryka 22, 96001 Zvolen, Slovakia. – 3 Dipartimento di Scienza del Suolo, delle
Piante e dell'Ambiente, Università di Napoli Federico II, Via Università 100, I-80055 Portici (NA),
Italy.
Keywords: Abies cephalonica; Humic substances; Maturation; Proliferation; Somatic embryogenesis.
Abies cephalonica embryogenic cell masses (ECMs) were grown on proliferation media in the presence
and absence of 100 microg/plate humic acids (HA) or fulvic acids (FA). Proliferation rate, proportion
of consecutive developmental stages of proembryogenic masses (PEMs) and some biochemical
parameters, such as cellular levels of ATP and glucose-6-phosphate (Glu-6-P), were detected. FA
increased significantly the proliferation rate, affected the proportion of PEMs and were able to invert
the negative effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB). It was also shown that the
proton pumping ATPase and PPase activities were decreased in microsomes obtained from PCIBtreated ECMs, while increasing when ECMs were grown in the presence of FA. The effects of humic
substances were also evaluated on the maturation phase, where both HA and FA, if present in the pretreatment media during the proliferation phase, induced a delay in somatic embryo formation. These
results suggest that humic substances could improve the proliferation of PEMs, thus affecting the
subsequent maturation process of A. cephalonica.
- 19 -
PI07
ADVENTITIOUS ROOT FORMATION IN TOMATO UNDER FLOODING.
M. .L. VIDOZ1, E. LORETI2, A. ALPI1 AND P. PERATA3.
1
Department of Crop Plant Biology, University of Pisa, Via Mariscoglio 34, 56124 Pisa, Italy. – 2
Institute of Biology and Agricultural Biotechnology- National Research Council (IBBA-CNR), Via del
Borghetto 80, 56100 Pisa, Italy. – 3 Plant and Crop Physiology Laboratory, Scuola Superiore
Sant'Anna, Via Mariscoglio 34, 56124 Pisa, Italy.
Keywords: tomato, flooding, adventitious root, ethylene, auxin.
Flooding is one of the most frequent and extensive abiotic stresses that affect plant growth. Although
tomato (Solanum lycopersicum) is known for its sensitivity to waterlogging, its ability to produce
adventitious roots (AR) increases plant survival when oxygen availability decreases in the root zone.
The aim of our study was to better understand ethylene and auxin function in tomato AR production
under flooding. Root primordia were observed in hypocotyls 48 h after the beginning of the
submergence treatment. However, when flooded plants were treated with aminoethoxyvinylglycine
(AVG), a compound that prevents the synthesis of the ethylene precursor, there was a hamper in AR
emergence. The use of 1-naphthylphthalamic acid (NPA), an auxin transport inhibitor, has also resulted
in a reduction of AR formation under waterlogging. Moreover, less AR were obtained in experiments
with tomato mutants characterized by a lower sensitivity to ethylene and auxin (Never ripe and
diagetropica, respectively), suggesting that perception and accumulation of ethylene and auxin below
the cotyledonary node are required to trigger this adaptive response to flooding.
PI08
PIT MEMBRANE PECTINS AND IONIC EFFECT IN FOUR LAURACEAE
SPECIES: ANATOMICAL AND BIOCHEMICAL BASES OF IONMEDIATED REGULATION OF XYLEM HYDRAULICS.
EMMANUELLE GORTAN1, STEVEN JANSEN2, ANDREA NARDINI1, SEBASTIANO SALLEO1.
1
Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italia.
Keywords: xylem, pectins, ionic effect, Lauraceae, wood anatomy.
The hydrogel behaviour of pit membrane pectins has been invoked as an explanation for the ionic effect
i.e. the ion-mediated regulation of xylem hydraulics (K). Until now there was little or no direct
evidence that pectins are present in mature pit membranes. Observations of the intervessel pit
membranes in 4 Lauraceae using electron microscopy showed significant variation in the presence of
pectins, which might be responsible for the different sensitivity of K to changes in sap ionic
concentration. Umbellularia californica and Laurus nobilis showed a large increase in K (+20%) in
response to increased ionic strength of the sap. Moreover, these species had more acidic pectins in their
pit membranes compared to methylesterified ones. The thickness of pit membranes did not vary among
the species studied. A positive relationship was found between the ionic effect and the vessel grouping
index, suggesting that grouped vessels result in more contact areas between vessels and a larger ionic
effect. Our data suggest that there is significant interspecific variation in the chemical nature of pit
membranes, which is related to the magnitude of the ionic effect.
- 20 -
PI09
STARCH-TO-SUGAR CONVERSION IN WOOD PARENCHYMA OF FIELDGROWING LAURUS NOBILIS PLANTS: A COMPONENT OF THE SIGNAL
PATHWAY FOR EMBOLISM REPAIR?
SEBASTIANO SALLEO1, PATRIZIA TRIFILÒ2, SARA ESPOSITO2, ANDREA NARDINI1 AND
MARIA ASSUNTA LO GULLO2.
1
Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italia. – 2
Dipartimento di Scienze della Vita "M. Malpighiâ" sezione Botanica, Università di Messina, Salita
Sperone 31, 98166 Messina S. Agata, Italia.
Keywords: Starch-to-sugar conversion, embolism repair, Laurus nobilis, transpiration, xylem
pressures.
The ability of stems of Laurus nobilis L. to refill embolised xylem conduits was studied in plants at
optimal water supply (W) and under conditions of soil drought inducing xylem pressures (Px) of -1.55
(S1) and -2.4 MPa (S2). Starch depolymerization in wood parenchyma was measured in percentage of
cells "with high starch content" (HSC) as counted microscopically. A direct relationship was found
between percentage of HSC and Px, with HSC between 65 and 75% of the total at Px â‰¥ -0.6 MPa at
which recovery from PLC (conductivity loss) was recorded. At low transpiration, starch re-appeared in
wood parenchyma cells but only in plants that showed diurnal stomatal opening. In S2 plants showing
diurnal stomatal closure and nocturnal opening with Px between -1.2 to -2.4 MPa, HSC were only 25%
and plants did not recover from PLC. This finding suggests that:1) the Px threshold for embolism repair
was â‰¥ -0.6 MPa and 2) impeded phloem loading limits starch content in wood parenchyma and
embolism repair. We conclude that starch depolymerization acts as a signal to phloem unloading sugars
to embolised conduits thus generating the necessary osmotic gradients driving refilling.
PI10
PHOSPHORUS AVAILABILITY AND INFLUENCES ON THE
MYCORRHIZAL DEVELOPMENT IN THE SOIL.
CATELLO DI MARTINO, VINCENZO MICHELE SELLITTO AND GIUSEPPE PALUMBO.
Department SAVA Università degli studi del Molise.
Keywords: Phosphorus, mycorrhizal, Durum wheat.
In the soil a reliable source of phosphorus and maintenance of cellular phosphorus homeostasis is
essenzal for the plant life.The role of (AMs) and vescicular- mycorrhizas (VAM) fungi in phosphate
production is not fully clear, but some reports suggest that hyphae of (AMs and VAM) fungi consist in
stimulation of exudation of root phosphatases, both acid and alkaline and in better uptake of Pi relased
after mineralization (recently has been identified genes encoding mycorrhiza-specific plant phosphate
transporters). The aim of this study was to evaluate the effects of mycorrhizal inoculation (Genus
Glomus) on durum wheat plants in different concentration of phosphorus in soil. By means microscopy
observation and strumental analisys in plant tissue, we have find that the P supply influences
mycorrhizal development. The largest extent of mycorrhizal colonization occurs when soil P
concentration is suboptimal for plant growth and by contrast the restriction of the symbiosis formation
is often deteted under high P availability. Furthermore close relation relationship evidence between
durum wheat growth in and Glomus mycorrhizal colonization also was observed.
- 21 -
PI11
HYDRAULIC ARCHITECTURE OF FOUR PTERIDOPHYTES
CORRELATES WITH THEIR ORIGINAL AND PRESENT HABITATS.
FABIO RAIMONDO1, ANTONIO LANZETTA1, SEBASTIANO SALLEO2, MARIA ASSUNTA LO
GULLO1 AND ANDREA NARDINI2.
1
Dipartimento di Scienze della Vita "M. Malpighi" sezione Botanica, Università di Messina, Salita
Sperone 31, 98166 Messina S. Agata, Italia. – 2 Dipartimento di Scienze della Vita, Università di
Trieste, Via L. Giorgieri 10, 34127 Trieste, Italia.
Keywords: Leaf hydraulics, gas exchanges, Pteridophytes.
Leaf hydraulic architecture and gas exchange were measured of four fern species growing in different
ecological habitats. Leaf hydraulic resistance was higher in Woodwardia radicans L. and Dryopteris
affinis (Lowe) Fraser-Jenkins, both adapted to shady and humid habitats than in Athyrium filix-foemina
(L.) Roth and Polystichum setiferum (Forssk.) T. colonizing sunny habitats with either wet (A. filixfoemina) or dry (P. setiferum) soils. The hydraulic resistance of the leaf rachis accounted for 70 and
50% of whole-leaf hydraulic resistance in the species from humid or sunny habitats, respectively. Such
differences in rachis hydraulic resistance were due to different conduit geometries and presence of an
endodermis outside the xylem. Minimum water potentials were similar in the four species studied. Gas
exchange varied by about twofold with higher values recorded in the species from sunny habitats and
were found to be inversely proportional to leaf hydraulic resistance, suggesting that hydraulic and
photosynthetic traits of ferns are correlated to each other on a functional and, possibly, evolutionary
basis.
PI12
THYLAKOID COMPLEXES ORGANISATION DURING BERRY
ONTOGENY IN ARUM ITALICUM MILLER.
PANTALEONI LAURA1, FERRONI LORENZO1, BALDISSEROTTO COSTANZA1, EVA-MARI
ARO2, SIMONETTA PANCALDI1.
1
Department of Biology and Evolution, University of Ferrara, Corso Ercole I D'Este 32, 44100 Ferrara,
Italy. – 2 Department of Biology, University of Turku, Tykistakatu 6A, 20014 Turku, Finland.
Keywords: Arum italicum, berry ontogeny, Photosystem II, BN/SDS-PAGE, spectrofluorimetry.
The peculiar development of Arum italicum berries occurs through maturation (from ivory to green
berry) and ripening (from green-yellow to red berry). An internal CO2 recycling photosynthesis occurs
in the ivory-green, green and green-yellow berries. The thylakoid system is mainly developed in the
green berry, showing the highest gross photosynthesis. The differences between the 3 stages cannot be
merely explained by different chlorophyll concentrations. We addressed the question whether they may
be due to the organisation of photosynthetic complexes in the thylakoid membranes. BN/SDS-PAGE,
77K and room temperature spectrofluorimetric analyses showed that: a) PSI was present only in traces
during berry ontogeny; b) PSII-LHCII supercomplexes were evidently detected only in the green berry;
c) LHCII trimers were abundant in the three stages analysed; d) an increase in ATP-synthase and other
non-photosynthetic protein complexes occurred with the progression of berry ontogeny. The proteomic
and fluorimetric analyses collectively suggest that the changes in PSII and LHCII organisation may
explain the observed trends in photosynthetic performance during A. italicum berry ontogeny.
- 22 -
PI13
EXPRESSION PATTERN OF POLYAMINE OXIDASES IN ARABIDOPSIS
THALIANA.
PAOLA FINCATO1, PANAGIOTIS N. MOSCHOU2, LUCIA POMETTINI1, RICCARDO
ANGELINI1, KALIOPI A. ROUBELAKIS-ANGELAKIS2, RODOLFO FEDERICO1, PARASKEVI
TAVLADORAKI1.
1
Department of Biology, University "Roma Tre", Rome, Italy. – 2 Biology Department, University of
Crete, Heraklion, Greece.
Keywords: Polyamine oxidase, Arabidopsis thaliana, expression pattern.
The until now best characterised plant polyamine oxidases (PAO), such as the apoplastic maize PAO
(ZmPAO), are involved in the terminal catabolism of spermine (Spm) and spermidine (Spd), conversely
to the animal PAOs which oxidise Spm and Spd through a polyamine back-conversion pathway. In
Arabidopsis thaliana, five PAO genes (AtPAO1-5) are present with a varying sequence homology to
ZmPAO and subcellular localization (cytosolic or peroxisomal). Sequence analysis indicated that
AtPAO2-4 derivate from a common ancestor and biochemical characterization of recombinant
AtPAO1, AtPAO2 and AtPAO4 showed that these enzymes oxidise the common polyamines Spd and
Spm and the stress-related uncommon polyamines norspermine and thermospermine through a backconversion pathway. In the present work, AtPAOprom::GUS transgenic Arabidopsis plants for
AtPAO1, AtPAO2 (representative member of the AtPAO2-4 subfamily) and AtPAO5 were analysed
and data suggest a distinct tissue-specific expression pattern for each AtPAO. Inducible expression
following various treatments was also evidenced. This study will contribute greatly towards elucidating
the physiological role of AtPAO gene family.
PI14
A VPE GENE IS UP-REGULATED DURING NUCELLUS PROGRAMMED
CELL DEATH IN SECHIUM EDULE SEED.
LOMBARDI L.1, BATTELLI R.2, PICCIARELLI P.2, LORENZI R.1, CECCARELLI N.2, ROGERS
H.J.3
1
Department of Biology, University of Pisa. – 2 Department of Crop Plant Biology, University of Pisa.
– 3 School of Biosciences, Cardiff University.
Keywords: Nucellus, programmed cell death, VPE, protease.
Nucellar degeneration during Sechium edule seed development occurs by means of programmed cell
death, a process that has been well characterized from the biochemical and cytological point of view.
Nucellar cell death is accompanied by DNA degradation and by an increase of activity of different
classes of proteinases: we reported the induction of caspase-like proteases characterized by an acidic
pH-optimum. This observation suggests a possible localization in the vacuole and their involvement in
the degradation of cellular contents inside acidic vesicles, through a series of events culminating in
autophagic cell death. Vacuolar processing enzymes have been demonstrated to be involved in many
examples of PCD and, in some cases, they have been recognized to play a role as caspase-like enzymes.
In this work the presence of a VPE proteolytic activity has been reported to be induced during nucellus
PCD; moreover, a cDNA fragment encoding a VPE protease has been isolated by the use of degenerate
primers. The amino acid sequence reveals that it is a homologue of other plant VPEs. The expression of
the VPE gene in Sechium nucellus increases during the cell death process.
- 23 -
PI15
PROTEIN DEGRADATION AND KDEL PROTEASE INVOLVEMENT
DURING TEPAL SENESCENCE IN LILIUM LONGIFLORUM.
BATTELLI R.1, LOMBARDI L.2, PICCIARELLI P.1, LORENZI R.2, CECCARELLI N.1, ROGERS
H.J.3.
1
Department of Crop Plant Biology, University of Pisa. – 2 Department of Biology, University of Pisa.
– 3 School of Biosciences, Cardiff University.
Keywords: Lilium longiflorum, flower senescence, protein, cysteine protease, immunogold.
During flower senescence the degradation of macromolecules allows resource re-allocation to the
developing ovary or to other plant organs. KDEL tailed cysteine peptidases have been demonstrated to
play a central role during protein remobilization in different plant organs. In this work, physiological
and biochemical changes during Lilium longiflorum senescence are described and a 1068 bp cDNA
encoding a cysteine protease has been isolated and sequenced. The predicted amino acid sequence
revealed that it encodes a cysteine protease belonging to the papain family. It is characterized by a Cterminal KDEL motif which acts as a retention signal for the endoplasmic reticulum and by the
catalytic residues cys-154 and his-289. The expression of the KDEL protease gene in lily increases in
conjunction with the senescence process and decreases in completely wilted tissues. Western blotting
identified homologous proteins in protein extracts of lily tepals and leaves. The immunogold-labelling
localized the peptidase in masses within the vacuole which likely plays a crucial role in cell
disassembly. The YFP fluorescent reporter has been used to localize the cysteine peptidase within the
cell.
PI16
ACCLIMATION OF PHOTOPROTECTION MECHANISMS IN
PHYSCOMITRELLA PATENS UNDER SALT AND OSMOTIC STRESS.
GHAZI AZZABI1-3, ALESSANDRO ALBORESI1, CATERINA GEROTTO2, TOMAS
MOROSINOTTO2, JEANNETE BEN HAMIDA3, AND ROBERTO BASSI1.
1
Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15, 37134 Verona,
Italy. – 2 Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58 B, 35131 Padova, Italy. – 3
Institut Superieur des Sciences Biologiques Appliquees de Tunis, Universitè Tunis El Manar, Tunisie.
Keywords: Acclimation, Drought stress , Physcomitrella patens, Non photochemical quenching, PsbS
and Li818.
Drought stress increases the production of reactive oxygen species (ROS) and oxidative damage in
plant cells. The moss Physcomitrella patens is well suited for the study of abiotic stress responses. Here
we studied the acclimative adaptation of the photoprotection function. To this aim P. patens plants were
grown for three days on control medium and then transferred either to the same substrate or to media
containing NaCl (100, 200 and 400 mM) or Sorbitol (200, 400 and 800 mM). Mosses can acclimate to
200 mM NaCl and 400 mM sorbitol and increase significantly their capacity for excess energy
dissipation (NPQ) with concomitant accumulation of photoprotective xanthophylls: lutein and
zeaxanthin. Immuno blots with specific antibodies, revealed that PsbS, elicitor of NPQ in land plants, is
down regulated while its algal counterpart (Li818) was slightly over-accumulated. The study is now
proceeding by generating specific k.o. mutants on VDE, PsbS and Li818 in order to verify which
component is essential for the generation of the resistant phenotype.
- 24 -
PI17
ROLE OF GIBBERELLINS IN TWO TOMATO AUXIN SIGNALLING
MUTANTS DURING THE EARLY STAGES OF FRUIT DEVELOPMENT.
F. MIGNOLLI, L. MARIOTTI, P. PICCIARELLI, N. CECCARELLI.
Dept Crop Plant Biology, University of Pisa via Mariscoglio, 34 56124 Pisa (Italy).
Keywords: Tomato mutants, auxin/gibberellin interaction, endogenous hormone levels, fruit
development.
Although it is well known that auxins and gibberellins play an important role during tomato fruit
development, the mechanism of their interaction in the regulation of this process is still unclear. In
order to understand the possible role of auxin signalling on gibberellin metabolism during fruit
development, we performed some experiments with two tomato mutants, diageotropica (dgt) and entire
(e), altered for genes involved in auxin signalling. Tomato dgt mutant is auxin insensitive and is
characterized by lower fruit set and fruit weight. Conversely, the tomato entire mutant, which lacks an
active auxin response repressor, may have strong effects on ovary growth even in absence of
fertilization. The present study uses these mutants as a tool to understand the effect of different auxin
perception on gibberellin metabolism during fruit development. Effects on fruits growth following
applications to ovaries of GA3 and LAB 198999 (an inhibitor GAs biosynthesis) in both mutants were
observed. Moreover, endogenous levels of auxin and gibberellins were monitored over the first stages
of ovary development after pollination.
PI18
LOW GLUTAMATE LEVELS MODULATE THE ACTIVITY OF
ARABIDOPSIS THALIANA Δ1-PYRROLINE-5-CARBOXYLATE
REDUCTASE.
SAMUELE GIBERTI AND GIUSEPPE FORLANI.
Department of Biology and Evolution, University of Ferrara, Italy.
Keywords: Proline biosynthesis, glutamate and ornithine pathways, amino acids, carbon flow.
Under normo-osmotic conditions and nitrogen availability, proline synthesis seems to proceed mainly
through ornithine, whereas under hyperosmotic stress and nitrogen deprivation it is produced directly
from glutamate. The two pathways share the last reaction, catalysed by a P5C reductase [EC 1.5.1.2].
As it occurs at the converging point of alternative routes, P5C reductase may be subjected to fine
regulation, despite not controlling the rate-limiting steps. The isolation of the enzyme was recently
achieved in our lab from suspension cultured cells of A. thaliana, a species in which a single gene for a
P5C reductase has been found. This allowed a thorough characterization of the protein with respect to
structural, kinetic, and biochemical properties. Here we report that both glutamate and arginine are able
to interfere with the catalytic rate of P5C reductase at concentrations at which other, non-related amino
acids are completely ineffective. Interestingly, glutamate levels in the range 10 -4 to 10-2 M are
inhibitory, whereas at concentrations exceeding 10 mM the effect is reverted. Results suggest a control
of proline synthesis when glutamate is depleted below optimal levels.
- 25 -
PI19
EFFECT OF POTASSIUM FERTILIZATION ON THE HYDRAULIC
CONDUCTANCE OF LAUREL PLANTS.
ODDO E.1, NARDINI A.2, LA BELLA F.1, GRISAFI F.1, SALLEO S.2
1
Dipartimento di Scienze Botaniche, Università di Palermo, via Archirafi 38, 90123 Palermo, Italy. – 2
Dippartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 5-9-10, 34127 Trieste, Italy.
Keywords: fertilization, hydraulic conductance, Laurus nobilis L., potassium, xylem.
