Società Italiana di Biologia Vegetale

Foggia 18th – 20

th September, 2013

Aula Magna

dell'Università degli Studi di Foggia

Via Romolo Caggese, 1 - Foggia

Società Italiana di

Biologia Vegetale

Fifth Congress

1

SCIENTIFIC PROGRAMME

Wednesday, September 18th

14:00 – 14:30 Participant registration

14:30 – 15:00 Opening ceremony

Academic Authorities greetings

Prof. Giuliano Volpe, Rector of the University of Foggia

Prof. Agostino Sevi, Director, Dept. of the Sciences of Agriculture, Food and Environment

15:00 – 17:30 Session 1 – Towards EXPO 2015: GENETICS AND PHYSIOLOGY OF BIOTIC STRESSES

(in collaboration with SIGA)

Chairpersons: Daniela Bellincampi and Maria Raffaella Ercolano

15:00 – 15:30 Invited lecture

Jean-Pierre Métraux

Department of Biology, Plant Biology, University of Fribourg (Switzerland)

Plant defenses to pathogens - more refined than expected!

15:30 – 17.00 Oral communications

Marone D., Russo M.A., Laidò G., De Vita P., Papa R., Blanco A., Gadaleta A., Cattivelli

L., Mastrangelo A.M. - Revealing the genetic architecture of powdery mildew resistance

in wheat by the construction of a consensus map and subsequent meta-QTL analysis

Pasqualina Woodrow, Ioannis Kafantaris, Federica Iannuzzi, Antonio Mirto, Amodio

Fuggi, Petronia Carillo - cDNA-NBS profiling of Hortensia RGA-like genes responsive to

leaf spot fungal infection.

Chiara Paparella, Lucia Marti, Daniel Savatin, Giulia De Lorenzo and Simone Ferrari -

An Arabidopsis thaliana LYSM receptor-like kinase regulates resistance to pathogens and

abscisic acid responses.

Pavan S., Schiavulli A., Zuluaga D.L., Marcotrigiano A.R., Zonno V., Ricciardi F.,

Bracuto V., Abdelaziz S., Zaza C., Bardaro N., Lotti C., Bouwmeester H., Ricciardi L. -

Characterization of the first pea (Pisum sativum L.) natural strigolactone-deficient mutant

resistant to crenate broomrape (Orobanche crenata Forsk.)

Mazzamurro V., Laviano L., Marcel C.T., Milc J., Francia E., Niks R.N., Vozabova T.,

Garvin D.F., Roncaglia E., Tagliazucchi Malagoli G., Bicciato S., Tagliafico E.,

Pecchioni N. - Transcriptome analysis in the interaction Brachypodium - Puccinia

brachypodii

Laura Bassolino, Yang Zhang, Henk-Jan Schoonbeek, Claudia Kiferle, Pierdomenico

Perata and Cathie Martin - Skin-specific accumulation of anthocyanins in tomato extends

shelf life.

17.00 – 17:30 General discussion

17:30 – 19:00 Coffee break and poster viewing (SIGA)

19:00 – 20.00 Get together party

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Thursday, September 19th

8:00 – 9:00 Poster mounting

09:00 – 13:00 Session 2 - Towards EXPO 2015: CLIMATE CHANGE, ABIOTIC STRESSES AND CROP

YIELD

(in collaboration with SIGA)

Chairpersons: Petronia Carillo and Nicola Pecchioni


Ronan Sulpice

Department of Botany and Plant Science, National University, Galway (Ireland)

Cold stress does not necessarily inhibit plant growth: it depends on when it occurs

during the day.

9:30 – 10:30 Oral communications

Longo C., Lupini A., Mercati F., Princi M.P., Miller A.J., Abenavoli M.R., Sunseri F. -

Nitrogen use efficiency in tomato involves a high affinity long-distance nitrate trasporter.

Daniela Trono, Maura N. Laus, Mario Soccio and Donato Pastore - How plant

mitochondria may contribute to cell resistance to abiotic stresses. The case study of durum

wheat.

Francesca Verrillo, Mariapina Rocco, Andrea Occhipinti, Massimo E. Maffei and Mauro

Marra - Plasmodesmata are involved in abiotic stress signalling.

Matteo Faà, Marco Biggiogera, Maria Elisa Sabatini, Mattia Donà, Alma Balestrazzi and

Daniela Carbonera - Understanding the dynamics of stress sensing within the plant

nucleolus: investigation of the role played by the MTTDP gene family encoding tyrosyl

DNA phosphodiesterase enzymes.

10:30 – 11.00 Coffee break

11:00 – 11:30

Lunardon A., Del Fabbro C., De Paoli E., Hardcastle T., Forestan C., Farinati S., Varotto

S. - Analysis of small RNA populations of maize leaves following abiotic stress

treatments.

Marconi G., Pace R., Raggi L., Traini A., Lutts S., Guiducci M., Falcinelli M., Benincasa

P., Chiusano M.L., Albertini E. - DNA methylation influences rapeseed (Brassica napus

var. oleifera Del.) gene expression in response to salt stress conditions.


Nese Sreenivasulu

Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben (Germany)

Systems biology perspectives to unravel global abiotic stress response.

12.00 – 12.30 General discussion

12.30 – 13.00 Closure of the SIGA Congress

13:00 – 14:30 Lunch and poster viewing

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14:30 – 16:30 Session 3 – PLANT NUTRITION Chairman: Sergio Esposito

14:30 – 15:30 Invited Lecture

Antje von Schaewen

Institut für Biologie und Biotechnologie der Pflanzen, Munster University (Germany)

Mechanisms contributing to enhanced stress tolerance by G6PDH isoenzyme

replacement.


Giovanna Salbitani, Vincenza Vona, Simona Carfagna - S-starvation induces oxidative

burst in Chlorella sorokiniana cells

Diana Simionato, Maryse A. Block, Nicoletta La Rocca, Juliette Jouhet, Eric Marechal,

Giovanni Finazzi and Tomas Morosinotto - Nitrogen limitation results in de novo

biosynthesis of tags and reorganization of the photosynthetic apparatus in the microalga

Nannochloropsis gaditana

Mirko Zaffagnini, Gaia Tioli, Roberto Orru, Samuel Morisse, Mariette Bedhomme,

Christophe H. Marchand, Stephane D. Lemaire, Paolo Trost - Mechanism of nitrosylation

and denitrosylation of cytosolic glyceraldehyde-3-phosphate dehydrogenase from

Arabidopsis

16:30 – 17:00 Coffee break

17:00 – 19:30 SIBV general assembly

SIBV election: President, Secretary, Executive committee

Rasi-Caldogno award

Eleonora Paparelli - Integration of chloroplast starch metabolism with

hormonal regulation of plant growth

20:30 Social dinner

Friday, September 20th

08:30 – 11:00 Session 4 – SIGNALLING Chairman: Massimo Maffei

8:30 9:30 Invited lecture

Klaus Palme

Department of Molecular Plant Physiology, Albert-Ludwigs-University, Freiburg

(Germany)

Technology drives insight – multiscale analysis of signalling networks in Arabidopsis

thaliana.

9:30- 10:20 Invited lecture

Christine Faulkner

Department of Biological and Medical Sciences, Oxford Brookes University (UK)

Plasmodesmata-located receptors control intercellular signalling during

pathogen perception.

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10:20 – 11:00 Oral communications

Cristian Cosentino, Sabrina Gazzarrini, Paolo Zuccolini, Gerhard Thiel, Giulia Romani

and Anna Moroni - A light-gated ion channel from phototropin.

Luisa Ederli, Emma Tedeschini, Giuseppe Frenguelli, Cecilia Del Casino, Giulia

Apostoli, Claudia Faleri, Mauro Cresti and Stefania Pasqualini - Cupressus arizonica

pollen signalling network triggers pollen tube elongation.

11:00 – 11:30 Coffee Break

11:30 – 14:00 Session 5 – ORGANELLE BIOLOGY Chairman: Alessandro Vitale

11:30 – 12:30 Invited Lecture

Enrique Rojo

Centro Nacional de Biotecnologìa, Consejo Superior de Investig. Cientìficas, Madrid

(Spain)

Intracellular trafficking in plant development


Joachim Scholz-Starke, Anna Boccaccio, Alex Costa, Margherita Festa and Armando

Carpaneto - The plant vacuole: a novel heterologous system for investigating the

functional properties of intracellular animal channels and transporters

Maria Cristina Bonza, Giovanna Loro, Smrutisanjita Behera, Andrea Wong, Jörg Kudla,

Alex Costa - Analyses of Ca2+

accumulation and dynamics in the endoplasmic reticulum

of Arabidopsis thaliana root cells using genetically encoded camaleon sensors

13:20 – 14:00 Short Communications

Francesca De Marchis, Andrea Pompa and Michele Bellucci - Expression of recombinant

proteins in transformed plant chloroplasts is strictly regulated by protein quality control

Paolo Longoni, Laura Pantaleoni, Sonia Accossato, Rino Cella, Michel Goldschmidt-

Clermont - Expression of a recombinant xylanase in chloroplasts of Nicotiana tabacum

and Chlamydomonas reinhardtii

14:00 Closing ceremony

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INVITED LECTURES

6

Session 1: Genetics and physiology of biotic stresses

L01

PLANT DEFENSES TO PATHOGENS - MORE REFINED THAN EXPECTED!

JEAN-PIERRE MÉTRAUX co-authors: Floriane L’Haridon, Matteo Binda, Mario Serrano, Eliane Abou-Mansour, Silke

Lehmann, Lehcen Benikhlef Department of Biology, Plant Biology, Université de Fribourg (Switzerland). Keywords: Innate immunity, cuticle, reactive oxygen species, mechanical stress. Plants are remarkably able to survive biotic stresses, such as those inflicted by pathogens.

They exhibit constitutive defenses that can protect them against a broad variety of

invaders. Besides, plants perceive invaders via molecules released by a pathogen or produced during the plant-pathogen interaction (named pathogen-associated molecular patterns, PAMPs) or molecules produced by the plant itself, for example during damage arising from an attempted infection (often termed damage-associated molecular patterns, DAMPs). The recognition of such molecules is made by specific receptors (termed pattern

recognition receptors, often but not always LRR-containing proteins) and leads to the induction of basal defenses that includes the production of antimicrobials, defense proteins or the induction of structural barriers. Well-adapted pathogens have however evolved molecules, so-called effectors, capable of interfering with the plant’s defense responses and gain access to the plant. But plants have also evolved the ability to recognize effectors or their effect using specific proteins (termed R or resistance proteins) again leading to a syndrome of resistance reactions that might be specific to the situation encountered.

Over the past years, we have examined if cuticular components liberated by fungi perforating cell walls during infection, might be recognized by the plant and act as DAMPs (for triggering innate immunity. In agreement with the model above, Arabidopsis thaliana plants impaired in the production of a functional cuticle, or plants treated with cutinase display enhanced resistance to the fungal pathogen Botrytis cinerea. Interfering with oxalate, an effector produced by B. cinerea affected the ability of the fungus to invade the

tissue. We have explored various alternative ways (wounding, touch) that affect the

surface of Arabidopsis and observed a resistance to B. cinerea. This presentation will review past, current and future experiments on innate immunity associated with the cuticle and the role of reactive oxygen species in this process.

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Session 2: Climate change, abiotic stresses and crop yield

L02

COLD STRESS DOES NOT NECESSARILY INHIBIT PLANT GROWTH: IT DEPENDS ON WHEN IT OCCURS DURING THE DAY.

RONAN SULPICE co-authors: Pyl E-T., Piques M., Ivakov A., Schulze W.X., Ishihara H., Stitt M.

Department of Botany and Plant Science, National University of Ireland, Galway, University Road, Galway, Ireland. Keywords: Temperature compensation, photosynthesis, growth, carbon partitioning. In the nature, plants are facing unstable and unpredictable growth conditions.

Environmental fluctuations occur over long periods of time but also within a diurnal cycle.

Temperatures vary diurnally, being typically lower during the night than during the light. Plants are growing during the full day but can assimilate carbon only during the light period. We reasoned that the response of plants to daytime and night temperature may vary; while daytime temperature affects photosynthesis, night temperature would affect the use of carbon that was accumulated in the light. Three Arabidopsis thaliana accessions

were grown in thermocycles under carbon-limiting conditions with different daytime or night temperatures (12 to 24 °C) and analyzed for biomass, photosynthesis, respiration, enzyme activities, protein levels, translation rate and metabolite levels. The data were used to model carbon allocation and growth rates in the light and dark. Low daytime temperature led to an inhibition of photosynthesis and growth. The inhibition of photosynthesis was partly ameliorated by a general increase in protein content, at the cost of growth efficiency. Unexpectedly, low night temperature had no effect on protein content,

starch turnover, or growth. It meant that plants were robust enough to fully compensate the physicochemical decrease in all the enzyme activities induced by lower temperatures. In a warm night, there is excess capacity for carbon use. We propose that use of this capacity is restricted by feedback inhibition, which is relaxed at lower night temperature, thus buffering growth against fluctuations in night temperature. As examples, the rate of starch degradation was completely temperature compensated against even sudden

changes in temperature, and polysome loading increases when the night temperature was

decreased. These results will be discussed in the context of the urgent need of new crop varieties exhibiting higher yields and robustness against increasingly unstable environmental conditions.

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L03 SYSTEMS BIOLOGY PERSPECTIVES TO UNRAVEL GLOBAL ABIOTIC

STRESS RESPONSE.

NESE SREENIVASULU

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben (Germany), and Grain Quality and Nutrition Center, International Rice Research Institute, DAPO Box 7777, Metro Manila 1301 (Philippines). Keywords: systems biology, transcriptome, drought tolerance, yield stability. Understanding the global abiotic stress response is an important stepping stone for the

development of universal stress tolerance in plants in the era of climate change. Although co-occurrence of several stress factors (abiotic) in nature is found to be frequent, current attempts are poor to understand the complex physiological processes impacting plant

growth under combinatory factors. The recent advances of reverse engineering approaches that led to seminal discoveries of key candi-date regulatory genes involved in cross-talk of abiotic stress responses will be highlighted. Such interactome networks help not only to derive hypotheses but also play a vital role in identifying key regulatory targets and

interconnected hormonal responses. To explore the full potential of gene network inference in the area of abiotic stress tolerance, we need to validate hypotheses by implementing time-dependent gene expression data from genetically engineered plants with modulated expression of target genes. Here, we summarize the current status of plant adaptation mechanisms and the strategies that we need to carve from systems biology strategies in model plants and extrapolating its relevance for cereals.

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Session 3: Plant nutrition

L04

MECHANISMS CONTRIBUTING TO ENHANCED STRESS TOLERANCE BY G6PDH ISOENZYME REPLACEMENT.

ANTJE VON SCHAEWEN Institut für Biologie und Biotechnologie der Pflanzen, Munster University (Germany).

In all eukaryotes, glucose 6-phosphate dehydrogenase (G6PDH) is the key enzyme of the oxidative pentose-phosphate pathway (OPPP), which is important for the provision of various sugar phosphates and reducing equivalents (NADPH) required for anabolic bio-synthesis (e.g. nucleotides, amino and fatty acids). From studying biochemical parameters of G6PDH isoenzymes that are sequestered in different plant cell compartments (cytosolic versus plastidic P1 and P2 isoforms), we designed a metabolic approach to improve plant

performance.

To this end, we replaced cytosolic G6PDH activity of Nicotiana tabacum cv. Xanthi with a heterologous P2 isoform of Arabidopsis roots (i.e. constitutive expression of cP2 followed by RNAi suppression of the cytosolic isoforms). We reasoned that elevated tolerance to NADPH feed-back inhibition in the cytosol (KiNADPH > KmNADP, typical for P2 enzymes of heterotrophic plastids; Wendt et al. 2000, Plant J. 23: 723-733) should promote oxidative

bursts at the plasma membrane upon activation of NADPH oxidases, which is characteristic for both, early stress responses and change of developmental program(s). First analyses revealed that the Xanthi cP2-cytRNAi transformants (T1 lines) are generally more tolerant to biotic and abiotic stress, and flower about one week earlier than the wild-type cultivar (Scharte et al. 2009, PNAS 106: 8061-8066). Detailed analyses of near-isogenic T4 lines confirmed stability of this isoenzyme-replacement strategy, resulting in about 10% higher biomass (including seed yield), elevated thousand grain weights, and altered storage

composition. Possible molecular mechanisms leading to these effects will be presented and discussed.

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Session 4: Signalling

L05

TECHNOLOGY DRIVES INSIGHT – MULTISCALE ANALYSIS OF SIGNALLING NETWORKS IN ARABIDOPSIS THALIANA.

KLAUS PALME 1,2,3,4 1 Institute of Biology II/Molecular Plant Physiology, Faculty of Biology, Albert-Ludwigs-

University of Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany; 2 Centre of Biological Systems Analysis (ZBSA), Albert-Ludwigs-University of Freiburg; Habsburgerstr. 49, D-79104 Freiburg, Germany; 3 Freiburg Institute of Advanced Sciences (FRIAS), Albert-Ludwigs-University of Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany; 4 Centre for Biological Signalling Studies (BIOSS), Albert-Ludwigs-University of Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany.

The study of root development relies on techniques for the accurate visualization of tissue

and organ structure to understand cell patterning and patterns of gene expression. Currently available techniques for three-dimensional (3D) imaging are limited with respect to the thickness of roots and the resolution that can be achieved. In order to achieve a detailed functional and quantitative understanding of roots, cellular features of roots must be quantified in the three-dimensional context of cells and tissue layers. We therefore

aimed, besides genetically, molecularly and functionally characterizing root development, at developing an intrinsic root coordinate system (iRoCS) as a reference model for analysis of the Arabidopsis root apical meristem. iRoCS has been used to rapidly parameterize image data within a single framework in a standardized way. It enables large cohorts of roots to be annotated, making statistical analyses accessible and giving an unbiased evaluation of previously hidden developmental phenotypes. iRoCS enabled novel insight into root patterning and even distinguished subtle changes in the distribution of cell

division in different cell layers in knock-out mutants. Future perspectives will be discussed.

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L06 PLASMODESMATA-LOCATED RECEPTORS CONTROL

INTERCELLULAR SIGNALLING DURING PATHOGEN PERCEPTION.

CHRISTINE FAULKNER

Department of Biological and Medical Sciences, Oxford Brookes University (UK). Chitin is one of several pathogen-associated molecular patterns (PAMPs) whose perception triggers a range of defense responses. In rice, chitin perception and signal transduction involves both the receptor-like protein OsCEBiP and the receptor-like kinase OsCERK1. Current data suggests both functions in Arabidopsis can be performed by AtCERK1 and the role of the OsCEBiP homologue AtLYM2 has been elusive. We show that AtLYM2 is a

plasmodesmata-located protein and, in the presence of chitin, mediates a reduction in molecular flux between cells via plasmodesmata. For this response, lym2-1 mutants are insensitive to the presence of chitin, but not flg22. Surprisingly, the chitin-recognition

receptor AtCERK1 is not required for chitin-induced changes to plasmodesmata flux suggesting that there are at least two chitin-activated response pathways in Arabidopsis. Correspondingly, AtLYM2 is not required for AtCERK1-mediated chitin-triggered defense

responses. Chitin-triggered regulation of molecular flux between cells is, however, required for defense responses against the fungal pathogen Botrytis cinerea but not the bacterial pathogen Pseudomonas syringae illustrating the specificity of AtLYM2 activity to chitin. We conclude that the regulation of symplastic continuity and molecular flux between cells is a vital component of chitin-triggered immunity in Arabidopsis. In a tangential project we have investigated the role of PDLPs, a plasmodesmata-located protein family, in defence responses against the oomycete pathogen Hyaloperanospora

arabidopsidis (Hpa). PDLPs have been shown to play a role in defence against bacterial pathogens, in herbivory responses and in intercellular movement of viruses. Our data indicates that PDLPs are also required for defence against Hpa. pdlp mutants are more susceptible to Hpa than wild-type plants and subcellular examination of infection sites illustrates that encasement of haustoria in mutant cells is impaired. Indeed, in mutant plants haustorial encasements are depleted in callose. PDLP1 expression is upregulated in haustoria containing cells PDLP1-GFP is located at the extrahaustorial membrane in these

cells. These data indicate that PDLPs are required for callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria is analogous to callose deposition at plasmodesmata.

