Scientific programme Book of abstracts of Abstracts Murcia... · O9 Manipulation of high value alkaloid products of opium poppy via metabolic engineering ... P7 Hypericum perforatum

Scientific programme

Book of abstracts

Murcia, Spain

17-18 November 2011

2

INDEX

ORGANIZERS ………………………………………………………………………………… 3 PROGRAMME ……………………………………………………………………………..….. 4 LIST OF ABSTRACTS ………………………………………………………………………... 7 ABSTRACTS Session 1: The status quo and road map

Conferences ……………………...…………………………………………………. 13 Posters ....……………………………………………………………………………. 18

Session 2: Molecular tools and approaches Conferences ……………………...…………………………………………………. 21 Posters ...…………………………………………………………………………….. 28

Session 3: System engineering approach Conferences ………………………...………………………………………………. 54 Posters ..…………………………………………………………………………….. 63

LIST OF AUTHORS ……………………………………………………………………. 84

3

Organizers

Mª Angeles Pedreño and Rosa Mª Cusidó

(University of Murcia & University of Barcelona)

Working group organizers

Fragiskos Kolisis (Greece) Michal Oren-Shamir (Israel)

Antonella Leone (Italy) Mariana Sottomayor (Portugal)

Heribert Warzecha (Germany) Paul D. Fraser (United Kingdom)

Organizing committee

Roque Bru (University of Alicante), Mª Purificación Corchete (University of Salamanca), Juan

Segura (University of Valencia) and Javier Palazón (University of Barcelona)

Local collaborators

Lorena Almagro, Sarai Belchí-Navarro, Ana Sabater-Jara, Laura Gómez-Ros, Francisco Fernández-

Pérez, Esther Novo-Uzal, Rosa Mª Cruz Ruiz, Begoña Miras, Ana Sánchez-Godínez, Federico

Pomar

Sponsors

Fundación CajaMurcia

Hero

Agilent Technologies

Oficina de Congresos de Murcia

Proquilab

4

PROGRAMME

Thursday, Nov. 17th 8:15-8:45 Registration and poster setup (Exhibition Hall, Sala de Exposiciones)

8:45-9:15 Heribert Warzecha– Welcome, Introduction to COST Action FA1006

9.15-13:00 SESSION 1: “THE STATUS QUO AND ROAD MAP” (Aula Multiusos)

Presentations covering ongoing existing EU FP7 networks

Session Chairs:Ludger Wessjohann & Heribert Warzecha

9:15-9:45 PLANT BIOTECHNOLOGY IN FP7

Tomasz Calikowski, European Commission, Scientific officer.

9:45-10:15 RATIONAL DESIGN OF PLANT SYSTEMS FOR THE SUSTAINABLE GENERATION OF VALUE-ADDED INDUSTRIAL PRODUCTS Kirsi-Marja Oksman-Caldentey, VTT Technical Research Centre of Finland

The EUFP7 SmartCell project (www.Smart-cell.org).

10:15-10:45 THE DEVELOPMENT OF TOOLS AND EFFECTIVE STRATEGIES FOR THE OPTIMIZATION OF USEFUL SECONDARY METABOLITE PRODUCTION IN PLANTA

Paul D. Fraser, Royal Holloway University of London

The EUFP7 Metapro project (www.metapro.com)

10:45-11:15 Coffee break and poster set up (Exhibition Hall, Sala de Exposiciones)

11:15-11:45 PRESENTATION OF THE TERPMED PROJECT

Alain Tissier, Leibniz Institute of Plant Biochemistry

The EUFP7 TERPMED project (www.terpmed.eu)

11:45-12:15 FLAVONOIDS IN FRUITS AND VEGETABLES: THEIR IMPACT ON FOOD QUALITY, NUTRITION AND HEALTH.

Stefain Martens, Edmund Mach Foundation, Italy

The EUFP6 FLAVO project (www.FLAVOproject.eu)

12:15-13:00 Discussion part I

13:00-14:30 Lunch and poster session

14:30-17:15 SESSION 2: “MOLECULAR TOOLS AND APPROACHES” (Aula Didáctica)

Session Chairs: Antonella Leone & Mariana Sottomayor

14:30-15:00 PHYTOPLUS –ADDED VALUE IN AND OUT OF PLANTS. PLANT ENZYMES AND SECONDARY METABOLITES FOR PHARMA AND FINE CHEMICAL PRODUCTION

Peter Welters, Phytowelt GreenTechnologies GmbH, Nettetal, Germany

5

15:00-15:30 COMBINATORIAL BIOCHEMISTRY OF TRITERPENE SAPONINS IN PLANTS

Alain Goossens, Plant Systems Biology, VIB, Ghent University, Belgium

15:30-15:45 THE USE OF TRANSIENT EXPRESSION AND METABOLOMICS TO EVALUATE METABOLIC ENGINEERING STRATEGIES OF TERPENOIDS

Harro Bouwmeester, Laboratory of Plant Physiology, Wageningen, The Netherlands

15:45-16:00 A NEW PROMOTER FOR SPECIFIC EXPRESSION OF TRANSGENES IN BIOSYNTHETIC CELLS OF GLANDULAR TRICHOMES

Peter Brodelius, Linnaeus University School of Natural Sciences, Kalmar, Sweden

16:00-16:30 Coffee break and poster session (Exhibition Hall, Sala de Exposiciones)

16:30-16:45 MANIPULATION OF HIGH VALUE ALKALOID PRODUCTS OF OPIUM POPPY VIA METABOLIC ENGINEERING AND GENOMICS APPROACHES

Turgay Unver, Cankiri Karatekin University, Faculty of Science, Department of Biology, Cankiri, Turkey

16:45-17:00 IDENTIFICATION OF A POTENTIAL BOTTLENECK IN BRANCHED CHAIN FATTY ACID INCORPORATION INTO TRIACYLGLYCEROL FOR LIPID BIOSYNTHESIS IN AGRONOMIC PLANTS

Brigitte Thomasset, CNRS, University of Technology of Compiègne, France

17:00-17:15 CHALLENGES AND BOTTLENECKS OF ELLAGITANNIN BIOSYNTHETIC PATHWAY GENE IDENTIFICATION

Antje Feller. Fondazione Edmund Mach, Istituto Agrario San Michele all’Adige –IASMA, Centro Ricerca e Innovazione, Italy

17:15-18:00 Discussion part II

19:00-20:30 Visit to Real Casino de Murcia

20:30-21:30 Go to the Tapas

Friday, Nov. 18th

9.00-13.15 SESSION 3: “SYSTEM ENGINEERING APPROACH” (Aula Multiusos)

Session Chairs: Michal Oren-Shamir & Fragiskos Kolisis

9:00-9:45 ELUCIDATING REGULATORY ASPECTS OF THE RESPONSE OF PLANT GENE EXPRESSION, METABOLISM AND CELL BIOLOGY TO STRESSFUL ENVIRONMENTS