The effect of potassium fertilization on hydraulic conductance was tested in 2-year-old potted laurel
seedlings, grown in a greenhouse at the Botanical Garden of Palermo. Plants were divided into a control
group (+K) and a potassium starved group (-K). A set of randomly chosen -K plants received irrigation
with 25 mM KCl (KCl plants) 24 hours before measurements, to test the short term effect of potassium
fertilization. Measurements were carried out in July and October 2008. Whole-plant hydraulic
conductance was measured by the evaporative flux method; root and shoot hydraulic conductance were
measured by the vacuum chamber. Potassium availability or season did not significantly affect leaf
water potential, leaf conductance to water vapour, whole plant hydraulic conductance or root hydraulic
conductance. In July, leaf specific conductivity (= hydraulic conductance divided by leaf area, kL) was
about 60 % lower in +K and -K plants than in KCl plants, but in October this significant difference
disappeared. The possible role of potassium availability on shoot and leaf specific hydraulic
conductivity is discussed in terms of the hydrogel effect in pit membranes of stem vessels.
PI20
GLYCINE BETAINE SYNTHESIS AS AFFECTED BY HIGH LIGHT AND
SALINITY.
CARILLO P.1, PARISI D.1, WOODROW P.1, SULPICE R.2, PONTECORVO G.1, MASSARO G.1,
FUGGI A.1
1
Dipartimento di Scienze della Vita, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta,
Italy. – 2 Max Planck Institute of Molecular Plant Physiology, Am Mahlenberg 1, 14424 PotsdamGolm, Germany.
Keywords: Glycine betaine, compatible solutes, salt stress, light intensity, durum wheat.
Glycine betaine (GB) not only acts as an osmoregulator, but interacts with both hydrophilic and
hydrophobic domains of macromolecules stabilizing their structures and activities and maintaining the
integrity of membranes against the damaging effects of abiotic stresses. In many halophytes, GB and/or
proline concentrations in leaves contribute to the osmotic pressure in the cell as a whole. In glycophytes
their concentrations are much lower but, if partitioned exclusively to the cytoplasm, they could generate
a significant osmotic pressure and function to balance vacuolar osmotic potential. In this study, we
determined the effects of both salinity and high light on durum wheat seedlings, with a special
emphasis on the potential role of GB in their protection. Very unexpectedly it appeared that high light
treatments inhibit the synthesis of GB, even in the presence of salt stress. In order to understand the
mechanisms underlying such result, we investigated the effects of different light intensities on the
transcripts encoding enzymes involved in its synthesis. Moreover, additional solutes susceptible to
compensate the decrease in GB were followed over this range of light treatments.
- 26 -
PI21
DIVERSE PHYSIOLOGICAL FUNCTIONS OF REDOX SENSITIVE BETAAMYLASE (BAM1) IN GUARD AND MESOPHYLL CELLS.
CONCETTA VALERIO1, ALEX COSTA2, EMMANUELLE ISSAKIDIS-BOURGUET3, PAOLO
PUPILLO1, PAOLO TROST1, FRANCESCA SPARLA1.
1
Department of Experimental Evolutionary Biology, University of Bologna, Via Irnerio 42, Bologna
40126, Italy. – 2 Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padua, Italy.
– 3 Institut de Biotechnologie des Plantes, Unitè Mixte de Recherche 8618, Centre National de la
Recherche Scientifique, Universitè Paris-Sud, 91405 Orsay cedex, France.
Keywords: starch, redox, disulfide, chloroplast, osmoregulation.
Among plastid-targeted beta-amylases from Arabidopsis, only BAM1 is specifically activated by
reducing conditions, being efficiently reduced by thioredoxin f1, m1, m2, y1, y2, m4 and by NADPHdependent thioredoxin reductase. Redox modulation of BAM1 activity suggests that BAM1 would be
active in the light rather than in darkness, in contrast with the timing of starch metabolism in mesophyll
cells. To elucidate this inconsistent behaviour, promoter activity of BAM1 was analyzed. Expression
signal was revealed in guard cells of young leaves. Accordingly, in comparison to wild type plants,
bam1 T-DNA mutants showed diurnal starch accumulation in guard cells. This suggests a role for
BAM1 in starch mobilization in guard cells, where starch is degraded in the light to sustain stomata
opening. After flowering, bam1 expression appeared in mesophyll cells, where it was also strongly
induced by osmotic and salt stress. Both total and redox-sensitive beta-amylase activity increased in
leaves of osmotically stressed plants, while leaf starch content decreased, indicating that a redoxdependent pathway of starch breakdown may be triggered in response to water deficit conditions.
PI22
STRIGOLACTONE INVOLVEMENT IN APICAL DOMINANCE IN PEA
(PISUM SATIVUM L.).
CARLO SORCE, ALESSANDRO LUISI, ROBERTO LORENZI.
Department of Biology, University of Pisa, via L. Ghini, 5, 56126 Pisa (Italy).
Keywords: apical-dominance, pea, strigolactone.
The new class of plant growth regulators strigolactones has been shown to be involved in the control of
axillary bud growth in mutants of pea (Pisum sativum L.) with reduced apical dominance. We are
currently investigating the role of these molecules in wild type pea plants, whose apical dominance is
suppressed by shoot apex removal. Application of the synthetic strigolactone analogue GR24 is
effective in inhibiting axillary bud outgrowth following decapitation, although wild type peas appear to
be less responsive to this molecule than mutant ones are. Treatments with three concentrations of GR24
have not yet allowed us to determine which is the highest effective dosage. Strigolactone changes in
intact and decapitated plants are currently being analyzed, to highlight their putative correlation with
apical dominance. Work is in progress to identify endogenous strigolactones of pea and to determine
the time course of their concentration in various plant parts following apex removal.
- 27 -
PI23
THE CP12 PROTEIN FAMILY.
LUCIA MARRI1, ALESSANDRO PESARESI2, ANA E. CARMO-SILVA3, PAOLO TROST1,
MICHAEL E. SALVUCCI3, PAOLO PUPILLO1, FRANCESCA SPARLA1.
1
40126, Italy. – 2 Istituto di Cristallografia, CNR, Area Science Park - Basovizza, S.S. 14, Km 163.5, I34012 Trieste, Italy. – 3 USDA-ARS, Arid-Land Agricultural Research Center, Maricopa, AZ 85238,
USA.
Keywords: Supramolecular complex, redox, disulfide, chloroplast, protein family.
In oxygenic photosynthetic organisms, two non-consecutive enzymes of Calvin cycle (glyceraldehyde3-phosphate dehydrogenase, GAPDH, and phosphoribulokinase, PRK) are regulated through different
mechanisms. Among these, the reversible formation of a supramolecular complex mediated by the
small protein CP12 has been identified by the isolation of GAPDH/CP12/PRK complex from both
Arabidopsis and tobacco leaves. Arabidopsis genome codes for three different CP12 isoforms, all
including transit peptides responsible for chloroplastic localization. Disorder predictors and CD spectra
classified all CP12 isoforms as natively unstructured proteins. Contrarily to their disordered nature, all
Arabidopsis CP12s contain four conserved cysteines, responsible for the formation of two internal
regulatory disulphide bridges with similar midpoint redox potentials. In agreement with their similar
redox properties, the three CP12 isoforms were equally able to assemble the ternary complex
GAPDH/CP12/PRK in vitro, with comparable inhibitory effects on the activities of both photosynthetic
enzymes, and unable to bind GapCp, a plastidial GAPDH isoform playing a role in organelle
glycolysis.
PI24
EVOLUTION OF PHOTOSYNTHETIC GLYCERALDEHYDE-3PHOSPHATE DEHYDROGENASE (GAPDH) REGULATION: A
STRUCTURAL PERSPECTIVE.
FRANCESCA SPARLA1, SIMONA FERMANI2, LUCIA MARRI1, ANTON THUMIGER1, PAOLO
PUPILLO1, GIUSEPPE FALINI2, PAOLO TROST1.
1
40126, Italy. – 2 Department of Chemistry, University of Bologna, Via Selmi 2, Bologna 40126, Italy.
Keywords: Calvin cycle, light/dark, protein structure, redox, co-crystallization.
The Calvin cycle includes a single reductive step catalyzed by glyceraldehyde-3-phosphate
dehydrogenase (GAPDH). In higher plants, two distinct isoforms of GAPDH are present in
chloroplasts: a major A2B2-GAPDH (specific of land plants) and a less abundant A4-GAPDH, similar
to its cyanobacterial ancestor. In A2B2-GAPDH, the C-terminal extension (CTE) of B-subunits is
responsible for the thioredoxin-mediated regulation. After formation of a disulfide, this protruding
regulatory domain is folded nearby the active site of the protein, interacting with essential residues for
coenzyme recognition and catalysis. Although A4-GAPDH does not contain CTE, it is regulated by the
small regulatory peptide CP12, also of cyanobacterial origin. The CTE of B-subunits and the Cterminal portion of CP12 have similar sequences, possibly reflecting a common evolutionary origin and
suggesting a similar regulatory function. Recent structural data (crystallization of A2B2-GAPDH and
co-crystallization of A4-GAPDH and CP12) indeed demonstrate that both CP12 and CTE occupy,
under oxidizing conditions, the same cleft into GAPDH tetramers thereby creating the conditions for
reversible inhibition of enzyme activity.
- 28 -
PI25
RESPONSES OF KOLIELLA ANTARCTICA TO LIGHT-INDUCED STRESS.
N. LA ROCCA, T. MOROSINOTTO, I. MORO, C. ANDREOLI, N. RASCIO.
Department of Biology, University of Padova, Via U. Bassi 58/b, Italy.
Keywords: Koliella antarctica, light stress, photosynthesis.
Cultures of Koliella antarctica, a green microalgae belonging to the Trebouxiophyceae, were grown at
a temperature of 4°C and at two continuous light intensities of 15 (low light=LL) and 150 (high
light=HL) µmol photons m-2 s-1. In comparison with the LL, the HL slightly decreased the cell growth,
but induced morphological and ultrastructural changes. In particular at LL, cylindrical cells (7-8 µm
length, 3-4 µm width) forming pseudo-filaments of 2-5 cells. At HL, instead, the filaments were longer
with cells smaller in size (4-5µm length, 2-2.5µm width). Ultrastructural analyses, evidenced in the
cells grown at the HL large storage bodies, chloroplast with a lower number of thylakoids and a thicker
cell wall. Pigment analyses showed that K. antarctica triggers a photoprotection mechanism involving
the synthesis of zeaxanthin under HL and luteine epoxide under LL. Photosynthetic apparatus showed
other acclimatory responses: in particular, HL acclimated cells had an enhanced capability to dissipate
excess energy as heat, thanks to the activation of a strong Non Photochemical Quenching.
PI26
VARIATIONS OF GLUCOSE, GLUCOSE-6-PHOSPHATE AND ATP IN
ARUM SP. DURING DIFFERENT PHENOLOGICAL STAGES.
VALENTINO CASOLO, ELISA PETRUSSA, FRANCESCO MACRÌ, ANGELO VIANELLO.
Plant Biology section Department of Biology and Plant Protection University of Udine Via delle
Scienze, 91 33100 - Udine Italy.
Keywords: Arum sp., ATP, glucose, glucose-6-P, fenology.
The linkage between energy metabolism in hypogeous storage organs and phenology is a topic not yet
fully elucidated. In this work, the variations of some energetic parameters were examined in Arum
italicum and A. maculatum, tubers. These organs were monthly collected and analyzed for glucose,
glucose-6-P and ATP. Proximal and distal portions of tuber, with respect to the shoot, were used. In
both species the level of metabolites was strictly linked to the phenological stage of the plant. Glucose
was low at the shoot emission and then increased till the fruit maturation, being always higher in the
distal zone. The level of glucose-6-P showed high amounts in the proximal part of the tuber during the
vegetative stages, followed by a slight decrease during anthesys and plant senescence. ATP level
increased in the proximal side during leaf development; it was maximal in all the tuber during
flowering while decreasing in senescence. These results indicate that the removal and accumulation of
storage starch is mainly localized in the peripheral parts of the tuber. Then, it is maintained high during
the plant grown and flowering, falling down only at the beginning of the fruit ripening.
- 29 -
PI27
EFFECTS OF ETHEPHON ON SYCAMORE CULTURED CELLS.
R. CERANA1, M. MALERBA2, P. CROSTI2.
1
Dept Environmental Sciences, Univ. Milan-Bicocca, Milan. – 2 Dept Biotechnology and Biosciences,
Univ. Milan-Bicocca, Milan.
Keywords: cell death, ethephon, ethylene, sycamore cells, stress responses.
The phytotoxin fusicoccin (FC), a well-known activator of the plasma membrane proton pump, induces
the synthesis of the stress-related hormone ethylene in sycamore cultured cells (Malerba et al., J. Plant
Physiol. 145: 93-100, 1995). In addition, FC induces a number of stress responses, i.e. cell death,
production of hydrogen peroxide and nitric oxide, release of cytochrome c from mitochondria,
accumulation of regulative 14-3-3 proteins in the cytosol and of the molecular chaperone Binding
Protein (BiP) in the endoplasmic reticulum (Malerba et al., Protoplasma 224: 61-70, 2004; Malerba et
al. Physiol. Plant. 133: 449-457, 2008). In this work we compared the effects of FC and ethephon (2chloroethane phosphonic acid), an ethylene-releasing compound, on the above parameters..
PI28
IODINE PHYSIOLOGY IN PLANTS.
M. LANDINI1, S. GONZALI1, M. TONACCHERA2, A. PINCHERA2, A. ALPI3, P. PERATA1.
1
PlantLab, Scuola Superiore Sant'Anna, Via Mariscoglio 34, 56124 Pisa, Italy. – 2 Dipartimento di
Endocrinologia e Metabolismo, Università di Pisa, Via Paradisa 2, 56124 Cisanello, Pisa, Italy. – 3
Dipartimento di Biologia delle Piante Agrarie Sez. Fisiologia vegetale, Università di Pisa, Via
Mariscoglio 34, 56124 Pisa, Italy.
Keywords: iodine, NIS symporter, Hol1 gene , biofortification, A. thathaliana.
Iodine is an essential element in human diet and strategies for food iodine fortification are being
developed. Iodine physiology in plants is largely unknown. The aim of this work is to expand our
knowledge on the physiology of this element in plants, also to increase iodine content in crops. Two
strategies have been developed, taking advantage of an A. thaliana T-DNA mutant for the Hol1 gene,
involved in the iodine volatilisation in the form of methyl halide, and an A. thaliana transgenic line
over-expressing the human Na-I symporter (NIS). The results suggest that Hol1 gene expression is
induced by iodine fed to the medium and its expression correlates with the amount of iodine taken up
by the plants. Furthermore, using radioactive iodine and ICP-MS analysis it was observed that both in
the hol1 mutant and in NIS over-expressing plants iodine uptake is enhanced. Moreover, in the hol1
mutant the amount of iodine accumulated after 4d of KI treatment is 114 mg/Kg (FW), 5 times higher
than in wild type and more than enough to cover the daily human requirement of 150 μg/Kg.
- 30 -
PI29
INTERACTION BETWEEN DNA POLYMERASE LAMDA OF
ARABIDOPSIS THALIANA AND PCNA IN TRANSLESION SYNTHESIS.
L. CONCIA1, A. AMOROSO2, E. CRESPAN2, C. MAGGIO1, C. RAYNAUD3, C. BERGOUNIOUX3,
R. CELLA1 AND G. MAGA2.
1
Dipartimento di Genetica e Microbiologia "A. Buzzati-Traverso", Università degli Studi di Pavia, Via
Ferrata 1, 27100 Pavia (Italy). – 2 Istituto di Genetica Molecolare IGM-CNR, Via Ferrata 1, 27100
Pavia (Italy). – 3 Institut de Biotechnologie des Plantes, Universitè Paris Sud - CNRS, Orsay, UMR
8618, F-91405 (France).
Keywords: Arabidopsis, bimolecular fluorescence complementation, DNA polymerase lambda,
oxidative DNA damage, translesion synthes.
The UV-B radiation and cell metabolism produce reactive oxygen species (ROS), which can damage
DNA with formation of 7,8-dihydro-8-oxoguanine (8oxoG) lesion. The presence of 8oxoG in the
replicating strand can lead to frequent misincorporation of A opposite the lesion (error prone synthesis).
In order to bypass the lesion in an error-free manner, a specialised DNA polymerase (pol) is required
that catalyses the correct incorporation of C opposite 8oxoG during the synthesis step. As in the case of
mammalian DNA pol l, we have shown that the Arabidopsis enzyme, which is the only member of the
X family of DNA pol present in plants, is very efficient in performing error-free translesion synthesis
past 8oxoG. Moreover, its fidelity and efficiency, as tested in vitro using a recombinant Arabidopsis
enzyme, is greatly enhanced by the presence of auxiliary human proteins PCNA and RP-A. Since
Arabidopsis possesses two PCNA-encoding genes, we asked the question of the specificity of the
interaction of these two proteins with DNA pol l. Using biochemical and cytological approaches
(Bimolecular fluorescence complementation), we have observed that only PCNA2 interacts with this
polymerase.
PI30
CHARACTERIZATION OF A GIBBERELLIN SENSITIVE DWARF
MUTANT OF SUNFLOWER (HELIANTHUS ANNUUS).
L. MARIOTTI, M. FAMBRINI, C. PUGLIESI, P.PICCIARELLI AND N.CECCARELLI
Department of Crop Plant Biology, University of Pisa, V.Mariscoglio 34, PI.
Keywords: Helianthus annuus – dwarf – gibberellin – mutanmutant.
A spontaneous dwarf mutant, dwarf2 (dw2), was isolated from a sunflower inbred line. The most
obvious alterations of the dw2 mutant are the lack of stem growth, reduced size of both leaves and
flower organs and retarded flower initiation. In particular, the stem of the dw2 mutant was 15-20 times
shorter than in the wild type plants. Pollen and ovules were produced but the filament failed to extrude
the anther from the corolla and dw2 plants never produced any seed. The phenotype of dw2 is mainly
because of reduced cell size. These characters are controlled by one gene in a pleiotropic way. The
mutant responded to gibberellin (GAs) applications, although some modified characters were not
completely reversed by treatments. Endogenous levels of 13-OH GAs are seven times higher in wild
type than in dw2 plants. The reduced level of ent-kaurene detected in dw2 plants also suggests that
early steps of hormone biosynthesis are impaired in the mutant. A molecular analysis of genes encoding
ent-copalyl diphosphate synthase and ent-kaurene synthase has been planned to clarify the nature of the
mutation.
- 31 -
PI31
OVEREXPRESSION OF AQUAPORIN VVPIP2;4 AMELIORATES GROWTH
PERFORMANCES BY MODIFYING WATER METABOLISM OF
GRAPEVINES IN ABSENCE OF WATER OR SALT STRESS, BUT NOT
UPON STRESS.
IRENE PERRONE1, GIORGIO GAMBINO2, WALTER CHITARRA1, IVANA GRIBAUDO2,
ANDREA SCHUBERT1, CLAUDIO LOVISOLO1.
1
DCA, University of Turin, Grugliasco. – 2 IVVV CNR - Grugliasco.
Keywords: water channel, hydraulics, embolism, assimilation.
We transformed grapevines to over-express VvPIP2;4, an aquaporin gene. We measured: gas
exchanges in irrigated conditions and under full stress, root hydraulic conductivity (Khroot) by High
Pressure Flow Meter (HPFM), embolism formation and recovery in petioles by HPFM differential
application as influenced by transient flushing pressure, proline content in leaf tissues of in vitro salt
stressed plants. In irrigated conditions, assimilation, stomatal conductance, transpiration and leaf
surface were significantly higher in transgenic plants than in wild types. Higher stomatal conductance
of transgenic plants was related to a higher Khroot. Transgenic plants embolized upon environmental
conditions not causing embolism in wild types, probably in relation to transpiration levels higher in
transgenic lines. In transgenic grapevines, proline content was not significantly higher than in controls
at 50 mM NaCl and 3 times higher at 0 mM NaCl. Overexpression of VvPIP2;4 ameliorated growth
performances by modifying water metabolism under no water restrictions and salt stress, whereas did
not induce a drought/salt stress resistance.
PI32
FRUCTANS METABOLISM DURING KERNEL GERMINATION.
PARADISO ANNALISA1, GRECO ELVIRA1, DE GARA LAURA1,2.
1
Dipartimento di Biologia e Patologia Vegetale, Università di Bari, via Orabona 4, 70125, Bari, Italy. –
Centro Interdisciplinare per le Ricerche Biomediche (CIR), Università Campus Biomedico, Via
Longoni 83, 00155 Roma, Italy.
2
Keywords: Fructans, kernel, germination.
Fructans are fructose polysaccharides occurring in some species of Monocots and Dicots. They are
considered prebiotics because they selectively promote the growth of gut beneficial bacteria such as
Lactobacilli and Bifidobacteria. Fructans metabolism was investigated in wheat kernels, for the
importance of this specie in agronomic and food field. It has been previously shown that mature kernels
(45 days after anthesis) contain a small amount of fructans (De Gara et al. (2003) J. Exp. Bot.54:249–
258), that are proobably used during kernel germination, as a source of sugars rapidly accessible for the
recovery of metabolic activities, before starch becoming the main source of sugars for the growing
seedling. In order to obtain information on fructan metabolism during germination, the contents of
fructan and the activities/expression of the enzymes of their metabolism were investigated during this
process in Triticum durum cv. Simeto. Our results suggest that fructans metabolism is particularly
active in the roots. The modulation of their degree of polymerization by synthesis and hydrolysis
probably contributes to optimize the osmotic potential for water uptake.
- 32 -
PI33
STUDIES ON THE ROLE OF THE E2F/RB PATHWAY IN POLLEN
DEVELOPMENT AND FUNCTION.
ROBERTA GIORDO1, LAURIAN ROBERT2, GIOVANNA BECCA1, DIEGO ALBANI1.