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Session 5: Organelle biology

L07

INTRACELLULAR TRAFFICKING IN PLANT DEVELOPMENT. ENRIQUE ROJO Centro Nacional de Biotecnologìa, Consejo Superior de Investig. Cientìficas, Madrid (Spain). Yeast has been the main model system for studying intracellular trafficking. Comparatively, much is less is known about trafficking in multicellular eukaryotes, and in particular how

this cell autonomous process is integrated into the development of complex organisms. In our lab we are interested in studying processes of protein trafficking that have special relevance for the physiology and development of plants. I will present data on two ongoing projects in the lab: (1) a genetic dissection of vesicular trafficking pathways to plant vacuoles, which are organelles with unique functions in these organisms, and (2) the characterization of the role of nuclear/cytosolic partitioning of the ART/IYO complex in

initiating stem cell differentiation.

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CONTRIBUTIONS FROM S.I.G.A.

14


S01

REVEALING THE GENETIC ARCHITECTURE OF POWDERY MILDEW

RESISTANCE IN WHEAT BY THE CONSTRUCTION OF A CONSENSUS MAP

AND SUBSEQUENT META-QTL ANALYSIS MARONE D.*, RUSSO M.A.*, LAIDÓ G.*, DE VITA P.*, PAPA R.*, BLANCO A.**, GADALETA A.**, CATTIVELLI L.***, MASTRANGELO A.M.* *) Consiglio per la Ricerca e la sperimentazione in Agricoltura - Cereal Research Centre, SS

673 km 25.2, 71122 Foggia (Italy) **) Department of Agro-Forestry and Environmental Biology and Chemistry, University of Bari, Via Amendola, 165/A, 70126 Bari (Italy) ***) Consiglio per la Ricerca e la Sperimentazione in Agricoltura - Genomic Research Centre, Via S. Protaso 302, 29017 Fiorenzuola d’Arda (Italy)

wheat, genetic map, powdery mildew resistance, meta-QTL, NBS-LRR The construction of a consensus map synthesizing the information provided by multiple segregating populations, offers the opportunity to map a larger number of loci than in most single crosses, in addition to providing opportunities to validate marker order. A high-density consensus map was developed for durum wheat by combining segregation data

from six mapping populations. All varieties are durum wheat cultivars, except for one accession of T. ssp. dicoccoides. The integrated map was composed of 1,898 loci distributed on all 14 chromosomes. The length and the marker density are 3.058.6 cM and 1.6 cM/marker, respectively. These characteristics make the map herein described a reference tool for wheat scientists, providing a more complete coverage of the durum wheat genome compared to previously developed maps. The DArT markers positioned on the A and B genomes of the consensus map were investigated in relation to their sequence

available in literature for redundancy, similarity search and correspondence with expressed sequences. Groups of DArT clones sharing the same genetic position are often characterized by a nearly identical sequence. An intriguing result of the sequence analysis

is that a high proportion of the DArT clones correspond to sequences related to disease resistance in plants, and in particular to NBS-LRR resistance genes and protein kinases, many of which containing a LRR domain. Such DArT in many cases mapped in the same

regions in which resistance genes have been previously identified. According to this results, a case study was to identify genomic regions in wheat involved in the control of powdery mildew resistance applying the meta-QTL analysis approach, thus verifying the position of DArT markers corresponding to sequences related to NBS-LRR genes. The MQTL analysis permits to collect QTL data from different published studies in order to obtain consensus QTL across different genetic backgrounds and provides a better definition of the regions responsible for the trait and the possibility to obtain molecular markers suitable for Marker-

Assisted Selection (MAS). As more than 80 independent QTL were identified from literature in different species of tetraploid and hexaploid wheats, the durum wheat consensus map was merged with other two maps of bread wheat for an optimal projection of resistance QTL/genes from all of the species considered. A total of 24 meta-QTL (MQTL) comprising 2-6 initial QTL with varying confidence intervals were found on 15 chromosomes. Many DArT clones associated to sequences related to disease responses have been found in the same regions of MQTL for powdery mildew resistance, providing a very useful tool for the

identification of candidate genes for the investigated trait.

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S02

CHARACTERIZATION OF THE FIRST PEA (PISUM SATIVUM L.) NATURAL

STRIGOLACTONE-DEFICIENT MUTANT RESISTANT TO CRENATE

BROOMRAPE (OROBANCHE CRENATA FORSK.) PAVAN S.*, SCHIAVULLI A.**, ZULUAGA D.L.*, MARCOTRIGIANO A.R.*, ZONNO V.*, RICCIARDI F.**, BRACUTO V.*, ABDELAZIZ S.*, ZAZA C.*, BARDARO N.*, LOTTI C.**, BOUWMEESTER H.***, RICCIARDI L.*

*) Department of Soil, Plant and Food Science, Section of Genetics and Plant Breeding, University of Bari, via Amendola 165/A, 70126 Bari (Italy) **) Department of Agro-Environmental, Chemistry and Crop Protection, University of Foggia, Via Napoli 25, 71100 Foggia (Italy) ***) Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB

Wageningen (The Netherlands) Orobanche crenata, resistance, strigolactones, breeding Crenate broomrape (Orobanche crenata Forsk.) is a devastating parasitic weed threatening the cultivation of legumes in the Mediterranean basin and in the Middle East, which can

lead to up to 100% yield losses. In pea germplasm, only moderate level of resistance has been found and all commercial cultivars are prone to broomrape infestation. As a result of a large germplasm screening, we identified a pea landrace showing high level of resistance to O. crenata, from which we developed a line which was named ROR12. The branching phenotype of ROR12 prompted us to quantify its content of strigolactones, a new class of plant hormones which is known to have a key role in the control of apical dominance as well as in stimulating the germination of Orobanche seeds. Notably, we determined a very

low amount of the three main pea strigolactones orobanchol, orobanchyl acetate and epoxy-orobanchol acetate in ROR12 root tissues and root exudates compared to three commercial cultivars. Orobanche seeds germination tests with root exudates from different

genotypes provided final prove that ROR12 is a strigolactone deficient line. Two years replicated trials in non infested fields indicated that the ROR12 line does not have markedly lower yield potential with respect to commercial cultivars, and therefore can be

conveniently used for breeding purposes. Two large segregant F2 population are being currently analyzed aiming to resistance gene/QTL mapping and cloning.

16


S03

TRANSCRIPTOME ANALYSIS IN THE INTERACTION BRACHYPODIUM –

PUCCINIA BRACHYPODII

MAZZAMURRO V.*, LAVIANO L.*, MARCEL C.T.**, MILC J.*,***, FRANCIA E.*,***, NIKS R. N.****, VOZABOVA T.****, GARVIN D.F.*****, RONCAGLIA E.***, TAGLIAZUCCHI MALAGOLI G.***, BICCIATO S.***, TAGLIAFICO E.***, PECCHIONI N.*,*** *) Department of Life Sciences, University of Modena and Reggio Emilia, Via Amendola 2 – Padiglione Besta, 42122 Reggio Emilia (Italy)

**) INRA-AgroParisTech, UR1290 BIOGER-CPP, Avenue Lucien Brétignières, 78850 Thiverval-Grignon (France) ***) Center for Genome Research - CGR, University of Modena and Reggio Emilia, Via Campi 287, 41125 Modena (Italy) ****) Laboratory of Plant Breeding, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen (The Netherlands)

*****) USDA-ARS, Plant Science Research Unit, 411 Borlaug Hall, University of Minnesota,

1991 Upper Buford Circle, St. Paul, MN 55108 (USA) Tiling array, Brachypodium distachyon, Puccinia brachypodii, transcriptome profiling, fine-mapping The model grass Brachypodium distachyon L (Brachypodium) has recently revealed its

potential for studying grass-pathogen interactions. In particular, the identification of genomic regions associated with resistance to the false brome rust fungus Puccinia brachypodii offered perspectives to elucidate the genetic and molecular basis of this trait. In this study, we aimed to: 1) provide an initial whole-genome expression dataset for Brachypodium-P. brachypodii interaction in the two inbred lines Bd3-1 (resistant) and Bd1-1 (susceptible), and 2) fine mapping and cloning Rpbq2 and Rpbq3: to increase the

resolution of QTL mapping and to reduce the number of candidate genes underlying QTL LOD curves. For the first, aim the two inbred lines have been characterized macroscopically and by confocal microscopy to follow the development of the fungus and the formation of

rust infection structures. The expression of six brachypodium genes, homologous to known wheat and barley defence-related genes, was monitored by qRT-PCR analysis in Bd3-1 and Bd1-1 at three time points (18, 24 and 72 hours post infection, hpi). The 18 hpi time point was selected for transcriptome profiling on the basis of the expression profiles of the

defence genes. The Affymetrix Brachypodium Tiling Array (BradiAR1b520742) revealed that expression levels of a set of genes (more than 100 in total) were altered in infected plants, mainly in the resistant line Bd3-1. At 18 hpi a significant re-programming of host metabolism occurred in infected leaves, with a modulation of genes involved in different metabolic networks such as defence, glycolysis, aminoacid and nitrogen metabolism. This study represents the first characterization of the functional genomic basis of resistance to a rust species in the model plant Brachypodium, and could be useful for translational

genomics to ‘complex’ cereals. For the second aim, fine mapping Rpbq2 and Rpbq3, a new large segregating RIL population has been developed for each QTL separately. Selection of Bd3-1 x Bd1-1 RILs heterozygous for the QTLs has been completed based on flanking marker haplotypes, with the target QTL in a heterozygous state, while the other two QTLs were selected to be homozygous for the susceptible allele. These marker-selected heterozygous RILs have been selfed to obtain large segregating populations for each QTL.

These results represent the first steps of a genetic approach towards the cloning of Rpbq2 and Rpbq3 determinants, and for their possible exploitation in cereals.

17


S04

NITROGEN USE EFFICIENCY IN TOMATO INVOLVES A HIGH AFFINITY

LONG-DISTANCE NITRATE TRASPORTER

LONGO C.*, LUPINI A.*, MERCATI F.*, PRINCI M.P.*, MILLER A. J.**, ABENAVOLI M.R.*, SUNSERI F.* *) Dipartimento di AGRARIA, Università Mediterranea di Reggio Calabria, Salita Melissari, 89124 Reggio Calabria (Italy) **) John Innes Centre, Norwich Research Park, Norwich, NR4 7UH (UK)

High affinity transport system (HATS), Solanum lycopersicum L., membrane potential, nitrate uptake

Nitrogen (N) availability is a major limiting factor for plant growth and productivity in both natural and agricultural environments. For this reason, over the past decades, N

fertilization has increased more than 20-fold causing massive environmental and health damage. Therefore, to minimize the N fertilizer input, it is crucial to identify and grow more N-use efficient genotypes.

Nitrogen use efficiency (NUE) is a complex trait that can be divided into the ability of plant to take up N from the soil (Nitrogen Uptake Efficiency, NUpE), and the ability of plant to transfer and use this element in plant organs, (Nitrogen Utilization Efficiency, NUtE) (Xu et al., 2012, Ann. Rev. Plant Biol. 63, 153-182). Plants have developed at least two uptake

systems to cope with nitrate availability in soils: high and low affinity transport systems (HATS and LATS) (Crawford & Glass, 1998, Trends Plant Sci. 3, 389-395). Since the nitrate concentrations in soils are often in the low millimolar ranges (Miller et al., 2007, J. Exp. Botany 58, 2297-2306), the high-affinity transport system (HATS) is particularly important for plant nutrition.

To compare N use efficiency (NUE) in tomatoes, several genotypes were grown at

different nitrate concentrations (ranging from 0.1 to 10 mM) in pots, for three weeks. Through “a well-defined response curve to nitrate” by using two indices determined as “the maximum yield at non limiting nutrient availability” () and “the nutrient concentration at

which half-maximum yield is achieved” (), nitrogen-efficient and -inefficient genotypes

were identified (Gourley et al. 1994, Plant Soil 158, 29-37). Applying the Gourley criteria, Regina Ostuni and UC82 were identified as nitrogen-efficient and inefficient genotypes, respectively. Gene expression analyses for two components of high-affinity nitrate uptake (SlNRT2.1/SlNar2) and long distance N translocation (SlNRT2.3) transporters were evaluated at low nitrate (NO3

-) supply (0.1 mM). Electrophysiological membrane potential responses to low nitrate supply were also assessed. In addition, for comparing nitrate uptake efficiency (NUpE) between genotypes, 15NO3

- influx and NO3- tissue concentrations,

in roots and leaves were measured. Electrophysiological measurements showed differences between genotypes in

membrane potential, but similar in response to nitrate. An observed higher 15NO3- influx in

UC82 suggested a different behaviour in NUpE between genotypes, confirmed also by a significantly higher level of NRT2.1 gene expression in UC82. However, a higher transcript level of the putative long-distance nitrate translocation gene

(SlNRT2.3) in NUE efficient genotype (Regina Ostuni) was observed, together with a higher shoot nitrate content. These results suggest that NRT2.3 nitrate transporter could play a

key role in NUE trait also in tomato as already reported in rice (Tang et al. 2012, Plant Physiol. 160, 2052-2063).

18


S05

ANALYSIS OF SMALL RNA POPULATIONS OF MAIZE LEAVES FOLLOWING

ABIOTIC STRESS TREATMENTS

LUNARDON A.*, DEL FABBRO C.**, DE PAOLI E.**, HARDCASTLE T.***, FORESTAN C.*, FARINATI S.*, VAROTTO S.* *) Department of Agronomy Food Natural resources Animals Environment - DAFNAE, University of Padova, Viale dell’Università 16, 35020 Legnaro (Italy) **) Institute of Applied Genomics, Via Jacopo Linussio 51, 33100 Udine (Italy)

***) Department of Plant Sciences, University of Cambridge, Downing Street, CB2 3EA Cambridge (UK) small RNA sequencing, microRNAs, siRNAs, abiotic stress, maize

Small RNAs (sRNAs), recognized as an important mechanism regulating gene

expression for many cellular processes, are classified upon their biogenesis in hairpin RNAs (hpRNAs) and small interfering RNAs (siRNAs), respectively derived from single-stranded and double-stranded RNA precursors. HpRNAs are microRNAs (miRNAs) that exert post-transcriptional regulation of their targets, or other sRNAs, derived by an imprecise processing of the hairpin. SiRNAs are classified in: trans-acting RNAs, that cleave specific target mRNAs; heterochromatic siRNAs required for the silencing maintenance of repetitive sequences through the deposition of repressive chromatin marks and DNA methylation;

natural antisense siRNAs, whose precursor is formed by the hybridization of complementary and independently transcribed RNAs (Axtell, 2013). Recently, Lish (2012) has proposed the presence of a class of sRNAs, containing both hpRNAs and siRNAs, transcribed from transposable element (TE) rearrangements that could trigger TE silencing in trans.

To investigate sRNAs role in maize abiotic stress response and in particular the

contribution of the heterochromatic siRNAs-generating pathway, we applied salt, drought and the combination of the two stresses to maize B73 wild-type plants and rmr6 mutant that contain a non-functional mutated Pol IV largest subunit. We sampled young leaf tissue

after ten days of stress condition and after seven days of recovery from the stress. Analysis of sRNA sequencing data started from conserved miRNAs: differentially

expressed miRNAs were identified employing a generalized linear model: salt and drought treatments caused slight up-regulation of five and one miRNAs, respectively, while the

interaction between the two stresses did not provoke any additional effect. We are currently working on putative novel miRNAs identification and on the investigation of the relationship between microRNAs abundances and the expression levels of the corresponding targets, through the comparison with RNA sequencing data produced in parallel. Analysis of siRNA population confirmed previous results that siRNAs unique sequences have low expression levels and differ between samples. We are currently comparing siRNA loci

abundances among samples, making distinction depending on the kind of genomic feature near to they align: genes, transposons or other repetitive sequences, whose expression levels will be obtained from RNA sequencing data. We aim to look for possible significant correlations between the expression levels of siRNA loci and those of their neighbouring elements.

19


S06

DNA METHYLATION INFLUENCE RAPESEED (BRASSICA NAPUS VAR.

OLEIFERA DEL.) GENE EXPRESSION IN RESPONSE TO SALT STRESS

CONDITIONS MARCONI G.*, PACE R.**, RAGGI L.*, TRAINI A.***, LUTTS S.****, GUIDUCCI M.**, FALCINELLI M.*, BENINCASA P.**, CHIUSANO M.L.***, ALBERTINI E.* *) Department of Applied Biology, University of Perugia, Borgo XX Giugno 74, 06121

Perugia (Italy) **) Department of Agricultural and Environmental Science, University of Perugia, Borgo XX Giugno 74, 06121 Perugia (Italy) ***) DiSSPAPA, University of Napoli, Via Università 100, 80055 Portici (Italy) ****) Groupe de Recherche en Physiologie végétale, Earth and Life Institute-Agronomy, Université Catholique de Louvain, Louvain-la-Neuve (Belgium)

salinity, DNA-methylation, MSAP, qRT-PCR Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an

integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salt-tolerant (Exagone) and salt-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in

Exagone and increased in Toccata. Eighteen bands showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes (Lacerata and trehalose-6-phosphatase synthase S4) involved in stress responses

and were chosen to better characterise the relationships between salinity stress, pattern of methylation, and level of gene expression. Our qRT-PCR data show that salinity stress influences the expression of the two stress-related genes we identified as being

differentially methylated in plants grown under salt stress and control conditions. Moreover, in order to verify that the changes in expression level of TPS4 corresponded to a change in level of trehalose accumulation, we extracted total soluble sugars and demonstrated that, in Exagone shoots, level of trehalose was correlated to gene expression and, therefore, to the level of DNA methylation.

20

Special communication

S07

RISK ASSESSMENT OF GENETICALLY MODIFIED PLANTS – AN EFSA

PERSPECTIVE

GENNARO A., DEVOS Y., MESTDAGH S., WAIGMANN E. European Food Safety Authority (EFSA), GMO Unit, Via Carlo Magno 1A, 43126 Parma (Italy)

GM plants, risk assessment, comparative approach, case-by-case

Genetically modified organisms (GMOs) and derived food and feed products are subject to a risk analysis before they can be released into the environment for commercial purposes or put on the market in the European Union (EU). In this risk analysis process, the role of the European Food Safety Authority (EFSA) is to independently assess and

provide scientific advice to risk managers on any possible risks of GMOs to human and

animal health and the environment. These risks are estimated by combining the likelihood and seriousness of harm to human and animal health or the environment that may arise following the cultivation or consumption of a particular genetically modified (GM) plant.

EFSA’s GMO Panel issued several guidelines describing elements and data requirements for the risk assessment (RA) of GM plants. In line with a number of internationally agreed RA principles, the guidelines require : (1) to use quantitative information where available; (2) to use a comparative approach whereby the level of risk is

estimated through comparison with an appropriately selected comparator and its associated farm management and cropping practices; (3) to be case-specific; (4) to be iterative and, in a transparent manner, examine previous conclusions in the light of new information; and (5) to follow a tiered approach.

An overview of elements and data requirements relevant to the RA of GM plants will be presented. In addition, an analysis of whether the existing RA guidelines are sufficiently

robust to cover plants/products obtained through new biotechnology-based plant breeding techniques (e.g., cisgenesis, intragenesis, site directed nucleases) will be presented. In the EU, relevant elements include amongst others: (1) the molecular characterisation of

the GM plant, which provides information on the structure and expression of the insert(s) and on the stability of the intended trait(s); (2) the comparative analysis of compositional, phenotypic and agronomic characteristics to identify intended and unintended changes in the GM plant; (3) the toxicological assessment of the genetic modification, which addresses

the impact on human and animal health of biologically relevant change(s) in the GM plant and/or derived food and feed resulting from the genetic modification; (4) the assessment of the allergenic potential of the novel protein(s); (5) the nutritional assessment, which aims to demonstrate that the food and feed derived from a GM plant is not nutritionally disadvantageous to humans and/or animals; (6) potential changes in the persistence (weediness) and invasiveness ability of the GM plant; (7) the potential for gene transfer; (8) interactions between the GM plant and target organisms; (9) interactions between the

GM plant and non-target organisms; (10) potential adverse effects on biogeochemical processes; (11) impacts of altered farm management practices associated with the cultivation of the GM plant; (12) potential interactions with the abiotic environment; and (13) the scientific quality of post-market (environmental) monitoring plans.