Gad Galili, Weizmann Institute of Science. Rehovot, Israel

9:45-10:15 COMPUTATIONAL MODELING OF INTRACELLULAR NETWORKS BEYOND FLUX BALANCE ANALYSIS

Kay Hamacher, AG Bioinformatics and theo. Biology Technische Universität Darmstadt, Germany

6

10:15-10:45 AN IN SILICO COMPARTMENTALIZED MODEL OF BRASSICA NAPUS CENTRAL METABOLISM

Eleftherios Pilalis, School of Chemical Engineering, NTUA, Greece

10:45-11:15 Coffee break sponsored by Agilent Technologies and poster session

(Exhibition Hall, Sala de Exposiciones)

11:15-11:30 REGULATION OF THE PRODUCTION OF TRANS-RESVERATROL IN ELICITED GRAPEVINE CELL CULTURES BY SIGNALLING COMPOUNDS

Sarai Belchi-Navarro, Faculty of Biology, University of Murcia and University of Alicante, Spain

11:30-11:45 1H-NMR BASED METABOLOMICS AS A NEW TOOL FOR VARIETY SELECTION OF FLAXSEED

François Mesnard, Faculty of Pharmacy and Faculty of Sciences, University of Picardie, Amiens, France

11:45-12:00 CAROTENOGENESIS IN THE POTATO TUBER

Mark Taylor, Cell and Molecular Sciences, James Hutton Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK

12:00-12:15 TEMPORARY IMMERSION SYSTEMS FOR CONTAINED PLANT PROPAGATION AND BIOMASS PRODUCTION

Christoph Wawrosch, Department of Pharmacognosy, University of Vienna, Austria

12:15-12:30 PROTEOMIC ANALYSIS OF CYCLODEXTRIN- AND METHYL JASMONATE-MEDIATED RESVERATROL ACCUMULATION IN GRAPEVINE CELL CULTURES

Roque Bru-Martínez, University of Alicante and University of Murcia, Spain

12:30-12:45 THE RELATIONSHIP BETWEEN TAXAN PRODUCTION AND THE EXPRESSION OF SEVERAL GENES INVOLVED IN TAXOL BIOSYNTHESIS IN DIFFERENT IN VITRO TAXUS SYSTEMS.

Miriam Onrubia, University Pompeu Fabra, Barcelona, Spain

12:45-13:15 Discussion part III

13:15-15:00 Lunch and poster session

15:00-18:00 Meeting of the Management Committee (Aula Didáctica )

21:00 Farewell dinner

7

ABSTRACTS

SESSION 1: THE STATUS QUO AND ROAD MAP

O1 Plant Biotechnology in European Bio-economy Calikowski, T.

O2 Plant cells are smart cells – an introduction to an EU-project SmartCell Oksman-Caldentey, K.-M.

O3 The development of tools and effective strategies for the optimisation of useful secondary METAbolite PROduction in planta

Fraser, P.D., Bock, R., Giuliano, G., Taylor, M., Hirschberg, J., Beyer, P., Giorio, G., Socaciu, C.

O4 The EUFP7 TERPMED project Tissier, A.

P1 Global approaches to P450 functions in plant metabolism

Ginglinger, J.F., Olry, A., Matsuno, M., Compagnon, V., Sauveplane, V., Bassard, J.E., Höfer, R., Ullmann, P., Ehlting, J., Pinot, F., Werck-Reichhart, D.

P2 Purification of the sesquiterpene lactone parthenolide with centrifugal partition chromatography Fischedick, J., Schulte, A.E.

SESSION 2: MOLECULAR TOOLS AND APPROACHES

O5 Phytoplus – added value in and out of plants. Plant enzymes and secondary metabolites for pharma and fine chemical production Welters, P.

O6 Combinatorial biochemistry of triterpene saponins in plants

Goossens, A.

O7 The use of transient expression and metabolomics to evaluate metabolic engineering strategies of terpenoids Bouwmeester, H.

O8 A new promoter for specific expression of transgenes in biosynthetic cells of glandular trichomes Wang, H., Han, J., Brodelius, P.E.

O9 Manipulation of high value alkaloid products of opium poppy via metabolic engineering and genomics approaches Unver, T.

O10 Identification of a potential bottleneck in branched chain fatty acid incorporation into triacylglycerol for lipid biosynthesis in agronomic plants Nlandu Mputu, M., Rhazi, L., Vasseur, G., Vu, T-D., Gontier, E., Thomasset, B.

O11 Challenges and bottlenecks of ellagitannin biosynthetic pathway gene identification Feller, A., Martens, S.

8

P3 Estimation of nuclear DNA content in various chemotypes of Thymus caespititius Brot. (Lamiaceae) by flow cytometry Bahcevandziev, K., Lima, A.S., Trindade, H.

P4 Cytochromes P450 involved in the biosynthetic pathway of saponins in Medicago spp

Carelli, M., Biazzi, E., Panara, F., Losini, I., Tava, A., Scotti, C., Piffanelli, P., Calderini, O.

P5 An omic approach to unravel the metabolism of the highly valuable medicinal alkaloids from Catharanthus roseus Carqueijeiro, I., Gardner, R., Duarte, P., Goossens, A., Sottomayor, M.

P6 Different approaches and plant materials to disentangle the control of biosynthesis of condensed tannins Passeri, V., Panara, F., Calderini, O., Paolocci, F., Damiani, F.

P7 Hypericum perforatum cells accumulate anthocyanins and flavonoids at the expense of xanthone biosynthesis during light adaptation Franklin, G., Dias, A.C.P.

P8 Promoter region analysis of a putative Arabidopsis thaliana peroxidase involved in lignification Gómez-Ros, L., Ros Barceló, A., Herrero, J., Esteban Carrasco, A., Zapata, J.M.

P9 A reverse genetics approach to identify cardoon (Cynara cardunculus) genotypes with improved polyphenolic content Ferro, A., Oliveira, M.M., Gonçalves, S.

P10 Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes Juárez, P., Presa, S., Espí, J., Pineda, B., Antón, M.T., Moreno, V., Buesa, J., Granell, A., Orzaez, D.

P11 Research activities on bioactive compounds from native Norwegian plants: Generation of yellow-colored poinsettia by engineering of the flavonoid biosynthetic pathway as an example Liu Clarke, J., Almvik, M., Martinussen, I.

P12 A modular cloning system for metabolic engineering in eukaryotes or prokaryotes Engler, C., Weber, E., Gruetzner, R., Ehnert, T.M., Werner, S., Marillonnet, S.

P13 Metabolic engineering of carotenoid synthesis in tomato fruit Nogueira, M.N., Enfissi, G., Bramley, P.M., Fraser, P.D.

P14 GoldenBraid: an iterative cloning system for standardized multigene assembly in plants

Sarrion-Perdigones, A., Falconi, E.E., Zandalinas, S.I., Juárez, P., Fernández-del-Carmen, A., Granell, A., Orzaez, D.