1
Dipartimento di Scienze Botaniche, Ecologiche e Geologiche, Università di Sassari, Via Piandanna,
07100 Sassari, Italy. – 2 Eastern Cereal and Oilseed Research Centre, Central Experimental Farm,
Ottawa, Ontario, K1A 0C6 (Canada).
Keywords: Pollen, Cell cycle regulation, E2F/RB pathway.
In Arabidopsis, the mature pollen grain comprises two sperm cells that are found within a larger
vegetative cell arrested in G1. Recent analyses of the Arabidopsis pollen transcriptome have revealed
that cell cycle arrest in pollen may result from the lack of expression of critical genes and/or the upregulation of potential repressors of cell cycle. In both plant and animal cells, regulation of cell cycle
largely depends on the E2F/RB pathway. Eight members of the E2F/DP family and one gene encoding
a Retinoblastoma-related protein (RBR) have been found and characterized in Arabidopsis. In this
report we describe a preliminary investigation of the role of the E2F/RB pathway in pollen
development and function. Arabidopsis plants were transformed with constructs driving pollen-specific
up-regulation of the activating E2Fs, AtE2Fa or AtE2Fb, or the atypical/repressive AtE2Ff factor. Plant
were also transformed with antisense constructs for the down-regulation of AtE2Fc, AtE2Ff and
AtRBR in mature pollen. Analyses of these transformants have revealed that overexpression of AtE2Fa
or down-regulation of AtRBR can lead to perturbation of pollen development and activity.
PI34
MODULATION OF PHOTOSYNTHETIC ACTIVITY DURING
CHLOROPLAST-CHROMOPLAST TRANSITION.
MATTEO BALLOTTARI1, LINDA BIANCO2, ALESSANDRO ALBORESI1, GAETANO
PERROTTA2, ROBERTO BASSI1.
1
Dipartimento di Biotecnologie Università di Verona. – 2 Centro Ricerche ENEA Trisaia.
Keywords: photosynthesis, LHC, chromoplast, carotenoids, photoprotection.
Chloroplast to chromoplast transition has been investigated in tomato (Solanum lycopersicum) fruits. In
particular modulation of photosynthetic machinery during fruit maturation has been characterized by
biochemical and spectroscopic analyses. Our results reveal that "green" fruits have photosynthetic
active tissues releasing oxygen, while lower photosynthetic activity was measured also at the "orange"
stage of development, but not in "red" fruits. Moreover, photoprotective mechanisms, as the "Nonphotochemical quenching" of chlorophylls excited states, are less induced in fruits than in leaves and
progressively reduced during chromoplast formation. This reduction of photoprotective mechanism is
accompanied by an oxidative burst in "orange-red" fruits. Interestingly, the early steps of
photosynthetic machinery reduction during chloroplast-chromoplast transition affect the same
photosynthetic proteins (LHCII, Lhcb6, PSI-LHCI) decreased in leaves during acclimation to high light
conditions. These evidences suggest common mechanisms of photosynthetic proteins turnover
modulation during chloroplast-chromoplast transition and high light acclimation.
- 33 -
PI35
PEROXIDASE DISTRIBUTION DURING POST HARVEST IN BRASSICA
RAPA.
G. MASSARO, M. G. ANNUNZIATA, F. IANNUZZI, P. CARILLO, A. FUGGI.
Seconda Università degli Studi di Napoli, Dip. di Scienze della Vita (Caserta, Italy).
Keywords: broccoli, Brassica rapa, peroxidase isoforms, browning, ascorbate.
Vegetables after harvesting activate defence and senescent processes that ultimately lead to tissue
death. The browning causing a loss of quality of such products occurs in the post-harvest storage
involves peroxidases and/or poliphenoloxidases. In this work peroxidases have been studied in broccoli
from Brassica rapa cv sylvestris in different storage conditions (10°C, 4°C in air and 4°C in a modified
atmosphere) up to 20 days from the harvest. The collected sample were divided in florets, stem and leaf
blade. Peroxidase activities increased strongly in inflorescence as well as in leaf blades and decreased
in stem when broccoli were stored at 10°C. The increase was slightly in those stored at 4°C and did not
change under modified atmosphere. Native isoelectrofocusing (pH 3–10) revealed numerous peroxidase
isoforms differently distributed in the plant tissues and whose activities changed in storage period. New
isoforms were also evidenced. In addition ascorbate, that inhibited some peroxidase isoforms, strongly
decreased in the post-harvest, mainly in the leaf blade near to the cut zone.The work was financed by
SUN and MIUR (Progetto PRIN 2006077008).
PI36
EFFECT OF HEAT STRESS ON GROWTH AND ANTIOXIDANT
METABOLISM IN TOBACCO BY-2 CELLS.
COSIMO GADALETA1, NUNZIO DIPIERRO1, SILVIO DIPIERRO1, LAURA DE GARA1,2, MARIA
CONCETTA DE PINTO1.
1
Dipartimento di Biologia e Patologia Vegetale, Università degli Studi di Bari, Via E. Orabona, 4, I70125 Bari, Italy. – 2 CIR, Università Campus Bio-Medico, V. Alvaro del Portillo 21, I-00128 Roma,
Italy.
Keywords: Ascorbate; cell cycle, glutathione; heat stress; reactive oxygen species.
Heat stress is a common stress factor for plants; it adversely affects plant growth and leads to the
overproduction of reactive oxygen species (ROS) within cells. ROS act as signalling molecules
involved in triggering defence responses against potentially damaging temperatures. In recent years we
characterized responses against non physiological heat stress able to induce programmed cell death(10
minutes at 55°C). The effects of a more physiological heat stress (exposure to 35°C for different times)
on cell growth and activation of defence mechanisms have been here studied in TBY-2 cells. Different
parameters related to redox homeostasis (ROS production and ascorbate - glutathione metabolism) as
well as the effects of the heat exposure on cell growth, mitotic index and cell viability have been
analysed. In particular, in order to clarify if different cell cycle progression is responsible for the
alterations in cell growth, the expression of cyclins and cyclin dependent kinases has also been also
analysed.
- 34 -
PI37
RESPONSE OF TOBACCO TO LEAD EXPOSURE: 2 - ACCUMULATION
OF LEAD IN VEGETATIVE E REPRODUCTIVE ORGANS OF THE PLANT.
M. ABET1, L. DEL PIANO1, C. SORRENTINO1, A. SALLUZZO2, M. SICIGNANO1, T. ENOTRIO1.
1
CAT Research Unit - Agricultural Research Council (CRA) - Via P. Vitiello n. 108, 84018 Scafati
(SA), Italy. – 2 Portici Research Center - ENEA (Italian Agency for New Technology Energy and
Environment) - Piazzale E. Fermi - 80055 Portici (NA), Italy.
Keywords: Lead, Tobacco, Distribution, Phytoremediation, Inductively Coupled Plasma Mass
Spectrometry (ICP MS).
Plant development, dry matter production and lead accumulation and distribution in vegetative and
reproductive organs of different tobacco varieties experimentally exposed to lead were studied. This
knowledge can be exploited either to select for low Pb genotype, as part of a crop improvement effort,
or to assess the possibilities of using tobacco for the purposes of phytoremediation. 60 day-old tobacco
plantlets were exposed to lead at the rates of 0 (Pb0), 4 g/kg (Pb1) and 8 g/kg (Pb2) supplied to the peat
as Pb(NO3)2. Plants were also exposed to potassium nitrate at rates calculated to contain the same
amount of nitrate as in Pb1 and Pb2. Plants were grown in pots in a greenhouse until the end of the
vegetative cycle. Lead concentration was determined on the following parts of plant: lamina and midrib
of five primings, upper, middle and lower stalk; root base (extension of the stalk), large roots (diameter
>2 mm) and small roots (diameter <2 mm). For two varieties lead content was also determined on
seeds, capsules, pedicels and flowers. In this paper results on lead concentration after quantification by
ICP MS are reported and discussed.
PI38
METABOLITE PROFILES IN CAULIFLOWER GROWN UNDER
DIFFERENT FARMING METHODS.
M.G. ANNUNZIATA1, G. MASSARO1, F. IANNUZZI1, P. CARILLO1, A. TROCCOLI2, A. FUGGI1.
1
Seconda Università degli Studi di Napoli, Dip. di Scienze della Vita (Caserta, Italy). –
Centro di Ricerca per la Cerealicoltura di Foggia.
2
CRA-CER
Keywords: brassica oleracea botrytis , metabolite profiles, , glucosinolates, ascorbate,glutathione.
The cauliflower, is a very appreciate vegetable crop for its nutritional and nutraceutic properties. In
particular it contains glucosinolates that have been reported to prevent some cancer diseases. The aim
of this work was to determined profiles of nitrogen and sulphur metabolites of the corimb of
cauliflower (Brassica oleracea subp. botrytis cv Atalaya) in plants grown under different farming
methods (traditional versus reduced/no work). The analyses were done on the immature flowers and the
corimb stem of plants harvested and stored at -80°C. The samples powdered in liquid nitrogen were
used to determine the content of protein, carbohydrates, pigments, ascorbate and glutathione, inorganic
and organic acids and glucosinolates. The comparative analyses of data evidenced that different
distributions of such metabolites occurred in the immature flowers and the corimb stem, but no
significant change seemed dependent on the applied farming methods. The work was financed by
Seconda Università di Napoli and MIUR (Progetto PRIN 2006077008).
- 35 -
PI39
PRELIMINARY CHARACTERIZATION OF LIPASE ACTIVITY IN OIL
BODY FROM OLEA EUROPAE.
SANTINA PANZANARO, ELIANA NUTRICATI, ANTONIO MICELI, LUIGI DE BELLIS.
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento Via per
Monteroni, 73100 Lecce
Keywords: Olea europaea, lipase activity, triolein, gum Arabic.
Lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is the first enzyme involved in the degradation of
stored triacylglycerols (TAGs). The lipase in olive fruits is involved in fatty acid production and it is
directly related to the acidity of the olive oil, an important index of olive oil quality. However, despite
the importance of olive oil in human health, few studies on mesocarp lipase activity and biochemical
properties in Olea europaea have been reported. Thus, to characterize olive oil lipase, fruits of cv.
Ogliarola cultivated in the Salento area, were harvested and collected at three stages of ripening
according to their skin color (green, spotted, purple). Lipase activity was detected in fatty layer,
obtained by centrifugation of an olive fruit mesocarp homogenate. The enzyme exhibited a maximum
activity (approximately 4.5 nmol acid oleic/mg protein/h) at pH 5.0. The addition of calcium to the
lipase assay medium leads to an increased activity, whereas the copper inhibited the activity of 80%;
the analysis of different metal ions is in progress. The data obtained suggest that the lipase activity
increases during olive development but goes down at senescence stage.
PI40
SIMPLE AGROBACTERIUM-BASED TRANSFORMATION PROCEDURE
USEFUL FOR GFP-FUSION.
RENATO RODRIGUES POUSADA1, ALESSANDRA TISI2, LAURA SPANÒ1.
1
Dipartimento Biologia di Base e Applicata, Università dell'Aquila, Via Vetoio snc, Coppito- 67010
L'Aquila, Italy. – 2 Dipartimento di Biologia, Università di Roma III, V.le Marconi ,00100 Roma, Italy.
Keywords: Transformation; GFP-fusion analysis; A.thaliana.
Agrobacterium based stable transformation procedures are well established in the plant science
community. In particular the floral dip method is a commonly used transformation protocol applied to
the model plant, Arabidopsis thaliana. Nevertheless, often the need exists for a simpler analysis of
gene-GFP fusions as control of the constructs that will be used in the creation of transgenic lines or to
just obtain data from them. Analysis of GFP-fusions has become an essential step in the
characterization of gene functional aspects related to tissue, cellular localization and associated protein
movement. The available methodologies though are often impaired by complex protocols and technical
pitfalls. Thus, a procedure based on a common and widely used system of in vitro Arabidopsis growth
could be very useful. We needed a quick and simple method for analysis of gene-product-GFP fusions.
Thus we decided to establish a simpler procedure for Agrobacteriummediated transformation of
Arabidopsis. Results demonstrating the effectiveness of this method using a 35S-CaMV promoter-GFP
construct and its validation for sub-cellular localization will be presented.
- 36 -
PI41
ACCLIMATION OF CHLAMYDOMONAS REINHARDTII TO DIFFERENT
LIGHT INTENSITIES.
YAKOV PAZ, GIULIA BONENTE.
Department of Biotechnology, University of Verona, strada Le Grazie, 15 37134 Verona, Italy.
Keywords: Acclimation, Chlamydomonas reinhardtii.
Previous studies have shown how plant species acclimate to low light intensity by increasing their Chl
content, decreasing the Chl a/b ratio, increasing their photosynthetic antenna size. In high light
conditions the opposite occurs. Here, we report the characterization of Chlamydomonas reinhardtii
cells acclimated to different light regimes. In high light conditions, we found a relevant accumulation of
carotenoids and an increase in the ratio of functional PSI/PSII as determined by the electrochromic shift
signal technique. These changes are accompanied by a strong activation for energy dissipation with
respect to cells grown in control light. In low light the xanthophyll loroxanthin content was maximal.
Surprisingly, however, we found no significant change in the Chl a/b ratio, and the functional PSII
antenna size remains unchanged irrespective from light intensity.These results are discussed by taking
in account the previous work on higher plants, where a strong modulation of PSII antenna size has been
consistently observed. We suggest that in c.r. acclimation occurs by the synthesis of low copy number
regulative proteins rather than by changing the structural antenna proteins.
PI42
PROTEOMIC ANALYSIS OF ENDOPLASMIC RETICULUM AND GOLGI
PROTEINS DURING TOMATO FRUIT RIPENING
FRANCESCO SPINELLI, DANIELA PONTIGGIA, BENEDETTA MATTEI, GIULIA DE
LORENZO
Dipartimento di Biologia vegetale, Università di Roma La Sapienza, Piazzale A. Moro 5, Roma
Keywords: DIGE, secretory pathway, protein trafficking, cell wall.
Tomato is a model organism for ripening of fleshy fruit. Our aim is monitoring the pattern of protein
expression along the secretory pathway during tomato fruit ripening, and identifying proteins of the
endoplasmic reticulum (ER) and Golgi apparatus that are differentially expressed. ER and Golgi are
crucial for the secretory pathway and play a central role in fruit ripening; in addition the Golgi is
responsible for the production of the non cellulosic component of cell wall. ER and Golgi microsomal
vesicles were separated by centrifugation through different types of density gradients. Assuming that an
organelle has a unique distribution pattern it is possible to identify ER or Golgi components by
comparing their enrichment using known markers of these compartments.
Profiling of protein distribution in the breaker stage tomato fruits was determined by pair-wise
comparison of two pooled gradient fractions, respectively enriched in the ER and Golgi microsomes,
using DIGE that employs fluorescent labelling of different samples to eliminate inter-gel variation and
facilitate quantitative evaluation of differentially expressed proteins.
- 37 -
PI43
RESPONSE OF TOBACCO TO LEAD EXPOSURE: 1 - INFLUENCE OF
LEAD ON PLANT BIOMASS.
L. DEL PIANO, M. ABET, C. SORRENTINO, M. SICIGNANO, T. ENOTRIO, E. COZZOLINO, A.
CUCINIELLO.
CAT Research Unit, Agricultural Research Council (CRA), Via P. Vitiello n.108, 84018 Scafati (SA),
Italy.
Keywords: Lead, Tobacco, Biomass, Tolerance, Phytoremediation.
Plant development, dry matter production and lead accumulation and distribution in vegetative and
reproductive organs of different tobacco varieties experimentally exposed to lead were studied. This
knowledge can be exploited either to select for low Pb genotype, as part of a crop improvement effort
or to asses the possibilities of using tobacco for the purposes of phytoremediation. 60 day-old tobacco
plantlets of 6 tobacco varieties were exposed to lead at the rates of 0 (Pb0), 4 g/kg (Pb1) and 8 g/kg
(Pb2) supplied to the peat as Pb(NO3)2. Plants were also exposed to potassium nitrate at rates calculated
to contain the same amount of nitrate as in Pb1 and Pb2. Plants were grown in pots in a greenhouse
until the end of the vegetative cycle. During tobacco growing plant height and time of flowering data
were collected. The dry weight of the following parts of plant was determined: leaves (5 primings);
upper, middle and lower stalk; root base, large roots and small roots; flowers, pedicels, capsules and
seeds of mature inflorescence. In the present paper results on dry matter production are reported and
discussed.
- 38 -
ABSTRACTS – Session II: Bioenergy
PII01
IMPROVED GROWTH IN PHOTOBIOREACTORS USING
CHLAMYDOMONAS REINHARDTII MUTANTS SELECTION FOR
REDUCED ANTENNA SIZE.
GIULIA BONENTE1, MANUELA MANTELLI1, CINZIA FORMIGHIERI1, CLAUDIA
CATALANOTTI2, TOMAS MOROSINOTTO3, GIOVANNI GIULIANO2 AND ROBERTO BASSI1.
1
Dip. di Biotecnologie-Univ. di Verona. – 2 Casaccia Reseacrh Center- ENEA Roma. – 3 Dip. di
Biologia-Univ. di Padova.
Keywords: microalgae, photobioreactors, photosynthesis, biomass, antenna.
Green algae are promising organisms for biofuels production, due to higher biomass yield and oil
content with respect to plants. However, the exploitation of mass algal cultures in photobioreactor
presents limitations due to adaptation of algae to low light intensity for optimal growth consisting into a
large antenna system for photosystems. The high number of Chls per Reaction Centre produces excess
light absorption in cell layers nearby surface and suboptimal illumination in deep layers leading to
energy dissipation into heat and energy consumption by respiration by inner layers, reducing the overall
productivity. We generated an insertion mutant library in C. reinhardtii, and screened for clones with a
reduced antenna. Two lines with strongly reduced antenna and lower chlorophyll content were isolated
(named az7 and u06). Phenotypical characterization shows that line u06 differs from line az7 for higher
sensitivity to photo oxidative stress and displays far lower productivity. Growth tests in multiple
overcast layers, simulating photobioreactors, showed that az7 productivity is strongly improved with
respect to Wt in terms of biomass yield.
PII02
AGRO-ENERGY: OLEAGINOUS CULTIVATION IN THE SALENTO AREA.
MICELI A., CERFEDA A.,TOMMASI L., NEGRO C., AND DE BELLIS L.
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali Via Provinciale Lecce-Monteroni
73100 LECCE.
Keywords: Agro-energy, plant oil, sunflower, rapeseed.
In Europe, obtaining energy from renewable sources is a central issue; in this contest the production of
biodiesel from vegetable oil is an important objective. The interest for the agro-energies has also
involved the Salento area (South Puglia), above all for the biofuel production that would be obtained
from high yield oleaginous plants. Therefore, we have studied the adaptation of different cultivars of
oleaginous plants, rapeseed (cv. Champlain, Exagone, Makila, Savannah, Toccata) and sunflower (cv.
Goleadord, Gloriasol, Mango, Panter, Starsol) to the typical pedoclimatic conditions of Salento. The
results indicated that all the studied oleaginous cultivars were not able to give a significant yield
nevertheless the average oil content in seeds was normal. This evidence is mainly related to the scarce
raining, a typical climatic condition in the Salento area.
- 39 -
PII03
SELECTION AND CHARACTERIZATION OF HIGH LIPID CONTENT
PLANT CELL CULTURES.
BISOGNO STEFANO1,2, PURELLI MARINA1, FABBRI ANDREA1, BALDAN BARBARA2.
1
Geneticlab Srl, via Corte Ferrighi,16/B Noventa Vicentina (VI), Italy. – 2 Dipartimento di Biologia,
UniPD, via U. Bassi 58/B 35131, Padova, Italy.
Keywords: biodiesel production, oil seeds, plant biomass, plant cell cultures, somatic embryogenesis.
The rise in global energy usage, together with the disappearance of fossil fuel reserves, has highlighted
the importance of developing technologies to harness new and renewable energy sources. Biodiesel
production from oil crops cannot realistically satisfy even a small fraction of the existing demand for
transportation fuel (Chang M, 2007, Curr Opin Chem Biol, 11: 677-684). Growing plant cell cultures in
bioreactors, production of food and other products derived from crops would not be compromised and
such a production system would eliminate the restrictions of seasons and climatic zones. In order to
obtain plant cell lines enriched in lipid content we set different photosynthetic or photomixotrophic
culture conditions modulating temperature, media composition, plant hormone concentration, sugar
content. We obtained calli and cell suspension cultures starting from high oil content seeds (sunflower,
canola, soia, peanut). Moreover we got somatic embryogenesis from A. hypogaea and H. annuus
embryogenic calli. In this way the storage lipid accumulation occurring in somatic embryo cotyledons
could be easily manipulated to increase the triglyceride amount.
PII04
BARLEY ROOTS CYT-G6PDH: PUTATIVE SEQUENCE AND RESPONSE
TO SALINITY.
DANIELA CASTIGLIA, MANUELA CARDI, DONATA CAFASSO, AND SERGIO ESPOSITO.
Dipartimento di Biologia Strutturale e Funzionale – Università di Napoli Federico II, Via Cinthia 80126 Naples – Italy.
Keywords: cytosolic glucose-6-phosphate dehydrogenase, G6PDH, OPPP, Hordeum vulgare.