21

POSTERS

22


PI01 PI02

CDNA-NBS PROFILING OF HORTENSIA RGA-LIKE GENES RESPONSIVE TO LEAF SPOT FUNGAL INFECTION. WOODROW P., KAFANTARIS I., IANNUZZI F., MIRTO A., FUGGI A. AND CARILLO P. Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università degli Studi di Napoli, via Vivaldi 43, 81100 Caserta, Italy.

Keywords: biotic stress, resistance genes, nbs profiling, leaf spot, hortensia. Hydrangeas are susceptible to leaf spot fungus Cercospora hydrangeae. Fungal spores germinate quickly and spread over the plant leaves under warm and rainy weather forming small purple or brown spots. Although Hydrangea plants are not killed by leaf spot, it reduces the flowering and plant vigor decreasing the market value of the plants. The aim of

our study was to isolate and characterize Hortensia resistance (R) gene transcripts under C. hydrangeae fungus infection and investigate their expression profile and evolutionary relationships. The majority of disease R-genes in plants encode nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins. A cDNA-NBS strategy was carried out using as template cDNAs isolated from control and infected plant leaves. The cDNA-NBS profiling gave an excellent bands reproducibility. Twenty new transcripts corresponding to NBS-LRR proteins were identified only in infected plants. Their phylogenetic analysis revealed that

most of them evolved through concerted evolution probably due to a selective pressure from a certain pathogen and/or genetic evolutionary events such as gene conversion, crossing-over, duplication and diversifying selection.

COMPARATIVE ANALYSIS OF CALCIUM INDICATORS AND

CAMELEON REPORTER PROTEINS UPON HERBIVORY. VERRILLO FRANCESCA1, NAGAMANGALA KANCHISWAMI CHIDANANDA2, MAFFEI MASSIMO E. 1 AND OCCHIPINTI ANDREA1.

1 Department of Life Sciences and Systems Biology, University of Turin, Via G. Quarello 15/A, 10135 Turin, Italy. 2 Fondazione Edmund Mach, Istituto Agrario San Michele, Via

Mach 1, 38010, San Michele all'Adige, Italy. Keywords: Calcium, confocal microscopy, biotic stress, Arabidopsis thaliana. Ca2+ is a key player in plant cell responses to herbivory. Changes in [Ca2+]cyt occur immediately after both biotic and abiotic damage. The fine tuning of gene regulation is strictly related to intensity, amplitude and spatio-temporal changes in [Ca2+]cyt.

Nowadays, the monitoring of Ca2+ is based mainly on luminescence/fluorescence techniques. Moreover, CFP/YFP-based Ca2+ reporters (yellow and cyan cameleons), chimeric proteins that rely on FRET as an indicator of Ca2+ changes, are also used. Here we

show a comparison between two Ca2+ indicators, Calcium Orange(TM) and Fluo-3, and a cameleon YC3.6 reporter protein expressed in Arabidopsis thaliana plants by using confocal laser scanning microscopy. A. thaliana leaves were challenged with mechanical damage,

herbivory by Spodoptera littoralis and mechanical damage plus application of S. littoralis oral secretions. Our results show that the two Ca2+ indicators behave differently: Calcium Orange is a specific indicator of wounding, whereas Fluo-3 has a lower specificity. On the other hand, cameleon YC3.6 allowed a clear distinction between mechanical damage and herbivore wounding and discriminated among different larvae instars.

23


PI03 PI04

AN ARABIDOPSIS THALIANA LysM RECEPTOR-LIKE KINASE REGULATES RESISTANCE TO PATHOGENS AND ABSCISIC ACID RESPONSES.

PAPARELLA CHIARA, MARTI LUCIA, SAVATIN DANIEL, DE LORENZO GIULIA AND FERRARI SIMONE.

Dipartimento di Biologia e Biotecnologie "Charles Darwin", Sapienza Università di Roma, Piazzale Aldo Moro 5 - 00185 Rome, Italy. Keywords: receptor-like kinases, plant immunity, abscisic acid, plant defense, pathogen resistance.

Transmembrane receptor-like kinases (RLKs) are important mediators of signalling in all

Eukaryotes. In plants, RLKs characterized by the presence of a LysM domain in the extracellular portion (LYK proteins) mediates recognition of symbiotic and pathogenic microorganisms. Here we have investigated the role a family of five Arabidopsis thaliana LYKs in innate immunity. Using reverse genetics, we have found that one of these genes, AtLYK3, negatively regulates expression of defense genes and resistance to pathogens. The

expression of AtLYK3 is strongly repressed by elicitors and fungal infection, while it is induced by the hormone abscisic acid (ABA), previously shown to have a negative role in resistance against some pathogens. Plants lacking a functional AtLYK3 show reduced sensitivity to this hormone, suggesting that this protein is important for the cross talk between signalling pathways activated by ABA and pathogens.

PECTIN METHYLESTERASE INHIBITORS LIMIT TOBAMOVIRUS

SPREADING IN TOBACCO AND ARABIDOPSIS. LIONETTI V.1, RAIOLA A.2, FABRI E.1, CERVONE F. 1 AND BELLINCAMPI D.1

1 Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome. 2 Department of Land, Environment, Agriculture and Forestry, University of Padua, Legnaro (PD).

Keywords: pectin methylesterase, pectin methylesterase inhibitors, Tobamovirus, plant resistance. Plant infection by a virus is a complex process influenced by virus-encoded factors and host components that support replication and movement. Critical factors for a successful infection are the movement proteins (MPs) that modify the size exclusion limit of

plasmodesmata during the cell-to-cell movement. Pectin methylesterases (PMEs) was shown to interact in vitro with the MP of different viruses and this interaction was proposed to be necessary for Tobacco mosaic virus (TMV) spreading as well as for its systemic

movement through the host vasculature. Here we report that the ectopic expression of a PME inhibitor from Actinidia chinensis in Nicotiana tabacum significantly delays the TMV cell-to-cell and systemic spreading. A reduced susceptibility against Turnip vein clearing

virus was also observed in Arabidopsis plants overexpressing a PME inhibitor from Arabidopsis. Overall, our results strongly support the important roles of PMEs in viral movement and indicate PME inhibitors as a useful tool to limit virus infection.

24


PI05 PI06

SKIN-SPECIFIC ACCUMULATION OF ANTHOCYANINS IN TOMATO EXTENDS SHELF LIFE. BASSOLINO LAURA1, ZHANG YANG2, SCHOONBEEK HENK-JAN2, KIFERLE CLAUDIA1, PERATA PIERDOMENICO1 AND MARTIN CATHIE2. 1 Plant Lab, Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy. 2 John Innes Centre, Norwich Research Park, NR4 7UH, Norwich, UK.

Keywords: anthocyanins, tomato, Aft/Aft atv/atv. Botrytis cinerea, shelf life. Shelf life is one of the most important traits for tomato industry. Two key factors, postharvest over-ripening and pathogen infection, determine tomato shelf life and different strategies have been employed to extend tomato shelf life during the past decade. In this

study we show that, skin-specific accumulation of anthocyanins in Aft/Aft atv/atv tomato, a cross between two anthocyanin-naturally enriched lines, results in extended fruit shelf life. Compared to ordinary, anthocyanin-less tomatoes, the fruits of Aft/Aft atv/atv display longer storage period and is less susceptible to Botrytis cinerea, the major tomato postharvest pathogen. Taken together these results suggest that the accumulation of anthocyanins in tomato fruits by traditional breeding can be an efficient way to extend tomato shelf life. Our finding has important agronomic and commercial implications, since

Aft/Aft atv/atv tomatoes are naturally enriched in anthocyanins thus having both nutraceutical properties and extended shelf life.

CHARACTERIZATION OF A RHIZOCTONIA ZEAE

POLYGALACTURONASE AND ITS INTERACTION WITH THE PLANT INHIBITOR PvPGIP2.

MAISTROU M.1, TRICARICO S. 1, CERVONE F.2 AND CAPRARI C.1.

1 Department of Bioscienze and Territory, University of Molise, Contrada Fonte Lappone, 86090 Pesche (IS) (Italy). 2 Department of Biology and Biotechnology "Charles Darwin", University of Rome "Sapienza", P.le A. Moro 5, 00185 Roma (Italy). Keywords: Polygalacturonase, Polygacturonase-inhibiting protein, Rhizoctonia zeae, pectin. The plant cell wall, the main structural element that a pathogen needs to overcome in

order to penetrate and colonize the plant tissue, is composed by four major complex polysaccharides (cellulose, hemicellulose, lignin, and pectin). The majority of pathogenic microorganisms produce cell wall degrading enzymes (CWDEs) that are essential for the invasion process. Among the different CWDEs produced by fungi polygalacturonases (PGs) play a critical role since their action on pectin makes other cell wall components more

accessible to other CWDEs and causes tissue maceration. To limit the aggressive potential of PGs, plants have evolved small gene families encoding polygalacturonase-inhibiting

proteins (PGIPs). The PG–PGIP interaction retards the hydrolysis of pectin but also favors the accumulation of oligogalacturonides (OGs) that activate the plant innate immunity system. Here we present the purification and characterization of a PG from R. zeae and its ability to interact with PvPGIP2.

25


PI07 PI08

DOES CITRUS LEAF MINER IMPAIR HYDRAULICS AND FITNESS OF CITRUS HOST PLANTS? RAIMONDO FABIO, TRIFILÒ PATRIZIA AND LO GULLO MARIA ASSUNTA. Dipartimento di Scienze Biologiche ed Ambientali, Università di Messina, Viale F. Stagno D'Alcontres 31, 98166 Messina S. Agata, Italy.

Keywords: Citrus spp., Phyllocnistis citrella Staiton, gas exchange, leaf hydraulics, photosynthesis. Gas exchange and hydraulic features were measured in leaves of three different Citrus species infested by Phyllocnistis citrella Staiton, with the aim to quantify the impact of this pest on leaf hydraulics and, ultimately, on plant fitness. Infested leaves were characterized

by the presence on the leaf blade of typical snake-shaped mines and, in some cases, of a crumpled leaf blade. Light microscopy showed that leaf crumpling was induced by damage to the cuticular layer. In all three Citrus species examined: a) the degree of infestation did not exceed 10% of the total surface area of infested plants; b) control and infested leaves showed similar values of minimum diurnal leaf water potential, leaf hydraulic conductance and functional vein density, and c) maximum diurnal values of stomatal conductance to water vapour, transpiration rate and photosynthetic rate (An) were similar in both control

leaves and the green areas of infested leaves. A strong reduction of An was recorded only in mined leaf areas. Our data suggest that infestation with P. citrella doesn't cause conspicuous plant productivity reductions in young Citrus plants, at least not in the Citrus species studied here.

ARABIDOPSIS SEPAL FUNCTIONS AS BIOTIC STRESS DEFENSE

BARRIER OF THE DEVELOPING FLOWER. PASQUALINI STEFANIA1, QUAGLIA MARA2, GEHRING CHRIS3, DAWE ADAM4 AND EDERLI LUISA1.

1 Department of Applied Biology, University of Perugia, I-06121 Italy. 2 Department of Agricultural and Environmental Sciences, University of Perugia, I-06121 Italy. 3 Division of

Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955–6900, Saudi Arabia. 4 South African National Bioinformatics Institute University of the Western Cape, Bellville 7535, South Africa. Keywords: sepal, biotrophic pathogen, salicylic acid, systems analysis. In Arabidopsis the sepals are modified green leaves like structure that enclose the

developing flower. A distinct feature of the sepals is that they contain cells of vastly different sizes and notably the polyploid giant pavement cells that have arisen through endo-reduplication, performing karyokinesis but not cytokinesis. While the function of

these pavement cells remains unclear, it has been speculated that they may play a role in the defense against insect predators, prevent water stress, and improve the mechanical properties of the organ, albeit by unspecified mechanisms. Here we undertake a systems

analysis and interpretation of the sepal transcriptome with a view to gain new insights into sepal function and propose that this organ functions as a highly effective biochemical defence structure of the developing reproductive organs. We also investigate the role for the defense regulator salicylic acid in the sepal and propose that the sepal acts as biotic-defense shield of the developing reproduction organs against the biotrophic pathogen G. cichoracearum.

26


PI09 PI10

THE TRIPLE MITOGEN-ACTIVATED PROTEIN KINASES ANPS REGULATE IMMUNITY BY ORCHESTRATING SIGNALING AND ROS FORMATION IN MITOCHONDRIA, PLASTIDS AND NUCLEUS.

SAVATIN DANIEL V., MARTI LUCIA, GIGLI-BISCEGLIA NORA, CERVONE FELICE AND DE LORENZO GIULIA.

Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza - Università di Roma, Piazzale A. Moro 5, 00185 Roma, Italy. Keywords: Plant Immunity, Danger signals, Biotic stresses. Representative danger signals (elicitors) belonging to the classes of the Pathogen- and

Damage- Associated Molecular Patterns (PAMPs and DAMPs, respectively) were utilized to

investigate the role in immunity of the Arabidopsis ANP gene family, which encodes triple mitogen-activated protein kinases. Mutant analyses show that ANPs are required for elicitor-triggered defense responses and protection against the necrotrophic fungus Botrytis cinerea. In physiological conditions, ANPs localize in mitochondria and in the cytoplasm, but, after elicitor perception, localize also into plastids and nucleus, revealing a dynamics

that is unique in plant cell biology. Sites of elicitor-induced ROS accumulation and ANP localization coincide, and ANPs are required both for ROS generation and ROS signaling. Our findings point to ANPs as key transduction elements that coordinate PAMP- and DAMP-triggered immunity and central hubs in the orchestration of ROS accumulation and signaling.

FUNCTIONAL CHARACTERIZATION OF XYLANASE INHIBITORS IN

WHEAT. MOSCETTI ILARIA1, TUNDO SILVIO1, KALUNKE RAVIRAJ1, JANNI MICHELA1,3, SELLA LUCA2, FAVARON FRANCESCO2 AND D'OVIDIO RENATO11.

1 Dipartimento di Scienze e tecnologie per l'Agricoltura, le Foreste, la Natura e l'Energia, (DAFNE), Università della Tuscia, Via S. Camillo de Lellis snc, 01100 Viterbo, Italy. 2 Dipartimento TeSAF, Università degli studi di Padova, viale dell'UniversitÃ 16, 35020 Legnaro (PD), Italy. 3 Present address: IGV-CNR Via Amendola 165/A, 70126 Bari, Italy. Keywords: Xylanase inhibitor protein, xylanases, wheat, transgenic plants, defence response. Cereals contain xylanase inhibitor proteins (XIs) which inhibit the activity of microbial

xylanases. Several observations suggests a role of XI in plant defense. We recently demonstrated a correlation between the capacity of TAXI-III, a Taxi-type XI, to inhibit the activity of Fusarium graminearum xylanases and the reduction of fusarium head blight

(FHB) disease symptom in wheat. We extended our analysis to the Xip-type XIs by over-expressing the Xip-I and Xip-III genes in transgenic wheat plants. Results showed that the Xip-I gene endows the transgenic wheats with new inhibition capacity, whereas the

transgenic plants containing the Xip-III transgene do not show any improvement in their capacity to inhibit xylanase activity. The molecular basis underlying the lack of XIP-III activity will be determined as well as the impact of the over-expression of Xip-I in wheat defence against fungal pathogens. Differences in the inhibition properties of TAXI-type and XIP-type inhibitors will be presented.

27


PII01 PII02

A COMPREHENSIVE ANALYSIS OF CU INDUCED CHANGES IN THE MINERAL PROFILES OF Cu SENSITIVE AND TOLERANT POPULATIONS OF SILENE PARADOXA L.

COLZI ILARIA1, PIGNATTELLI SARA1, BUCCIANTI ANTONELLA2, CATTANI ILENIA3, BEONE GIANMARIA3, SCHAT HENK4 AND GONNELLI CRISTINA1. 1 Department of Biology, Università di Firenze, via Micheli 1, 50121 Firenze, Italy. 2 Department of Earth Science, University of Florence, via La Pira 4, 50121 Firenze, Italy. 3 Institute of Agricultural and Environmental Chemistry, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, Piacenza, Italy. 4 Department of Genetics, Faculty of Earth and Life Sciences, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. Keywords: mineral profile, copper tolerance, Silene paradoxa, compositional data analysis.

This work investigates the Cu induced changes in element profiles in contrasting ecotypes of Silene paradoxa L. A copper tolerant population and a sensitive population were grown in hydroponics and exposed to different CuSO4 treatments. Shoot and root concentrations of Ca, Cu, Fe, K, Mg, Mn, Mo, Na, P, S and Zn were evaluated through ICP-OES. In the tolerant population Cu treatment induced a higher element accumulation in roots and had minimal effects on the shoot element profile, thus resulting in a progressively decreasing shoot-to-root ratio for each element. In the sensitive population element concentrations in

root and shoot were much more affected and without a consistent trend. Cu exposure strongly disturbed element homeostasis in the sensitive population, but barely or not in the tolerant one, probably mainly due to a higher capacity to maintain proper root functioning under Cu exposure. Differences in element profiles were also observed in the absence of toxic Cu exposure, reflecting divergent population-specific adaptations to differential nutrient availability levels prevailing in the populations' natural environments

PHYSIOLOGICAL RESPONSE OF DROUGHT-TOLERANT AND

DROUGHT-SENSITIVE TOMATO GENOTYPES TO WATER STRESS. RIGANO MARIA MANUELA1, ARENA CARMEN2, DI MATTEO ANTONIO1 AND BARONE AMALIA1.

1 Dipartimento di Agraria, Università degli Studi di Napoli Federico II, Via Università 100, 80055, Portici (Napoli). 2 Dipartimento di Biologia, Università degli Studi di Napoli Federico

II, Via Cintia 21, 80126, Napoli. Keywords: tomato, introgression lines, water stress. Water stress is an increasing environmental constraint in Mediterranean areas affecting tomato growth and yield. It is reported that Solanum pennellii, a wild species from the driest environments, showed a higher water use efficiency compared to cultivated S.

lycopersicum. In our laboratory a S. pennellii line (IL 9-2-5) was identified as more tolerant to water deficit in terms of yield losses. In the present work, the tolerant genotype IL 9-2-5 and the susceptible genotype (M82) were subjected to three different water regimen:

irrigation with 100% (V1), 50% (V2) and 25% (V3) restoration of water evapotranspiration. In order to evaluate the physiological response of IL 9-2-5 to water deficit, leaf functional traits were measured together with photosynthetic pigments,

phenolic compounds and ascorbic acid content. The higher tolerance to water deficiency of IL 9-2-5 was likely linked to the development of a better antioxidant system. In addition, in IL 9-2-5 higher values of leaf schlerophylly and leaf dry matter content were registered, especially in treatment V3. These represent important properties that ensure a better resistance of leaves to limited water supply.

28


PII03 PII04

UNDERSTANDING THE DYNAMICS OF STRESS SENSING WITHIN THE PLANT NUCLEOLUS: INVESTIGATION OF THE ROLE PLAYED BY THE MTTDP GENE FAMILY ENCODING TYROSYL DNA

PHOSPHODIESTERASE ENZYMES. FAÀ MATTEO, BIGGIOGERA MARCO, SABATINI MARIA ELISA, DONÀ MATTIA, BALESTRAZZI ALMA AND CARBONERA DANIELA. Department of Biology and Biotechnology 'L. Spallanzani', Via Ferrata 1, 27100 Pavia, Italy.