P15 Pathway to polyketide derived 2-pyrones in Gerbera Pietiäinen, M., Teeri, T.H.

P16 Is metabolic engineering the best approach to modify the monoterpene profile in spike lavender (Lavandula latifolia Med) essential oil? Mendoza I, Muñoz-Bertomeu J, Arrillaga I, Segura J

9

P17 Expression and characterization of two isoformes of cis-prenyltransferase AtCPT6 and AtCPT7 in Arabidopsis thaliana Surmacz, L., Swiezewska, E.

P18 The molecular basis for chemotypes in Thymus caespititius Lima, S., Novak, J., Mendes, M., Figueiredo, A.C., Pedro, L., Barroso, J., Trindade, H

P19 Dalmatian sage extracts: aromatic profile and antioxidant capacity

Ortiz, V., Carrasco, A., Martinez-Ruiz, J., Parra, M., Martinez, F.J., Sanchez, M., Tudela, J.

P20 In vitro cultures for production of Plantago major and P. lanceolata secondary metabolites Dziadczyk, P., Sochacka-Pi�tal, M., Tyrka, M., Bocian, A., Ruman, T., Semik, M., Buczkowicz, J.

P21 Boosting the biosynthesis of bioactive abietane diterpenoids by overexpressing the AtDXS or AtDXR genes in Salvia sclarea hairy roots Vaccaro, M.C., Ocampo, V.E., Malafronte, N., De Tommasi, N., Leone, A.

P22 Production of aspartic proteases in arthichoke (Cynara scolymus L.) suspension cell cultures

Celikkol Akcay, U.

P23 Terpene transport issues van der Krol, S., Ting, H-M., Bouwmeester, H.

P24 Isoflavone synthase in legumes and non-leguminous plants Pi�manová, M., R�ži�ka, P., Honys, D.

P25 Different conversion routes of L-methionine into aroma volatiles in melon fruit Gonda, I., Bar, E., Sikron, N., Burger, J., Schaffer, A.A., Tadmor, Y., Katzir, N., Fait, A., Lewinsohn, E.

P26 A role for protein acetylation in modulating the carbon flux towards isoprenoids and other metabolic pathways? Xing, S., Poirier, Y.

P27 Pregnane-modifying enzymes in plants: occurrence, catalysis and function Müller-Uri, F., Kreis, W.

SESSION 3: SYSTEM ENGINEERING APPROACH

O12 Elucidating regulatory aspects of the response of plant gene expression, metabolism and cell biology to stressful environments Galili, G.

O13 Computational modeling of intracellular networks beyond flux balance analysis Hamacher, K.

O14 An in silico compartmentalized model of Brassica napus central metabolism Pilalis, E., Chatziioannou, A., Kolisis, F.N.

O15 Regulation of the production of trans-resveratrol in elicited grapevine cell cultures by signalling compounds. Belchi-Navarro, S., Almagro, L., Miras-Moreno, B., Fernández-Pérez, F., Bru, R., Pedreño, M.A.1

10

O16 1H-NMR based metabolomics as a new tool for variety selection of flaxseed

Ramsay, A., Molinie, R., Fliniaux, O., Jousse, C., Guillot, X., Roscher, A., Grand, E., Kovensky, J., Gontier, E., Mesnard, F.

O17 Carotenogenesis in the potato tuber Taylor, M.A.

O18 Temporary immersion systems for contained plant propagation and biomass production

Wawrosch, C.

O19 Proteomic analysis of cyclodextrin- and methyl jasmonate-mediated resveratrol accumulation in grapevine cell cultures

Martinez-Esteso, M.J., Vilella-Antón, M.T., Sellés-Marchart, S., Morante-Carriel, J., Vera-Urbina, J.C., Pedreño-García, M.A., Bru-Martínez, R.

O20 The relationship between taxane production and the expression of several genes involved in taxol biosynthesis in different in vitro Taxus systems Onrubia, M., Bonfill, M., Moyano, E., Cusidó, R.M., Goossens, A., Palazon, J.

P28 Early signalling network in tobacco cells elicited with methyl jasmonate and cyclodextrins Almagro, L., Pugin, A., Pedreño, M.A.

P29 Metabolic profiling of citrus peel tissues reveals an important role of phenylpropanoids in induced resistance

Ballester, A.R., Lafuente, M.T., de Vos, R., Bovy, A., González-Candelas, L.

P30 Resveratrol production in grapevine suspension cultured cells and evaluation of its antitumoral activity

Belchi-Navarro, S., Fernández-Pérez, F., Bru, R., Pedreño, M.A.

P31 Phospholipid signalling in Silybum marianum elicited cell cultures Corchete, P.

P32 Psoralen: Bioproduction and Regulation in Bituminaria bituminosa Del Río, J.A., Díaz, L., Pérez, I., Correal, E., Dabauza, M., Walker, D., Ortuño, A.

P33 Ethylene signalling drives the accumulation of antioxidants in tomato fruit Di Matteo, A., Ruggieri, V., Sacco, A., Carriero, F., Rigano, M.M., Frusciante, L., Barone, A.

P34 Rosmarinic acid production in salicylic acid-treated Thymus membranaceus shoots

López-Orenes, A., Martínez-Pérez, A., Calderón, A.A., Ferrer, M.A.

P35 Volatile phytochemicals production by hairy root cultures Figueiredo, A. C., Pedro, L. G., Barroso, J.G.

P36 Towards reduced acrylamide potato products: Analysis of precursor accumulation pathways using 13C-labelling in conjunction with GC/MS

Pont, S.D.A., Ponte, A., Shepherd, L.V.T., Davies, H.V., Hancock, R.D.

P37 A multidisciplinary approach to understand the control of flavonol synthesis in grape berry

Malacarne, G., Coller, E., Heppel, S., Vrhovsek, U., Czemmel, S., Bogs, J., Moser, C.

11

P38 Activity of plant quinones against oxylipin biosynthesis in-vitro – lipoxygenase and cyclooxygenase inhibition Marsik, P., Landa, P.

P39 Flavonoids levels and mycotoxins expression as part of the defence mechanisms of Citrus sp. fruits against Alternaria alternate

Ortuño, A., Díaz, L., González, J., García-Lidón, A., Porras, I., Lacasa, A., Del Río, J.A.

P40 New method for the identification of flavonoids, ginkgolides and bilobalide in Ginkgo biloba extracts by HPLC-ESI-MS-TOF Hernández-Ruiz, J., Sánchez-Godínez, A., Youssef, S.M., Sabater-Jara, A.B., Cruz, R.M., Novo-Uzal, E., Pedreño, M.A.

P41 Identification and the developmental formation of carotenoid pigments in the yellow/orange Bacillus spore-formers Perez-Fons, L., Steiger, S., Khaneja, R., Cutting, S.M., Sandmann, G., Fraser, P.D.