A full-length cDNA coding for cyt-G6PDH (cytosolic Glucose-6-phophate dehydrogenase) was
sequenced from barley roots (Hordeum vulgare,cv.Nure); cyt-G6PDH expression in response to salinity
was investigated by Real Time PCR. G6PDH catalyzes the first step of the oxidative pentose-phosphate
pathway (OPPP), providing NADPH for biosyntheses. OPPP is responsive to different stresses, as
nutrient starvation, drought, salinity. cyt-G6PDH sequence was 1767 bp long and contained an ORF
coding for 510 amino acid residues with a MW=57.5 kDa. The polyadenilation signal was found at
nucleotide 1727; a Rossman Fold motif, a putative active region and NADP-binding site were found.
The deduced amino acid sequence exhibited 66-94% and 48-50% identity with the known cytosolic and
plastidic G6PDH sequences from higher plants, respectively. The addition of 0.15 M NaCl to
hydroponically grown plants on 5mM of NH4+ and NO3- medium caused fast decrement of G6PDH
trascripts in the first 6h (40%-53%, respectively), after 24h the transcripts returned to steady state
levels; in the following period a further decrement in the transcript levels was probably induced by
cellular death caused by salinity.
- 40 -
PII05
PROTEOMIC ANALYSIS OF BARLEY ROOTS GROWN UPON SALT
STRESS.
STEFANIA MONTANARI1, MYRIAM FERRARA1, TEJA SIREC2, SERGIO ESPOSITO1.
1
Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II, Vi Via Cinthia,
80126 Naples , Italy. – 2 Department of Bioology, Biotechnical Faculty, University of Ljubljana,
Slovenia.
Keywords: Salt Stress, 2D electrophoresis, Hordeum vulgare.
The protein patterns and new transcripts from barley roots (Hordeum vulgare) were examined in plants
grown under different nutritional conditions (ammonium or nitrate as N source), and then exposed to
salt stress. These conditions are often present in agricultural fields, exposed to irrigation and
fertilization. In particular, fields are often fertilized with ammonium salts. In neutral and basic pH
conditions nitrification bacteria convert ammonium in nitrate, which is absorbed by the roots. Only in
the acid soils, where nitrification bacteria canâ€™t operate, ammonium is absorbed. Salt excess in the
soil can disturb physiological mechanisms which leads to reduced crop productivity. We used the twodimensional electrophoresis as a principal technique. The results show different patterns of proteins in
ammonium-grown and nitrate grown plants. Interestingly, salt stress induced visible changes in several
spots, increasing with time and salt levels. The identification of these proteins is under investigation.
We focused on the optimization of 2D-PAGE and on the problematics of reproduction of good 2D-gels,
useful for objective comparative analysis.
PII06
SEQUENCING, CLONING AND CHARACTERISATION OF P2-G6PDH
FROM BARLEY ROOTS.
MANUELA CARDI1, DANIELA CASTIGLIA1, DONATA CAFASSO1, NICOLAS ROUHIER2,
JEAN-PIERRE JACQUOT2 AND SERGIO ESPOSITO1.
1
Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli Federico II Via Cinthia,
80126 Naples, Italy. – 2 Unitè Mixte de Recherche INRA-UHP 1136, Interactions Arbres/Microorganismes, Universitè Henri Poincarè, IFR 110, Facultè des Sciences, BP 239 54506, Nancy,
Vandoeuvre Cedex, France.
Keywords: Plastidic glucose-6-phosphate dehydrogenase, G6PDH, OPPP, Hordeum vulgare.
Glucose-6-phosphate dehydrogenase (G6PDH - EC 1.1.1.49) represents the regulatory enzyme of the
OPPP (oxidative pentose phosphate pathway) because of its tight regulation and redox dependence. The
main function of OPPP in plants is the production of NADPH for biosyntheses of nitrogen compounds
and lipids. In this study, we report the cloning of a plastidic G6PDH of the P2 type from Hordeum
vulgare cv Nure. Bioinformatic analyses showed the presence of a conserved NADP+ binding domain,
of a conserved active site sequence and of a sequence corresponding to the Rossman fold. In addition,
the sequence contains a putative N-terminal plastidic transit peptide. Hence, in order to express the
recombinant protein in its mature form in E. coli, this targeting sequence (99 amino acids) was removed
during the cloning step in pET 3d. Although very well overexpressed, the protein is produced as
inclusion bodies as confirmed by western blotting with antibodies directed against potato P2-G6PDH.
A denaturation/renaturation strategy is used to extract some folded protein and then study its
biochemical properties.
- 41 -
PII07
EXTRINSIC PSII SUBUNITS AND 2-CYS PEROXIREDOXIN FROM
THYLAKOIDS OF EXTREME HALOPHYTES.
ANDREA TROTTA1, SUSANNA REDONDO-GOMEZ2, CRISTINA PAGLIANO3, ENRIQUE
FIGUEROA2, NICOLETTA RASCIO4, NICOLETTA LA ROCCA4, FLORA ANDREUCCI1,
ROBERTO BARBATO1.
1
Dipartimento di Scienze dell'Ambiente e della Vita, Università del Piemonte Orientale, viale Teresa
Michel 11, 15121 Alessandria, Italy. – 2 Departamento de Biologia Vegetal y Ecologia, Universidad de
Sevilla, Apartado 1095, 41080, Sevilla, Spain. – 3 Dipartimento di Scienze dei Materiali e di Ingegneria
Chimica, Politecnico di Torino, viale Teresa Michel 5, 15121 Alessandria, Italy. – 4 Dipartimento di
Biologia, Università di Padova, via Bassi 58b, 35131 Padova, Italy.
Keywords: Photosystem II , PsbQ , PsbP , Halophytes , Peroxiredoxin.
PSII in extreme halophytes (Salicornia veneta and Arthrocnemum macrostachyum) was characterized.
The following results were obtained: i) in S. veneta PsbQ was absent and PsbP was present at a strongly
reduced amount; the same pattern was observed for respective mRNAs; other PSII subunits, as well as
PSI and cytochrome b6/f polypeptides were unaffected; ii) functional activity of PSII was not
significatively modified; iii) when plants (A. macrostachyum) were grown in different NaCl
concentrations (i.e. from 60 g/L to 0 g/L), the level of PsbP was only marginally affected, whereas
PsbQ, again, was not present; iv) electron transfer between Qa and Qb, as well as chlorophyll content
and a/b ratio, was decreased in low salt conditions; furthermore, SDS-PAGE of thylakoids stained by
TMBZ/H2O2, revealed the presence of several bands, whose intensities were more pronounced in low
salt samples; a band with an apparent molecular weight of 40 kDa was identified, by ESI/Q-TOF, as 2cys peroxiredoxin.
PII08
METABOLIC ENGINEERING IN MICROALGAE FOR BIOENERGY
PRODUCTION.
FERRANTE P., CATALANOTTI C., BRANDTNER D., SARACENI P., MINI P., GIULIANO G.
ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Roma, Italy.
Keywords: Microalgae, bioenergy production, metabolic engineering.
Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for
biotechnological applications, such as molecular farming or biological hydrogen production. Using the
Chlamydomonas metal-responsive CYC6 promoter, we have worked out a chemically regulated gene
expression system that can be finely tuned to produce temporally controlled "waves" in gene expression
(Ferrante et al (2008) Plos ONE 3, e3200). This system is being used in our laboratory to achieve
temporally controlled production of biohydrogen. In collaboration with the group of Roberto Bassi,
both metabolic engineering and insertional mutagenesis approaches are being used to modulate the size
of Light Harvesting antennas in Chlamydomonas. In a different approach, we are working to optimize
the growth parameters and lipid profiles of different microalgal strains (both sea- and freshwater).
- 42 -
PII09
TOBACCO CHLOROPLAST TRANSFORMATION: SYSTEM FOR PLANT
"BIO-FACTORY".
P. LONGONI1, S. LEELAVATHI2, V. S. REDDY2, R. CELLA1.
1
Dept. of Genetics and Microbiology, University of Pavia, via Ferrata 1, 27100 Pavia, Italy. – 2
International Centre of Genetic Engineering and Biotechnology, New Delhi. Aruna Asaf Ali Marg,
India.
Keywords: biolistic, cell-wall degrading enzymes, lignocellulosic bioethanol, tobacco, transplastomic
plants.
The expression "plant bio-factory" describes a transgenic organism able to express a recombinant
protein of interest. As plant biomasses can be produced in bulk at a very low cost, transgenic plants can
be can be a convenient way of producing economically valuable proteins and enzymes. Nuclear and
chloroplast transformation strategies allow to accumulate differing amounts of recombinant protein
within several sub-cellular compartments. Available evidence suggests that chloroplast transformation
gives several advantages: a) hundred of chloroplasts are present in a single mesophyll cell, and each of
them contains tens of copies of the plastid genome: as a result, a gene integrated within the chloroplast
genome will be present in thousand copies for each cell; b) chloroplast tolerate high concentration of a
single protein; c) transgenes are integrated by site-specific recombination; d) tobacco plastid are
maternally inherited and thus intrinsically more biosafe with respect to gene dispersal. Aim of this work
is the production of transplastomic plants characterized by high accumulation of enzymes to be used for
the biodegradation of ligno-cellulosic biomasses.
PII10
USE OF MICROALGAE GROWING IN PHOTOBIOREACTORS FOR
WASTEWATERS REMEDIATION.
DORIA E., LONGONI P., RADAELLI G., NAUDI A., CELLA R. AND NIELSEN E.
Department of Genetics and Microbiology, Università Degli Studi di Pavia, via Ferrata 1 27100 Pavia,
Italy.
Keywords: wastewater treatment, Scenedesmus obliquus, Chlorella p.
Decisions adopted jointly by the European parliament establish that the maximum amount of nitrogen
from farm wastewater that can be spread in the environment is 170 Kg / ha. Thus nowadays there is a
renewed interest for microalgae not only as biomass and oils producers, but also as organisms useful for
phytoremediation. Our research focused on individuation of algal cultures able to grow in specific
wastewater. Previously we isolated a Chlorella strain from piggery wastewater demonstrating that it is
able to grow and to decrease remarkably wastewater NH 4+. More recently we set up a system using a
Scenedesmus strain for urban wastewater treatment aimed to reduce nitrates. We used two
photobioreactors: the first for growing microalgae in their optimal culture medium, monitoring light,
temperature, air bubbles dimension and flux, CO2 level and pH; the second to decrease NO3 content of
the urban waste upon inoculating algae grown in the first bioreactor. Data obtained showed a complete
nitrate consumption within 3 days, and a contemporary six fold increase in microalgae cell number.
- 43 -
PII11
PECTIN MODIFICATION TO IMPROVE PLANT BIOMASS UTILIZATION.
FEDRA FRANCOCCI, VINCENZO LIONETTI, SIMONE FERRARI, ROBERTA GALLETTI,
GIULIA DE LORENZO, DANIELA BELLINCAMPI, FELICE CERVONE.
Dipartimento di Biologia Vegetale, Università di Roma “La Sapienza”, Piazzale Aldo Moro, 5 00185
Rome, Italy.
Keywords: biofuels, plant cell wall, pectin, saccharification, homogalacturonan.
A key process for the production of ethanol from plant biomass is the degradation of the cell wall
polysaccharides into fermentable sugars (saccharification). The major bottleneck for the industrial
scale-up of saccharification is the recalcitrance of plant cell walls to digestion and the need of harsh
pre-treatment. Intermolecular links of pectin influence wall plasticity and that the acidic form of
homogalacturonan (HGA) is cross-linked by calcium ions to form rigid “egg-box” structures. We
hypothesized that saccharification could be improved. To test this hypothesis we used Arabidopsis and
tobacco plants expressing a polygalacturonase from Aspergillus niger (PG plants) and Arabidopsis
plants overexpressing an inhibitor of pectin methylesterases (PMEI plants). PG plants have reduced
levels of HGA, while PMEI plants have reduced pectin methylesterase activity and increased HGA
methylation. Cellulose degradation is favoured transgenic plants and does not require pre-treatments.
We concluded that the reduction of the de-esterified HGA regions of pectin in crop plants or dedicated
energy plants through the expression of PGs and PMEIs may be used to improve biomass utilization.
- 44 -
ABSTRACTS - Session III: Signalling
PIII01
THE LOTUS JAPONICUS AMT1;3 HIGH AFFINITY AMMONIUM
TRANSPORTER CONTROLS PRIMARY ROOT ELONGATION IN
RESPONSE TO AMMONIUM EXTERNAL CONDITIONS,
INDEPENDENTLY BY ITS TRANSPORT FUNCTION.
D'APUZZO ENRICA1, ROGATO ALESSANDRA1, BARBULOVA ANI1, OMRANE SELIM1,
PARLATI AURORA1, RICCARDI MIRIAM1, CARFAGNA SIMONA2, ESPOSITO SERGIO2,
COSTA ALEX3, LO SCHIAVO FIORELLA3, CHIURAZZI MAURIZIO1.
1
Institute of Genetics and Biophysics, A. Buzzati-Traverso, CNR, Via P. Casyellino 111, 80131,
Napoli. – 2 Università degli Studi Federico II, Via Cinthia, 80126. Napoli, Italy – 3 Università degli
Studi di Padova, Via U. Bassi 58/B, I-35131, Padova, Italy.
Keywords: Ammonium toxicity, Signalling, Transporter, root architecture.
Nitrogen is often a limiting resource for plants and an efficient uptake and utilization of this nutrient is
a critical function of the roots response to environmental changes. Sizes and locations of plant-available
N nutrients pools in soils are often highly variable as well as conditions for nutrient uptake, hence a
sensing and signalling system for the scanning of the external substrate concentration in the rooted area
and for the communication of this information to the plant machinery controlling the root
developmental program, can be crucial for an efficient plant response. We identified a novel root
signalling pathway governed by the local ammonium concentration in the model legume Lotus
japonicus. We provide genetic and molecular evidences about the involvement of the ammonium
transporter AMT1;3 in the control of this ammonium-mediated pathway, demonstrating that this role is
independent by an uptake activity. Thus, AMT1;3 could be considered a member of the rapidly growing
"Transceptor" plant family, which include "hybrid" proteins with transport and reception functions.
PIII02
PLASTID TO NUCLEUS RETROGRADE SIGNALLING: ISOLATION AND
CHARACTERISATION OF A GUN4 MUTANT OF THE UNICELLULAR
GREEN ALGA CHLAMYDOMONAS REINHARDTII.
CINZIA FORMIGHIERI 1, MANUELA MANTELLI1, MAURO CEOL2, GIULIA BONENTE1,
JEAN-DAVID ROCHAIX2 AND ROBERTO BASSI1.
1
Dipartimento di Biotecnologie, Università di Verona. – 2 Departement de Biologie Moleculaire,
Universite de Geneve.
Keywords: retrograde signalling, gun mutant, Chlamydomonas reinhardtii, GUN4, Mg-chelatase.
Arabidopsis thaliana has so far the leading role in retrosignalling research. Genomes uncoupled (gun)
mutants of A. t., failing in the communication from plastid to nucleus, carry mutations that perturb or
interact with the tetrapyrrole biosynthetic pathway. Here we report the isolation of the first gun4 K.O.
mutant of the model alga Chlamydomonas reinhardtii, through random insertional mutagenesis and
large scale phenotypic screening (1). A crucial role of Mg-Protoporphyrin IX in retrosignalling has
been proposed, based on studies with model organisms. The gun4 mutants in A. t. and Synechocystis
allowed identification and characterisation of a novel porphyrins binding protein, GUN4, with a
stimulating action on the first committed reaction in chlorophyll biosynthesis through its physical
interaction with Mg-chelatase. GUN4 could participate in retrosignalling by regulating Mg-Proto IX
synthesis and trafficking. The present work describes a gun4 C. r. mutant displaying a reduction in
chlorophyll content and high photosensitivity. We show that absence of GUN4 protein has a strong
effect in the level of accumulation of chlorophyll binding proteins under limited chlorophyll supply.
- 45 -
PIII03
DOES THE ANIMAL PROAPOPTOTIC PATHWAY WORK IN PLANT
CELLS?
ANNA MANARA, BARBARA SOTTOCORNOLA, MASSIMO DELLEDONNE AND MASSIMO
CRIMI.
Dept. of Biotechnology, University of Verona – Italy.
Keywords: Bcl-2 proteins, Bid, apoptosis.
Bid is a BH3-only member of the Bcl-2 family that regulates cell death at the level of mitochondrial
membranes. Full length Bid protein (flBid) becomes activated after a proteolytic cleavage catalyzed by
apical caspases (tBid). The cleaved protein then re-locates to mitochondria and promotes membrane
permeabilization, presumably by interaction with mitochondrial lipids and other Bcl-2 proteins. The uncleaved Bid (flBid) also has pro-apoptotic potential in vivo, when ectopically expressed in cells, or in
vitro. Although no homologues of Bcl-2 proteins have been identified in plant genomes to date, recent
evidence suggests that both Bcl-2 and Bax can fulfill similar roles when introduced into plant cells
(Jones, 2000). In this study both full length and truncated Bid proteins were expressed in plant.
PIII04
CHLORELLA SACCHAROPHILA CYTOCHROME F
CHARACTERIZATION: IS THIS PROTEIN INVOLVED IN HEAT SHOCKINDUCED PROGRAMMED CELL DEATH?
ANNA ZUPPINI, CATERINA GEROTTO, ROBERTO MOSCATIELLO, ELISABETTA
BERGANTINO AND BARBARA BALDAN.
Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B, 35131 Padova, Italy.
Keywords: cell death, Chlorella saccharophila, chloroplast, cytochrome f, heat shock.
We present cloning and characterization of a novel partial cDNA (ChspetA) encoding cytochrome f, an
essential component of the major redox complex of the thylakoid membrane, in the psychrophile
unicellular green alga Chlorella saccharophila and analyse its involvement in HS-induced PCD.
Semiquantitative reverse transcriptase PCR analysis showed that ChspetA expression is up-regulated in
heat-shocked cells and the protein profile of cytochrome f highlighted a dual localization (in the
thylakoid membranes and cytosol) depending on the time lapse from the HS. Evans blue assay, analysis
of chromatin condensation and chloroplast alterations showed the induction of cell death in cell
suspensions treated with cytosolic extracts from heat-shocked cells. The data suggest that cytochrome f
fulfils its role through a modulation of its transcription and translation levels, together with its
intracellular localization. This work focuses on a possible role of cytochrome f into PCD-like process in
a unicellular chlorophyte and suggests the existence of chloroplast-mediated PCD machinery in an
organism belonging to one of the primary lineages of photosynthetic eukaryotes.
- 46 -
PIII05
ANALYSIS OF ARABIDOPSIS THALIANA AUCSIA MUTANTS.
MOLESINI B.1, PII Y.2, PANDOLFINI T.2, SPENA A.1.
1
Dipartimento di Biotecnologie- Università di Verona-Strada Le Grazie 15 - 37134 Verona. – 2
DiSTeMeV-Università di Verona -Villa Lebrecht Via della Pieve 70 - 37029 San Floriano (VR).
Keywords: IAA, Aucsia mutants, auxin sensitivity.
Tomato Aucsia genes have been shown to control fruit initiation and affect auxin-related processes. The
Aucsia homologues of Arabidopsis, AtAucsia-1 and AtAucsia-2 are expressed in all plant organs and
preferentially in inflorescences. Reporter gene studies have been performed with either Aucsia-1p:GUS
or with Aucsia-2p:GUS to visualize the expression pattern of the two genes during seedlings and plant
development. Furthermore, we have analysed two Arabidopsis Salk lines: SALK_117986 a T-DNA
insertion AtAucsia-1 mutant and SALK_065659 a T-DNA insertion AtAucsia-2 mutant. In plants
homozygous for the insertion in AtAucsia-2, AtAucsia-2 mRNA level is 50% lower than in wild-type.
Homozygous ataucsia-1 mutant shows no detectable expression of AtAucsia-1 mRNA. Homozygous
ataucsia-1 mutant seedlings show no visible differences in growth compared with wild-type, but when
cultivated with growth-inhibiting concentrations of IAA, they are less sensitive indicating that ataucsia1 mutants might be affected in auxin response. Homozygous ataucsia-2 mutant seedlings display a
slight reduction in primary root growth compared to wild-type, whereas their sensitivity to auxin is not
altered.
PIII06
HORMONAL CONTROL OF FRUIT DEVELOPMENT: AUCSIA GENES AS
NEW PLAYERS IN AUXIN-MEDIATED FRUIT INITIATION?
MOLESINI B.1, PANDOLFINI T.2, ROTINO G.L.3, DANI V.3, SPENA A.1
1
Dipartimento di Biotecnologie - Università di Verona-Strade la Grazie 15, 37134, Verona. – 2
Dipartimento di Scienze Tecnologie e Mercati della Vite e del Vino-Università di Verona-Villa
Lebrecht, Via della Pieve 70, 37029, San Floriano (Verona). – 3 CRA-ORL-Unità di ricerca per
l'orticoltura, Via Paullese, 28, 26836 - Montanaso Lombardo (Lodi).
Keywords: parthenocarpy, aucsia genes, auxin biology, fruit initiation.
Auxins play a crucial regulatory role in several plant developmental processes, including fruit initiation.
Fruit originates from the ovary after pollination and fertilization. The uncoupling of fruit development
from fertilization (parthenocarpy) can be achieved by genetic manipulation of auxin synthesis, auxin
sensitivity and auxin or gibberellin signal transduction pathway. We have identified a novel gene
family consisting of two genes encoding small peptides named AUCSIA, that regulate fruit initiation in
tomato and affect other auxin-related processes (Molesini et al., 2009). Aucsia-silenced tomatoes
exhibited parthenocarpic fruit development and alterations in leaf morphology. Total indole-3-acetic
acid content of pre-anthesis Aucsia-silenced flower buds was 100 times higher than wt. Aucsia-silenced
plants displayed a reduced polar auxin transport (PAT) in roots, and showed an increased sensitivity to
1-naphthylphthalamic acid, a PAT inhibitor. Aucsia genes are present in chlorophytes and streptophytes
and encode peptides distinguished by a 16-amino-acid-long AUCSIA motif, a lysine-rich carboxylterminal region, and a conserved tyrosine-based endocytic sorting motif.