Keywords: DNA repair, abiotic stress, nucleolus, tyrosyl-DNA phosphodiesterase. The nucleolus, required for ribosome biogenesis, is also implicated in cell cycle control,

telomere homeostasis and aging. The role of nucleolus as a sensor for cellular stress has been largerly investigated in animals. Information are still scanty in plants, although the possible occurrence of a nucleolar checkpoint for stress sensing has been suggested by Donà et al. 2013 (J. Exp. Bot. 64: 1941-1951) in a recent study on the DNA repair enzyme

Tyrosyl-DNA Phosphodiesterase 1a (Tdp1alpha). Down-regulation of the MtTdp1a gene in Medicago truncatula resulted in altered nucleolar morphology and impaired ribosome biogenesis. The Tdp enzyme family also includes Tdp2ï•¡ which in animal cells repairs the topoisomerase II-mediated DNA lesions but also acts as a signaling component. In the attempt to unravel the role of MtTdp2a gene in relation to nucleolar function, transgenic M. truncatula cell suspension lines overexpressing MtTdp2a were obtained and characterised.

Preliminary TEM analyses revealed the effects of MtTdp2a overexpression on the nucleolar organization, evidencing the presence of nucleolar cavities. Molecular and ultrastructural investigations are currently in progress.

METABOLISM IS SHUT DOWN BY OSMOTIC STRESS IN LOTUS

JAPONICUS ROOTS, LIKELY TO ALLOW STRESS-INDUCED ABA ACCUMULATION.

LIU JUNWEI1, HE HANZI 2, VITALI MARCO1, CHARNIKHOVA TATSIANA2, HAIDER IMRAN2, SCHUBERT ANDREA1, RUYTER-SPIRA CAROLIEN2,3, BOUWMEESTER HARRO2, VISENTIN IVAN1, LOVISOLO CLAUDIO1 AND CARDINALE FRANCESCA1.

1 Dept. of Agricultural, Forest and Food Sciences (DISAFA), University of Turin, via Leonardo da Vinci 44–10095 Grugliasco (TO), Italy. 2 Laborattory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, NL–6708 PB Wageningen, The Netherlands. 3 Business Unit Bioscience, Plant Research International, Droevendaalsesteeg 1, 6708 PB

Wageningen, the Netherlands.

Keywords: Abscisic acid (ABA); Lotus japonicus; osmotic stress, phosphate starvation; strigolactones.

Strigolactones (SLs) are plant hormones proposed as mediators of environmental stimuli in resource allocation processes, for which purpose they must be responsive to environmental conditions. We quantified SLs in tissues and exudates of Lotus japonicus roots stressed

osmotically and/or P starved. The transcripts of SL-related genes were quantified. Also,

root ABA and physiological performances of SL-depleted plants were studied under drought, and the effect of excess SLs on ABA was recorded in stressed WT roots. Results showed that osmotic stress rapidly and strongly decreases SL abundance in root exudates and extracts, independently of P abundance. This was associated with less transcript of most SL-related genes. While SL-depleted Lotus plants showed only a subtle phenotype under drought, pre-treatment with exogenous SLs inhibited the osmotic stress-induced

ABA increase in WT roots, down-regulating the transcription of the biosynthetic gene LjNCED2. In conclusion, we hypothesize that fast SL decrease under osmotic stress allows the physiological increase of ABA, and that SLs are likely to affect plant acclimation by modulating ABA metabolism and possibly, sensitivity.

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PII05 PII06

ACCUMULATION AND TOLERANCE IN POPULUS ALBA L. UNDER CADMIUM STRESS.

CHIARA ROMÈ, ALESSANDRA FRANCINI AND LUCA SEBASTIANI. Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, I-56127 Pisa, Italy. Keywords: cadmium, chlorophyll, growth analysis, heavy metals, screening.

This work aims to study the effects of different doses of Cd on growth dynamics and metal accumulation in poplar. Plantlets of clone Villafranca (Populus alba L.), were grown under controlled conditions in sandy substrate. The study was performed for 6 weeks with 0, 50, 75 and 100 μM Cd. Using the leaf plastochron index (LPI) as classification system, the leaves were separated into apical (LPI>7), median (14<LPI>7) and basal (LPI<14). Cd did not affect poplar growth in terms of dry weight, but necroses occur at 75 μM Cd on basal

leaves. Cd concentrations in leaves, stems and roots was related to Cd added in the

substrate with the translocation factor>1 from roots to apical leaves and stem. The highest Cd concentration (5.55 mg kg-1) was recorded in stems at 100 μM Cd and the lowest (0.99 mg kg-1) in the median leaves at 50 μM Cd. Measurements of chlorophyll fluorescence did not indicate significant differences, but pigments analyses performed by HPLC at 75 μM Cd showed an increase of Chl a (+56%) in basal leaves and a reduction (-58%) in apical leaves. These results suggest that Villafranca can modify the Cd uptake to preserve the leaves to stress.

HYDRAULIC CONDUCTIVITY OF DETACHED SHOOTS OF VITIS

VINIFERA L. VINES OVEREXPRESSING THE GRAPE AQUAPORIN GENE VVPIP2;4N.

VITALI MARCO, FERRANDINO ALESSANDRA, SCHUBERT ANDREA AND LOVISOLO CLAUDIO.

University of Turin, Dept Agricultural, Forest and Food Sciences, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy. Keywords: drought, grapevine, aquaporin, embolism, abscisic acid. Hydraulic conductivity of detached grapevine shoots overexpressing VvPIP2;4N was assessed during a 30-minutes period either submerging the basal cut of the shoot under

water or letting the shoot to transpire in air. We used detached shoots to avoid both root and root-to-shoot signalling effects on water transport, due to VvPIP2;4N aquaporin root activity. The measurement in free air caused embolism, reducing hydraulic conductivity. When shoots were detached from irrigated plants, both embolism extent and shoot transpiration, the cause of embolism formation in air-transpiring shoots, were higher in

transgenic shoots than in wild type controls. However, when measurements were performed on in-vivo embolized shoots (i.e. detached from droughted plants), the in-air

embolism formation of transgenic shoots decreased as in wild type controls. In addition, hydraulic conductivity of the droughted shoots absorbing water increased more in transgenic than in wild type shoots, showing a VvPIP2;4N -dependent embolism repair during water absorption. These results show that a drought-derived signal controls drought embolism and activates embolism repair in VvPIP2;4N overexpressing shoots.

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PII07 PII08

HOW PLANT MITOCHONDRIA MAY CONTRIBUTE TO CELL RESISTANCE TO ABIOTIC STRESSES. THE CASE STUDY OF DURUM WHEAT. TRONO DANIELA1, LAUS MAURA N.2, SOCCIO MARIO2 AND PASTORE DONATO2. 1 Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per la Cerealicoltura, S.S. 16, Km 675 - 71122 Foggia, Italy. 2 Dipartimento di Scienze Agrarie,

degli Alimenti e dell'Ambiente, Università di Foggia, Via Napoli, 25 - 71122 Foggia, Italy. Keywords: Mitochondria, Hyperosmotic stresses, Plant mitochondrial potassium channel, Plant uncoupling protein, Phospholipase A2. In the last years, some proteins and regulation pathways involved in the control of harmful

ROS production under salt and water deficit have been shown by studying durum wheat mitochondria (DWM). The ATP-inhibited Plant Mitochondrial Potassium Channel (PmitoKATP), the Plant Inner Membrane Anion Channel (PIMAC), the Alternative Oxidase (AOX) and the Plant Uncoupling Protein (PUCP) are well active in DWM and may significantly control KCl influx (PmitoKATP and PIMAC) as well as membrane potential and, as a consequence, ROS production (PmitoKATP, AOX and PUCP). In particular, PmitoKATP and PUCP are activated by ROS and in turn may control large scale ROS production under

stress according to a feed-back mechanism. A second modulation pathway is due to activation of the recently discovered mitochondrial Phospholipase A2, that releases free fatty acids from membrane phospholipids, thus strongly activating PmitoKATP and PUCP. Interestingly, activation of PmitoKATP (but not of AOX and PUCP) does not cause uncoupling, loss of ATP synthesis and worsening of cell bioenergetics. These proteins are of potential interest in the future breeding programs for stress resistance in durum wheat.

THE LHCBM9 SUBUNIT OF THE MAJOR LIGHT-HARVESTING

COMPLEX LHCII HAS A UNIQUE PROTECTIVE ROLE WITHIN THE FAMILY OF LHC PROTEINS OF CHLAMYDOMONAS REINHARDTII

UPON SULFUR STARVATION.

BALLOTTARI MATTEO1, GREWE SABRINA2, DOEBBE ANJA2, MUSSGNUG JAN H.2, ALCOCER MARCELO3, D'ANDREA COSIMO3 HANKAMER BEN4, BASSI ROBERTO1 AND KRUSE OLAF2.

1 Dipartimento di Biotecnologie, Università di Verona, I-37134 Verona, Italy. 2 Algae Biotechnology & Bioenergy Group, Department of Biology, Center for Biotechnology,

Bielefeld University, D-33615 Bielefeld, Germany. 3 INF-CNR, Dipartimento di Fisica, Politecnico di Milano, P.za L. da Vinci 32, 20133 Milano, Italy. 4 Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland Qld 4072, Australia.

Keywords: sulfur starvation, hydrogen, algae, photosynthesis.

Photosynthetic organisms have developed several strategies to balance sun light-harvesting and intracellular energy utilization to be able to prosper in continuously varying

environmental conditions. Plants and algae possess gene families encoding for highly

homologues proteins which can form light-harvesting pigment protein complexes (LHC) of types I and II. In this work, we describe the detailed analyses of the LHCII subunit LHCBM9 of the microalga Chlamydomonas reinhardtii: the expression of this subunit is very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g. in nutrient deficiency. In particular Lhcbm9 is strongly expressed in sulfur starvation, a condition in which C. reinhardtii produces hydrogen, that could be

potentially used as a biofuel: in this condition knockdown cell lines with 60-70% reduced amounts of Lhcbm9 showed a reduced growth and reduced hydrogen production. Functional analyses, performed in vitro and in vivo demonstrated that LHCBM9 is peculiar amongst the family of LHCII proteins and serves an important protective function during stress condition.

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PII09 PII10

INTERACTION BETWEEN CHLOROPLAST RELOCATION AND XANTHOPHYLL CYCLE IN REGULATION OF PHOTOSYNTHESIS AND PHOTOPROTECTION OF ARABIDOPSIS THALIANA. DALL'OSTO LUCA1 CAZZANIGA STEFANO1 KONG SAM-GEUN2 WADA MASAMITSU2 AND BASSI ROBERTO1. 1 Università di Verona, Verona Italy. 2 Kyushu University, Hakozaki, Japan.

Keywords: chloroplast, photoprotection, avoidance response, xanthophylls. Photoprotective mechanisms include chloroplast avoidance and NPQ. We evaluated their relative importance in regulating excitation pressure on Photosystem II by comparing photosensitivity of genotypes impaired in chloroplast avoidance response (phot2),

synthesis of zeaxanthin (npq1) or the activation of non-photochemical quenching (npq4). Suppression of avoidance response resulted in oxidative stress under excess light, while removing either zeaxanthin or PsbS had a milder effect. The double mutants phot2 npq1 and phot2 npq4 showed the highest sensitivity to photooxidative stress, implying additive effects. The kinetics at different light intensities of phot2 lacked a component, here named as qM, linearly correlated with the leaf transmittance changes due to white light-induced chloroplast relocation which was absent when red light was used as actinic source. On

these basis we conclude that a component due to a decrease in leaf optical density affects the apparent fluorescence decay kinetic. Thus, excess light-induced fluorescence decrease is in part due to avoidance of photon absorption rather than to a genuine quenching process.

cAMP DEPLETION NEGATIVELY AFFECTS HEAT STRESS TOLERANCE

IN TOBACCO BY-2 CELLS. SGOBBA A.1, PARADISO A.1, SABETTA W.2, VIGGIANO L.1, BLANCO E.2, DE PINTO M.C.1.

1 Department of Biology, University of Bari "Aldo Moro", Bari, Italy. 2 Institute of Plant Genetics, CNR, Bari, Italy.

Keywords: Antioxidants, cAMP, Heat stress, Heat shock proteins, Tobacco BY-2 cells. Cyclic AMP is an important second messenger in all living organisms; however its existence and its role in plants has been debated for long time. Nowadays it is clear that also in plants, cAMP is involved in several physiological processes, including cell cycle regulation, seed germination, growth and reorientation of the pollen tube. It has also been reported that cAMP is involved in the activation of defence mechanisms in response to biotic and

abiotic stresses. In order to verify the involvement of cAMP in plant stress response the effect of moderate heat stress (35°C) in wild type and transgenic tobacco BY-2 cells, with reduced intracellular level of cAMP (cAS cells), has been studied. The obtained results show

that heat stress negatively affects cell growth inhibiting cell cycle progression and cell expansions and increasing cell death. cAMP depletion makes tobacco BY-2 cells more susceptible to heat stress determining an increase in cell death. The involvement of heat

shock proteins and antioxidants in determining the high susceptibility of cAS cells to heat stress has been also studied and discussed.

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PII11 PII12

DIFFERENCES IN MOLECULAR AND MORPHOLOGICAL FEATURES INDUCED BY SALT STRESS IN TWO ITALIAN VARIETIES OF RICE SHOWING CONTRASTING SALT SENSITIVITY. FORMENTIN ELIDE, BARIZZA ELISABETTA, ZOTTINI MICHELA AND LO SCHIAVO FIORELLA. Department of Biology, University of Padova, via U. Bassi 58/B, 35131 Padova, Italy.

Keywords: Italian rice, salinity, ion transporters, nitric oxide, root architecture. Soil salinity is a global environmental challenge affecting crop production in particular in coastal areas. Italy is the largest rice-producing country in Europe and high quality Italian rice varieties are cultivated in the Delta of the Po river. In the frame of the RISINNOVA research project, we are studying the salinity response of Baldo and Vialone nano, two

varieties showing contrasting salt sensitivity. At molecular level, we compared the two genotypes by RNA sequencing to find out useful markers for the selection of Italian rice cultivars resistant to salinity. As there are many indications that the understanding of the salt resistance mechanisms in rice might be achieved by studying ion transporters, we analysed some of these proteins. Moreover, to identify some of the components involved in the signalling pathway induced by high salt, we investigated the role of nitric oxide in response to such a stress, both in plant roots and in cultured cells. As root architecture is

known as largely affected by salinity, we analysed changes in root morphology under mild and severe salt stress. These latter data are the first ones reported on root architecture of Baldo and Vialone nano.

PLASMODESMATA ARE INVOLVED IN ABIOTIC STRESS

SIGNALLING. VERRILLO FRANCESCA1, ROCCO MARIAPINA2, OCCHIPINTI ANDREA1, MAFFEI MASSIMO E.1, MARRA MAURO3.

1 Department of Life Sciences and Systems Biology, University of Turin, Innovation Centre, Via Quarello 15/A, 10135 Turin, Italy. 2 Department of Biological and Environmental

Sciences, University of Sannio, Via Port'Arsa 11, 82100 Benevento, Italy. 3 Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 00133 Rome, Italy. Keywords: Plasmodesmata; calcium signalling; ROS; NO; Membrane potential; K channels. Plants exhibit a variety of responses to abiotic stresses enabling them to tolerate and survive adverse conditions. It is becoming clear that a signaling network is interlinked at

many levels. We made the surprising observation that an Arabidopsis line (pdko3) mutated in genes encoding plasmodesmal proteins responds differently with respect to wild type plants to biotic stress (Bricchi et al., 2013, Plant J. 73, 14–25). Heere we show the

responses of plasma transmembrane potential (Vm) depolarization, voltage gated K+ channel activity, cytosolic calcium ([Ca2+]cyt) and reactive oxygen and nitrogen species (ROS, NO) upon treatment with different chemicals: Paraquat, an inducer of oxidative

stress; Chitosan, a fungal cell wall plant elicitor; Benzothiodiazole, an synthetic analog of Salicylic acid. Unlike wild type plants, pdko3 mutants always showed an almost complete loss of voltage gated K+ channel activity, no Vm depolarization, and a decreased accumulation of [Ca2+]cyt, NO and H2O2 levels. Our results confirm a major role of the regulation of plasmodesmata as a crucial part of coordinated control of cell-to-cell communication and signaling also in abiotic stress responses.

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PII13 PII14

CHARACTERIZATION OF STRESS-RESPONSIVE CONSERVED AND NOVEL miRNAS IN GRAPEVINE.

PAGLIARANI C., VITALI M., FERRERO M., ALBERTO D. AND SCHUBERT A. Department of Agricultural, Forest and Food Sciences (DISAFA), University of Turin, Via Leonardo da Vinci, 44 - 10095 Grugliasco (TO) - Italy. Keywords: small RNAs, drought stress, gene silencing.

We investigated the effects of abiotic stress on miRNA populations working on two grapevine genotypes, Vitis vinifera cv. Cabernet Sauvignon (CS) and M4 rootstock, and on the related grafts (CS/M4 and M4/CS). Experiments were performed on grapevines subjected to a water stress treatment. Samples were collected from both irrigated and droughted plants of the two genotypes at the moment of maximum stress. Low molecular

weight RNA was extracted and used for sequencing and expression analyses. Real Time

PCR analyses were performed on a group of conserved miRNAs typically involved in stress response and on novel miRNAs, to investigate expression changes related to stress, genotype or tissue. Upon drought, expression data showed significant differences induced by stress on both known and novel miRNA populations. Analyses on grafted plants showed the specific activation of some novel miRNAs in only one genotype, independently of the treatment. Expression profiles of putative target transcripts were also determined, in order to gain evidence of the effects of the cognate miRNAs on their abundance.

ACCLIMATION OF KOLIELLA ANTARCTICA TO DIFFERENT LIGHT

INTENSITIES. LA ROCCA N., MORO I., SCIUTO K., RASCIO N. AND MOROSINOTTO T.

Department of Biology, University of Padova, Via U. Bassi 58/b, Italy. Keywords: Koliella antarctica, light changes, NPQ.

Antarctic algae are capable of growing both in sea ice and polar water and adapt to very low light and higher irradiation typical of these two environments. Elucidating the molecular mechanisms of photosynthesis acclimation in these species can help understanding environmental factors controlling distribution and productivity of major phytoplankton taxa in the Southern Ocean and predict the impact of climatic changes on their populations. With this aim we studied the green microalgae Koliella antarctica,

isolated from Ross Sea, by analyzing its acclimation response to different light intensities, low (LL, 15 μE m-2 s-1) and high (HL, 150 μE m-2 s-1). In comparison with LL, HL exposure led to a slight decrease of the cell growth and of total chlorophyll content, while no

changes in Chl a/b ratios occurred. HL acclimated cells showed high capability to dissipate the energy excess as heat, through the rapid activation of a strong Non Photochemical Quenching. This ability was rapidly acquired (4-8 h) when algae were shifted from LL to HL.

Involvement of xanthophylls and/or LHCSR protein in this process will be discussed.

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PII15 PII16

ANTIOXIDANT ACTIVITY AND QUERCETIN CONTENT IN CEREALS AND PSEUDOCEREALS GRAINS: STRESS-INDUCED AND NATURAL VARIABILITY. DE SANTIS GIUDITTA, FRAGASSO MARIA GIOVANNA, PLATANI CRISTIANO, RINALDI MICHELE AND RASCIO AGATA. Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per la

Cerealicoltura, S.S. 673 km 25,200 – 71122 Foggia. Keywords: Quinoa, Wheat, antioxidant activity, flavonoids, quercetin. Environmental stresses induce defence responses in plants with activation of metabolic processes able to modify the content of secondary metabolites with antioxidant action, with

potential health benefits. Flavonoids, natural antioxidants, are considered to be effective substances for the prevention of diseases of the higher age (atherosclerosis, cancer, etc.) and are highly gastro protective too. Quercetin flavonoid is ubiquitously found in nature and particularly in quinoa (Chenopodium quinoa Willd.), a pseudo-cereal of Chenopodiaceae family that is widely used in the production of cereal-based foods. Quercetin could be a constituent of chemotherapeutic drugs because it shows a growth inhibitory effect to tumorigenic cells (prostate and skin cancer treatment). The

identification of natural sources of antioxidants and/or environmental conditions that may elicit the accumulation of healthy substances in foods are the subject of numerous studies. In this paper, we present the preliminary results of studies conducted to evaluate the effect of abiotic stress on the antioxidant activity of wheat grain and the variability for the content of quercetin in several genotypes of quinoa.