P42 Carotenoid diversity in the genus Citrus: regulation of the biosynthesis and accumulation Rodrigo, M.J., Alquézar, B., Alós, E., Zacarías, L.

P43 Enhancement of the production of phytoesterols in Daucus carota cell cultures Sabater Jara, A.B. Almagro, L., Belchí-Navarro, S., Pedreño, M.A.

P44 Stability and degradation of anthocyanins in fruit and ornamentals Oren-Shamir, M., Bar-Akiva, A., Sinilal, B., Ovaida, R., Weiss, D., Perl, A.

P45 Transcriptome profiling in Catharanthus roseus Pomahacova, B., Körbes, A.P., Mustafa, N.R., van Verk, M.C., Schulte, A.E.

P46 Pyrrolizidine alkaloids in Senecio sp. – development of standard references for food safety assessment Mustafa, N.R., Pomahacova, B., Schulte, A.E., Verhagen, L.C., Luijendijk, T.J.C., Lund, K.

LATE SUBMITTED ABSTRACTS

P47 Phenotypic space of floral volatiles in the Antirrhinaceae

Weiss, J., Mühlemann, J., Orlova, I., Dudareva, N., Egea-Cortines, M.

P48 Metabolic profiling of Catharanthus roseus cell lines

Saiman, M.Z., Mustafa, N.R., Schulte, A.E., Choi, Y.H., Verpoorte, R.

Session 1: The status quo and road map

Session 1: The status quo and road map 13

��

��

O-1

Plant Biotechnology in European Bio-economy

Calikowski, T.

Scientific Officer, Unit "Biotechnologies", Directorate General for Research and Innovation, Directorate "Food,

Agriculture and Biotechnologies", European Commission

*Corresponding author, e-mail: [email protected] http://cordis.europa.eu/fp7/kbbe/home_en.html

Topic: The status quo and road map

Building the European Bio-economy is an intrinsic part of the Europe 2020 strategy. In 2010, the European

Commission has presented seven flagship initiatives including "Innovation Union" to improve framework

conditions and access to finance for research and innovation, so as to ensure that innovative ideas can be

turned into products and services that create growth and jobs. The major objectives of the Bio-economy

strategy are moving to a low carbon economy and building competitive bio-based industries. Innovation for

sustainable growth is reflected in the Framework Programmes, which are the main tool of research funding

in the European Union. Framework Programme 7 (FP7) supports building of the Bio-economy through its

Theme 2 "Food, Agriculture and Fisheries, and Biotechnologies", with the budget of 1,935 billion euro, over

the 7 year duration of FP7. The specific priorities for this theme address ‘Grand challenges’ such as primary

production mitigating and adapting to climate change, advancing sustainable, eco-efficient ('green') and

competitive industries, contributing to food security and safety for Europe and beyond, and to socially

inclusive and healthy Europe, and to the concept of the 'Oceans for the future' and 'a zero waste society'.

Among the aims to be achieved is the production of food, feed and industrial goods such as fuels, chemicals,

lubricants, biopharmaceuticals in a more sophisticated and environmentally friendly manner by life sciences

and biotechnology. The process of building the European Bio-economy is assisted by European Technology

Platforms (e.g. ETP "Plants for the Future"), which are the forums linking industrial, academic and civil

society stakeholders.

The FP7 activity with the specific focus on Plant (green) Biotechnology is Activity 2.3 "Life Sciences and

biotechnology for sustainable non-food products and processes", and also Activity 2.1 "Sustainable

production and management of biological resources from land, forest, and aquatic environments".

Concretely, from 2007 to 2010 some 27% of all projects (17/63) in the portfolio of projects from Activity's

2.3 were related to this field.


��

��

Some on-going activities from the 2007-2011 calls include:

• Energy crops: Sweet sorghum. Jatropha. Poplar. Improving plant cell walls

• Plant-produced vaccines

• High-value products/Secondary Metabolites/ Small Molecules1: Plant terpenoids, Carotenoids

• Industrial products: Improved oils, Rubber and latex crops, forest-based composites

• Biomass availability for industrial applications

• Plant photosynthetic efficiency: C3 to C4

• Biomass from perennial grasses

• India Partnership Initiative biomass/biowaste.

There is a significant industrial participation in the selected projects, both as large industry and as SMEs.

The 2012 call (deadline on 15 November 2011) includes enabling-research related to agriculture, increased

sustainability of all production systems (agriculture, forestry, fisheries and aquaculture), plant health and

crop protection, as well as biotechnology projects on e.g. multipurpose crops, fiber crops, improved water

stress tolerance of plants etc.

The 2013 call is currently in preparation, and its key elements will be an increased support offered to SMEs,

emphasis on demonstration activities and increased impact on Bio-economy (through scientific excellence

and commitment to exploitation of results) together with full use of the discovery-innovation chain. This will

support a transition to the Common Strategic Framework Horizon 2020 (2014-2020).

1 – including projects SMARTCELL, TERPMED on improvements of metabolic engineering of terpenoids for potential application in pharmaceutical industry (against cancer and neurological diseases), project METAPRO on optimisation of production of isoprenoids (carenoids) for application in industrial and health sectors, and project AGROCOS on biodiversity-based discovery of small molecules with interest in agriculture and cosmetic industry.


��

��

O-2

Plant cells are smart cells – an introduction to an EU-project SmartCell

Oksman-Caldentey, K.-M.

Cell Factory, VTT Technical Research Centre of Finland, P.O.Box 1000, 02044 VTT (Espoo), Finland

*Corresponding author, e-mail: [email protected]


Molecules derived from plants make up a sizeable proportion of the drugs currently available on the market.

These include a number of secondary metabolite compounds the monetary value of which is very high.

However, they are often too complex to be economically manufactured by chemical synthesis, and many

times the isolation from naturally grown or cultivated plants is not a sustainable option. Therefore the

biotechnological production of high-value plant secondary metabolites in cultivated cells is potentially an

attractive alternative. However, advanced metabolic engineering and exploitation of plants as Green

Factories has been prevented due to poorly understood metabolic pathways in plants and the regulation

thereof. We have created a novel concept for rationally engineering plants towards improved economical

production of high-value compounds for pharmaceutical use. A systems biology approach using

metabolomics and transcriptomics has been taken to move beyond the state of the art. Gene discovery,

multigene transfer technologies and suitable advanced analytical tools are essential towards screening, and

functionally categorizing genes at structural, regulatory and transport levels.