- 47 -
PIII07
REACTIVE OXYGEN SPECIES SIGNALLING IN THE ARABIDOPSIS
THALIANA MUTANT NPQ1LUT2, IMPAIRED IN XANTHOPHYLL
BIOSYNTHESIS.
ALESSANDRO ALBORESI1, LUCA DALL'OSTO1, ALESSIO APRILE2, PETRONIA CARILLO3,
GIOVANNI GIULIANO4, LUIGI CATTIVELLI2 AND ROBERTO BASSI1.
1
Dipartimento di Biotecnologie, Università di Verona, Strada Le Grazie 15, 37134 Verona, Italy. – 2
CRA, Genomic Research Centre, Via S. Protaso 302, 29017 Fiorenzuola d'Arda (PC), Italy. – 3
Dipartimento di Scienze della Vita, Seconda Universita degli Studi di Napoli, Via Vivaldi 43, Caserta,
Italy. – 4 Italian National Agency for New Technologies, Energy and the Environment (ENEA),
Casaccia Research Center, Rome, Italy.
Keywords: ROS, zeaxanthin, transcriptome, singlet oxygen, retrograde signal.
Photosynthetic apparatus proteins are encoded by both plastid and nuclear genes. Therefore under
environmental stress, when photosystem II (PSII) is photoinhibited, a fine tuning of the expression of
the two genomes is required. The molecular mechanism of this cross-talk is only partially understood.
By previous work, the transcription of nuclear-encoded photosynthetic genes correlates to the redox
state of plastoquinone pool (PQ). However, a barley mutant with constitutively reduced PQ was
recently shown to have unaffected transcription rate. Here we verify the alternative hypothesis that
transcriptional regulation is controlled by reactive oxygen species (ROS) accumulation. The
Arabidopsis thaliana npq1lut2 mutant that lacks zeaxanthin and lutein shows overproduction of 1O 2 as
compared to the wild-type. Upon light stress, nuclear encoded genes were strongly down-regulated,
implying the presence of a chloroplast signal regulating their expression under stress conditions. The
comparison with other transcriptomes revealed a overlapping response with other ROS species. The
importance of the emerging central role of 1O2 in ROS signaling cascades in plants is discussed.
PIII08
TOWARDS A ROLE FOR (P)PPGPP IN CHLAMYDOMONAS.
LEONARDO MAGNESCHI1, FLORENCE MUS2, ELENA LORETI3, AMEDEO ALPI4,
PIERDOMENICO PERATA1, ARTHUR GROSSMAN2
1
PLANT Lab, Scuola Superiore Sant'Anna, Via Mariscoglio 34, Pisa (Italy). – 2 Department of Plant
Biology, Carnegie Institution for Science, 260 Panama Street, Stanford (CA, USA). – 3 Istituto di
Biologia e Biotecnologie Agrarie CNR, Via Moruzzi 1, Pisa (Italy). – 4 Dipartimento di Biologia delle
Piante Agrarie, Università di Pisa, Pisa (Italy).
Keywords: Chlamydomonas, (p)ppGpp, abiotic stresses.
Guanosine tetra- and pentaphosphate (ppGpp and pppGpp) are effector nucleotides whose levels
elevate during nutritional stress in eubacteria by means of RelA/SpoT activity, leading to the bacterial
stringent response. Chloroplasts possess bacterial-type systems for transcription and translation, and a
nuclear-encoded chloroplast-localized Chlamydomonas reinhardtii RelA/SpoT Homologue (CrRSH)
has been discovered. CrRSH is promptly induced by dark anoxia, nutritional (sulphur and nitrogen
starvation) and other abiotic (salt and high light) stresses. To elucidate the role of (p)ppGpp in
Chlamydomonas, we generated a Cr-rsh knock-down insertional mutant showing gene down-regulation
under normal growth condition and the inability to induce CrRSH under sulphur starvation. The mutant
grows less but has an higher chlorophyll content compared to the wild type. Under nutrient or abiotic
stress conditions, Cr-rsh colonies are greener and recover better than the genetic background. Our
preliminary data suggest that (p)ppGpp influences stress responses in Chlamydomonas as it does in
eubacteria.
- 48 -
PIII09
THE USE OF A NEW GENETICALLY ENCODED PROBE ALLOWS IN
VIVO DETECTION OF H2O2 AT PEROXISOMAL LEVEL IN YOUNG AND
SENESCENT ARABIDOPSIS LEAVES, UNCOVERING A CA2+-DEPENDENT
SCAVENGING SYSTEM.
ALEX COSTA1, ILARIA DRAGO2, SMRUTISANJITA BEHERA1, MICHELA ZOTTINI1, PAOLA
PIZZO2, JULIAN I SCHROEDER3, TULLIO POZZAN2,4 AND FIORELLA LO SCHIAVO1.
1
Dipartimento di Biologia, Università degli Studi di Padova, Via U. Bassi 58/B, 35131 Padova, Italy. –
Dipartimento di Scienze Biomediche e Istituto di Neuroscienze CNR, Università degli Studi di
Padova, Via G. Colombo 3, 35121 Padova, Italy. – 3 Division of Biology, Cell and Developmental
Biology Section, and Center for Molecular Genetics, University of California San Diego, CA 920930116 La Jolla, USA. – 4 Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy.
2
Keywords: peroxisomes, calcium, H2O2, catalase.
In this study, we have approached the problem of H 2O2 metabolism by plant cells and their modulation
by Ca2+ signalling. To this end we have developed and characterized two novel genetically encoded
probes targeted to the peroxisomal lumen (and to the cytoplasm) to monitor directly in intact living
cells H2O2 and Ca2+ (HyPer and D3cpv). We have also developed Arabidopsis plants stably expressing
such probes. We here demonstrate, through in vivo imaging analyses, that H2O2 content into the leaf
peroxisomes is modulated during the life cycle of Arabidopsis, in particular the Ca2+ dependent H2O2
catabolism is greatly accelerated when pre- and post-bolting phases are compared. In addition we have
investigated intraperoxisomal Ca2+ dynamics in plant cells subjected to stimuli that are known to
increase cytoplasmic Ca2+ levels and we demonstrate that peroxisomal Ca2+ concentration rapidly
equilibrates with that in the cytosol. Last, but not least, we demonstrate that the peroxisomal Ca 2+
increases potently stimulates the scavenging of H2O2 and provide compelling evidence supporting that
this is mediated by Ca2+ activation of peroxisomal CAT3 activity
PIII10
SILENCING OF THE RECEPTOR-LIKE PROTEIN KINASE CRK11
REDUCES PATHOGEN SUSCEPTIBILITY IN ARABIDOPSIS
STEFANIA PASQUALINI1, LAURA MADEO1, FRANCESCO PAOLOCCI2, ORNELLA
CALDERINI2, CHIARALUCE MORETTI3, ROBERTO BUONAURIO3 E LUISA EDERLI1
1
Dipartimento di Biologia Applicata, Università di Perugia. – 2 Istituto di Genetica Vegetale, CNR,
Perugia. – 3 Dipartimento di Scienze Agrarie e Ambientali, Università di Perugia.
Keywords: CRKs, Arabidopsis, Pseudomonas, ozone.
In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cystein-rich
repeat in their extracellular domains. Genes encoding many of these cystein-rich RLKs (CRKs) are
induced by pathogen infection and by ozone, suggesting a possible role in plant defence response. To
analyze the role of CRK11 under abiotic (ozone) and biotic (Pseudomonas syringae pv tomato DC3000
Pst) stresses, we identified a T-DNA insertion mutant for CRK11 (Salk_122172). Disruption of CRK11
expression resulted in a decreased susceptibility to P. syringae infection, whereas ozone sensitivity did
not change with respect to WT Col-0. In this genetic background we observed an early induction of
CRK11 by salicylic acid (SA), Pst and ozone (300 ppb). In stark contrast, in Ws-4 (Wassilewskija)
Arabidopsis plants CRK11 expression was neither expressed constitutively nor induced in plants
challenged with Pst, ozone and SA. Since Ws ecotype presents a natural genetic mutation in FLS2 gene
and is insensitive to flg22 (flagellin) treatment we hypothesize a cross talk between FLS2 and CRK11
receptors.
- 49 -
PIII11
BIOCHEMICAL CHARACTERIZATION OF THE ATYPICAL
CHLOROPLASTIC GLUTAREDOXIN S12.
ZAFFAGNINI MIRKO1, BEDHOMME MARIETTE2, COUTURIER JEREMY3, ROUHIER
NICHOLAS3, LEMAIRE STEPHANE D2, TROST PAOLO1.
1
Laboratory of Molecular Plant Physiology, Department of Evolutionary Experimental Biology,
University of Bologna, Via Irnerio 42, Bologna, 40126, Italy. – 2 Institut de Biotechnologie des
Plantes, UMR 8618, Batiment 630, Orsay cedex, France. – 3 Unitè Mixte de Recherches 1136 UHPINRA Interaction Arbres-Microorganismes, IFR 110 GEEF, Nancy Universitè, Facultè des Sciences,
54506 Vandoeuvre Cedex, France.
Keywords: glutaredoxin, glutathione, cysteine, redox signaling .
Glutaredoxins (Grxs) are small disulfide oxidoreductases particularly efficient for the reduction of
protein-glutathione mixed disulfides, using glutathione or thioredoxin reductases for their regeneration.
We used structural analyses coupled with thiol titrations, fluorescence measurements, site-directed
mutagenesis and mass spectrometry to determine the structural and biochemical properties of poplar
GrxS12, an atypical chloroplastic Grx possessing a monothiol 28WCSYS32 active site. We show that
the only oxidation state of GrxS12 is a glutathionylated derivative of the active site cysteine (Cys29)
and that this Grx is able to catalyze deglutathionylation of both artificial and protein substrates through
a monothiol mechanism requiring only the most N-terminal cysteine of the enzyme. Protein
deglutathionylation by GrxS12 proceeds by a ping-pong mechanism in which rate enhancement is
mainly attributed to the low pKa of the active site cysteine. The results suggest that GrxS12 could play
an important role in redox sensing and signal transduction in chloroplasts.
PIII12
ANALYSIS OF PGIP EXPRESSION ON TWO POPLAR CLONES TREATED
WITH CADMIUM.
MONIA LABELLA1, CRISTINA GIANCOLA1, GIUSEPPE IANIRI2 AND CLAUDIO CAPRARI1.
1
Dipartimento di Scienze e Tecnologie per l'Ambiente e il Territorio (DiSTAT), Università degli Studi
del Molise, Contrada Fonte Lappone snc, 86090 Pesche (IS), Italy. – 2 Dipartimento di Scienze
Animali, Vegetali e dell'Ambiente (Dip. SAVA), Università degli Studi del Molise, Via De Sanctis
s.n.c. - 86100 Campobasso, Italy.
Keywords: PGIP, poplar, cadmium, PCR.
Polygalacturonase-inhibiting proteins (PGIPs) are extracellular proteins that belong to the leucine-rich
repeat (LRR) protein superfamily PGIPs are plant defence glycoproteins associated with the cell wall
that interact with fungal PGs and modulate their activity, favouring the accumulation of
oligogalacturonides that serve as elicitors of plant defence responses. In this study, reverse
transcriptase-polymerase chain reaction (RT-PCR) was used to analyze the expression profiles of
PdPGIP2 and PdPGIP4 genes. RT-PCR analysis was carried out on cDNA synthesized from leave and
roots of woody cuttings of two poplar clones [Nigra poli (Populus nigra) and Lux (Populus deltoides)]
after treatments with two different cadmium concentrations. Cadmium is one of the most phytotoxic
metal pollutants and produces several toxic symptoms in plants. The aim of this work is to understand
whether the stress response to cadmium includes induction of PGIPs.
- 50 -
PIII13
THE HSP20-LIKE CHAPERONE P23 OF ARABIDOPSIS IS A SPECIFIC
TARGET OF CK2, INVOLVED IN SALICYLIC ACID SIGNALLING
PATHWAY.
ALEX COSTA1, KENDRA TOSONI2, STEFANO D'ALESSANDRO1, GIORGIO ARRIGONI2,
FIORELLA LO SCHIAVO1, MARIA RUZZENE2 AND MICHELA ZOTTINI1.
1
Department of Biology, University of Padova, Padova, Italy. – 2 Department of Biological Chemistry,
University of Padova, Padova, Italy.
Keywords: Salicylic acid, casein kinase2, nitric oxide, HSP chaperones.
Salicylic acid (SA) plays a key role in various physio-pathological processes in plants. Its action
mechanism involves many signalling molecules such as H2O2 and nitric oxide (NO). We recently
demonstrated that, in Arabidopsis seedlings, SA-induced NO production is mediated by the activity of
casein kinase 2 (CK2). Comparing the phosphorylation pattern of protein extracts obtained from
Arabidopsis seedlings treated with SA and/or a specific inhibitor of CK2, we identified a 23KDa
protein (P23) that was further investigated. P23 cDNA was cloned in a prokaryotic expression vector
and the recombinant protein was obtained. In an in vitro assay it has been observed that the
recombinant P23 is highly phosphorylated by the human CK2 and by cytoplasmic protein extracts from
Arabidopsis. In order to confirm that CK2 is the major protein kinase responsible for P23
phosphorylation, an in gel kinase assay was performed. Transient transformation experiments
performed on Arabidopsis mesophyll protoplasts allow to determine the localization of P23 at nuclear
and cytoplasmic level. This subcellular localization is in agreement with the CK2 localization already
reported for plant cells.
PIII14
COMPARATIVE PROTEOMIC ANALYSIS OF H2O2-INDUCED PCD IN
TOBACCO BY-2 CELLS.
M. MARSONI1, C. CANTARA1, V. LOCATO2, L. DE GARA2, M. BRACALE1, C. VANNINI1
1
Department Environment, Health, Security, University of Insubria, via G. B. Vico, 21100 Varese,
Italy. – 2 Department of Biology and Plant Pathology, University of Bari, via E. Orabona 4, 70125 Bari,
Italy.
Keywords: TBY-2 cells, H2O2, PCD,proteome, ProteoMiner.
ROS play a crucial role in many physiological and pathological processes in plants. In particular H 2O2
is implicated as a second messenger which can orchestrate the plant hypersensitive disease resistance
by initialing a series of reactions and by mediating systemic signaling in the establishment of plant
immunity.Our ultimate goal is to dig deep into a tobacco (TBY-2) proteome in order to identify
proteins differentially accumulated in H2O2 treated cells. However, when large proteomes consisting of
thousands of proteins are analyzed, the dynamic resolution is generally limited and only the most
abundant proteins are detected. So, despite sophisticated protein fractionation strategies, the number of
proteins normally detected is surprisingly low considering the number of expected proteins in a
cell.Therefore, in order to have access to the "hidden" proteome, we used the ProteoMiner™ technique
which employs a large, highly diverse beaad-based library of combinatorial peptide ligands and it is
able to simultaneously reduce high-abundance proteins and enriche low-abundance ones.
- 51 -
PIII15
OZONE INDUCES A HR-LIKE RESPONSE IN THE O3-SENSITIVE
POPULUS DELTOIDES X MAXIMOWICZII ERIDANO CLONE.
G. BARTOLI1, L.M.C. FORINO2, A.M. TAGLIASACCHI2, R. BERNARDI1, M. DURANTE1.
1
Department of Agricultural Plant Biology, Genetics Section, University of Pisa (Italy). – 2 Department
of Biology, University of Pisa (Italy).
Keywords: ozone stress, poplar hybrids, hypersensitive response (HR), PAL2, PR proteins.
The effects induced by a realistic short high-peak of O3 on sensitive and resistant poplar hybrids (the
O3-sensitive Populus deltoides x maximowiczii Eridano clone and the O3-resistant Populus x
euoramericana I-214 clone) were investigated, in order to verify whether a hypersensitive response, like
that which results from the incompatible plant-pathogen interaction, could take place in sensitive poplar
clones. Within 24-48 hrs from O3-fumigation end, only the leaves of the sensitive clone showed typical
necrotic areas, symptoms of ozone injury. In the most affected mesophyll tissues we evidenced a
combination of cytological markers and structural changes (highly localized cell death, nucleus
degeneration and chromatin condensation, cell walls collapse and incomplete degeneration of cell
remnants, autofluorescent compounds and callose deposition) referable to a HR response. A
concomitant change in gene expression of phenylalanine ammonia-lyase (PAL2), polyphenoloxydase,
glutathione S-transferase, metallothioneins and other pathogenesis-related proteins were also evidenced.
These results suggest that the ozone may trigger a hypersensitive response in our plant system.
PIII16
STUDY OF THE OLIGOGALACTURONIDE SIGNALLING PATHWAY
USING PROTEOMIC STRATEGIES AND IN VITRO AND IN VIVO
AFFINITY FISHING USING DERIVATIZED ELICITORS.
LORENZO MARIOTTI, FRANCESCA SICILIA, DANIELA PONTIGGIA, FRANCESCO
SPINELLI, BENEDETTA MATTEI, GIULIA DE LORENZO.
Dipartimento di Biologia Vegetale, Università di Roma La Sapienza, piazzale A. Moro 5, Roma.
Keywords: innate immunity, confocal microscopy, phospho-proteomic, Surface Plasmon Resonance.
During the infection of plant tissue by pathogens, homogalacturonan is broken down in lower size
fragments, called oligogalacturonides (OGs), with a degree of polymerization from 10 to 15 that trigger
the plant defence response. Although the effects of OGs in plant defence are well recognised, the
perception/transduction mechanisms of these elicitors are still not completely understood. A phosphoproteomic approach on apoplastic, plasma membranes and cytosolic proteins is being used to study the
role of protein phosphorylation in the OG signalling pathway. We have also prepared biologically
active fluorescent (f-OGs) and biotinylated OGs (b-OGs). f-OGs have been used to study in vivo the
dynamic of OG perception in tobacco and Arabidopsis leaves by confocal microscopy. b-OGs have
been used to isolate and identify putative OG high-affinity binding sites. Moreover, using b-OGs and
proteins that have OG-binding sites in Surface Plasmon Resonance (SPR) analysis, we are developing a
highly sensitive assay to determine levels of OGs in plant tissues.
- 52 -
PIII17
POLY ADP-RIBOSE POLYMERASE INVOLVEMENT IN THE
PROGRAMMED CELL DEATH OF TOBACCO BY–2.
V LOCATO1, S CIMINO1, MC DE PINTO2, CH FOYER3, L DE GARA1,2.
1
CIR University Campus Bio-Medico of Rome , Italy. – 2 Dep. Plant Biology & Pathology University
of Bari, Italy. – 3 School of Agriculture, Food and Rural Development, Newcastle University– UK
Keywords:.
Programmed cell death (PCD) is a genetically controlled death process required for the differentiation
of specific tissues and organs as well as for the responses against stress conditions. Different stressors
inducing PCD have been identified and the involvement of ascorbate and glutathione (GSH)
metabolism have been characterized in tobacco BY-2 cells undergoing PCD. A correlation between
variation in GSH content/cellular localization and changes in the expression/activity of nuclear poly
ADP-ribose polymerase (PARP) seem to occur through the different phases of Arabidopsis cultured cell
growth. These results further underline the interplay between cellular redox regulation and nuclear
metabolism. PARP involvement in apoptosis is largely documented in animals, but little is known about
its role in plant PCD. Here a first characterization of PARP behaviour in tobacco BY-2 cells
undergoing PCD is reported.
- 53 -
ABSTRACTS - Session IV: Biodiversity
PIV01
NON-PHOTOCHEMICAL QUENCHING IN SHADE-ADAPTED
PTERIDOPHYTES.
LORENZO FERRONI, LAURA PANTALEONI, COSTANZA BALDISSEROTTO, SIMONETTA
PANCALDI.
Department of Biology and Evolution, University of Ferrara, C.so Ercole I D'Este, 32, 44100 Ferrara Italy.
Keywords: Pteridophytes, chlorophyll fluorescence, non-photochemical quenching, photosystem II.
Many Pteridophytes, i.e. phylogenetically divergent non-flowering vascular plants, live in shaded,
humid environments. We addressed the question whether shade pteridophyte species had a convergent
or divergent ability of energy dissipation in photosynthesis. Using a PAM fluorometer, the ability of
non-photochemical quenching (NPQ) of chlorophyll fluorescence was compared in six species of
shade-adapted divergent Pteridophytes and the Angiosperm Piper nigrum cultivated in a warm, humid
greenhouse of the Botanical Garden of Ferrara. Induction kinetics showed no obvious relation with
phylogenesis. The highest and lowest NPQ were developed by Selaginella sp. and Adiantum sp.,
respectively. The conspicuous difference between the two species was mainly due to the fast-relaxing
NPQ component, qE. The two species were further analysed through irradiance-response curves of
potential and actual PSII quantum yield, NPQ, qL, q0 and by the quantification of the partitioning of
excitation energy between photochemistry and dissipative processes. Collectively, the two
Pteridophytes show peculiar properties of light-energy management compared to each other and to P.
nigrum.
PIV02
THE IMPORTANCE OF ANTENNA PROTEINS FOR LAND
COLONIZATION: LIGHT HARVESTING AND PHOTOPROTECTION IN
GREEN ALGAE, MOSSES AND HIGHER PLANTS.