MEDITERRANEAN GREEN ROOFS: SELECTING FOR PROPER PLANT

SPECIES AND SOIL TYPE. TRIFILÒ PATRIZIA1, NARDINI ANDREA2, RAIMONDO FABIO1, ANDRI SERGIO AND LO GULLO MARIA ASSUNTA1.

1 Dipartimento di Scienze Biologiche ed Ambientali, Università di Messina, Viale F. Stagno D'Alcontres 31, 98166 Messina S. Agata, Italy. 2 Dipartimento di Scienze della Vita,

UniversitÃ di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy. Keywords: green roof, Mediterranean region, sage, arbutus, drought stress. Recent studies have highlighted the ecological, economical and social benefits of green roof technology to urban areas. Green roofs are potential hostile environments for plant growth because of limited water availability. For this reason, this technology is still uncommon in

hot and arid regions like Mediterranean countries. Moreover, relatively few studies have explored the possibility to use plant species alternative to the traditional Sedum sp. for development of green roof modules, despite the floristic richness (and drought adaptation)

of native species in Mediterranean biomes. This study provides experimental evidences for the possibility to use two Mediterranean native species (i.e. Arbutus unedo L., arbutus and Salvia officinalis L., sage) in green roof systems. Our data show that both arbutus and sage

plants face environmental stresses imposed by green roof technology with success. However, much attention must be paid to soil type and an irrigation system seems to be advisable, especially for S. officinalis plants.

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PII17 PII18

IN VITRO RESPONSE OF MELISSA OFFICINALIS TO OXIDATIVE STRESS IMPOSED BY OZONE.

USAI GABRIELE1, D'ANGIOLILLO FRANCESCA, TONELLI MARIAGRAZIA1, NALI CRISTINA1, LORENZINI GIACOMO1, PISTELLI LUISA2 AND PISTELLI LAURA1. 1 Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa - Italy. 2 Department of Pharmacy, University of Pisa, via Bonanno 6, 56126 Pisa - Italy.

Keywords: air pollution, lemon balm, tissue culture, ROS, secondary metabolites. Tropospheric O3 is an important air pollutant that causes negative effects on photosynthetic machinery and other crucial processes in plants. A multitude of ROS-generating systems during the O3-induced oxidative burst have been reported; however plants display efficient

enzymatic and non-enzymatic antioxidant systems. Gas exposure of in vitro culture is an easy, cost-effective way of treating plants to air pollutants for experimental purposes. The

present work focuses on testing the ability of in vitro Melissa officinalis to respond to O3 stress (200 ppb, 3 h). No foliar symptoms are observed in fumigated plants. Nevertheless membrane integrity and fast kinetics of chl a fluorescence are significantly affected by O3

(+17% of TBARS levels and -25% of Fv/Fm, respectively). An overproduction of O2- and H2O2 (+31% and +64%, respectively) is also assessed. PAL, CAD and SKDH activity significantly increases (+74, +54 and +90%) twinned to a pronounced increase of total

phenols, lignins and tannins (+45, +46 and +82%). In particular a 3-fold raise of rosmarinic acid concentration is detected. Physiological and pharmacological implications of these findings are worthy of further attention.

PROTECTION EFFECT OF TUBER BORCHII VITT. MYCORRHIZA ON HEAVY METALS TOXICITY IN CISTUS CRETICUS SPP. SABELLA ERIKA, NUTRICATI ELIANA, APRILE ALESSIO AND DE BELLIS LUIGI.

Department of Biological and Environmental Sciences and Technologies, University of

Salento, prov.le Lecce - Monteroni, Lecce, Italy. Keywords: Tuber borchii Vitt., mycorrhizal fungi, heavy metals, Cistus creticus spp., comet assay. Heavy metal contamination of soil is a widely recognized problem. Heavy metals cause toxicity in plants, generally inhibiting normal physiological processes. The major effects

include changes in chloroplast ultrastructure and pigment composition. Heavy metals generally reduce the content of leaf pigments. Mycorrhizal fungi are able to alleviate the stress caused in plants by heavy metal contamination of soil. To investigate the possible effects that mycorrhizal fungus Tuber borchii Vitt. (that is capable to produce valuable ascomata) induces in host plants, mycorrhized/non mycorrhized seedlings of Cistus

creticus spp. were treated with a mixture of heavy metals (Zn, Cr and Pb) and Quantitative

Real-time PCR was performed to analyze changes in expression of genes (DXS, DXR, GGPPS1, GGPPS2, HMGR) coding for protein involved in biosynthetic pathway of leaf pigments. The DNA damage induced by heavy metals was detected using comet assay. Expression analyses indicated that mycorrhizal plants have significantly increased tolerance to toxicity induced by heavy metals compared with non mycorrhizal plants. The comet assay showed that mycorrhization reduced the DNA damage of plants.

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PII19 PII20

THE ROLE OF ß-AMYLASE 1 (BAM1) AND ß-AMYLASE 3 (BAM3) IN STARCH DEGRADATION IN RESPONSE TO MILD OSMOTIC STRESS.

ZANELLA MARTINA1, BORGHI GIANLUCA1, TROST PAOLO1, SANTELIA DIANA2, COSTA ALEX3 AND SPARLA FRANCESCA1. 1 Department of Pharmacy and Biotechnology, University of Bologna, Italy. 2 Institute of Plant Biology, University of Zurich, Switzerland. 3 Departmente of Bioscience, University of Milan, Italy.

Keywords: starch, proline, drought stress. Because of their immobility, plants face various kinds of environmental stresses that limit plant productivity. Overcoming drought and soil salinity is one of the challenges for agriculture. Soluble carbohydrates and proline accumulation play a key role in osmotic

regulation. Starch is the major non-structural carbohydrate in plants and primary starch is

accumulated in green tissues following a daily rhythm. Here we investigated the role of the starch degrading enzymes BAM1 and BAM3 in the response of Arabidopsis plants to mild osmotic stress (150 mM mannitol). BAM1 and BAM3 are hydrolases that release maltose from the non-reducing end of starch. BAM3 is the principal ß-amylase involved starch degradation during the night. BAM1 is a thioredoxin-regulated enzyme, mainly expressed in guard cells under normal growth condition. Analysis of soluble and insoluble sugars, promoter activity, real-time PCR and ß-amylase activity performed on wild type plants and

single mutants lacking either BAM1 or BAM3 demonstrated that in response to mannitol treatments BAM1 replaces BAM3, catalyzing a diurnal starch degradation correlated to an increase of soluble sugars and proline.

UNCOVERING THE ROLE OF GLUTATHIONE IN THE ANTIOXIDATIVE

DEFENSE RESPONSE TO SALT STRESS IN RICE. BERTAZZINI MICHELE AND GIUSEPPE FORLANI.

Department of Life Science and Biotechnology, University of Ferrara, L. Borsari 46, 44121 Ferrara – Italy.

Keywords: rice, salt stress, Reactive Oxygen Species, glutathione, ThioBarbituric Acid Reactive Substances. About 6% of the Earth’s total surface is currently affected by salts, and soil salinity is one of the main factors limiting crop yield. This is particularly true for rice (Oryza sativa L.), a staple food for one half of the world’s population, for which a damage threshold as low as 1.9 dS m-1 electrical conductance has been reported. Plants can cope with moderate

salinity by maintaining a balance between the salt-stimulated production of reactive oxygen species and their scavenging to keep them at signaling level. Because of this, many studies focused to date on the salt-induced activation of antioxidative systems. However

very little is known, if any, concerning Italian rice germplasm. We previously showed the occurrence of a significant variability in the natural tolerance to salt stress among 17 Italian rice cultivars. Here we report on glutathione homeostasis in rice seedlings grown in

the presence of increasing salt concentrations. Both the absolute content and the redox state were influenced by the treatment, and a differential behaviour was found between salt-sensitive and resistant genotypes. This work was supported by Progetto AGER, grant n° 2010-2369.

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PII21

PII22

EVALUATION AND MOLECULAR CHARACTERIZATION OF CORN HYBRID LINES FOR THEIR USE AS BIOMASS CROPS.

VITA F.1, LUCAROTTI V.1, POMPEIANO A.1, KIFERLE C.2 AND ALPI A.1

1 Department of Agriculture, Food and Environment (DAFE), University of Pisa, Via Mariscoglio 34, 56124, Italy. 2 PlantLab, Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy. Keywords: NDF, qPCR, 2-DE, Corn, Brown Midrib.

The corn world production is used primarily for feeding livestock (almost two thirds). Among others, there is a growing use for the production of energy. To evaluate the potential as biomass crop, a set of 6 corn hybrids were selected from CRA, Bergamo. The

analysis were carried out to measure their content in Neutral Detergent Fiber (NDF) and their adaptation to different abiotic stress (cold, salinity, ipoxic) through Proteomic (2-DE) and Trascriptomic (qPCR) approaches. Different protein extraction protocols have been evaluated through 2DE coupled with bioinformatic analysis using specific software

(Progenesis SameSpots). Efforts were focused on assessing performance of key genes expression related to stress response and biomass production. The results showed a different plant adaptation and NDF content in the analyzed lines.

NMR IMAGING, MICROSCOPIC CHARACTERISTICS AND WATER

FLOW OF WHEAT LEAVES MUTAGENIZED FOR WATER BINDING STRENGTH.

RASCIO A.1, RASCIO N.2, DE SANTIS G.1, VALENTINI M.3.

1 CRA- Centro di Ricerca per la Cerealicoltura, S.S.16 Km 675, 71122 Foggia, Italy; 2 Dipartimento di Biologia, Università degli Studi di Padova, viale Colombo 3, 35121, Padova, Italy; 3 CRA- Centro di Ricerca per lo Studio delle Relazioni tra Pianta e Suolo, Centro Strumentale di Tor Mancina, Monterotondo, Roma, Italy. Keywords: Wheat mutant, water binding, NMR imaging, hystochemical, water flow.

Water-biomolecule surfaces interaction is the basis of important physiological processes for the survival of vegetative organs to dehydration stresses. In this poster results of microscopic and functional properties of a water-mutant leaves (Triticum turgidum L. subsp. durum ) that bind water very strongly, are summarized. The mutant had enlarged

extension of the lignified area of schlerenchyma tissue and altered staining of mesophyll pectins. Leaf cross section microscopy and NMR analysis showed greater discontinuities in

T1 spin-density and lower T2 intensity imaging of protons. Mutation affected water biophysical properties and would have inhibited the expansive growth of the cells, rather than the proliferative growth. The micromorphology and hystochemical features of this water-mutant may explain the altered NMR imaging and the lower water flow rate of its leaves.

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PIII01 PIII02

S-STARVATION INDUCES OXIDATIVE BURST IN CHLORELLA SOROKINIANA CELLS.

SALBITANI GIOVANNA, VONA VINCENZA AND CARFAGNA SIMONA. Dipartimento di Biologia, Università di Napoli Federico II, Via Foria 223, I-80139 Napoli, Italy. Keywords: Chlorella sorokiniana, reactive oxygen species, S-deprivation, ascorbate

peroxidase. Plant responses to S-deprivation is well described, but an understanding how plants sense and signal changes (at the cellular or whole-plant level) in the availability of S-nutrient is lacking. Most studies on reactive oxygen species (ROS) have shown that roots undergo an oxidative burst due to deprivation of nitrogen and phosphorus. Direct evidence showing

that ROS are a signalling in S-starved plant cell is not yet available. The involvement of

ROS in S-signalling may be more complex than that of other nutrient deprivation because the ascorbate-glutathione cycle that is downstream of S-assimilation is involved in the removal of H2O2. In the microalgae the imposition of nutrient deprivation can be rapid. As medium become S-depleted, Chlorella sorokiniana cells will not be immediately deficient because of their ability to remobilize sulphur from glutathione. Glutathione pools become depleted after only 2 h of S-starvation; so, we suppose that the anti-oxidative mechanism could be limited by decreased glutathione availability. A significant production of H2O2 and

increase in APX activity in the microalgae Chlorella sorokiniana in conditions of S-deprivation is shown and discussed.

MINERAL NUTRITION IN GRAPEVINE: A TRASCRIPTOMIC

APPROACH. BARIZZA ELISABETTA, TELATIN ANDREA, LO SCHIAVO FIORELLA, VALLE GIORGIO AND ZOTTINI MICHELA.

Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova.

Keywords: Mineral nutrition, Vitis vinifera, potassium deficiency, hydroponic cultures. Mineral nutrition of the grapevine is one of the most important factors affecting the quality of the wine. In particular potassium is essential for grapevine growth and yield, being the most abundant cation in the berries. In fact, it helps to regulate the sugar content and the perfect ripening of berries and improves the taste and the flavor of the wine. However the key role of potassium is shown also on the entire metabolism of the plant. Besides it is

well-known the importance of mineral nutrition on grapevine, however little is known on the molecular mechanisms involved. In this view, we studied the effects of potassium deficiency on growth, development and gene expression in plants of V. vinifera, varieties

Glera, grown in hydroponic cultures. In particular we conducted a physiological and molecular characterization of the stress responses triggered by potassium deficiency.

39


PIII03

EFFECTS OF BORON EXCESS ON GENE EXPRESSION OF BOR AND NIP GENE FAMILIES OF GRAFTED, UNGRAFTED AND SELF GRAFTED

TOMATO PLANTS. APRILE ALESSIO1, NUTRICATI ELIANA1, SABELLA ERIKA1, DI GIOIA FRANCESCO2, SANTAMARIA PIETRO2 AND DE BELLIS1 LUIGI1

1 Dipartimento di Scienze e Tecnologie Ecologiche ed Ambientali, Università del Salento, Prov.le Lecce-Monteroni, Lecce, Italy. 2 Dipartimento di Scienze Agro Ambientali e

Territoriali, Università di Bari, Via Orabona 4, Bari, Italy. Keywords: boron, tomato, gene expression, NIP, BOR. Boron is an essential micronutrient for plants. Understanding the molecular mechanisms of

boron (B) transport has been greatly advanced in the last decade. Plants have evolved an efficient system of boron uptake and transport, using a range of efflux carriers named BOR

proteins (Leaungthitikanchana et al., 2013) and NIP protein, aquaporins identified in Arabidopsis as boric acid channel gene, responsible for the B uptake into roots and essential for B distribution to young leaves (Mlwa et al., 2010). In this work we isolated a cDNA encoding the boron transporter (BOR) and two cDNAs encoding for NIP protein in tomatoes (Solanum lycopersicum L. cv. "Ikram"). The experiment was carried out on ungrafted, self-grafted and grafted onto "Arnold" cv., an inter-specific tomato hybrid rootstock (S. lycopersicum x S. habrochaites), grown at different B concentrations (0, 5,

10, or 15 mg L-1 of B). After 28 days of boron treatments, root tips, distal leaves and leaf petioles were sampled and stored in liquid nitrogen. Three biological replicates were collected. The expression levels were quantified by real-time PCR and described in relation to boron concentrations and rootstocks.

40


PIV01 PIV02

A LIGHT-GATED ION CHANNEL FROM PHOTOTROPIN. COSENTINO CRISTIAN1, GAZZARRINI SABRINA1, ZUCCOLINI PAOLO1, THIEL GERHARD2, ROMANI GIULIA3 AND MORONI ANNA1,3.

1 Dept of Biosciences, University of Milan, Italy. 2 TU-Darmstadt, Germany. 3 Institute of Biophysics, CNR, Italy. Keywords: optogenetics, blue-light, phototropin. Optogenetics is a booming technique that utilizes light-activated microbial proteins for the

remote control of cellular functions [Christie et al, Mol Plant. 2012 5(3):533-44]. We have engineered from scratch a light-activated potassium (K+) channel that will add to the palette of the optogenetic tools. The light-gated channel BLINK (Blue Light Induced K+ channel) was obtained by fusing the LOV2 domain of the plant protein phototropin to the

viral channel Kcv. We selected functional BLINK channels after several rounds of library screening in yeast. A protocol for differential growth (ligh-dark) was adapted to the yeast complementation assay based on ΔTRK, a S. cerevisiae mutant utilized in Kcv studies

[Chatelain et al, PLoS One. 2009 4(10)]. BLINK channels, characterized by heterelogous expression, show a large increase in current (>100%) upon blue light illumination. We present a full characterization of the light-induced currents, including kinetics, dose-response, selectivity and pharmacology. We are presently extending the study to chimeric channels obtained by substituting Kcv with other viral K+ channels displaying improved response kinetics to light.

ARABIDOPSIS P23 CO-CHAPERONE IS INVOLVED IN ROOT

GROWTH. D'ALESSANDRO STEFANO1, ZANIN SOFIA2, RUZZENE MARIA2, ZOTTINI MICHELA1 AND LO SCHIAVO FIORELLA.

1 Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35100, Padova, Italy. 2 Department of Biomedical Science, University of Padova, Via Ugo Bassi 58/B, 35100,

Padova, Italy. Keywords: HSP90, Arabidopsis, nitric oxide, auxin, root. Homologues of the p23 co-chaperone of HSP90 have been found in all eukaryotes, suggesting conserved functions for this protein throughout evolution. While p23 has been well studied in animals, little is known about its function in plants. Arabidopsis owns two

isoforms of p23 and their expression pattern was analysed in planta. The expression profile of the two isoforms was characterized and redundant functions in the analysed pathway identified. In order to determine the function of the two p23 genes, knockout insertional

mutant lines and overexpressing transgenic lines for both genes were selected. The analysis of knockout mutants and overexpressing lines showed these proteins as involved in Nitric oxide production both in physiological and in stress-induced conditions. All these

lines showed alterations in root growth parameters. In order to understand the molecular mechanism underlying this phenotype, different hormone reporters, such as DII-Venus, DR5::GUS and SHY2::YFP, were analysed. The results allow us to suggest an involvement of p23 in hormone regulation through regulation of reactive nitrogen species. Last, kinase CK2 was identified as modulator of p23 function.

41


PIV03

CUPRESSUS ARIZONICA POLLEN SIGNALLING NETWORK TRIGGERS POLLEN TUBE ELONGATION.

EDERLI LUISA1, TEDESCHINI EMMA1, FRENGUELLI GIUSEPPE1, DEL CASINO CECILIA2, APOSTOLI GIULIA2, FALERI CLAUDIA2, CRESTI MAURO2 AND PASQUALINI STEFANIA1

1 Dipartimento di Biologia Applicata, Università di Perugia, 06121 Perugia (Italia). 2Dipartimento di Scienze della Vita, Università di Siena, Siena (Italia). Keywords: nitric oxide, reactive oxygen species, pollen, Cupressus arizonica.

NO and ROS play essential roles in many biotic and abiotic stresses and in plant development procedures, including pollen tube growth. Although the role of these molecules has been well elucidated in the angiosperm pollen, the crosstalk between NO

and ROS still remains to be elucidated in the gymnosperm pollen. Here, NO and ROS localization during tube germination and effect of NO and ROS scavengers/inhibitors on germination and growth and actin filament organization were investigated. Tip and nucleus localized ROS and NO were detected in growing pollen tubes; to determine whether their

endogenous presence is required for pollen tube growth, we incubated pollens with NO quencher (cPTIO)/inhibitor (L-NNA) or with DPI, an inhibitor of NAD(P)H oxidase. ROS and/or NO inhibition dramatically decreased pollen germination and influenced tube elongation: pollens are bifurcated, with diverted growth trajectory or ballooned tip. The assembly of actin was perturbed by chemicals, suggesting that the ROS and/or NO play a critical role for integrity of actin filament which drives pollen tube elongation. A model for

the crosstalk between signalling molecules and actin organization was proposed.

42


PV01 PV02

ROLE OF ENDOPLASMIC RETICULUM CHAPERONE ENDOPLASMIN IN THE SYNTHESIS AND ACTIVITY OF CLAVATA3. KLEIN EVA MARIA1, DE MARCHIS FRANCESCA2, POMPA ANDREA2 AND VITALE ALESSANDRO1. 1 Consiglio Nazionale delle Ricerche; Istituto di Biologia e Biotecnologia Agraria. 2 Consiglio Nazionale delle Ricerche; Istituto di Genetica Vegetale.