An introduction is given to the large integrated EU-project called “SmartCell - Rational Design of Plant

Systems for Sustainable Generation of Value-Added Industrial Products” which has now continued for 2.5

years. SmartCell brings together 14 leading European academic laboratories and four industrial partners in

order to create a comprehensive knowledge base of how secondary metabolite biosynthetic pathways operate

in plants. Our focus is on terpenoids, the largest class of secondary metabolites, which exhibit extremely

diverse biological and pharmaceutical activities. However, all knowledge and tools are generic and broadly

applicable to engineer any plant biosynthetic pathway. The case study component i.e. manufacturing a

valuable terpenoid in an optimized large-scale system gives SmartCell a unique opportunity to directly make

transition from fundamental science to application. For long-term exploitation an integrated database,

compound library, cell culture collection and a genebank available for academic and industrial communities

are established. The presentation will also shortly highlight the application possibilities of this approach both

for improving the production levels in cultivated cells as well as increasing chemical diversity of plant-based

molecules by our combinatorial chemistry approach.


��

��

O-3

The development of tools and effective strategies for the optimisation of useful secondary

METAbolite PROduction in planta

*Fraser, P.D.1, Bock, R.2, Giuliano, G.3, Taylor, M.4, Hirschberg, J.5, Beyer, P.6, Giorio, G.7, Socaciu, C.8 1Centre for Systems and Synthetic Biology (CSSB), School of Biological Sciences, Royal Holloway University of

London, Egham Hill, Egham, Surrey, Tw20 OEX. UK. 2Max-Planck Institute of Molecular Plant Physiology, 1 Am

Muehlenberg, Potsdam, 14476, Germany. 3Casaccia Research Center, Ente Per Le Nuove Tecnologie, L’Energia E

L’Ambiente, Via Anguillarese 301, Roma, 00123, Italy. 4James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK. 5Department of Genetics, Institute of Life Sciences, Edmond J. Safra Campus, Givat Ram, 91904. Israel. 6Insitüt für

Biologie II, Albert-Ludwigs-Univerität Freiburg, Schaenzlestr. 1, Freiburg, 79104, Germany. 7Cell Biology Unit,

Metapontum Agrobios S.R.L. Strada Statale Ionica, 106 km. 448,2-Frazione di Metaponto, SNC, Bernalda, 75012,

Italy. 8Proplanta S.R.L., St Budai Deleanu 28, Cluji-Napoca, 400474, Romania.

*Corresponding author, e-mail; [email protected]


Plant secondary metabolites can confer important agronomic traits, they are often essential components of

the human diet, and in some cases have been used as phytomedicines, industrial raw materials and high-

value fine chemicals. These important properties have made many plant secondary metabolites industrially

valuable. In the FP7 EU funded METAPRO project (www.isoprenoid.com) we aim to optimise the

production of several high-value secondary plant metabolites in order to demonstrate the tools and strategies

developed for the generic production of useful secondary metabolites in plants.

The METAPRO project has targeted isoprenoids, these compounds represent one of the largest classes of

natural products know. Many isoprenoids have industrial relevance with global markets in the range of $ 1

billion per annum. A contributing factor to the high cost of these molecules resides in the fact that they are

produced in low yields by slow growing plant species. It is therefore not surprising that total or semi-

synthetic chemical synthesis is presently the method of choice for obtaining many of these isoprenoid

molecules. However, their structural complexity makes chemical synthesis expensive and difficult. Given the

high value and the lack of amenable plant based sources, a genetic engineering approach provides a logical

solution to the creation of renewable bio-sources of these molecules with improved economic and

environmental potential.

Activities in the METAPRO project are focusing on; (i) utilising systems biology to provide a better

understanding of isoprenoid formation and its interaction with intermediary metabolism, (ii) elucidate and

utilise mechanisms of post-transcriptional regulation, (iii) alter organelle parameters to optimise

sequestration and synthesis, (iv) improved the stability of the engineered metabolites in the plant cell and (v)

implement novel engineering approaches. The demonstration molecules chosen in METAPRO are

astaxanthin and crocin, these are both high-value compounds predominantly used in the feed and food sector.

Acknowledgments- The authors are grateful to the EU FP7 programme for METAPRO funding grant number 244348.


��

��

O-4

Presentation of the TERPMED project Tissier, A.

Leibniz-Institute of Plant Biochemistry, Weinberg 3, 06120-Halle (Saale), Germany



TERPMED is a small collaborative project funded by the EU under the FP7program KBBE-2008-3-1-01

"Plant natural products: Alternative sources for the synthesis of bioactive or industrial added value

products". Since mid- 2009, 8 participants from Germany, Greece, the Netherlands, Serbia, Spain and the

USA, including one SME from the Netherlands, have started to investigate the biosynthesis of two classes

of plant natural products with health promoting activities. The first class is that of the sesquiterpene lactones

(SLs), which are frequently encountered in the Asteraceae. As a model SL, parthenolide, produced in

Tanacetum parthenium, and related compounds were selected. Parthenolide and a number of related SLs

have shown, among others, interesting anti-cancer activities. The second group of compounds is that of the

phenolic diterpenes (PDs), which include carnosic acid (CA) and rosmanol. These PDs are produced in

several Lamiaceae species like rosemary and sage (Salvia fruticosa). They have high anti-oxidative

properties, and in addition CA was recently shown to have neuro-protection activities. A combination of

metabolic profiling, diversity screening and RNA sequencing is being implemented to elucidate the

biosynthetic pathways for these compounds. Interestingly, both SLs and PDs are produced in glandular

trichomes, thus providing a rich source of potential biosynthetic pathway genes. A collection of purified

compounds both for the SL and PD class is also being assembled for testing on various cancer and neuron

cell lines. Transgenic approaches are also implemented both for pathway elucidation and reconstruction as

well as the development of new sources for some of these compounds. The overall organization, the

scientific strategy and recent highlights will be presented.


��

��

P-1

Global approaches to P450 functions in plant metabolism

*Ginglinger, J.F., Olry, A., Matsuno, M., Compagnon, V., Sauveplane, V., Bassard, J.E., Höfer, R.,

Ullmann, P., Ehlting, J., Pinot, F., Werck-Reichhart, D.

Dept. Plant Metabolic Networks, Institute of Plant Molecular Biology of CNRS UPR2357, University of Strasbourg,

France *Corresponding author, e-mail: [email protected]

Topic: Status quo and road map

Cytochromes P450 catalyze the slow and rate-limiting steps in all branches of the secondary metabolism.

The sequencing initiatives have recently revealed an unexpected number of P450 genes in the plant genomes.

Most of them cannot be associated to any function. This points to a very poor understanding of the plant

metabolism. We therefore designed complementary strategies to track the overlooked aspects of the

secondary metabolism. The first is the construction of yeast strains for expression of P450 enzymes in an

optimized environment, expressing plant P450 reductases and, when required, engineered to accumulate

GPP or FPP precursors of terpenoid metabolism. The second is a collection of > 200 yeast-expressed

enzymes and the optimization of a method for medium throughput screening of their catalytic functions

based on the common property to consume molecular oxygen (Olry et al., 2007). The method is shown to be

suitable for the detection, real-time monitoring, and quantitative evaluation of enzyme activity. It allows

large-scale screenings for identification of substrates and inhibitor, determination of catalytic and inhibition

parameters.