SILVIA DE BIANCHI, ALESSANDRO ALBORESI, MATTEO BALLOTTARI, GIULIA BONENTE
AND ROBERTO BASSI.
Dipartimento di Biotecnologie, Università di Verona, Strada Le Grazie 15, 37134 Verona, Italy.
Keywords: Photoprotection, Lhc - Lhc-like proteins.
Light harvesting complexes (Lhc) form a gene family highly represented in all photosynthetic
eukaryotes acting in light harvesting and photoprotection. An inspection of the Chlamydomonas
reinhardtii, Physcomitrella patens and Arabidopsis thaliana genomes shows a number of genes
encoding Lhc and Lhc-like proteins. Lhcb3, Lhcb6 and Lhcb7 were found only in land plants and in a
moss, Physcomitrella patens, but not in green algae. Since land environment is characterized by far
more stressing conditions with respect to water, this suggests their special role in photoprotection.
Concerning LHC-like proteins, PsbS and Li818 R2 have been shown to be essential for thermal
dissipation of excess energy respectively in plants and algae (Li, X. P. et al. 2000, Peers, G. et al. 2007)
where these gene products are respectively expressed; lower plants such as the moss Physcomitrella
patens accumulate both gene products (Alboresi, A. et al. 2008). In this work we discuss the importance
and the evolution of this different proteins analyzing their role in three model organisms,
Chlamydomonas reinhardtii, Physcomitrella patens and Arabidopsis thaliana.
- 54 -
PIV03
EVOLUTION OF FLOWERING TIME AND FRUIT QUALITY TRAITS IN
TOMATO.
FALCONE G.*, FANTINI E.*, AND GIULIANO G.
ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Roma, Italy *These authors
contributed equally to this work.
Keywords: wild tomato species, flowering photoperiodic response, carotenoid biosynthetic pathway.
The genome of cultivated tomato (S. lycopersicum) has a limited sequence variation due to bottlenecks
during domestication and breeding; however, the study of genetic diversity between tomato and related
wild species could provide useful tools for the breeding of agronomically useful characters. We took a
candidate gene approach to identify genetic differences responsible for the variability of two traits: the
photoperiodic flowering response and the different colour of ripe berries. S. lycopersicum ecotypes and
closely related wild tomato species were selected. The CRYPTOCHROME and CONSTANS gene
families, controlling flowering time in Arabidopsis, have been completely sequenced, but up to now no
mutations discriminating day-neutral from short-day species have been identified. Concerning berry
colour, we studied the carotenoid biosynthetic pathway. The sequencing of genes from PSY down to
LCY-e (α-branch) and CHY1/CHY2 (β-branch) is complete. The sequence analysis has highlighted the
presence of numerous mutations that differentiate the colour-fruited species from the green-fruited
ones. Some non-synonymous substitutions are candidate to be hypomorphic alleles.
PIV04
PHYSIOLOGICAL RESPONSES OF PHAEODACTYLUM TRICORNUTUM
AND CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE) TO
DIFFERENT N-REGIMES.
ALESSANDRA NORICI, STEFANO MATTIONI, MARIO GIORDANO.
Dip. Scienze del Mare, Università Politecnica delle Marche, Brecce Bianche, 60131 Ancona, Italia.
Keywords: diatom, C to N ratio, growth, N utilization.
Growth parameters, N and C allocation into cell constituents, N and C assimilation rates, internal CO 2
pools of Phaeodactylum tricornutum and Cylindroteca closterium grown at 0.01, 1 and 10 mM either
NO3-or NH4+, were measured in order to understand N cycling through macromolecular reservoirs in
cells. At 0.01 mM N, growth of both species was limited, more C was allocated into structural or
storage lipids and Si deposition increased. Higher photosynthetic inorganic C affinity was shown
depending on the N chemical form, consistent with enhanced CCM activity and improved resource-use
efficiency. Growth of P. tricornutum was highest at 10 mM NH4+. In C. closterium, growth was
saturated at 1 mM NO3-, same growth rate was reached at 1 mM NH4+. Growth was inhibited by 10
mM NH4+ and proteins were accumulated in the cells for detoxification mechanisms. C. closterium was
able to achieve, on average, higher C assimilation rates than in P. tricornutum, despite similar C to N
ratios. Different storage capacity for inorganic and organic N, due to different cell sizes, may be
responsible. N utilization strategy is discussed in terms of primary productivity and resource
competion.
- 55 -
PIV05
TOMATO GENOME SEQUENCING.
PIETRELLA M.1, FALCONE G.1, FANTINI E.1, GONZALEZ, M.1, ERCOLANO MR.2, BARONE
A.2, CHIUSANO M.L.2, D'AGOSTINO N.2, TRAINI A.2, FRUSCIANTE L.2, PERROTTA, G.3,
VEZZI A.4, VALLE G.4 AND GIULIANO G.1.
1
Department BAS, ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Roma, Italy. – 2
Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples
"Federico II", Via Università 100, 80055 Portici, Italy. – 3 ENEA, Trisaia Research Center, S.S. Ionica Km 419.5, 75026 Rotondella (MT), Italy. – 4 CRIBI Biotechnology Centre and Department of Biology,
Univ. of Padova, via U. Bassi 58/B, 35131 Padova, Italy.
Keywords: Tomato, chromosome 12 sequencing.
Tomato (Solanum lycopersicum) is an economically and nutritionally valuable crop and constitutes a
model plant for the Solanaceae family. Its genome encodes approx. 35.000 genes, which are largely
grouped in contiguous euchromatic regions corresponding to approx. 25% of the 950 Mb genome. An
international project is under way to sequence the euchromatic DNA on a BAC-by-BAC strategy
(http://sgn.cornell.edu/about/tomato_sequencing.pl). Italy is sequencing chromosome 12 and providing
mapping and bioinformatic tools to the international effort. Seed BACs are selected for the presence of
genetic markers and validated via genetic (Introgression Line) and cytogenetic (FISH) mapping. Each
sequenced seed BAC is then extended into a minimum tiling path. Approx 41% of the euchromatic
portion is publicly available (43% on Chromosome 12). A bioinformatic platform has been built to
provide a preliminary annotation of the genome. An additional effort, to obtain a draft WGS sequence
with Next Generation sequencing, is under way.
PIV06
MOLECULAR ANALYSIS OF TOMATO MUTANTS PRODUCING
ANTHOCYANINS IN THE FRUIT.
GIOVANNI POVERO1, SILVIA GONZALI2, ANDREA MAZZUCATO2, GIAN PIERO SORESSI1,
LAURA BASSOLINO1, PIERDOMENICO PERATA1.
1
PlantLab, Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy. – 2
Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, 01100 Viterbo, Italy.
Keywords: Anthocyanin, tomato, antioxidants.
Anthocyanins are high value plant antioxidants which are not present in the fruits of cultivated tomato.
However, both the dominant gene Anthocyanin fruit (Aft) and the recessive gene atroviolacea (atv),
introgressed into domesticated tomato from two different wild Solanum species, stimulate a limited
anthocyanin pigmentation. Surprisingly, double mutant atv/atv Aft/Aft tomatoes can be distinguished by
the presence of intensely pigmented fruit. In our study, the expression pattern of 18 genes involved in
the anthocyanin pathway, including those encoding the Myb transcription factors ANT1 and AN2 (both
displaying nucleotide and aminoacid polymorphism between Aft line and cultivated genotypes), was
analyzed by qRT-PCR in wild type plants and in single and double Aft and atv mutants. Anthocyanin
levels and expression of the genes involved in the anthocyanin biosynthetic pathway were higher in the
double mutant than in the single parental lines in the peel of tomato fruit, while they were negligible in
the flesh (in all the genotypes). The characterization of the mutations Aft and atv could contribute to the
production of tomatoes with a higher nutritional value.
- 56 -
PIV07
DIVERSITY IN THE RESPONSE TO LOW TEMPERATURE IN A SET OF
REPRESENTATIVE BARLEY GENOTYPES CULTIVATED IN EUROPE.
C.LAGO1, D.PAGANI1, M.GUT2, C.CROSATTI1, A.TONDELLI1, A.M.STANCA1,F.RIZZA1.
1
1 CRA-GPG Centro Genomica e Postgenomica Animale e Vegetale, 29017 Fiorenzuola d'Arda (PC),
Italy. – 2 IHAR Institute, Department of Quality Evaluation and Cereals Breeding Methods, Zawila 4,
30 - 423 Krakow, Poland.
Keywords: Biodiversity,barley,frost tolerance,vernalization.
In winter cereals freezing tolerance is associated with the occurrence of a cold hardening adaptive
process, that occurs when plants are exposed to low non-freezing temperatures. The capacity for a fast
induction of hardening can be an evolutionary advantage under field conditions of progressively falling
temperatures because resistant plants can prepare for subzero temperatures well before the susceptible
ones. This work was aimed to investigate to which extent the plant response in early stage of
acclimation (short hardening treatment at +3°C or suboptimal hardening temperature e.g.10°C) plays a
critical role in determining the potential frost hardiness in barley. This aspect has been analysed in a
biodiversity study on 55 winter and spring cultivars of different origin. Early hardening treatments were
effective in improving frost tolerance, as estimated by chlorophyll fluorescence and survival analyses.
The implication of the vernalization requirement and the accumulation of a cold induced protein
determining barley frost tolerance is discussed.
- 57 -
ABSTRACTS - Session V: Crops for the future
PV01
HEAVY METALS HOMEOSTASIS IN SOLANUM LYCOPERSICUM CV.
M82 AND S. LYCOPERSICUM X S. PENNELLII IL 6-4.
DE BIASI MARGHERITA-GABRIELLA1, SALLUZZO ANTONIO2, LIPPO SILVIA1, CHIAIESE
PASQUALE1, FILIPPONE EDGARDO1
1
Department of Soil, Plant, Environmental and Animal Production Sciences, School of
Biotechnological Science, University of Naples "Federico II", Via Università 100, 80055 Portici (NA)
Italy. – 2 ENEA (Italian National Agency for New Technology Energy and the Environment) Portici
Research Center, Via Vecchio Macello - 80055 Portici (NA) Italy.
Keywords: Ionomic, Solanum licopersycum, Introgression Line, ICP-MS, heavy metals accumulation,
food safety.
Genetic linkage map was constructed for Solanum lycopersicum and currently there are several
molecular maps based on crosses between the cultivated and various wild tomato species. However,
until now, little information is available on QTL for ions accumulation in tomato. Moreover, as our
knowledge, effects of new gene combinations in introgressed lines on ions uptake related to food safety
have not been extensively studied. We are studying two tomato genotypes: Solanum lycopersicum cv
M82 and IL 6-4. The latter is an introgression line of the tomato wild-relative species S. pennellii in the
cv M82; on the basis of bioinformatic approach, genes involved in ions uptake, translocation and
accumulation should have introgressed. All plants were grown in hydroponic in the presence of nontoxic concentration of Cd (10 microM), Pb (3 μM) and Zn (100 μM) given separately or combined. The
ionome modifications induced by treatments were revealed by ICP-MS performed on tomato leaves.
The analysis of all leaves showed that IL 6-4 accumulated more ions than M82 and that both excluded
toxic metals, at least in the presence of combined stress. This project was granted by GenoPom MIUR
program.
- 58 -
ABSTRACTS - Session VI: Plants-Microbe Interactions
PVI01
IDENTIFICATION OF TRANSCRIPTION FACTORS INVOLVED IN THE
SYMBIOSIS BETWEEN LOTUS JAPONICUS AND AM FUNGUS.
VERONICA VOLPE, MIKE GUETHER, PAOLA BONFANTE.
Department of Plant Biology and IPP-CNR, University of Torino, Viale Mattioli 25, 10125 Torino,
Italy.
Keywords: arbuscular mycorrhizas, transcription factors, lotus japonicus, RNA interference.
Arbuscular mycorrhizas (AMs) are very common symbioses established between land plants and soil
fungi, where both partners benefit by nutritional exchanges. The establishment of AMs requires deep
cellular and molecular changes in both the symbionts. Plant development and differentiation are
programmed primarily at a transcriptional level: however, the role of transcription factors (TFs) in
mycorrhization process has never been faced. The aim of this work is to provide an overview of TF
genes expressed during AM symbiosis. Transcriptome analysis of L. japonicus using a microarray has
identified 23 TFs whose expression is altered in mycorrhizal roots (Guether et al. 2009). Four of them,
3 LjSCR and 1 LjMYB, were analyzed in qRT-PCR to confirm their differential expression and were
selected for a functional analysis. We first developed mutant lines of LjSCR and LjMYB using RNAi
technology. Silencing was then confirmed by qRT-PCR for LjMYB expression, while the impact of
mycorrhization on this silenced line is still under validation, as well as the analysis of other LjSCR and
LjMYB overexpressing lines.
PVI02
DOES MYCORRHIZATION INFLUENCE TOMATO FRUIT QUALITY? A
TRANSCRIPTOMIC APPROACH.
SALVIOLI, A.1 , ZOUARI, I.1, LACOURT, I.2 AND BONFANTE, P.1
1
Dipartimento di Biologia Vegetale dell'Università and Istituto per la Protezione delle Piante – CNR,
Viale Mattioli 25, 10125-I, Torino, Italy. – 2 Sotral SpA, via Livorno 60, 10144 Torino, Italy.
Keywords: Arbuscular mycorrhizal fungi, Mycorrhizas, tomato fruit quality, gene expression , Real
time RT-PCR.
Soil fertilization is considered one of the most important practices influencing plant productivity as well
as fruit quality. In this framework, the role of arbuscular mycorrhizal fungi (AMF), i.e. the symbiotic
microbes which live in association with plants roots, may be crucial. While the positive effect of AMF
as bio-fertilizers on plant physiology and productivity has already been reported, little is known about
their effect on fruit quality traits. In particular, the potential effect of mycorrhization on fruit gene
expression has never been considered so far. The aim of the investigation was therefore to assess the
potential long-distance effect of AM fungi on tomato fruit transcriptomics.Using cDNA microarray,
transcription profiles of tomato fruit harvested from plants inoculated with Glomus mosseae and from
non inoculated plants were compared. Twenty genes revealed an up- or down-regulation under the
mycorrhizal versus the control condition. Real time RT-PCR analysis confirmed the expression profiles
of 58% of the selected genes. The data suggest that fruit ripening process, lipid, sugar and aroma
metabolisms are modulated by mycorrhization at the level of gene expression.
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PVI03
MOLECULAR AND MORPHOLOGICAL DETERMINATION OF FUNGAL
ASSOCIATION IN THE ORCHID SPIRANTHES SPIRALIS.
ALESSANDRA TONDELLO1,2, ELENA VENDRAMIN2, MARIACRISTINA VILLANI1,
BARBARA BALDAN1, ANDREA SQUARTINI2.
1
Dip. di Biologia, UniPD Viale G. Colombo 3, 35131 Padova, Italy; - 2 Dip. di Biotecnologie Agrarie,
UniPd, viale dell'Università 16, 35020 Legnaro, Padova, Italy.
Keywords: confocal and electron microscopy, orchid mycorrhizas, Spiranthes spiralis, PCR
amplification of ribosomal ITS.
Mycorrhizas are widespread intimate symbioses between members of fungal phyla and a vast majority
of plants. Orchid mycorrhiza is a category with important peculiarities as fungi often sustain the life of
these small-seeded plants during the delicate offspring stage and begin receiving carbon only once the
plant reaches its established form. S. spiralis (L.) Chevall. is a sub-mediterranean species; it has a
remarkable phenology in western Europe, where is the latest-blooming native species of orchid.
Rosettes appear in summer and stalks flower in September. We isolated mycorrhizal fungi from plants,
and in order to investigate their taxonomical identity, we extracted total DNA both from the root tissues
and from isolated fungi. The latter were cultured from inner root tissues upon surface sterilization.
Selective PCR amplification using ITS1-ITS4, ITS1F-ITS4 primers was carried out. In parallel, using
confocal microscopy on acridine orange-stained freehand sections we demonstrate the presence of a
profuse cortical colonization by intracellular hyphal pelotons. Electron microscopy examination of
isolated fungi allowed to observe the details of this hyphal ultra-structure.
PVI04
DOES MYCORRHIZATION INFLUENCE TOMATO FRUIT QUALITY? A
TRANSCRIPTOMIC APPROACH.
SALVIOLI, A.1 , ZOUARI, I.1, LACOURT, I.2 AND BONFANTE, P.1
1
Dipartimento di Biologia Vegetale dell'Università and Istituto per la Protezione delle Piante – CNR,
Viale Mattioli 25, 10125-I, Torino, Italy. – 2 Sotral SpA, via Livorno 60, 10144 Torino, Italy.
Keywords: Arbuscular mycorrhizal fungi, Mycorrhizas, tomato fruit quality, gene expression , Real
time RT-PCR.
Soil fertilization is considered one of the most important practices influencing plant productivity as well
as fruit quality. In this framework, the role of arbuscular mycorrhizal fungi (AMF), i.e. the symbiotic
microbes which live in association with plants roots, may be crucial. While the positive effect of AMF
as bio-fertilizers on plant physiology and productivity has already been reported, little is known about
their effect on fruit quality traits. In particular, the potential effect of mycorrhization on fruit gene
expression has never been considered so far. The aim of the investigation was therefore to assess the
potential long-distance effect of AM fungi on tomato fruit transcriptomics.Using cDNA microarray,
transcription profiles of tomato fruit harvested from plants inoculated with Glomus mosseae and from
non inoculated plants were compared. Twenty genes revealed an up- or down-regulation under the
mycorrhizal versus the control condition. Real time RT-PCR analysis confirmed the expression profiles
of 58% of the selected genes. The data suggest that fruit ripening process, lipid, sugar and aroma
metabolisms are modulated by mycorrhization at the level of gene expression.
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PVI05
NITRIC OXIDE ACCUMULATION DURING THE EARLY STAGES OF THE
ARBUSCULAR MYCORRHIZAL SYMBIOSIS.
CALCAGNO C., LANFRANCO L., NOVERO M., GENRE A., BONFANTE P.
Dipartimento di Biologia Vegetale, Università degli Studi di Torino.
Keywords: nitric oxide, arbuscular mycorrhizal symbiosis, cofocal microscopy, mycorrhizal mutants,
Medicago truncatula.
Nitric oxide (NO) is a signalling molecule involved in developmental processes and in response to
several abiotic and biotic stresses. The aim of this work is to verify the involvement of NO in early
plant responses to arbuscular mycorrhizal (AM) fungi.The accumulation of NO in root tissues was
visualized by confocal microscopy on Medicago truncatula roots treated with fungal exudates of
<Gigaspora margarita> germinated spores.Experiments were performed with A. tumefacienstransformed roots derived from the wild type (WT) and the non-mycorrhizal mutants dmi1, dmi2 and
dmi3.WT root fragments treated with fungal exudates showed an increase of fluorescence during the
first ten minutes.The dmi1 and dmi2 mutants did not respond while a weak increment was recorded for
dmi3. M. truncatula roots treated with Nod factor and the non host plant Arabidopsis thaliana treated
with fungal exudates showed no increase in fluorescence. These data suggest that NO accumulation
occurs down-stream Dmi1 and Dmi2 functions but up-stream Dmi3.In conclusion, genetic and cellular
evidences suggest that NO accumulation is a novel component in the signalling pathway leading to AM
symbiosis.
PVI06
INTRACELLULAR CALCIUM CHANGES IN THE SYMBIOTIC SIGNALLING
PATHWAY ACTIVATED BY FLAVONOIDS IN RHIZOBIA.
MOSCATIELLO ROBERTO1, ROMANELLI MARIA GIOVANNA1, SQUARTINI ANDREA2,
MARIANI PAOLA1, NAVAZIO LORELLA1.
1
Dipartimento di Biologia, Università di Padova, Via U. Bassi 58/B, 35131 Padova, Italy. – 2
Dipartimento di Biotecnologie Agrarie, Università di Padova, Viale dell'Università 16, 35020 Legnaro,
Padova, Italy.
Keywords: calcium signalling, rhizobium, legume, symbiosis, flavonoids.
Rhizobia are Gram-negative bacteria that form nitrogen-fixing symbiosis with legume host plants.
Nodulation requires a complex dialogue between the interacting partners, based on the exchange of
signalling molecules of both plant and microbial origin. A messenger role for calcium in rhizobium,
parallel to that well-known to be played in the host plant, had not yet been considered. Transgenic
rhizobium cell cultures expressing aequorin were generated and used in Ca2+ measurement assays after
challenge with plant symbiotic signals. Flavonoids known as active inducers of expression of
nodulation genes in R. leguminosarum bv. viciae were found to activate intracellular Ca2+ changes,
whereas non-inducing flavonoids failed to trigger any detectable Ca 2+ rise. The transient elevation in
Ca2+ was shown to be required for activation of nodulation genes. A rhizobium strain cured of the
symbiotic plasmid responded to inducers with an unchanged Ca2+ response, showing that the
transcriptional regulator NodD plays its role downstream the Ca 2+ signal. These data shed light on a
previously uninvestigated facet (bacterial Ca2+ signalling) of the two-way partner signal exchange and
transduction.
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PVI07
SALICYLIC ACID DIFFERENTIALLY AFFECTS SUSPENSION CELL
CULTURES OF LOTUS JAPONICUS AND ONE OF ITS NON-SYMBIOTIC
MUTANTS.
FIORENZA BASTIANELLI1, ALEX COSTA1, ENRICA D'APUZZO2, MICHELA ZOTTINI1,
MAURIZIO CHIURAZZI2 AND FIORELLA LO SCHIAVO1.