Keywords: Endoplasmic Reticulum; chaperone; endoplasmin; Clavata. Regulation of root and shoot apical meristems is tightly regulated by CLAVATA (CLV) genes. These genes encode for three proteins that form an active complex on the cell surface: CLV1 and CLV2 form a plasma membrane receptor complex, activated by an

extracellular short peptide derived from the soluble secretory protein CLV3. Endoplasmin/GRP94 is the endoplasmic reticulum (ER)-located member of the HSP90 protein class, but little is known about its function and its protein clients in plants. It has been shown that mutated Arabidopsis thaliana plants expressing highly reduced amounts of endoplasmin show a Clavata-like phenotype, indicating a role for endoplasmin in the formation of the CLV complex. By transient expression in tobacco protoplasts, we show that the synthesis of a functional fusion between CLV3 and GFP is supported by

endoplasmin and that this support is stronger than the one performed by the chaperone BiP, the ER-located HSP70. This provides biochemical evidence that CLV3 is an endoplasmin client. We also suggest that the proteolytic maturation of CLV3, leading to the formation of the active peptide, starts early in the cell, possibly within the ER.

TRANSMEMBRANE DOMAINS TO ACCUMULATE RECOMBINANT

PROTEINS IN PLANTS. VIRGILI-LOPEZ GORETTI1,2,3, LANGHANS MARKUS2, BUBECK JULIA2, PEDRAZZINI EMANUELA1, GOUZERH GUILLAUME 3, NEUHAUS JEAN-MARC3, ROBINSON DAVID G.2 AND VITALE ALESSANDRO1.

1 Istituto di Biologia e Biotecnologia Agraria, CNR, via Bassini 15, 20133 Milano, Italy. 2 Dept. Plant Cell Biology, Centre for Organismal Studies, University of Heidelberg, D-69120 Heidelberg, Germany. 3 Laboratory of Cell and Molecular Biology, University of Neuchatel, Rue Emile-Argand 11, CH-2000 Neuchatel, Switzerland. Keywords: endomembrane system; protein targeting; HIV p24; transgenic plants; membrane proteins.

Fusion of the cytosolic HIV antigen p24 to transmembrane domains of different lengths and topology were tested to accumulate recombinant proteins in transgenic plants. Fusions to a domain of the maize seed storage protein gamma-zein were also used, as a reference

strategy that leads to very high stability via the formation of protein bodies in the endoplasmic reticulum (ER) lumen. Although in transgenic tobacco all the membrane-anchored constructs accumulated to lower levels compared to the zein fusion, residence at

the ER membrane, either as a type I integral membrane fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments of the secretory pathway. These results extend to the ER membrane previous findings that pointed to the ER lumen as a compartment of choice to produce recombinant proteins in plants. Supported by the EU Framework VI project "PharmaPlanta" (LSHBCT-2003-503565) and the Italian Ministry of Education, Universities and Research

(PRIN2010CSJX4F).

43


PV03 PV04

EXPRESSION OF RECOMBINANT PROTEINS IN TRANSFORMED PLANT CHLOROPLASTS IS STRICTLY REGULATED BY PROTEIN QUALITY CONTROL.

DE MARCHIS FRANCESCA, POMPA ANDREA AND BELLUCCI MICHELE.

Institute of Plant Genetics (IGV), Research Division of Perugia, CNR via Madonna Alta 130, 06128 Perugia, Italy. Keywords: acyl carrier protein, disulfide bonds, phaseolin, signal peptide, thylakoids. Chloroplast DNA (plastome) transformation is a well established method for the expression of recombinant proteins in higher plants. Several evidences indicated that the expression of

genes located in the plastome, both endogenous or heterologous genes, is mainly

regulated at the posttranslational level and recombinant protein stability is the key aspect for heterologous protein accumulation. Many factors have to be considered for successful expression of foreign proteins in plastids, like for example proper folding, posttranslational modifications, and targeting to different organelle compartments. We want to show, as case studies, two examples of foreign soluble proteins expressed in tobacco chloroplasts

and the different strategies adopted to increase their accumulation. One protein to be expressed is the olive acyl carrier protein (ACP), which is a small soluble acidic protein of 12-14 kDa. The accumulation of recombinant small proteins in plastids is empirically known to be very difficult. The other is a bean storage protein, the homotrimeric phaseolin, with a subunit molecular mass of 45 to 50 kDa, whose expression is down-regulated in the chloroplasts.

TRAFFIC REPORT FOR THE ROUTES AND SIGNALS TO THE

TONOPLAST. PEDRAZZINI EMANUELA1, KOMAROVA NATALIYA Y.2, RENTSCH DORIS2 AND VITALE ALESSANDRO1

1 Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Milano, Italy. 2 Institute of Plant Sciences, University of Bern, Bern, Switzerland.

Keywords: endoplasmic reticulum quality control, membrane proteins, protein sorting, plant vacuole, tonoplast, vesicle traffic. Bringing together a relevant amount of information, mostly published in recent years, we have designed a first comprehensive map of the routes for protein sorting and trafficking to the tonoplast, which includes at least five distinct pathways. This traffic involves a number

of checkpoints: (i) approval by the ER quality control, (ii) recognition of ER exit motifs (iii) recognition of motifs that promote sorting to the tonoplast. Recent evidence suggests that the different pathways are protein-specific and could also reflect vacuole specialization for

lytic or storage function. The routes can be distinguished based on their sensitivity to drugs that interfere with specific traffic steps as well as using mutant plants that are defective in adaptor proteins of vesicle coats, or dominant-negative mutants of Rab GTPases. We finally

list the known motifs involved in tonoplast sorting, which most probably promote interactions with specific adaptor proteins of vesicle coats. Supported by the Italian Ministry of Education, Universities and Research (PRIN2010CSJX4F).

44


PV05 PV06

PROTEIN BODY FORMATION IN CEREAL SEEDS AS AN EVOLUTION OF PROTEIN SORTING TO STORAGE VACUOLES. MAINIERI DAVIDE, MORANDINI FRANCESCA, MAITREJEAN MARIE, SACCANI ANDREA, PEDRAZZINI EMANUELA, VITALE ALESSANDRO. Istituto di Biologia e Biotecnologia Agraria, CNR Via Bassini 15 20133 Milano Italy.

Keywords: S seed storage proteins, prolamins, endoplasmic reticulum,vacuol. The seed storage proteins present in all plants accumulate in storage vacuoles. Prolamins, which are the major seed storage proteins in cereals and are present only in these plants, instead accumulate within the endoplasmic reticulum (ER) lumen as very large insoluble polymers held by disulfide bonds, termed protein bodies. The model prolamin gamma-zein

of maize contains seven cysteine residues involved in interchain bonds. We show that progressive substitution of these amino acids with serine residues leads to similarly progressive increase in solubility and availability to traffic from the ER along the secretory pathway. Total substitution results in very efficient secretion, whereas the presence of a single cysteine is sufficient to promote partial sorting to the vacuole via a pathway that is sensitive to brefeldin A and wortmannin, similarly to the normal traffic pathway of vacuolar storage proteins. We propose that the mechanism leading to accumulation of prolamins in

the ER is a further evolutionary step of the one responsible for accumulation in storage vacuoles. Supported by the 2006 Accordo Quadro CNR-Regione Lombardia.

THE SMALLEST PROTEIN DISULFIDE ISOMERASE-LIKE PROTEIN

FROM ARABIDOPSIS THALIANA. CASAZZA ANNA PAOLA, GRASSO ALDO AND ALDO CERIOTTI.

Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Via Bassini 15, 20133 Milano, Italy.

Keywords: endoplasmic reticulum oxidoreductases, oxidative protein folding, protein disulfide isomerases. Thiol-disulfide oxidoreductases are the principal actors of oxidative protein folding in the endoplasmic reticulum (ER). Due to the presence of at least one thioredoxin (TRX) domain containing the catalytic active CXXC motif, these proteins belong to the TRX superfamily. As ER resident proteins they also possess an N-terminal signal sequence and a C-terminal

ER retention signal. The most extensively studied member is PDI, Protein Disulfide Isomerase, which catalyzes the formation, reduction and isomerization of disulfide bonds and assists polypeptide folding. The main differences among the PDI-like (PDIL) proteins

essentially lie in the number and the position of TRX modules, in the active site sequence and in the presence of additional protein domains. To date, only few studies have been performed to understand the physiological role of plant ER oxidoreductases, with the most

documented function being their involvement in seed germination and development. Preliminary results regarding the characterization of the smallest PDIL protein from Arabidopsis thaliana will be presented.

45


PV07 PV08

EXPRESSION OF RECOMBINANT OF A RECOMBINANT XYLANASE IN CHLOROPLASTS OF NICOTIANA TABACUM AND CHLAMYDOMONAS REINHARDTII

LONGONI PAOLO1, PANTALEONI LAURA, ACCOSSATO SONIA1, CELLA RINO1 AND GOLDSCHMIDT-CLERMONT MICHEL2.

1 Department of Biology and Biotechnology, University of Pavia, Pavia, Italy. 2 Departments of Plant Biology and of Molecular Biology, University of Geneva, Geneva, Switzerland. Keywords: Chlamydomonas reinhardti, Nicotiana tabacum, plastome transformation, recombinant enzymes, xylanase.

The setup of new platforms for the production of recombinant enzymes is crucial for the

improvement of biomass-based processes or for new applications. Compare to plants, unicellular green microalgae as cell factories, might have several advantages since they are characterized by a relatively low cost of production and a shorter time span from transformation to production (weeks vs. months). Moreover, the availability of photosynthetic mutants avoids the use of antibiotic-resistance genes. In order to compare

the possibility of chloroplast transformation for molecular farming, a GH 10 xylanase gene was introduced into the plastome of C. reinhardtii and of N. tabacum. Although both systems allowed expressing the protein of interest, we noticed a difference in its accumulation. In fact, accumulation in the microalga appears to be highly dependent on the growth phase, with a higher level of accumulation during the early exponential phase. On the contrary, the same enzyme is stable in tobacco leaves during the entire leaf developmental phase, increasing its amount in old leaves. This suggests that C. reinhardtii

chloroplasts have a more stringent control of protein homeostasis.

COMPARATIVE PROTEOMIC ANALYSIS OF TOMATO FRUIT

MEMBRANE PROTEINS DIFFERENTIALLY EXPRESSED DURING RIPENING.

CLAUDIA FABBRI, FRANCESCO SPINELLI, DANIELA PONTIGGIA, FELICE CERVONE, GIULIA DE LORENZO AND BENEDETTA MATTEI.

Dipartimento di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy. Keywords: Solanum lycopersicum, Fruit ripening, Proteomics, Membrane, Label-free quantification.

Tomato (Solanum lycopersicum) is one of the most important vegetable crops and a model system for fleshy fruit development. A label-free quantitative proteomic approach was performed to identify differentially expressed proteins in the microsomal fraction at two stages of tomato fruit ripening (mature-green, red). Total microsomes were prepared by differential centrifugation, and proteins were analyzed by 1DE-LC-MS/MS. Mass

spectrometric data from three independent replicates were processed in the MaxQuant environment using stringent criteria leading to the identification of 1317 proteins, among

which 583 were quantified by label-free quantitation of ion chromatograms. The data indicate differential expression of several classes of proteins, including those involved in cell wall biogenesis and metabolism, signalling, photosynthesis and energy metabolism, sugar, aminoacid and lipid metabolism, protein synthesis and degradation, protein trafficking and folding, secondary metabolite production, response to stress. Altogether, the data provide new insights on the ripening process in tomato fruit and deepen our

knowledge of the plant membrane proteom.

46


PV09 PV10

PROTEOMIC ANALYSIS OF COLD STRESSED ARABIDOPSIS THALIANA CHLOROPLASTS. LUCAROTTI V.1, VITA F.1, BALLOTTARI M.2 AND ALPI A.1. 1 Department of Agriculture, Food and Environment, University of Pisa. 2 Department of Biotechnologies, University of Verona.

Keywords: chloroplast, cold stress, pigment analysis, proteomics. Low temperature is one of the major abiotic stress limiting productivity and distribution of many species. Cold stress affects chloroplast especially at photosynthetic apparatus level. For this reason our aim is to analyze the change in level of expression of A. thaliana chloroplast proteins during stress. 13-days old plants were acclimatized at 4°C (1 week)

and treated at -10°C (12 h). Freezing treatment produced stress phenotypes characterized by rolling leaves and a decrease in pigments content. Moreover plants exhibited severe oxidative detectable stress. In acclimated plants, which accumulate over twice as much zeaxanthin as the control, the above mentioned parameters were significantly ameliorated. Changes of total proteins in chloroplasts were examined using two-dimensional electrophoresis. Mass spectrometry analysis allowed the identification of differentially expressed proteins, including well-known cold-responsive proteins. Among those,

photosynthetic proteins showed enhanced degradation during freezing stress (Rubisco large subunit and Rubisco activase). Those proteins might work cooperatively to reach an homeostatic equilibrium to overcome stress condition.

ANALYSES OF Ca2+ ACCUMULATION AND DYNAMICS IN THE ENDOPLASMIC RETICULUM OF ARABIDOPSIS THALIANA ROOT

CELLS USING GENETICALLY ENCODED CAMALEON SENSORS. BONZA MARIA CRISTINA1, LORO GIOVANNA1,2, BEHERA SMRUTISANJITA3, WONG ANDREA4, KUDLA JÖRG3 AND COSTA ALEX1. 1 Department of Biosciences, University of Milan, Via G. Celoria 26, 20133 Milan, Italy. 2Department of Biology, University of Padua, Italy, Via U. Bassi 58/B, 35131 Padua, Italy. 3Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 4,

48149 Münster, Germany 4Department of Biomedical Sciences, University of Padua, Padua, Italy Via U. Bassi 58/B, 35131 Padova .

Ca2+ is a versatile second messenger in plants that triggers developmental programs and translates environmental stimuli into specific cellular responses. Cytoplasmic Ca2+ signals are the results of a precised orchestration of Ca2+ influxes and effluxes occurring at

different cellular membranes (e.g. PM, ER and tonoplast). In planta, limited information is

available about how the ER contributes to cellular Ca2+ dynamics and homeostasis. We report the generation of a new ER-targeted Cameleon reporter protein (CRT-D4ER) for suitable analysis of Ca2+ accumulation and dynamic in the lumen of the ER in plant cells. Using stably transformed Arabidopsis plants expressing the CRT-D4ER we observed a transiently enhanced accumulation of Ca2+ in the ER in response to stimuli inducing cytosolic Ca2+ rises in root tip cells. In all experimental conditions ER Ca2+ dynamics were

substantially different from those monitored in the cytosol. A pharmacological approach allowed us to evaluate the contribution of the ER resident Ca2+-ATPase in the regulation of the ER Ca2+ homeostasis. Our results identify a critical role of the ER as a Ca2+ buffering system.

47

Poster Session 6: Plant metabolism

PVI01 PVI02

OVEREXPRESSION OF AN OLIVE ACYL CARRIER PROTEIN (ACP) IN TOBACCO TISSUES.

DE MARCHIS FRANCESCA, POMPA ANDREA, CULTRERA NICOLÒ, MARIOTTI ROBERTO,

ALAGNA FIAMMETTA, BALDONI LUCIANA AND BELLUCCI MICHELE. Institute of Plant Genetics (IGV), Research Division of Perugia, CNR, via Madonna Alta 130,

06128 Perugia, Italy. Keywords: chloroplast transformation, nuclear transformation, fatty acids.

Olive (Olea europaea) is an oil crop which accumulates large amount of triacylglycerols (TAGs) in the fruit mesocarp. Due to the economic relevance of olive oil, the study of the key steps in TAG biosynthesis, including the early step of fatty acid (FA) elongation, are extremely important. Acyl carrier protein (ACP) is the central cofactor protein for fatty acid

synthesis, and recently several olive ACP cDNAs have been isolated and their expression has been characterized in different olive tissues. To clarify the role played by ACP in the

regulation of FA biosynthesis, one olive ACP cDNA has been overexpressed in tobacco plants, in order to alter the levels of ACP and analyze the possible consequences on lipid biosynthesis. Two series of tobacco transformants have been obtained: one formed by transgenic plants with the ACP gene inserted in the nucleus, the other including transplastomic plants harbouring the ACP gene in their plastome. Tobacco transformants expressing high amounts of olive ACP in different tissues have been selected. The phenotypic analysis of these plants is currently in progress.

MECHANISM OF NITROSYLATION AND DENITROSYLATION OF

CYTOSOLIC GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM ARABIDOPSIS.

ZAFFAGNINI MIRKO1, TIOLI GAIA1, ORRU ROBERTO1, MORISSE SAMUEL2, BEDHOMME MARIETTE1,2, MARCHAND CHRISTOPHE H.2, LEMAIRE STÈPHANE D.2 AND TROST PAOLO1.

1 Laboratory of Plant Redox Biology, Department of Pharmacy and Biotechnology, University of Bologna, Via Irnerio 42, 40126 Bologna, Italy. Laboratoire de Biologie Molèculaire et Cellulaire des Eucaryotes, FRE3354 Centre National de la Recherche Scientifique, Universitè Pierre et Marie Curie, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France.

Keywords: denitrosylation, glutathione, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nitrosylation, redox signaling. Nitrosylation is a reversible redox modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms including plants. The extent of nitrosylation of a given cysteine is governed by the equilibrium between nitrosylation and

denitrosylation reactions. Here, we have employed glycolytic GAPDH (GapC) from Arabidopsis as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using several approaches including activity assays, the biotin switch technique, site-directed mutagenesis and mass spectrometry. GapC activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated by GSNO/NO-donors. GSNO was found to trigger both nitrosylation and glutathionylation although nitrosylation was widely

prominent. GapC was found to be denitrosylated by GSH but not by plant cytosolic thioredoxins. GSH fully converted nitrosylated GapC to the reduced, active enzyme, without forming any glutathionylated GapC. Interestingly, GSH-dependent denitrosylation of GapC was found to be linked to the GSH/GSNO ratio and not to the GSH/GSSG ratio. The importance of the redox regulation of Arabidopsis GapC in vivo is discussed.

48


PVI03 PVI04

ENZYMATIC BLEACHING OF HEALTH-PROMOTING CAROTENOIDS IN DURUM WHEAT. GIBERTI SAMUELE AND FORLANI GIUSEPPE. Department of Life Science and Biotechnology, University of Ferrara, L. Borsari 46, 44121 Ferrara – Italy.

Keywords: wheat, carotenoid content, lypoxygenase, co-oxidation, metabolism of hydroperoxydienoic acids. A significant loss of lutein usually occurs in durum wheat (Triticum turgidum L. ssp. durum) semolina during pasta processing, leading to a reduction of key quality parameters including colour and product health value. Carotenoid bleaching is thought to rely on the

activity of lypoxygenases (LOXs, linoleate:oxygen oxidoreductases, EC 1.13.11.12), non-heme iron-containing dioxygenases that catalyse peroxidation of polyunsaturated fatty acids (PUFAs) to form conjugated hydroperoxydienoic acids. LOXs indeed cause in vitro co-oxidation of carotenoids in the presence of PUFAs, and possibly affect phytonutrient content in either fresh vegetables or processed foodstuffs. However, we previously showed that carotenoid bleaching by two LOX isoforms purified from wheat cultured cells proceeds at a much lower rate than linoleate peroxidation. Here we report on the occurrence in

wheat extracts of hydroperoxyde-metabolizing enzyme(s) whose products cause stoichiometric co-oxidation of β-carotene and lutein. This work was supported by MiPAAF in the frame of the ALISAL Project.

CAROTENOID CLEAVAGE DIOXYGENASE 4 IN CHROMOPLASTS OF

WHITE AND YELLOW-FLESHED PEACH GENOTYPES. FORLANI GIUSEPPE AND GIBERTI SAMUELE. Department of Life Science and Biotechnology, University of Ferrara, L. Borsari 46, 44121 Ferrara – Italy.

Keywords: peach, carotenoid content, carotenoid cleavage dioxygenases, activity assay, polyclonal antibodies.