The third is a predictive map of P450 functions in plant metabolism. We assembled public expression data

from more than 1,800 microarray experiments and generated an extensive expression profile of the CYP

superfamily in Arabidopsis. Expression data from a complete set of genes annotated to be involved in

metabolic pathways were added to the expression matrix. Annotations were curated manually in order to

obtain complete and correct functional annotations. These annotated expression data were then used to

perform co-expression analyses using each CYP as a bait and, based on the pathway annotation of co-

expressed genes, numerous CYPs were placed into new metabolic pathways. Data for all CYPs have been

made available using a web interface “CYPedia” (http://www-ibmp.u-strasbg.fr/~CYPedia/).

CYPedia has provided grounds for targeted analysis of novel CYPs using reverse biochemistry and genetics.

It proved extremely successful. Some examples of P450 functions revealed by genomic approaches in the

phenolic, oxylipin and terpenoid metabolism will be described.

References:

Olry et al. (2007) Plant J. 51: 331-340.

Ehlting et al. (2008) BMC Plant Biology 8:47.

Sauveplane et al. (2009) FEBS J. 276(3):719-735.

Matsuno et al. (2009) Science, 225:1688-1692.

Ginglinger JF, PhD thesis 2010


��

��

P-2

Purification of the sesquiterpene lactone parthenolide with centrifugal partition

chromatography *Fischedick, J., Schulte, A.E.

PRISNA BV, Einsteinweg 55 PO Box 9502, 2300RA, Leiden, The Netherlands.



Parthenolide is a germacranolide sesquiterpene lactone found in Tanacetum parthenium, commonly known

as feverfew. Parthenolide has numerous biological activities including anti-tumor, anti-viral, anti-

leishmanial, and anti-inflammatory action. The goal of the following study was to develop a centrifugal

partition chromatography (CPC) method for the purification of parthenolide. CPC is a bi-phasic liquid-liquid

chromatographic technique where one layer is held in place by centrifugal force to act as a stationary phase

while the other layer is pumped through the system to act as a mobile phase. 250 grams of T. parthenium

dried flower heads were extracted 3 times with ethanol. The crude extract was partitioned between

ethylacetate and water. The ethylacetate layer was collected rinsed 2 additional times with water and the

ethylacetate removed under reduced pressure at 40 °C to yield 17.3 gram of a crude extract. The crude

extract was processed in 3 CPC runs using heptane: ethylacetate: methanol: water in a 1:1:1:1 ratio with the

lower layer serving as a stationary phase and the upper layer serving as the mobile phase. Fractions were

analyzed by TLC, combined as appropriate, evaporated under reduced pressure, and analyzed by HPLC for

analysis of parthenolide purity. Parthenolide eluted in fractions 40-70 yielding 2.3 grams of 98% pure

parthenolide. In conclusion CPC is a useful technique for the purification of parthenolide from T.

parthenium.

This work is part of the EU-FP7 project TERPMED

Session 2: Molecular tools and approaches

Session 2: Molecular tools and approaches 21

��

��

O-5

Phytoplus – added value in and out of plants. Plant enzymes and secondary metabolites for

pharma and fine chemical production Welters, P.

Phytowelt GreenTechnologies GmbH, Nettetal, Germany


Topic: Molecular tools and approaches

Plants have been used by mankind within living memory for food, feed, fiber and medical purposes. The

secondary metabolites of plants have been especially useful for medical purposes and plant extracts

constituted an important source of drugs. At the end of the last century their importance seemed to be

diminished due to the development of protein drugs and the difficulty to identify the active principle in a

plant extract. Furthermore, the complex structure of most natural compounds demanded a costly chemical

multi-step synthesis. Only recently with advances in analytical methods, chemistry and especially the

growing knowledge about enzymatic reactions, natural compounds regain interest in medicinal applications.

Terpenoids with more than 40000 different structures exhibit a special versatility. Phytowelt is tapping this

rich source of active compounds in two ways. With the platform technology phytodiversity (mass cell

electrofusion), plants producing terpenes such as mint plants can be improved. The probability to find

enzymes for the production and derivatization of plant derived compounds is highest in organisms producing

these compounds naturally, hence in plants. Thus, phytomining, another platform technology of Phytowelt, is

used to identify plant derived enzymes for the production of APIs in microorganisms and biocatalysis in

order to conduct even rare and difficult reactions necessary for the introduction of chiral structures. The

potential and recent results of these technologies will be shown.


��

��

O-6

Combinatorial biochemistry of triterpene saponins in plants

Goossens, A.

Department of Plant Systems Biology, VIB, and Department of Plant Biotechnology and Bioinformatics, Ghent

University, B-9052 Gent, Belgium



There is an ever increasing demand for novel compounds to feed drug discovery programs, due to, amongst

others, the growing drug tolerance and resistance in microorganisms and newly emerging diseases. In

microorganisms, combinatorial biosynthesis is a widely used tool to increase structural variation in several

classes of natural products. Combinatorial biosynthesis, also called combinatorial biochemistry, comprises a

series of methods that establish novel enzyme-substrate combinations in vivo and, in turn, lead to the

biosynthesis of new natural product-derived compounds that can be used in drug discovery programs.

Because of the more complex genetic makeup of plants and the lack of tools to manipulate them,

combinatorial biosynthesis in plants is still in its infancy. The recent technical advances in gene discovery

and functional genomics have brought plants within the scope of combinatorial biosynthesis. Here, we

demonstrate the establishment of a combinatorial biosynthesis platform in plants, with the metabolite class of

the triterpene saponins as the target.

Transcript and metabolite profiles, (transgenic) tissue culture collections and gene platforms from five

different plant species, all producing triterpene saponins, are being generated, characterized and exploited.

The plants of particular interest are the model plant Medicago truncatula and the medicinal plants

Glycyrrhiza glabra, Panax ginseng, Bupleurum falcatum and Maesa lanceolata, all producing saponins with

potential therapeutic activities. Combinatorial biosynthesis is pursued by piling up potential saponin

biosynthetic genes in transgenic M. truncatula and M. lanceolata hairy roots, and in the yeast

Saccharomyces cerevisiae, with the final aim of creating novel triterpene saponins with novel or superior

biological activities.


��

��

O-7

The use of transient expression and metabolomics to evaluate metabolic engineering strategies

of terpenoids Dong, L.1, Liu, Q.1, Ting, H. M.1, Cankar, K.1, Van Herpen, T.1, Majdi, M.1,3, Goedbloed, M.1, Beekwilder,

J.1,2, Van der Krol, S.1 and *Bouwmeester, H.1

1Laboratory for Plant Physiology, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, the Netherlands 2Plant Research International, Wageningen UR, P.O. Box 16, 6700 AA Wageningen, the Netherlands 3Plant Breeding and Biotechnology Department, Faculty of Agriculture, Tarbiat Modares University, P. O. Box 14115-

336, Tehran, Iran



Plants produce a wealth of biologically active compounds and a particularly interesting class of metabolites

are the terpenoids. Examples of terpenoid metabolites with medicinal properties are the anti-malarial drug

artemisinin, extracted from Artemisia annua, the anti-cancer indole alkaloids (half terpenoid, half alkaloid)

vincristine and vinblastine, extracted from Cataranthus roseus and other sesquiterpene lactones, such as

parthenolide.