1
Department of Biology, Padova University, Via U. Bassi 58/B, I-35131 Padova, Italy. – 2 Institute of
Genetics and Biophysics "A. Buzzati Traverso", Via P. Castellino 12, 80131 Napoli, Italy.
Keywords: Lotus japonicus cell cultures, salicylic acid, cell death, H 2O2, NO.
Salicylic acid (SA) is known to play an important role in the interaction between plant and microorganisms, both symbiotic and pathogen. In particular, high levels of SA block nodule formation and
mycorrhizal colonization in plants. A mutant of Lotus japonicus, named Ljsym4-2, was characterized as
unable to establish positive interactions with Rhizobium and fungi; in particular, it does not recognize
signal molecules released by symbiotic micro-organisms so that, eventually, epidermal cells undergo
PCD at the contact area. We have performed a detailed characterization of wild-type and Ljsym4-2
cultured cells by taking into account several parameters characterizing cell responses to SA. In the
presence of 0.5 mM SA, Ljsym4-2 suspension-cultured cells reduce their growth and eventually die,
whereas in order to induce the same effects in wt suspension cells, SA concentration must be raised to
1.5 mM. An early and short production of nitric oxide (NO) and reactive oxygen species (ROS) was
detected in wt-treated cells. In contrast, a continuous production of NO and a double-peak ROS
response, similar to that reported after a pathogenic attack, was observed in the mutant Ljsym4-2 cells.
PVI08
IDENTIFICATION OF THE EFFECTS OF FUSARIUM VERTICILLIODES ON
MAIZE TRANSCRIPTOME IN RELATION WITH HOST RESISTANCE.
LANUBILE A., PASINI L. AND MAROCCO A.
Institute of Agronomy, Genetics and Crop Sciences, Università Cattolica del Sacro Cuore, Via Emilia
Parmense 84, 29100 Piacenza, Italy.
Keywords: Fusarium ear rot, microarray analysis, PR genes, oxydative stress.
Fusarium ear rot is found wherever corn is grown. Fusarium ear rot is caused by F. verticillioides. We
have identified genes expressed in kernels and silks of maize resistant and susceptible lines during F.
verticilliodes infection. Ears infected with F. verticilliodes were harvested 48 hours after infection and
also from uninfected ears. RNA was extracted and cDNAs, labelled with fluorescent nucleotides, were
used for arrays hybridisation. In the F. verticilliodes-treated plants, 280 differentially accumulating
maize gene transcripts were detected, compared with untreated plants. Functional annotation of the
transcripts revealed the activation and the repression patterns in both lines in comparison with not
infected controls. The differentially expressed genes were validated in qPCR. Also silks were infected
and harvested 12, 24, 48 and 72 hours after infection and from uninfected ears. The main PR genes
identified in the array experiment and involved in pathogen response were tested using qPCR. The
presence and the activity of the fungus were monitored in seeds and silks using qPCR and the
expression of β-tubulin and the fumonisin biosynthetic gene Fum 21.
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PVI09
PHYTOPLASMA DISEASE IN APPLE: INVOLVEMENT OF REDOX
ACTIVITIES DURING THE RECOVERY.
BRAIDOT, E., ZANCANI, M., PATUI, S., BERTOLINI, A., DEL LINZ, N., MACRÌ, F.,
VIANELLO, A.
Sezione di Biologia Vegetale, Dipartimento Biologia e Protezione delle Piante, UniversitÃ di Udine,
Via delle Scienze, 91, I-33100 Udine, Italy.
Keywords: Lotus phytoplasm; recovery; NADH oxidase/peroxidase; salicylic acid; oxylipin pathway.
The phytoplasms are prokaryotic plant pathogens, responsible for deterioration of several trees. In
apple, the recovery phenomenon is a spontaneous process implying the disappearance of symptoms,
associated to hydrogen peroxide production in the phloem. To better characterize the recovery, leaves
from healthy, diseased and recovered plants were assayed for plasma membrane NAD(P)H
oxidase/peroxidase, lipoxygenase and hydroperoxide lyase activities, the two latter being key enzymes
of the oxylipin pathway. In diseased plants, the NAD(P)H peroxidase activity was more sensitive to
KCN, indicating a higher ability to produce hydrogen peroxide, while lipoxygenase activity was lower
with respect to the healthy and recovered plants. Salicylate, a signal molecule involved in plant
systemic response to pathogen, has been also determined. The concentration of salicylate was higher in
diseased leaves, when compared to healthy and recovered leaves, where the levels were comparable.
These results suggest that the recovery process in apple tree involves the activation of surface/plasma
membrane redox systems, does not require salicylic acid and is mainly dependent on the oxylipin
pathway.
PVI10
ENHANCEMENT OF THE WHEAT DEFENCE RESPONSE TO FUNGAL
PATHOGENS BY INCREASING THE DEGREE OF METHYL
ESTERIFICATION OF CELL WALL PECTINS.
CHIARA VOLPI1, MICHELA JANNI1, VINCENZO LIONETTI2, DANIELA BELLINCAMPI3 AND
RENATO D'OVIDIO1.
1
Department of Agrobiology and Agrochemistry, University of Tuscia, Viterbo, Italy. – 2 Department
of Plant Biology, University of Rome "La Sapienza", 00185 Rome, Italy. – 3 Department of Chemistry,
University of Rome "La Sapienza", 00185 Rome, Italy.
Keywords: cell wall, pectin methylesterase inhibitor, wheat, fungal pathogen, plant defence.
Plant cell wall represents the main barrier for several microbial patogens. Because of this, its
reinforcement should increase plant resistance. One way to reach this goal and test this hypothesis is the
modification of the pectin component of the cell wall. Pectin is secreted in a highly methylesterified
form and is demethylesterified in muro by pectin methylesterase (PME). The activity of PME is
regulated by specific protein inhibitors (PMEIs). Since highly methylesterified pectin can be less
susceptible to hydrolysis by enzymes such as fungal endo-PGs, the inhibition of endogenous PME by
PMEI might increase pectin resistance to degradation by fungal PGs. We have produced a number of
wheat lines expressing the pectin methylesterase inhibitors AcPMEI showing a reduced PME activity.
No obvious phenotypic differences between the transgenic lines and the wild type plants have been
observed, however, the degree of methylation is higher in the transgenic plants. Moreover, transgenic
tissue is more resistant to digestion by fungal PGs and transgenic plants showed a significant reduction
of symptoms caused by Bipolaris sorokiniana and Fusarium graminearum.
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PVI11
LIPOPEROXIDATIVE EVENTS INFLUENCE OCHRATOXIN A
BIOSYNTHESIS IN ASPERGILLUS OCHRACEUS DURING THE
INTERACTION WITH TRITICUM DURUM SEEDS.
REVERBERI M.1, SCARPARI M.1, PUNELLI F.1, ZJALIC S.1, RICELLI A.2, FABBRI A.A.1, AND
FANELLI C.1.
1
Department of Plant Biology, Università Sapienza, L.go Cristina di Svezia 24 00165, Roma, Italy;. – 2
ICB-CNR, P.le Aldo Moro 5 00185, Roma, Italy.
Keywords: ochratoxin A, oxylipins, lipoxygenase, aspergillus ochraceus, triticum durum.
In different Aspergilli, lipoperoxidative events stimulate mycotoxin biosynthesis, conidiogenesis and
sclerotia formation. A lox-like gene sequence (AoloxA-like; DQ087531) has been found in Aspergillus
ochraceus which presents an high homology with a 15-arachidonate lox gene of A. fumigatus
(XP_746844). To study the effect of oxylipins on morphogenesis and OTA biosynthesis and to
investigate the interaction between wheat seeds and A. ochraceus during fungal colonization, an
AoloxA null mutant [AoloxA(-)] has been generated. AoloxA(-) displays a different colony
morphology, delay in conidia formation and development of sclerotia. The reduced lipoxygenase
activity and oxylipins formation in AoloxA(-) lead to a strong reduction of OTA biosynthesis and
modify the linoleic acid-derived oxylipins profile in the mycelium. Further, the seeds of T. durum cv
ciccio contaminated with AoloxA(-) did not accumulate 9-hydroperoxyoctadecadienoic acid and did
not express the pathogenesis-related PR1 mRNA. The results obtained show that lipoperoxidation, also
driven by AoloxA, modulates morphogenesis, OTA biosynthesis and the interaction with the wheat
seeds in the mycotoxigenic A. ochraceus.
PVI12
THE STRIGOLACTONE BIOSYNTHETIC PATHWAY OF LOTUS
JAPONICUS.
JUNWEI LIU1, IVAN VISENTIN2, MIKE GATHER3, PAOLA BONFANTE3, CLAUDIO
LOVISOLO1, ANDREA SCHUBERT1, FRANCESCA CARDINALE1.
1
Dip. Colture Arboree, via L. da Vinci 44, 10095 Grugliasco (TO). – 2 DiVaPRA, via L. daa Vinci 44 –
10095 Grugliasco (TO). – 3 Dip. Biologia Vegettale, V.le Mattioli 25 – 10125 Torino, Università deglii
Studi di Torino, Italy.
Keywords: strigolactones, Lotus japonicus, biosynthetic genes, mycorrhizas, stress.
Strigolactones (SL) are a group of sesquiterpene lactones, initially identified in root exudates as the
seed-germination stimulants for the parasitic weeds Striga and Orobanche. Later, they were proven to
act as branching factors for arbuscular-mycorrhizal fungi (AMF), increasing the chances of a successful
host colonization. SL were also shown to be the root-produced shoot branching inhibition factor; this is
the first endogenous function assigned to SL, which can be seen as new plant hormones. SL derive
from the carotenoid pathway and belong to the apocarotenoids group; in their synthesis, they may be
competing with ABA for precursors. Our aim is to study the regulation of SL biosynthesis by AMF and
environmental stress in Lotus japonicus. To this purpose, we plan to: 1) identify L. japonicus genes in
the biosynthetic pathway of SL 2) validate their role in SL biosynthesis 3) generate and characterize
silenced plants unable to produce SL. Data will be presented on the in silico analysis of known and
predicted biosynthetic genes in other plants (Arabidopsis, pea, Medicago, rice), and on the cloning of
full-length sequences of putative orthologues in L. japonicus.
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PVI13
DISSECTION OF THE VIROID MOLECULAR DETERMINANT INDUCING
PEACH CALICO DISEASE.
NAVARRO B.1, DELGADO S.2, RODIO M.E.1, FLORES R.2 AND DI SERIO F.1.
1
Istituto di Virologia Vegetale, Consiglio Nazionale delle Ricerche, Via Amendola 165/A, 70126 Bari,
Italy (2) Instituto de Biologia Molecular y Celular de Plantas, CSIC/Universidad Politècnica de
Valencia, Ciudad Politècnica de la Innovaciòn - Edificio 8E, c. Ingeniero Fausto Elio, s/n 46011
Valencia.
Keywords: Non-protein-coding RNA, Pathogenesis, Plastid, Molecular evolution.
Viroids, infectious non-protein-coding RNAs, may cause plant diseases by a mechanism still unknown.
Peach latent mosaic viroid (PLMVd), which replicates and accumulates in the chloroplast, has been
proposed as a model for studying viroid pathogenesis. We recently showed that peach calico (PC), a
severe chlorosis exclusively elicited by PLMVd variants containing a specific insertion of 12-13 nt, is
due to the impairment of plastid ribosomal RNA maturation at a very early developmental stage. Here,
we show that the nucleotide composition and conformation of the insertion strongly affect the
pathogenic properties of PLMVd infecting variants. This information can be relevant for identifying
host factor(s) involved in symptom elicitation interacting directly with the pathogenic determinant, and
for exploring the involvement of RNA silencing in viroid pathogenesis as recently suggested in other
viroid-host combinations. In the present study we have also observed that new PLMVd variants, most
likely non-pathogenic, emerge from the inoculated PC-inducing variants, further supporting the use of
PLMVd as a model system for studying pathogenesis and evolution of non-protein-coding RNAs.
PVI14
REQUIREMENTS OF TRANS-ACTING FACTORS IN THE REPLICATION OF
TOMBUSVIRUS SATELLITE RNAS.
LUISA RUBINO AND MARCELLO RUSSO.
Istituto di Virologia Vegetale del CNR, Unità Organizzativa di Bari Via Amendola, 165/A 70126 Bari,
Italy.
Keywords: tombusvirus, satellite RNA, virus replication, replicase, RNA silencing.
Satellite RNAs (satRNAs) are subviral RNAs totally dependent on a helper virus-encoded factors for
their replication. satRNAs share no or little sequence homology with the helper virus genome and are
dispensable for virus propagation. Three satRNAs associated with members of the genus Tombusvirus
have been described so far: they are linear single-stranded non-coding RNAs c. 600-800 nt in size, with
no sequence homology with the helper, except for a 50 nt region in the satRNA and in the 5' non-coding
region of the helper virus genome. The 621 nt satRNA associated with Cymbidium ringspot virus
(CymRSV) is not able to attenuate virus-induced symptom expression, is not replicated in protoplasts
from transgenic N. benthamiana plants expressing the viral replicase and is target of virus-induced
RNA silencing. satB10, a different satRNA associated to Tomato bushy stunt virus in natural
infections, attenuates viral symptom expression. It was shown that satB10 replication is supported by
the expression of viral replicase proteins only and that satB10 is target of a satRNA-triggered RNA
silencing, suggesting that different strategies are required for the replication of different satRNAs.
- 65 -
PVI15
DEEP SEQUENCING OF VITIS VINIFERA SHORT RNAS REVEALS NONCONSERVED MIRNAS AND A NUMBER INFECTIOUS AGENTS-DERIVING
SIRNAS.
V. PANTALEO1, G. SZITTYA2, L. MOZZI1, S. MOXON2, T. DALMAY2, J. BURGYAN1.
1
Istituto di Virologia Vegetale del CNR, Strada delle Cacce 73 10135 Torino (Italy). – 2 School of
Biological Sciences, University of East Anglia, Norwich, (United Kingdom).
Keywords: miRNAs, siRNAs, plant development, Vitis vinifera, RNA silencing.
Grapevine (Vitis vinifera) is one of the most ancient and important fruit crops. V. vinifera is an host of a
large number of viral and virus-like agents including phytoplasmas, viroids and viruses either
associated with diseases or at a latency state. In late 2007 a major achievement has been reached in
V.vinifera biology since the sequence of the grapevine genome has been published and it is available
for public research. Thus it prompted us to use the grapevine as a natural system to investigate some
molecular aspects of plant-virus interactions involved in the plant development. In this work we present
the results of deep sequencing analysis of short RNAs in grapevine clone ENTAV15. Infectious agentderiving siRNAs either from viruses or viroids were cloned and located along their originating genome
sequences, thus revealing siRNAs site- and strand-dependent distribution. Moreover, 21 novel miRNAs
and their star molecules were cloned; bioinformatics analysis either of their precursors and predicted
targets, and northern blot analysis for their expression in different tissues likely suggest the
identification of novel non-conserved miRNAs.
PVI16
HOST-DERIVED SIGNALS ACTIVATE PLANT INNATE IMMUNITY.
ROBERTA GALLETTI, DANIEL SAVATIN, SIMONE FERRARI, GIULIA DE LORENZO.
Dipartimento di Biologia Vegetale, Università di Roma “La Sapienza”, Piazzale Aldo Moro, 5 00185
Rome, Italy.
Keywords: elicitors, oligogalacturonides, innate immunity, signal transduction, plant defense.
Oligogalacturonides (OGs) protect Arabidopsis against fungal infection and this protection is
independent of ethylene, jasmonate and salicylic acid. Although OGs have been studied for years, little
is known about the elements involved in the signal transduction pathways triggered by these elicitors.
Genes strongly and significantly induced upon treatment with OGs were selected from microarray data
obtained from Arabidopsis plants treated with the elicitors, and knockout lines in these genes were
obtained. Mutants are currently under characterisation and results on their possible involvement in OGmediated signalling will be presented. Transcript profiling experiments show that responses triggered
by OGs and the bacterial PAMP flg22 largely overlap in Arabidopsis. In contrast to flg22, OGmediated signalling does not require the co-receptor BAK1/SERK3. Our results suggest a possible role
played by other members of the SERK family. Similarly to flg22, OG-triggered responses are blocked
by the bacterial effector AvrPto, suggesting that this protein may physically interact with either the OGreceptor(s) or co-receptor(s).
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PVI17
A CHIMERIC APPROACH TO STUDY THE ROLE OF WAK1 AS
RECEPTOR OF OLIGOGALACTURONIDES
ALEXANDRE BRUTUS1, FRANCESCA SICILIA1, ENZO MARCHETTI2, ALBERTO MACONE3,
FELICE CERVONE1, GIULIA DE LORENZO1
1
Dipartimento di Biologia Vegetale, Università di Roma “Sapienza” Piazzale Aldo Moro 5, 00185
Roma (Italia). – 2 Dipartimento di Genetica e Biologia Molecolare “C. Darwin”, Università di Roma
“Sapienza” Piazzale Aldo Moro 5, 00185 Roma (Italia). – 3 Dipartimento di Biochimica “A. Rossi
Fanelli”, Università di Roma “Sapienza” Piazzale Aldo Moro 5, 00185 Roma (Italia).
Keywords: plant cell wall, receptors, innate immunity, oligogalacturonides.
Pectin determines maintainance of the wall integrity and cohesion of the cells. This characteristic is due
to the polyanionic nature of its backbone, i.e. homogalacturonan. These structures may be fragmented
by hydrolases and release oligogalacturonides (OGs) that have elicitor and regulator activity. In
Arabidopsis, OGs induce the expression of defense genes and protect the plant against fungal diseases.
The perception system of OGs is still unknown. Since the response of Arabidopsis to OGs largely
overlaps that of the flagellin and the elongation factor-Tu, it has been hypothesized that the receptors of
OGs are similar to their corresponding receptors. On the other hand, candidate receptors of OGs are the
Wall-Associated Kinase (WAK). WAKs are receptor-like kinases (RLKs) that display the Ser/Thr
kinase signature and an extracellular domain containing epidermal growth factor like repeat. WAK1 is
the most characterized: it binds in vitro to non-methylesterified homogalacturonans and to elicitor
active OGs. However, the detailed role of single WAK receptors remains largely unknown. In order to
define the function of WAK1, we have used an approach based on chimeric LRR-RLKs.
PVI18
ALTERATIONS OF THE TURNIP VEIN CLEARING VIRUS SPREADING IN
ARABIDOPSIS THALIANA OVER-EXPRESSING PECTIN
METHYLESTERASE INHIBITORS
IBRAHIM ELMAGHRABY1, VINCENZO LIONETTI2, FELICE CERVONE2, FRANCESCO
FAVARON1, ALESSANDRO RAIOLA1 AND DANIELA BELLINCAMPI3
1
Departimento Territorio e Sistemi Agro-Forestali, Università degli Studi di Padova, Legnaro, Padova.
– 2 Dipartimento di Biologia Vegetale, Università di Roma "La Sapienza", Roma. – 3 Dipartmento di
Chimica, Università di Roma "La Sapienza", Roma.
Keywords: Virus, MP, PME, PMEI, Arabidopsis.
The ability of viruses to cross the plant cell wall to propagate infection throughout a plant is based on
interactions among viral and host factors. The cell wall located pectin methylesterase enzyme (PME) is
one of these factors. It is established that PME can act as host-cell receptor of Tobacco mosaic virus
movement protein (TMV-MP) and also that the PME-MP interaction is required for cell-to-cell
movement of the virus through plasmodesmata. Plant PMEs have also been shown to interact with MP
from other viruses such as Turnip vein clearing virus (TVCV) and Cauliflower mosaic virus (CaMV)
suggesting this interaction as possibly being involved in the cell-to-cell movement of the virus.
However, the role of the PME-MP interaction to virus movement has not been elucidated. PME activity
in plant is regulated by several factors including the apoplastic pectin metylesterase inhibitors (PMEIs).
With the aim to better clarify the mechanism of virus movement in planta and to possibly counteract the
viral infection we have generated Arabidopsis plants over-expressing PMEI. In these plants we have
analyzed the changes of TVCV spreading in comparison to untransformed control plants..
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ABSTRACTS – Session VII: Membrane dynamics and functions
PVII01 ARABIDOPSIS THALIANA PLASMA MEMBRANE CA2+-ATPASE
ISOFORM 8: MOLECULAR ANALYSIS OF THE AUTOINIBITORY
MECHANISM USING CHIMERAS.
M. CRISTINA BONZA1, LAURA LUONI1 AND SABINA VISCONTI2.
1
Department of Biology, University of Milano, Milano – 2 Department of Biology, University of Roma
"Tor Vergata", Roma Italy.
Keywords: plasma membrane, Ca2+-ATPase, H+-ATPase.
ACA8 is a plasma membrane (PM) Ca2+-ATPase of A. thaliana which has an extended N-te containing
both an autoinhibitory domain and a calmodulin (CaM) binding site: CaM binding suppresses
autoinhibition. This regulatory mechanism is similar to that of animal PM Ca2+ ATPase and plant PM
H+-ATPase, but in these proteins the regulatory site is localised in the C-te. To analyse in details the
autoinbitory mechanism, mutants in which the N-te of ACA8 is inserted at the C-te of the protein have
been constructed using the cDNA of CaM insensitive mutant ï„74-ACA8. Moreover, chimeras in
which ï„74-ACA8 is fused to the C-te domains of AHA1 (isoform 1 of A. thaliana PM proton pump)
or of PMCA4b (isoform 4b of human PM Ca 2+-ATPase) have been produced. All mutants expressed in
the S. cerevisiae strain K616 are functional. Results show that repositioning the N-te region of ACA8 to
the C-te of the protein does not interfere with its ability to autoinhibit the pump, thus the regulatory
function of the terminal domain is independent from its position in ACA8. A detailed characterisation
of the chimeras analysing their activity in presence of 14-3-3 and fusicoccin or CaM is in progress.