Emerging data suggest a main role of carotenoid-bleaching enzymes in determining the level of these phytonutrients in plant-derived food. In peach, carotenoid accumulation in the mesocarp causes the difference between yellow and white genotypes. The latter are generally characterized by a peculiar and more intense aroma, because of the formation of volatiles deriving from the breakdown of the carotenoid skeleton. Dioxygenases appear to be key factors causing volatile release in fruits, and a differential expression of carotenoid

cleavage dioxygenase (CCD) 4 gene was found in yellow vs. white-fleshed isogenic peach

genotypes. However, despite gene cloning and heterologous expression of the protein from Arabidopsis, apple and safran, a direct experimental evidence supporting the ability of CCD4 to cleave β-carotene is still missing. We succeeded in setting up conditions for in vitro measurement of β-carotene bleaching by chromoplasts isolated from peach fruit. In order to link activity with the presence of CCD4, polyclonal antibodies were raised against

synthetic peptides corresponding to conserved protein regions. This work was supported by MiPAAF in the frame of the ALISAL Project.

49


PVI05 PVI06

FRUCTANS DETERMINATION AND IDENTIFICATION IN SOME TYPICAL SOUTHERN ITALY EDIBLE PLANTS, AND THEIR POSSIBLE ROLE AS PREBIOTICS. MARCO LENTINI, MANUELA CARDI AND SERGIO ESPOSITO. Dipartimento di Biologia - Università di Napoli "Federico II" - Complesso Universitario di Monte Sant'Angelo - Via Cinthia - 80129 Napoli - ITALY.

Keywords: Fructoligosaccharides, FOS, Prebiotics, Probiotics, inulin. Fructans are a complex class of biological molecules, where one or more fructosyl-fructose bonds constitutes a majority of the linkages. Generally they are defined as polymeric material or oligomers as small as the disaccharide inulobiose; these macromolecules may

or may not contain glucosyl substituents. Fructans are widely distributed in the plant kingdom, being present in monocotyledons, dicotyledons and in green algae. Fructans differ in molecular structure and in molecular weight so may be classified into three groups: the inulins, the levans and the branched fructans. In this work we measured the occurrence of total fructans in some edible species of Southern Italy, namely Campania Region. Fructans were extracted, isolated and identified from asparagus (Asparagus officinalis), quince (golden apple, Cydonia oblonga), onion (Allium cepa) and other typical

plants used in traditional southern Italy cousin. These molecules can be tested on different bacterial strains in order to establish their positive effects as prebiotics on human gut probiotic flora. Possible applications of a balanced diet and /or fructans to improve the positive effects on human health are discussed.

ENZYMATIC CHARACTERIZATION AND PROPOSAL FOR A

CATALYTIC MECHANISM OF A RECOMBINANT PLASTIDIAL GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM BARLEY

(HORDEUM VULGARE CV. NURE) ROOTS. CARDI MANUELA, PIZZO ELIO AND ESPOSITO SERGIO.

Dipartimento di Biologia – Università di Napoli "Federico II" – Complesso Universitario di Monte Sant'Angelo - Via Cinthia - 80126 Napoli – ITALY. Keywords: OPPP, glucose-6-phosphate dehydrogenase, barley roots.

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, as both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity, it has been difficult to describe accurately the properties of these isoforms. An extensive comparative bioinformatic analysis has carried out to identify the P2-G6PDH

encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long

plastidial transit peptide. The recombinant P2-G6PDH shows moderate stability, allowing a complete kinetic analysis versus different substrates and artificial reductants. Intriguingly, all results collected agree with values known for other P2-G6PDH previously purified from barley, and determined for other plant P2-type G6PDHs. Overall, these data shed light on the catalytic mechanism, summarized in a proposed model in which the sequential mechanism is very similar to mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further

kinetic and structural studies of this peculiar plant isoform.

50


PVI07 PVI08

DISTINCT REGULATION OF TWO DURUM WHEAT SECRETORY PHOSPHOLIPASES A2 UNDER DIFFERENT METABOLIC CONDITIONS. VERLOTTA ANGELO AND TRONO DANIELA. Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per la Cerealicoltura, S.S. 16, Km 675 - 71122 Foggia, Italy.

Keywords: Durum wheat, Phospholipase A2, regulation of gene expression, modulation of enzymatic activity. Phospholipase A2 (PLA2) hydrolyses membrane phospholipids to yield free fatty acids and 1-acyl-2-lysophospholipids, that are known to have a role in plant growth, development

and stress responses. Two genes encoding putative secretory PLA2s (sPLA2s) were isolated from durum wheat and designated as TdsPLA2II and TdsPLA2III in analogy with the corresponding genes in rice. TdsPLA2III gene was up-regulated in durum wheat seedlings grown under cold, mannitol, salt, heavy metal stress and by herbicide treatment; contrarily, TdsPLA2II gene was constitutively expressed under all of the conditions examined. Moreover, the activity of the recombinant TdsPLA2II and TdsPLA2III isoforms reached a plateau, respectively, at micromolar and millimolar Ca2+ concentrations, thus

suggesting that the former may be fully activated when secreted, while the latter is likely to be only partially activated depending on the specific Ca2+ levels, that are known to be modulated under stress conditions. Altogether these results suggest a constitutive role for TdsPLA2II and an involvement of TdsPLA2III in plant cell response to adverse conditions through a regulation at both molecular and biochemical level.

MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF THE

TOMATO R2R3-MYB PROTEINS AN2 AND ANT1 PLAYING A REGULATORY FUNCTION IN THE ANTHOCYANIN SYNTHESIS. BASSOLINO LAURA1, GONZALI SILVIA1, POVERO GIOVANNI1, KIFERLE CLAUDIA1 , QUATTROCCHIO FRANCESCA2, KOES RONALD2, SPELT CORNELIS2, FANTINI ELIO3, GIULIANO GIOVANNI3 AND PERATA PIERDOMENICO1. 1 Plant Lab, Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy. 2 Vrije Universiteit Amsterdam, De Boelelaan 1085-1083, 1081 HV Amsterdam,

Netherlands. 3 Utagri, Enea C.R. Casaccia, via Anguillarese, 301, 00123 Roma, Italy. Keywords: anthocyanin; transcription factors; Solanum lycopersicum; AN2; ANT1. Anthocyanins are water-soluble polyphenolic compounds with a high nutraceutical value. Despite the fact that the cultivated tomato does not synthesize anthocyanins, it has been previously demonstrated that the biosynthetic pathway is fully present in the fruits of this

plant. However little is known about the regulatory mechanisms involved in anthocyanin

synthesis in tomato. In this work, we carried out a molecular and functional characterization of two genes, SlAN2 and SlANT1, encoding two R2R3-MYB transcription factors and we showed that are both positive regulatory proteins of anthocyanin synthesis in tomato fruits or vegetative tissues. In addition, we isolated a gene (SlAN1) encoding a bHLH TF, and a gene encoding a WD40 (SlAN11) protein, which are both homologs of

Petunia genes involved in anthocyanins regulatory network. We suggested that the two MYBs, AN2 and ANT1, and the others proteins here identified, AN1 and AN11, are components of MYB-bHLH-WD40 complexes involved in the regulation of anthocyanin pathway in different tomato tissues.

51


PVI09 PVI10

PROFILE OF THE ENZYMES INVOLVED IN FRUCTAN SYNTHESIS AND HYDROLYSIS DURING DURUM WHEAT KERNEL MATURATION.

LOCATO V.1, CIMINI S.1, PARADISO A.2, D'EGIDIO M.G.3, VAN DEN ENDE W.4 AND DE GARA L.1 1 CIR, Università Campus Bio-Medico University, Rome, Italy. 2 Department of Biology, University of Bari, Bari, Italy. 3 Experimental Institute of Cerealcoltures, Rome, Italy. 4 KU Leuven, Heverlee, Belgium.

Keywords: durum wheat, kernel maturation, fructan metabolism, nutritional value, functional food. Wheat has been recently suggested as an interesting source of fructans, mainly of graminan type containing β-(2,1) and β-(2,6) linkages. We showed that fructan level is

higher in the kernels during the first period of maturation (15-20% of kernel dry weight in

milky phase) while it only reaches 2% of kernel dry weight at the end of the process.How fructan biosynthesis and degradation are regulated during kernel maturation is still unknown. Here, the metabolism of fructans has been studied during kernel maturation of Triticum durum cv Neolatino. The kernels were collected at different stages of maturation (from 7 days after anthesis to harvesting). Changes in fructan content, activity and gene expression of the main enzymes involved in their biosynthesis and hydrolisis were analyzed. The enzymes involved in fructan biosynthesis had the highest activities in the

first 20 days after anthesis when fructans were accumulated. These results increase the knowledge of fructan metabolism in wheat kernels and the identification of maturation stages in which kernels have the highest nutritional value in terms of fructan level can have interesting implications in functional food production.

IN VIVO AND IN VITRO PROPAGATION OF SALVIA SPECIES FOR

THE PRODUCTION OF BIOACTIVE METABOLITES. BASSOLINO LAURA1, GIACOMELLI EMANUELA2, BISIO ANGELA3 AND RUFFONI BARBARA1.

1 CRA-FSO Sanremo. 2 Università di Genova - Dipartimento di Medicina Sperimentale (DIMES). 3 Università di Genova - Dipartimento di Farmacia (DIFAR).

Keywords: Salvia, diterpenoids, vivo and vitro propagation. Plant exudates derived from the secretory structures of cultivated Salvia species are a valuable source of biologically active metabolites, such as flavonoids, tannins and terpenoids that have been widely used by traditional medicine. Because of the potential pharmacological use, the aim of this project is to increase the relative concentration of diterpenoids compounds in planta through a molecular farming approach. In this study we

developed in vivo and in vitro propagation protocols for S. cinnabarina and S. haenkey, to enhance biomass yield and to assess qualitative and quantitative diterpenoid accumulation. Our data showed that different Salvia spp. have specific responses to sterilization

procedure and media hormonal composition. Further, key enzymes leading to diterpene production will be over-expressed in sage to metabolically engineer their biosynthetic pathway.

52


PVI11

POLYPHENOLS DISTRIBUTION IN FRUIT OF SWEET CHERRY LANDRACES OF CAMPANIA REGION (ITALY).

IANNUZZI FEDERICA, MIRTO ANTONIO, WOODROW PASQUALINA, CARILLO PETRONIA AND FUGGI AMODIO. Dept. Environmental Biological and Farmaceutical Sciences and Technologies, Second University of Naples.

Keywords: Antocianins, free amino acids, sugars, Prunus avium L. Polyphenols have been recognized to have an important role in the prevention of various illness associated with oxidative stress, such as cancer and cardiovascular and neurodegenerative diseases. They are among the most abundant micronutrient in the human diet, mainly in the Mediterranean one, being fruits and vegetables the main source

of such nutrients. Polyphenol concentrations in plant organs vary according to species and

numerous genetic and environmental factors. South Italy is a large source of fruit biodiversity in which to look for polyphenols rich plant varieties. In this view the distribution of polyphenols and other metabolites have been determined in fruits of about forty landraces of sweet cherry germoplasm cultivated in the "CRAA-Azienda Agricola sperimentale Improsta" of Campania Region. The landraces differed for their polyphenol contents and distribution patterns. Such data, togheter with their sugar and free aminoacid contents, also allowed to improve their overall characterization. Financial support was

obtained by "Regione Campania - Italy, PSR 2007/2013, Misura 214, Azione f2, progetto Agrigenet".

53

Poster Session 7: Plant biotechnology

PVII01

PVII02

NITROGEN LIMITATION RESULTS IN DE NOVO BIOSYNTHESIS OF TAGS AND REORGANIZATION OF THE PHOTOSYNTHETIC APPARATUS IN THE MICROALGA NANNOCHLOROPSIS GADITANA.

SIMIONATO DIANA1; BLOCK MARYSE A.2,3,4,5; LA ROCCA NICOLETTA1; JOUHET JULIETTE2,3,4,5; MARÈCHAL ERIC2,3,4,5; FINAZZI GIOVANNI2,3,4,5; MOROSINOTTO TOMAS1.

1 Dipartimento di Biologia Università di Padova, Padua, Italy. 2 Laboratoire de Physiologie Cellulaire et Vègètale, Unitè Mixte Recherche 5168, Grenoble, France. 3 Centre National Recherche Scientifique, Universitè Grenoble 1, Grenoble, France. 4 Institut National Recherche Agronomique, Grenoble, France 5 Commissariat à l'Energie Atomique et Energies Alternatives, Institut de Recherches en Technologies et Sciences pour le Vivant,

Grenoble, France.

Keywords: Nannochloropsis, biodiesel, photosynthesis, lipids biosynthesis, microalgae electron transport.

Exploiting oleaginous microalgae of the genus Nannochloropsis for biodiesel production is an

interesting perspective as new energy source. However, several studies are still needed to make the process competitive. One of the major aspects to consider is the optimization of algae light use efficiency. Light, in fact, provides energy supporting algae growth and must be exploited with the highest possible efficiency to achieve sufficient and sustainable productivity and make their cultivation competitive. We here investigated the connection between

photosynthesis and lipids biosynthesis by analyzing N. gaditana exposed to nitrogen (N) limitation. Cells showed an extensive accumulation of triacylglycerols, that were mainly originated from freshly synthesized fatty acids with a smaller fraction coming from the recycling of membrane glycerolipids. This intense fatty acids biosynthesis was shown to depend from the photosynthetic activity. Also, photosynthetic apparatus was demonstrated to respond to N starvation by decreasing linear electron transport and inducing cyclic mode and this response

allowed cells to maintain enough photosynthetic activity to sustain fatty acids biosynthesis.

CHARACTERIZATION OF THE PHOTOSYNTHETIC APPARATUS FROM

THE EUSTIGMATOPHYCEA NANNOCHLOROPSIS GADITANA: EVIDENCES OF CONVERGENT EVOLUTION IN PHOTOSYSTEM I

SUPRAMOLECULAR ORGANIZATION. STEFANIA BASSO, DIANA SIMIONATO, CATERINA GEROTTO, ANNA SEGALLA, GIORGIO M. GIACOMETTI AND TOMAS MOROSINOTTO.

Dipartimento di Biologia - Università degli Studi Padova, via U. Bassi 58 B 35131 Padova Italy. Keywords: biofuels, photosystem I, Nannochloropsis, photosynthetic apparatus. Nitrosylation is a reversible redox modification of protein cysteines playing a major role in Nannochloropsis gaditana is a seawater microalga belonging to Eustigmatophyceae which

is receiving a growing attention thanks to a possible application in biofuels production. Nannochloropsis species have a particular photosynthetic apparatus with the presence of only Chlorophyll a and with violaxanthin and vaucheriaxanthin esters as the major

carotenoids. We characterized the photosynthetic apparatus of this species by purifying thylakoid membranes and isolating different pigment binding complexes upon mild detergent solubilization. Results from the biochemical and spectroscopic analyses showed

that Photosystem II antennas are loosely associated to the reaction center, while in Photosystem I the association is stronger and antenna-reaction center super-complexes survive purification. Sequence analysis of genes encoding for light harvesting complexes in Nannochloropsis and its comparison with other photosynthetic eukaryotes suggest this is not the result of subunits conservation but rather due to a convergent evolution. Hypotheses on the selective advantage in having a different structural organization of PSI / PSII antenna complexes will be proposed.

54


PVII03

PVII04

EXPRESSION OF OSTEOGENIC FACTORS IN TRANSGENIC TOBACCO PLANTS.

CERESOLI VALENTINA,1,2, MAINIERI DAVIDE1, DEL FABBRO MASSIMO2 AND PEDRAZZINI

EMANUELA1. 1 Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Milano, Italy. 2 Centro di Ricerca per la Salute Orale, Dip. Scienze Biomediche, Chirurgiche e Odontoiatriche UniversitÃ degli Studi di Milano, Istituto Ortopedico Galeazzi, Milano, Italy.

Keywords: bone morphogenetic proteins, osteogenesis, plant made pharmaceuticals, molecular farming, recombinant proteins. Bone morphogenetic protein 2 (BMP2), a secreted dimer belonging to the TGF-beta superfamily, stimulates bone formation. Recombinant human BMP2 (rhBMP2) synthesized

in E.coli or CHO cells is successfully used in orthopedics, but its production is very expensive. Our purpose is to develop new strategies to obtain functional rhBMP2 in

transgenic plants that can be accepted as a safe and effective system to produce pharmaceuticals. To increase accumulation of rhBMP2, we have produced a fusion (termed ER-BMP2) between the last 114 amino acids of BMP2, corresponding to the active peptide, and the N-terminal domain of maize gamma-zein, which promotes high protein accumulation within the Endoplasmic Reticulum (ER) lumen via assembly into protein bodies. A thrombin cleavage site was also inserted, to allow the isolation of pure BMP2 peptide. The accumulation level of ER-BMP2 was compared with that of native BMP2 (N-

BMP2) by transient expression in tobacco protoplasts. ER-BMP2 accumulated in the ER at higher levels than N-BMP2. Unexpectedly, the latter was not secreted and was probably targeted to the vacuole, where it was degraded. Production of transgenic plants is in progress.

THE NEW LOX/RNO METHOD ALLOWS MEASUREMENT OF HIGH

ANTIOXIDANT ACTIVITY OF FREE AND BOUND PHENOLS IN SEEDS OF CEREAL SPECIES.

LAUS MAURA N., SOCCIO MARIO AND PASTORE DONATO. Department of Agriculture, Food and Environmental Sciences, University of Foggia, Via Napoli, 25 – 71122 FOGGIA.

Keywords: Antioxidant activity, soybean lipoxygenase, phenolic compounds, cereals, quinoa. The new LOX/RNO method for measuring antioxidant activity (AA) is based on the secondary reaction between the soybean lipoxygenase (LOX)-1 isoenzyme and 4-nitroso-N,N-dimethylaniline (RNO). In particular, cooxidation of RNO due to some physiological

radical species produced by the enzyme may be followed at 440 nm and may be prevented by antioxidants. The advantage of the LOX/RNO method is to simultaneously detect different antioxidant mechanisms thus highlighting high AA and synergism among

phytochemicals. In this study, the new method was used to evaluate AA of free-soluble (FSP) and insoluble-bound (IBP) phenolic extracts from whole grains of six different cereal species and from seeds of the pseudocereal quinoa. All the tested species showed high

total (FSP+IBP) AA values ranging from about 300 to 2300 Âµmol Trolox/g dry whole flour; in particular, AA was higher in the IBP extracts, thus showing predominance of the phenolic fraction bound to insoluble polymers of cell wall. As for the ratio AAFSP/AAIBP, that may be of interest for nutritional purposes, interestingly, data resulted not related to total AA and to vary remarkably up to 20 times among the different tested species.

55


PVII05

PVII06

THE REACTION BETWEEN SOYBEAN LIPOXYGENASE–1 ISOENZYME AND FLLUORESCEINE AS A NOVEL TOOL TO ASSESS ANTIOXIDANT ACTIVITY IN FOODS. SOCCIO MARIO, LAUS MAURA N. AND PASTORE DONATO. Department of Agriculture, Food and Environmental Sciences, University of Foggia, Via Napoli, 25 – 71122 FOGGIA.

Keywords: Soybean lipoxygenase-1 isoenzyme, fluorescein, antioxidant activity, ORAC. Fluorescein (FL) is the probe utilized by the Oxygen Radical Antioxidant Capacity (ORAC) assay widely used to assess antioxidant activity (AA). In this study, an improved ORAC assay (ORACLOX) was developed based on the enzymatic reaction between soybean

lipoxygenase (LOX)-1 isoenzyme and FL in the presence of linoleic acid (LA). Hydroperoxidation of LA is coupled to anaerobic secondary reactions generating some physiological radicals, including LOO•, LO•, OH• and Âand Â¹O2, that we observed to quench FL fluorescence. Kinetics of LOX/FL reaction were studied with Vmax and Km obtained equal to 0.28 ΔA485/min and 1.53 μM, respectively. In this ORACLOX assay, antioxidant compounds were found to induce a decrease of FL quenching rate and calibration was carried out using Trolox as standard antioxidant. The assay resulted

suitable to evaluate AA of dietary phenolic compounds and, in the light of the capability to simultaneously detect many antioxidant functions, it resulted able to highlight synergism among phenols from different sources. The method is also able to reveal increase of AA in the blood after dietary phenols ingestion.