We study the biosynthesis of these pharmaceutically active terpenoids in plants and isolate and characterize

the genes that are involved in their biosynthesis. Hereto we use state-of-the-art technologies such as

metabolomics, transcriptomics, high-throughput sequencing and bioinformatics. We use these genes for

metabolic engineering in plants where we aim to increase or modify the production of terpenoids in

homologous or heterologous plant hosts. We use our metabolic engineering work also to obtain new insights

into the function of enzymes, the role and importance of subcellular compartmentation and subcellular

transport and the impact that endogenous enzymes of the engineered host may have on the results of the

engineering. In this presentation we will discuss two crucial developments for a rapid evaluation of the

consequences of metabolic engineering strategies. These are the use of transient expression, in Nicotiana

benthamiana, and the use of untargeted metabolomics. Transient expression in N. benthamiana allows for

the rapid evaluation of the effects of new constructs (within two weeks) and the simultaneous expression of

many genes, without the necessity to introduce all of them in one construct. The benefit of the use of

untargeted metabolomics is that rather than detecting only the expected products we will also see unexpected

products. Unexpected products seem to be rule rather than exception and some examples in which

metabolomics was key to new discoveries will be discussed.

Acknowledgements: We acknowledge funding by Dafra Pharma R&D, the Netherlands Organisation for Scientific

Research (NWO; the Integration of Biosynthesis and Organic Synthesis (IBOS) programme) and the European

Commission (projects SmartCell (222716) and TerpMed (227448)


��

��

O-8

A new promoter for specific expression of transgenes in biosynthetic cells of glandular

trichomes Wang, H., Han, J., *Brodelius, P.E.

Section for Biomaterials and Medicinal Chemistry, School of Natural Sciences, Linnaeus University, SE-39182

Kalmar, Sweden



Today malaria is successively treated with derivatives of the sesquiterpenoid artemisinin isolated from

Artemisia annua L. in combination with another antimalarial drug (artemisinin combinatory therapy; ACT).

We are involved in studies on the biosynthesis of artemisinin in A. annua. As part of these studies, we have

cloned a number of promoters of genes encoding enzymes of terpene metabolism in A. annua. The activity

of these recombinant promoters has been studied by the expression of the β-glucoronidase (GUS) reporter

gene. We have shown that the promoters of genes encoding enzymes of artemisinin biosynthesis are

specifically active in glandular trichome cells (Figure 1). Using qPCR we could show that the recombinant

promoters show different activity from the wild-type promoters. We have observed both decreased and

increased activity. One of the recombinant promoters (CYP71AV1) showed 100- to 500-fold increased

activity in tissues involved in artemisinin biosynthesis (i.e. young leaves, flower buds and flowers). We have

also shown that the CYP71AV1 promoter is specifically active in secretory cells of transformed tobacco

trichomes. Furthermore, we have transformed A. annua with a number of genes under the control of this

highly trichome-specific promoter. Transgenic plants have been obtained and are now being analyzed. Some

of our preliminary results will be presented.

Figure 1. Expression of GUS under the control of the CYP71AV1 promoter in transgenic plants of A. annua. A: Young

leaf of A. annua; B: Close-up of young leaves of A. annua; C: Stained glandular trichome on a flower bud of A. annua.

GST: glandular secretory trichome; TST: T-shaped trichome.


��

��

O-9

Manipulation of high value alkaloid products of opium poppy via metabolic engineering and

genomics approaches Unver, T.

Cankiri Karatekin University, Faculty of Science, Department of Biology, Cankiri, Turkey



The opium poppy, Papaver somniferum L., produces several types of benzylisoquinoline alkaloids including

the narcotic analgesic morphine, the cough suppressant codeine, the muscle relaxant papaverine, and the

anti-microbial agents sanguinarine and berberine. To discover mechanism and pathway of the alkaloid

biosynthesis the plant is mostly used. Morphine biosynthesis is started with the conversion of the tyrosine

into S-reticuline, a crucial intermediate produced and converted by the two enzymes encoded by S-

Reticuline–4-O- methyltransferase and 7-O-methyltransferase (4-OMT, 7-OMT) genes, respectively.

We have started to investigate the roles of the 4-OMT and 7-OMT genes in the alkaloid biosynthesis by

utilizing both metabolic engineering and functional genomics approaches. Briefly, 4-OMT and 7-OMT were

cloned into vectors appropriate for gene silencing and over-expression. The alkaloid content changes upon

Virus Induced Gene Silencing (VIGS) and over-expression will be measured by HPLC and TOF LC/MS

analyses in stem and capsules. The mRNA and protein products of the genes related with alkaloid

biosynthesis of the silenced plant tissue samples and over-expressed with 4-OMT and 7-OMT genes will be

compared and measured via immuno-bloting and Real Time qRT-PCR. These experiments that will enable

us to understand the molecular mechanisms underlying alkaloid synthesis pathway, will be able to

manipulate levels of medicinally important alkaloids such as codein.


��

��

O-10

Identification of a potential bottleneck in branched chain fatty acid incorporation into

triacylglycerol for lipid biosynthesis in agronomic plants Nlandu Mputu, M. 1, Rhazi, L. 2, Vasseur, G. 3, Vu, T-D. 3, Gontier, E.3, *Thomasset, B1. 1UMR CNRS 6022, Université de Technologie de Compiègne, 60200 Compiègne, FR ; 2Institut Polytechnique LaSalle Beauvais, 19 rue Pierre Waguet, 60026 Beauvais, FR ; 3EA 3900 Biologie des Plantes et contrôle des Insectes ravageurs, Université de Picardie Jules Verne, 33 rue St Leu,

80039 Amiens, FR



Context: In plants, unusual fatty acids are produced by a limited number of species. The industrial

potentiality of these unusual structures has led several groups to work on their production in transgenic

plants. Unfortunately, their research generated disappointing results and lead to very modest accumulation in

seeds. This is largely due to our limited knowledge concerning the substrate specificity and selectivity of

acyltransferases which are required for the incorporation of such unusual fatty acids into storage

triacylglycerols.