PVII02
MUTATION IN THE ACTUATOR OF ACA8, A CA-ATPASE OF A.
THALIANA, GENERATE PARTIALLY DEREGULATED PUMPS.
TIZIANA FUSCA, MARIA CRISTINA BONZA, LAURA LUONI, SILVIA MENEGHELLI,
CLAUDIA MARRANO, MARIA IDA DE MICHELIS.
Dip. Biologia, UNI Milano, Istituto di Biofisica del CNR, Sezione Milano, via Celoria 26, 20133
Milano, Italy.
Keywords: plasma membrane Ca ATPase, autoinhibition, Calmodulin.
ACA8, a type 2B Ca ATPase, has a regulatory N-terminus with autoinhibitory action suppressed by
binding of calmodulin (CaM). ACA8 N-terminus binds a region of the small cytoplasmic loop
connecting transmembrane domains 2 and 3. To define the role of this interaction in autoinhibition we
have analysed a number of single point mutants of ACA8 E252-N345 sequence. Mutation to Ala of any
of 6 acidic residues (E252, D273, D291, D303, E302, D332) originates an enzyme with normal activity
in presence of CaM, but less CaM-stimulated. These results highlight the relevance in autoinhibition of
a negative charge in the small cytoplasmic loop of ACA8. The most deregulated mutant is D291A,
which is less activated also by controlled proteolysis or by acidic phospholipids; moreover, the
phenotype of the D291A mutant is stronger than that of D291N suggesting a more direct involvement
of this residue in autoinhibition. Of the other mutants (I284A, N286A, P289A, P322A, V344A,
N345A), only P322A has a basal activity higher than that of the WT. These results provide the first
evidence that the small cytoplasmic loop of a type 2B Ca ATPase plays a role in the attainment of the
autoinhibited state.
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PVII03 MOLECULAR MECHANISMS OF VIOLAXANTHIN DE-EPOXIDASE PH
DEPENDENT ACTIVITY.
GIORGIA SAGA1,2, PASCAL ARNOUX3, DAVID PIGNOL3, GIORGIO GIACOMETTI1,
ROBERTO BASSI2, TOMAS MOROSINOTTO1.
1
Department of Biology, University of Padova, via U.Bassi 58B, 37 37121 Padova, Italy. – 2
Dipartimento Biotecnologie, Università di Verona, strada le Grazie 15, Verona, Italy. – 3 LBC, IBEB,
CEA Cadarache, Saint Paul le Durance, France.
Keywords: photoprotection, carotenoid, monotopic membrane proteins, pH switch, conformational
change.
Violaxanthin de-epoxidase (VDE) is the enzyme responsible for zeaxanthin production. The synthesis
of this carotenoid in plants exposed to high light conditions is an important photoprotection mechanism,
enhancing excess energy dissipation and reactive oxygen species scavenging. The inactive enzyme is
soluble but, upon activation by low pH, it binds to the thylakoids membrane, where its substrate is
found. We recently obtained the first structural data on this enzyme at both acidic and neutral pH. At
neutral pH, VDE is monomeric and its active site is occluded. Acidification induces a conformational
change and dimerization of the enzyme. Structural data opened the possibility to investigate deeper this
enzyme and further work allowed the identification of its active site as well as residues involved in the
pH dependent conformational change. A key step in VDE activation is its binding to the thylakoids
membrane, where the substrate violaxanthin is found. We identified protein regions fundamental for
membrane association and we can now propose a model for the its pH dependent interaction with the
thylakoids.
PVII04
BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF A
CHLOROPLAST-LOCATED PLANT GLUTAMATE RECEPTOR.
ENRICO TEARDO1, ELIDE FORMENTIN1, ANNA SEGALLA1, MANUELA ZANETTI1, ORIANO
MARIN2, GIORGIO MARIO GIACOMETTI1, FIORELLA LO SCHIAVO1, MARIO ZORATTI3
AND SZABÒ ILDIKÒ 1.
1
Department of Biology. – 2 CRIBI Biotechnology Center. – 3 CNR Institute of Neuroscience and
Department of Biomedical Sciences, University of Padova, Italy.
Keywords: chloroplast, glutamate receptor, localization, electrophysiology.
Bioinformatic approaches have allowed the identification in Arabidopsis thaliana of twenty genes,
grouped into three subfamilies, encoding for homologues of animal ionotropic glutamate receptors
(iGLRs) Indirect evidences suggests that plant iGLRs function as non-selective cation channels. In the
present work we provide multiple evidence for the chloroplast localization of one of these receptors,
GLR3.4 in Arabidopsis cells and in spinach. Western blot analysis locate this protein to the inner
envelope membrane. Purified inner envelope vesicles display a cation-selective electrophysiological
activity which is inhibited by DNQX, an inhibitor of animal iGLRs known to act in plants as well.
Pharmacological experiments point to an involvement of chloroplast-located glutamate receptors in the
regulation of oxygen evolution. These results identify for the first time a glutamate receptor in
chloroplasts and its activity in native membranes at single channel level.
- 69 -
PVII05 CALCIUM PERMEATION IN PLANT CATION CHANNELS DETERMINED
BY A NOVEL FLUORESCENCE/PATCH-CLAMP APPROACH.
PAUL VIJAY KANTH GUTLA, ANTONELLA GRADOGNA, AND ARMANDO CARPANETO.
Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini 6, 16149 Genova, Italy.
Keywords: plant vacuole, SV channel/TPC1, calcium potassium, patch-clamp, fluorescence.
Calcium, in plant cells, is typically mediated by non-selective cation channels. The patch-clamp
technique combined with optical detection of calcium-dependent fura-2 fluorescence changes is suitable
to investigate calcium fluxes. Here we used the excised patch configuration and focused the
photomultiplier to the tip of the recording pipette where the fluorescent dye was present (FLuoresence
combined with Excised Patch = FLEP). Upon voltage stimulation of tonoplast patches, sustained and
robust fluorescence signals indicated the permeation of calcium through the Slow Vacuolar channel.
We determined the fractional calcium currents at different cytosolic calcium and potassium
concentrations. Calcium permeation could be recorded simultaneously with oppositely directed
potassium fluxes. As FLEP is very efficient for measuring small calcium currents, we propose the
FLEP technique for the study of divalent ion-selective channels or transporters that may be difficult to
access using conventional electrophysiological approaches. Ref: Gradogna A, Scholz-Starke J, Gutla
PV, Carpaneto A (2009) The Plant Journal, 58:175-182.
PVII06
THE MOSS PHYSCOMITRELLA PATENS SHARES NON
PHOTOCHEMICAL QUENCHING MECHANISMS WITH BOTH GREEN
ALGAE AND HIGHER PLANTS.
CATERINA GEROTTO1, ALESSANDRO ALBORESI2, ANNA SEGALLA1, ROBERTO BASSI2,
GIORGIO M. GIACOMETTI1 AND TOMAS MOROSINOTTO1.
1
Dipartimento di Biologia, Università di Padova, Via Ugo Bassi 58 B, 35131 Padova, Italy. – 2
Dipartimento Scientifico e Tecnologico, Università di Verona, Strada Le Grazie 15, 37134 Verona,
Italy.
Keywords: Non Photochemical Quenching, Physcomitrella patens, PsbS, Li818, photoprotection.
Photosynthetic organisms evolved several photoprotective processes to survive in a variable
environment. The fastest one, called Non Photochemical Quenching (NPQ), is activated by the
generation of a pH gradient across thylakoid membranes and leads to the dissipation of excess energy
as heat. Green algae and plants are all able to induce NPQ, whose activation, however, depends from
two different proteins: Li818 and PsbS, respectively. The moss Physcomitrella patens is the only
organism where both PsbS and Li818 have been found in the genome and expressed as polypeptides.
Consistently, P.patens is able to induce a strong NPQ. Both psbsko and li818ko mutants showed a
decrease NPQ capacity, showing that both higher plants and green algae NPQ mechanisms are active in
P.patens. We then complemented these KO mutants to over-express PsbS protein in li818ko mosses and
vice-versa. The phenotype of the complemented mosses showed that PsbS and Li818 are able to induce
an NPQ response independently. This work thus suggests that plants upon land colonization evolved an
alternative mechanism to activate NPQ and successively lost the one typically found in green algae.
- 70 -
PVII07 A NOVEL FLAVONOID CARRIER IN WHITE GRAPE BERRIES:
LOCALISATION AND FUNCTION.
ALBERTO BERTOLINI, ELISA PETRUSSA, CARLO PERESSON, ENRICO BRAIDOT,
FRANCESCO MACRÌ AND ANGELO VIANELLO.
Sezione di Biologia Vegetale, Dipartimento Biologia e Protezione delle Piante, UniversitÀ di Udine,
Via delle Scienze, 91, I-33100 Udine, Italy.
Keywords: bilitranslocase, flavonoid transport, Vitis vinifera, white berry, seed.
Up to date, it has been suggested that in grapevine (Vitis vinifera L.) berry the flavonoid transfer into
the vacuole or to the cell wall requires primary (e.g. ABC-protein) or secondary (e.g. MATE-protein)
active transporters. Recently, a putative flavonoid carrier, homologue to mammalian bilitranslocase
(BTL) and able to perform a secondary active transport, was found in carnation petals and red grape
berries. In this work, a BTL homologue has been shown in white berries from cv Tocai/Friulano.
Similarly to that found in the red grape, the transporter exhibited a molecular mass of ca. 31 kDa and
was expressed in the last maturation stages in both skin and pulp tissues. Immunohistochemical
analysis showed that it was localised in the first layers of the epidermal tissue, in the vascular bundle
cells of the mesocarp, in the placental tissue and in peripheral integuments of the seed. The transport
activity of the carrier exhibited higher values for both K M and Vmax, in comparison to the red cultivar,
and was inhibited in an uncompetitive manner by quercetin and eriodictyol. These results confirm that
BTL homologue acts as a carrier involved in the transport of colourless flavonoids.
PVII08
BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF A
WHEAT MITOCHONDRIAL POTASSIUM CHANNEL.
VANESSA CHECCHETTO1, UMBERTO DE MARCHI2, MANUELA ZANETTI1, MARIO
SOCCIO3, MARIO ZORATTI2, DONATO PASTORE3, GIORGIO MARIO GIACOMETTI1 AND
SZABÒ ILDIKÒ 1.
1
Department of Biology, University of Padova, viale G. Colombo 3. 35121 Padova, Italy. – 2
Department of Biomedical Sciences, University of Padova, viale G. Colombo 3. 35121 Padova, Italy. –
3
Department of Enviromental Sciences, University of Foggia, Via Napoli, 25 - 71100 Foggia, Italy.
Keywords: channels, patch clamp.
The study of plant mitochondrial channels is still at a preliminary stage. Activities compatible with the
presence of a potassium (K +) channel in Triticum durum mitochondria have only recently been
identified by classical bioenergetics. However, a biochemical and electrophysiological characterization
of plant mitochondria as well as the molecular identification of the K + channel is still missing. Using
classical biochemical techniques we carried out an evaluation of the purity of isolated mitochondria
using specific antibodies against mitochondrial and other organellar proteins, demonstrating that our
mitochondria preparation had little contaminations. In order to gain information about the molecular
identity of the K+ channel, we used a specific antibody against the highly conserved pore region of all
K+ channels. The functional characterization of the channel activities in wheat mitochondria was
performed by using the patch clamp electrophysiological technique, never used before on plant
mitochondria. Preliminary experiments reveal the presence of a K +-selective channel with a
conductance of 100 pS in the inner mitochondrial membrane.
- 71 -
PVII09 LIGHT INDUCED DISSOCIATION OF AN ANTENNA HETEROOLIGOMER IS NEEDED FOR ACTIVATION OF GRANA MEMBRANE
DYNAMIC AND TRIGGERING OF NON-PHOTOCHEMICAL QUENCHING.
BETTERLE NICO1, BALLOTTARI MATTEO1, SILVIA DE BIANCHI1, LUCA DALL'OSTO1,
SIMONE ZORZAN1, STEFANO CAZZANIGA1, TOMAS MOROSINOTTO2 AND ROBERTO
BASSI1.
1
Laboratorio di Fotosintesi, Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada Le
Grazie 15, 37134, Verona. – 2 Dipartimento di Biologia, Università degli Studi di Padova, Via U.Bassi
58/B, 35121, Padova.
Keywords: photosynthesis, NPQ, membranes re-organization.
The photoprotection mechanism NPQ dissipates chlorophyll excited states exceeding the capacity for
photosynthetic electron transport, and it's activated by the PsbS protein. Here, we show that PsbS
controls the association/dissociation of a five-subunit membrane complex, composed of Lhcb4, Lhcb6
and the trimeric LHCII-M antenna proteins. Dissociation of this supercomplex, called B4C, is
indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly
suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex,
thus not available for interaction with PsbS. Electron microscopy shows that B4C dissociation leads
into re-organization of PSII distribution within grana membranes consisting into lateral segregation of
the outer antenna away from PSII core complex. We interpret these results as the dissociation of B4C
(i) reducing the light harvesting capacity of PSII and (ii) making available quenching sites.These
changes are reversible, allowing for both changes in PSII antenna size, and quenching of excited states
thus allowing for adaptation to rapidly changing environmental conditions.
PVII10
MODELLING OF PROTEIN-PROTEIN INTERACTIONS WITHIN THE
PHOTOSYSTEM II CORE COMPLEX AND ANTENNA SYSTEM IN GRANA
MEMBRANES OF PLANT CHLOROPLASTS.
SIMONE ZORZAN, SILVIA DE BIANCHI, NICO BETTERLE, MATTEO BALLOTTARI E
ROBERTO BASSI.
Dipartimento di Biotecnologia, Università di Verona, Strada Le Grazie 15, I-37134 Verona, Italy.
Keywords: dynamic simulation photosystem membrane.
Oxygenic photosynthesis occurs in chloroplasts, the first step consisting in water oxidation by
Photosystem II in stacked grana membranes. Recent studies have elucidated the atomic structure of
PSII core complex while evidences of dynamic association/dissociation of supercomplex components
during photoprotection and repair events have been obtained. Nevertheless, the dynamic these
phenomena is not yet clear. In this work the use the simulation package Meredys to simulate the
formation and disruption of proteic complexes and supercomplexes on grana surfaces, at a mesoscopic
level during activation of energy dissipation mechanisms: each element represents a protein at a
nanoscopic level of detail and the attention is mainly focused on the bond formation and breakage with
rates consistent with experimental evidences. The process of formation of the more abundant
configuration (C2S2M2) of the supercomplexes, where two cores, 4 major antennas trimers and 6
minor antennas are regularly combined, is simulated starting from PSII cores and light harvesting
complexes (LHC) monomers, and its dynamic is discussed.
- 72 -
PVII11 TWO GATING MODALITIES IN THE PORE OF THE MINIATURE K+
CHANNEL KCV.
MATTIA DIFRANCESCO1, ALESSANDRA ABENAVOLI1, INDRA SCHROEDER2, ULF PETER
HANSEN2, STEFAN M KAST3, GERHARD THIEL3, ANNA MORONI1.
1
Università degli Studi di Milano, Italia. – 2 University of Kiel, Germany. – 3 TU-Darmstadt, Germany.
Keywords: K+ channel, gating, pore.
Kcv is a viral protein that forms functional K+ channel in heterologous systems. Because of its
miniature size (94 amino acids) we use Kcv as a model system to study and manipulate basic properties
of the K+ channel pore. By analysing single-channel recordings we obtained evidence that two voltagedependent modalities of gating are found in Kcv: a slow and a fast gating. The presence of a "slow
gating" is revealed by the very low (in the order of 1-3%) mean open probability. Slow gating is not
related to the presence of a bundle crossing, as shown by accessibility of the cavity to MTS reagents.
Fast gating, analyzed by beta distributions, is responsible for the negative slope conductance in the
single-channel I/V curve at extreme potentials and can be explained by depletion-aggravated instability
of the filter region. Channel opening involves the transient formation of salt bridges between residues at
the N and C termini of the channel, as confirmed by mutational experiments inspired by our running
molecular dynamics simulation of Kcv.
PVII12
SYNTHESIS OF THE ARABIDOPSIS AtKCO3 POTASSIUM CHANNEL.
GIUSEPPE GRASSI1, ALEXANDRA GRIPPA1, ALESSANDRO VITALE1, KATRIN
CZEMPINSKI2, EMANUELA PEDRAZZINI1.
1
Istituto di Biologia e Biotecnologia Agraria – Connsiglio Nazionale delle Ricerche - Via Bassini 15,
20133 Milano, Italy, EU. – 2 Institute of Biochemistry and Biology - Molecular Biology Dept,
University of Potsdam, Golm, Germany, EU.
Keywords: tonoplast, protein synthesis, protein turnover, protein sorting.
AtKCO3 is a single pore channel with two transmembrane domains and the N- and C-terminal domains
exposed in the cytosol. An AtKCO3::GFP fusion was previously found to be located in the tonoplast by
transient expression. By subcellular fractionation, we confirmed the tonoplast localization of wild type
AtKCO3 and the AtKCO3::GFP fusion in Arabidopsis transgenic plants. We determined that
AtKCO3::GFP forms dimers, and not tetramers as predicted, indicating that the C-terminal GFP could
interfere with the channel biogenesis. We identified a putative PDZ-binding motif of class 1 (-X-S/T-Xhydrophobic) at the C-terminus of AtKCO3 (-ATSV). PDZ proteins act as adaptors that facilitate
signalling or determine localization of various proteins. Preliminary experiments show that deletion of
this motif enhances the stability of AtKCO3. This suggests a role of PDZ proteins in determining the
turnover and half-life of AtKCO3. Supported by the EU Marie Curie Research Training Network
"Vacuolar Transport Equipment for Growth Regulation in Plants" (MRTN-CT-2006–035833.
- 73 -
PVII13 SEARCHING FOR MITOCHONDRIAL POTASSIUM CHANNELS IN
CEREALS USING A GENE CANDIDATE STRATEGY.
ELIDE FORMENTIN1, MARIO SOCCIO2, DONATO PASTORE2 AND FIORELLA LO SCHIAVO1.
1
Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, 35131 Padova, Italy. – 2
Dipartimento di Scienze Agroambientali, Chimica e Difesa Vegetale, Università di Foggia, Via Napoli
25, and Centro di Ricerca Interdipartimentale BIOAGROMED, Via Napoli 52, 71100 Foggia, Italy.
Keywords: rice, endomembranes, mitochondria, potassium channels, subcellular localization.
Mitochondria play a key role in mediating death signals during biotic and abiotic stresses. A
mitochondrial K+ channel, whose activity increased under hyperosmotic stress, has been reported in
durum wheat (Pastore et al., 1999 and 2007). To date, no K + channel has been identified yet in plant
mitochondria. Our aim is the isolation and characterization of such a class of proteins using rice as a
model system. We analyzed the rice genome using the K+ selectivity filter TTXGYGD as a bait. Then,
by using web tools such as Predotar and MitoP, a Shaker like channel bearing a putative mitochondrial
signal peptide has been identified. Since a signal peptide is unnecessary for mitochondrial localization,
a subcellular analysis will be extended to all of the proteins identified. The genes have been fused to a
reporter gene (YFP) and the constructs have been used to transform tobacco leaves by Agrobacterium
mediated transformation. Leaves have been analyzed under a confocal microscope to monitor the
fluorescence. The setting up of this protocol has been achieved and the first two channels have been
localized: at the tonoplast and endosomal vesicles, respectively.
PVII14
PLANT CELLULAR DYNAMICS ASSOCIATED TO ROOT
COLONIZATION BY ARBUSCULAR MYCORRHIZAL FUNGI.
A. GENRE1, S. IVANOV2, M. CHABAUD3, A. FACCIO1, E. FEDOROVA2, D. BARKER3, T.
BISSELING2, P. BONFANTE1.
1
DBVUniversità di Torino/IPP-CNR, Torino, Italy. –
CNRS/INRA Toulouse, France.
2
MOLBI, Wageningen, Nederlands. –
3
LIPM
Keywords: mycorrhiza, symbiosis, cell biology, live cell imaging.
Over the last years, the introduction of in vivo confocal microscopy and fluorescent protein expression
has provided powerful tools to the study of plant cell biology. In the field of arbuscular mycorrizas this
has led to the identification of plant the cell mechanisms involved in the construction of the intracellular
compartment where the symbiotic fungi are hosted. Our previous researches have illustrated how root
cells predict fungal colonization by organizing cytoplasmic aggregations that include cytoskeleton and
endoplasmic reticulum. We are currently investigating the membrane dynamics associated to the
formation of the perifungal membrane and interface compartment that envelope all intracellular hyphae.
We will present here the cell responses observed in the different cell types colonised by the fungus, by
using new GFP constructs that label secretion-related cell components and specific membrane proteins
(Golgi stacks, aquaporin and SNAREs). Our observations suggest the existence of cell mechanisms that
are common to different processes (cell division, apical growth, pathogen-elicited and symbiont-elicited
responses), and are finely adapted to each one of them.
- 74 -

Società Italiana di Biologia Vegetale

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