PRODUCTION OF CYTOTOXIC METABOLITES IN BITUMINARIA

BITUMINOSA L IN VITRO CULTURES. D'ANGIOLILLO FRANCESCA1, NOCCIOLI CECILIA2, RUFFONI BARBARA3, PIAGGI SIMONA4, SCARPATO ROBERTO4, PISTELLI LAURA1, PISTELLI LUISA2.

1 Department of Agriculture, Food and Environment, University of Pisa, via del Borghetto 80, 56124 Pisa - Italy. 2 Department of Pharmacy, University of Pisa, via Bonanno 6, 56126

Pisa - Italy. 3 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, CRA FSO Ornamental Plants Research Unit , corso degli Inglesi 508, 18038 Sanremo (IM) – Italy. 4 Department of Biology, University of Pisa, via Derna 1, 56126 Pisa - Italy. Keywords: Bituminaria bituminosa - in vitro cultures - HPLC-DAD –cytotoxicity test - pterocarpans.

Bituminaria bituminosa (L.) C.H. Stirt. Fabaceae) is mediterranean perennial species characterized by a strong smell of bitumen. The plant is used for different purposes: as hay or forage, as phytostabilizers, and as producers of several compounds with considerable

pharmaceutical interest (flavonoids, furanocoumarins, and pterocarpans). The aim of the present work (supported by the Interreg IT-FR Marittimo Project "PYRGI") was to evaluate the secondary metabolites produced by in vitro shoots, roots, callus and also hairy roots.

The profile of the different plant extracts was evaluated by HPLC-DAD, and then also tested for the cytotoxic activity in HeLa cell culture. All extracts contained the isoflavone daidzein, while the pterocarpans erybraedin C and bitucarpin A were found only in the extracts of the in vitro shoots and roots. In the cytotoxicity assay the majority of the extracts tested showed a dose-dependent decrease in cell survival. The highest level of cytotoxicity was found in 40-days-old in vitro shoot and root extracts, while it was very low in hairy roots. The phytocomplexes seems to have different cytotoxic properties then the single isolated

compounds.

56


PVII07

RHIZOSECRETION OF RECOMBINANT ANTIBODIES BY TRANSGENIC TOBACCO PLANTS.

BARTOLUCCI ALESSANDRA, MAGGIO CATERINA, GUARNIERI FRANCESCO, BOEDDU PAOLA, CELLA RINO. Department of Biology and Biotechnology, University of Pavia, Pavia, Italy. Keywords: molecular farming, nuclear trasformation, rhizosecretion, recombinant

antibodies, tobacco. Plants have been shown to be suitable host for producing recombinant proteins, due to the ease of genetic transformation and to the simplicity of cultivation. Although several proteins were produced and targeted to different subcellular compartments (cytoplasm, endoplasmic reticulum or apoplast, their extraction and purification is expensive on a large-

scale. On the contrary, root secretion can be a successful option for the following reasons:

a) lower biochemical complexity of the secretome, b) continuous production of recombinant proteins in hydroponic cultures, a process named "rhizosecretion". Following the successful production of transgenic tobacco plants producing bacterial and fungal cellulases, we decided to apply the same strategy to produce human scFvs (functional antibody fragments) of pharmaceutical interest. The scFv used is able to recognize and to inactivate a membrane receptor involved in a signaling pathway that induces tumor formation. We obtained a total of 154 independent transgenic lines, which are currently under screening.

The results reached so far indicate that plants express the recombinant protein and that the antibody is still able to bind the antigen.

57

Poster Session 8: Plant development

PVIII01

PVIII02

STUDY OF THE ROLE OF A PUTATIVE ACETYLORNITHINE DEACETYLASE ON FRUIT DEVELOPMENT IN ARABIDOPSIS THALIANA.

MOLESINI B. AND PANDOLFINI T.

Department of Biotechnology, University of Verona, Strada Le Grazie, 15 37134 Verona. Keywords: fruit development, Arabidopsis thaliana, polyamine, arginine. Comparative transcriptomic analysis carried out on preanthesis parthenocarpic and wild-type tomato flower buds permitted the identification of 260 differentially expressed cDNA clones. One of these parthenocarpy-related genes was annotated as a putative N-acetylornithine

deacetylase (NAOD). NAOD catalyzes the terminal step of ornithine (Orn) biosynthetic

pathway. Orn is a precursor of Arginine (Arg); Orn and Arg, are involved in polyamine biosynthesis. The aim of this work is to investigate the role of this gene in fruit development. We identified a putative NAOD gene in Arabidopsis thaliana. To study its biological role, we have generated silenced lines by transforming Arabidopsis plants with a hairpin construct and searched for T-DNA null mutants. We obtained two silenced lines that showed approximately

90% reduction in NAOD transcript level in comparison with wild type. The phenotypical analysis of these lines revealed a reduced growth and a lower number of siliques in comparison with control plants. A T-DNA insertional line was obtained from the SALK collection. The expression of the gene was almost completely abolished in the homozygous mutant line.

THE FOUR LYSINE-SPECIFIC HISTONE DEMETHYLASES OF

ARABIDOPSIS DIFFERENTIALLY CONTRIBUTE TO THE CONTROL OF FLOWERING TIME AND DEFENCE RESPONSES. MARTIGNAGO D., ALABDALLAH O., GHUGE S., ANGELINI R., TAVLADORAKI P. Department of Science, University "Roma Tre", Viale Marconi 446, 00146 Roma, Italy. Keywords: Flowering, stress, epigenetics, histone demethylases.

Individual histone modifications, or a specific combination of them, act as signals ('histone code') that are 'read' by other proteins able to influence chromatin structure and gene expression. Among them, histone methylation is an important epigenetic modification controlled by a balance between methylation and demethylation. In Arabidopsis, four lysine-specific histone demethylases (AtLSDs) are present. To determine their physiological roles, loss-of-function mutants and 35S::AtLSD transgenic plants were obtained and analysed under physiological and stress conditions. Furthermore, to determine the AtLSD

specific targets, the expression levels of flowering regulators, as well of senescence and

stress marker genes were assessed in the transgenic plants. The expression pattern of the four AtLSDs was also analysed in detail by histochemical staining of beta-glucuronidase (GUS) in AtLSD::GUS transgenic plants. Data indicate involvement of the various AtLSDs in the control of flowering time, as well as in defence responses to salt stress. Overall, this study highlights functional similarities, but also important differences among the four

members of the AtLSD gene family.

58


PVIII03

ROLE OF PROLINE IN GAMETOPHYTE DEVELOPMENT. LONOCE C.1, BIANCUCCI M.1, RECCO I.1, FRUGIS G.2, MATTIOLI R.1, COSTANTINO P.1, AND TROVATO M. 1

1 Dipartimento di Biologia e Biotecnologie, Sapienza Università di Roma, Italy. 2 CNR - Istituto di Biologia e Biotecnologia Agraria (IBBA), Monterotondo Scalo, Italy. Keywords: Proline, Pollen, P5CS. We recently found that in Arabidopsis mutants homozygous for p5cs1 and heterozygous for

p5cs2 pollen grains carrying mutations in both p5cs1 and p5cs2 are sterile and hampered in normal development. These pollen grains appear wrinkled, shriveled and lacking nuclei. Because p5cs1 and p5cs2 encode the rate–limiting enzyme of prooline synthesis in plants, these data suggest that proline is required for pollen development and viability.

Accordingly, we detected only traces of proline in aberrant pollen grains, and were able to complement the functional and developmental defect of mutant pollen with exogenous proline. However, it is not clear, to date, the role of proline in pollen development and

function. As a step forward in characterizing the role of proline in pollen development, we are studying the relative expression of the genes involved in proline synthesis and degradation in anther development. In addition, to address the problem of the relative contribute of proline synthesis in sporophytic tissues compared to gametophytic tissues, we are preparing proline synthesis mutants expressing p5cs2 (or RNAi-p5cs2) either in sporophytic or gametophytic tissues.

59

Late submissions

PIX01 PIX02

ANALYSIS OF THE CROSS-TALK BETWEEN MAIZE AND FUSARIUM VERTICILLIOIDES REVEALED BY RNA-SEQUENCING. LANUBILE A.1, MASCHIETTO V.1, FALASCONI I.1, BELLIN D.2, FERRARINI A.2, DELLEDONNE M.2 AND MAROCCO A.1 1 Institute of Agronomy, Genetics and Crop Sciences, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29100 Piacenza, Italy, 2 Department of Biotechnology , University

of Verona, Strada le Grazie 15, 37134 Verona, Italy. Keywords: RNA-Seq, Fusarium ear rot, candidate genes, Zea mays.

Fusarium verticillioides is a plant pathogen able to produce fumonisin in maize kernels. To clarify the molecular processes undergoing in maize upon infection, the expression profiles of resistant and susceptible genotypes after F. verticillioides inoculation have been

characterized using RNA-Seq technology. More than 100 million sequence reads were generated for condition (inoculated/uninoculated). The sequence reads were analyzed to measure gene expression levels, to detect alternative splicing events and single nucleotide polymorphisms. We observed 2,296 and 2,578 differentially expressed genes (DEGs) 72 hours after inoculation for the resistant and susceptible genotypes, respectively, of which 1018 were in common and showed 5,342 SNPs variants. About 320,000 and 175,000 reads mapped on Fusarium genome in the susceptible and resistant genotypes, respectively, and

129 fungal genes were differentially regulated in both lines. The identification of plant DEGs that interact with fungus will produce useful tools for the identification of candidate genes, the development of molecular markers and their use for selection of resistant maize genotypes by means of marker assisted selection.

THE PLANT VACUOLE: A NOVEL HETEROLOGOUS SYSTEM FOR

INVESTIGATING THE FUNCTIONAL PROPERTIES OF INTRACELLULAR ANIMAL CHANNELS AND TRANSPORTERS.

SCHOLZ-STARKE JOACHIM1, BOCCACCIO ANNA1, COSTA ALEX2, FESTA MARGHERITA3 AND CARPANETO ARMANDO1.

1 Institute of Biophysics – CNR, Via De Marini 6 16149-Genova, Italy. 2 Department of Biosciences, University of Milan, Via Celoria 26, 20133-Milano, Italy. 3 Department of Pharmacy and BioTechnology (FaBiT), Alma Mater Studiorum - University of Bologna, Via Irnerio 42, 40126-Bologna, Italy.

Animal intracellular transporters and channels play pivotal role in many cellular processes and they are involved in important human genetic diseases. However, this large class of proteins is still largely unexplored and there is a lack of knowledge on their basic properties such as voltage dependence, selectivity, turnover rate, single channel conductance and

pharmacology. This situation is mainly due to the impossibility to directly apply standard electrophysiological techniques to intracellular organelles from animal cells because of their

small size. Here we propose the use of plant vacuoles as a convenient expression system for intracellular channels and transporters. Plant vacuoles are easy to isolate and because of their large dimension (up to 40 μm diameter) they can be successfully and conveniently studied using the patch-clamp technique. We have previously characterized a mammalian chloride/proton antiporter (CLC-7, see Costa et al., 2012, J. Phys. 590:3421-30). In this context we present data on the functional properties of the lysosomal human channel TPC2

and compare with the plant homologue TPC1. Supported by MIUR project PRIN2010CSJX4F.

60

Late submissions

PIX03 PIX04

THE INVOLVEMENT OF TWO PLASTIDIAL ARABIDOPSIS THALIANA ABC1 KINASES IN IRON DISTRIBUTION AND OXIDATIVE STRESS RESPONSE.

MANARA A., DALCORSO G. AND FURINI A.

Dipartimento di Biotecnologie, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona. Keywords: Abc1 kinases, chloroplast, iron, oxidative stress. The Abc1 protein kinases are a large family of proteins involved in respiration in bacteria and mitochondria, even though, knowledge about their putative functions in chloroplasts is

still limited. We carried out a functional characterization of AtSIA1, a chloroplast

Arabidopsis thaliana Abc1-like protein, focusing on its potential redundancy with its homolog AtOSA1: A. thaliana knock-out mutant lines for AtSIA1 and AtOSA1 were considered and the double mutant was created. A reduction in content of plastidial iron and in the abundance of components of the Cytb6f complex were observed in mutants, suggesting that both AtSIA1 and AtOSA1 affect iron distribution within the chloroplast.

Moreover, mutant plants accumulate more ferritin and superoxide than wild type plants, and their enhanced sensitivity to oxidative stress was supported by the upregulation of the antioxidant networks. Mutants produce altered levels of prenylquinones, but only atsia1 plants develop larger plastoglobules, containing higher levels of VTE1. Taken together, these data suggest that AtSIA1 and AtOSA1 may act as protein kinases in signaling pathways that influence plastidial responses to oxidative stress.

DIVERSE CIS-REGULATION OF A VACUOLAR METAL TRANSPORTER

IN ARABIDOPSIS THALIANA AND ARABIDOPSIS HALLERI. FASANI E.1, DALCORSO G.1, VAROTTO C.2, LI M.2 AND FURINI A.1

1 Department of Biotechnology, University of Verona, Strada Le Grazie 15, 37134 Verona. 2 EcoGenomics Group, Edmund Mach Foundation, Via Mach 1, 38010 San Michele all'Adige (TN). Keywords: metal hyperaccumulation, VMT, expression, promoter, GUS.

The vacuolar metal transporter VMT is involved in metal hyperaccumulation. VMT is expressed at higher levels in metal hyperaccumulators Arabidopsis halleri and Noccaea caerulescens than in non-accumulator species, such as Arabidopsis thaliana. To gather information about the different transcriptional cis-regulation of VMT, we performed an in silico comparison of promoter sequences amplified from a variety of Brassicaceae species, including both hyper- and non-accumulators, and three putative motifs were identified and

chosen for further in vivo analysis. Three different sequences corresponding to the VMT

promoter were amplified in A. halleri. Truncated forms were generated isolating the core sequence and deleting two of the motifs, while site-directed mutagenesis was performed on the third motif. The cloned sequences were fused to the GUS reporter gene and the obtained constructs were used for A. thaliana transformation (AtVMT promoter was used as control). Promoters of both A. thaliana and A. halleri VMT are active in roots, guard cells

and hydathodes, but only Ah-pVMT seems to be specific for leaf mesophyll and trichomes.

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Late submissions

PIX05 PIX06

CHARACTERIZATION OF MEMBRANE PROTEINS CTP1 AND CTP2 OF ARABIDOPSIS THALIANA. PIASENTIN S., NEGRI S., DALCORSO G. AND FURINI A. Dipartimento di Biotecnologie, Università degli studi di Verona, strada le Grazie 15, 37134 Verona.

Keywords: Arabidopsis, Zip transporters, AtCTP1, AtCTP2, Mn transport. In green plants, uptake, root-to-shoot transport and cellular concentration of metals are finely regulated, but little is known about micronutrient transporters. The aim of this research is the characterization of CTP1 and CTP2 (putative Cation Transporter Protein), two membrane transporters in A. thaliana considering their localization, expression and

function. Both proteins are putatively involved in the transport of divalent cations and their ability to complement yeast mutant defective in Zn and Mn uptake has been recently demonstrated. The upregulation of CTP1 and CTP2 was found in shoots and roots of A. thaliana under Zn deficiency, while a higher expression of the two orthologs was found in the hyperaccumulator species Arabidopsis halleri suggesting their involvement in Zn hyperaccumulation. Preliminary results conducted on the A. thaliana ctp1ctp2 knock-out double mutant showed Mn insensitivity. These data points to the idea of CTP1 and CTP2 as

Zn and Mn transport proteins. Subcellular localization studies, promoter analysis and accumulation assay are now in progress to gather more information about the role of both transporters in A. thaliana.

EFFECT OF HARVEST TIME (HT) ON QUALITY OF TABLE GRAPES

(CV. ITALIA). PIAZZOLLA F., AMODIO M.L. AND COLELLI G.

Dip.to di Scienze Agrarie, degli Alimenti e dell’Ambiente -Università di Foggia, Via Napoli 25, 71122 Foggia.

Storing grapes on the wine is a very common practice of South Italy, so the aim of this work was to evaluate the effect of this practice on quality of Italia table grapes . Grapes cultivated with ‘Apulia tendone’ system and covered with a plastic film, were harvested from October 8, 2010 (I HT) and after 11 (II HT), 27 (III HT), and 48 days (IV HT). With the progressing of the harvest, respiration rate increased, and color turned from green to

light yellow, as observed by changes in Hue angle. Soluble solids did not change over time, whereas titratable acidity decreased. Phenols and antioxidant activity, did not follow a linear trend; grapes of I HT presented the highest antioxidant activity (279 mg/100g),

followed by the IV HT, and then by II and III HT (about 180 mg/100g). From the sensorial point of view, crunchiness and sourness decreased with the progress of the time, whereas for berry appearance and cluster score, grapes of III HT and IHT were better evaluated

than grapes from other harvests. Results of this experiment showed that delaying the harvest time could increase sensorial quality of table grapes and did not impact in a sensible way other quality attributes.

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Late submissions

PIX07 PIX08

RESPONSES OF FIELD GROWN TOMATO PLANTS TO ROOT COLONIZATION BY THE ARBUSCULAR MYCORRHIZAL FUNGUS GLOMUS MOSSEAE UNDER P LIMITING SOIL CONTENT

CONDITIONS DI MARTINO C.,1 DI SANTO P.1 AND DI CILLO P.2 1 Università degli studi del Molise. 2 SACOM Spa Larino (CB).

The effects of root colonization by the arbuscular mycorrhizal (AM) fungus Glomus mosseae on growth, flower, fruit production and fruit quality were studied in field-grown tomato plants exposed to limiting P soil content conditions (2 ppm) after greenhouse

germination.Inoculated (M+) and non-inoculated (M−) tomato seedlings were treated under field condition by natural water irrigation. Mycorrhizal plants had significantly higher organic nitrogen and phosphate compounds in both roots and shoots compared to No Mycorrhizal plants.AM inoculation also significantly increased shoot dry matter and the

number of flowers and fruits.The fruit yields of M+ plants were higher than M− plants by 40%. Furthermore, M+ plants produced tomato fruits that contain significantly higher quantities of licopene, glutathione, ascorbate and total A.A. than M− plants. Mycorrhizal colonization than its effects increased with decreasing P soil content.The overall results suggest that mycorrhizal colonization affects host plant nutritional status, and growth under field conditions and thereby, increasing the reproductive activity, fruit production

and quality of fruits under P limiting soil content conditions.

ANTIOXIDANT AND ANTIPROLIFERATIVE PROPERTIES OF

EXTRACTS FROM SPINACH GROWN UNDER LOW OXYGEN AVAILABILITY.

FORNACIARI S.1, MILANO F.2, MUSSI F.C.3, PINTO-SANCHEZ L.C.3, BUSCHINI A.B.2, AND ARRU L.3

1 Interdept. Research Centre Biogest-Siteia, University of Modena and Reggio Emilia. 2 Dept. of Life Sciences, University of Parma. 3 Dept. of Life Sciences, University of Modena and Reggio Emilia.

A fruit and vegetable rich diet plays a protective role on human health, reducing the risk of major chronic diseases including cancer. Cancer cells are known to have an unbalance in their redox state, with a higher amount of ROS, thus being more susceptible to any additional oxidative stress. We studied the possible antiproliferative and antioxidant activity of spinach leaf juice from plants subject to hypoxic stress on the HT29 human colorectal

adenocarcinoma cell line. The antiproliferative activity was evaluated on a counting chamber of a hemocytometer in order to evaluate the number of cells in active

proliferation. The Trypan blue exclusion method was used to determine cytotoxicity. The antioxidant activities were assessed through the alkaline version of the Comet assay, evaluating the DNA damage on 24 h pre-treated HT29 cells. We found that hypoxic spinach leaf extract have a significant antioxidant and protective effect on HT29 cell line when cells are treated with H2O2. The extract exhibits also an antiproliferative effect on the same cells, markedly enhanced when coming from 24 hours hypoxic spinach leaves.

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Società Italiana di Biologia Vegetale