Aim: In order to fill this gap, we have compared the incorporation of Oleoyl-CoA and Branched Chain

Acyls-CoAs into Glycerol-3-Phosphate (G3P), Oleoyl Lysophosphatidic Acid (LPA) and diacyglycerol

(DAG) by the enzymes of the Kennedy pathway (G3PAT, LPAAT, DAGAT) from developing seeds of

agronomic plants (flax (Linum usitatissimum) and rape (Brassica napus)) and from a plant able to produce

high amounts of hydroxy fatty acids (castor bean (Ricinus communis)).

Methods: Transcriptomic (microarrays), metabolomic (mass spectrometry) and biochemical (labeling

metabolites) approaches were used to study the specificity and selectivity of LPAATs.

Results: Our assays demonstrated that LPAATs and DAGATs of the three studied cultivars (1) incorporated

preferentially oleyl-CoA, (2) could incorporate cyclopropane acyl-CoA when added alone as substrate,

however weakly for rapeseed and castor bean seed, (3) presented a low capacity to incorporate methyl

branched acyl-CoA when added alone as a substrate (4) weakly incorporated cyclopropane acyl-CoA and

was unable to incorporate methyl branched acyl-CoA when presented with an equimolar mix of oleyl-CoA

and branched chain acyl-CoA. In all cases, the LPAAT had a low affinity for branched chain acyl-CoA.

These results shown that LPAAT and DAGAT activities from agronomic plants constitute a bottleneck for

the incorporation of branched chain acyl-CoA into TAG. In this work, we have also identify some LPAAT

enzymes capable of integrating unusual fatty acids at the sn-2 position of triglycerides.


��

��

O-11

Challenges and bottlenecks of ellagitannin biosynthetic pathway gene identification

*Feller, A., Martens, S.

Fondazione Edmund Mach, Istituto Agrario San Michele all’Adige – IASMA, Centro Ricerca e Innovazione, Area

Alimentazione, Via E. Mach 1, 38010 San Michele all’Adige (TN), Italy.



Ellagitannins are polyphenolic antioxidants found in certain fruits, trees, tea and medicinal plants. They are a

subclass of hydrolyzable tannins and derived from an intermediate of the shikimate pathway. In many fruits,

such as raspberries, strawberries, blackberries or pomegranate, ellagitannins, besides anthocyanins, are the

most abundant antioxidants. The high amount of antioxidants present in these fruits have been associated

with a reduced risk of cardiovascular disease, Diabetes mellitus (type 2) or cancer and these properties,

together with the pleasant taste, have made berries one of the favorite fruits on the fresh food market but also

as source for nutraceuticals or functional foods.

Some ellagitannins and intermediate phenolic compounds of the pathway have been biochemically

investigated and their structure has been determined. In addition, some of the biosynthetic enzymes

catalyzing the reactions in the biosynthetic pathway have been purified from pedunculate oak (Quercus

robur), staghorn sumac (Rhus typhina) and the weed Tellima grandiflora. However, compared to related

pathways such as proanthocyanidins (condensed tannins), anthocyanins or carotenoids which are also present

in many fruits, the overall knowledge on genetics, chemistry and molecular biology is lacking behind. In

consumable foods such as raspberries and strawberries, the ellagitannin biosynthetic pathway is only

characterized on the metabolite level.

Due to the described bioactivities associated with ellagitannin studies at the molecular level and

identification of genes involved in this biosynthetic pathway are mandatory for further engineering strategies

in planta to modulate the amount of metabolites (transgenic strawberry/raspberry; breeding programmes) but

also in microbial system to establish more efficient production of this valuable compounds. An important

step towards the identification of biosynthetic genes was the sequencing of the strawberry (Fragraria vesca)

genome and in addition our lab has sequenced and annotated a cDNA library from different developmental

fruit stages from the raspberry “Tulameen” variety. Compared to the previously investigated plant species

the use of berries which are well characterized on the genetic and genome level will overcome some of the

bottlenecks found in recent studies. Using genome wide homology searches, we have identified putative

shikimate dehydrogenase encoding genes and we are in the process of functionally characterizing these

enzymes in E.coli. Available E.coli and Arabidopsis mutants will be used for complementation analysis.

More challenging will be the identification of other pathway genes since no putative candidate genes or

mutants are available. Strategies and bottlenecks on how to achieve this goal will be discussed here.


��

��

P-3

Estimation of nuclear DNA content in various chemotypes of Thymus caespititius Brot.

(Lamiaceae) by flow cytometry Bahcevandziev, K.1,2, Lima, A.S.3, *Trindade, H.3 1Escola Superior Agrária de Coimbra, Bencanta, 3040-316 Coimbra, Portugal 2Centro de Estudos e Recursos Naturais, Ambiente e Sociedade, Bencanta, 3040-316 Coimbra, Portugal 3Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, Instituto de Biotecnologia e Bioengenharia, Centro

de Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisboa, Portugal



Thymus caespititius is an aromatic and medicinal plant, which has been traditionally used due to the anti-

septic, anti-spasmodic and diuretic properties of its essential oil. This species is characterized by the

existence of chemotypes.

The biosynthesis of essential oils, although genetically controlled, is strongly affected by the environmental

influences of a particular growing region, and also by the agronomic conditions, harvesting time and the type

of processing.

The flow cytometry was used to evaluate the nuclear DNA content of the seeds selected from various

chemotypes of T. caespititius, originated from different regions of Azores. This technique permits fast

evaluation of the DNA and RNA contents, expression of transgenes and cell counting and is very important

of genetic cytomas. T. caespititius seeds were analyzed together with a standard, Lycopersicum esculentum

leaf (DNA standard :2C=1,96 pg) for nucleus isolation. Nuclei coloration was performed using propidium

iodate. The evaluated T. caespititius chemotypes showed different nuclear DNA contents, varying between

2C = 1.149 and 2C = 1.20 pg.

Essential oil composition was analyzed by GC and GC/MS revealing a difference in the thymol content (12

to 27%) in the chemotypes, while the carvacrol content varied from 31 to 57%. Sabinene was found only in

high amounts in one chemotype (23%).

The relative and absolute DNA content of a genome (C value) can present different values, in the same

species, which can be the result from various genetic and environmental factors. We expect, by studying the

genome of the T. caespititius chemotypes to contribute for a better knowledge of the species and the

variability observed. We also expect to find out if there is a correlation between the morphological characters

and the levels of ploidy observed.

Acknowledgments: FCT - PTDC/AGR-GPL/101334/2008.


��

��

P-4

Cytochromes P450 involved in the biosynthetic pathway of saponins in Medicago spp

Carelli, M.1, Biazzi, E.1, Panara, F.2, Losini, I.3, Tava, A.1, *Scotti, C.1, Piffanelli, P.3, Calderini, O.2 1Consiglio per la Ricerca e la sperimentazione in Agricoltura (CRA), Centro di Ricerca per le Produzioni Forraggere e

Lattiero Casearie, 26900 Lodi, Italy. 2Consiglio Nazionale delle Ricerche (CNR) Istituto di Genetica Vegetale (IGV), 06128 Perugia, Italy. 3Fondazione Parco Tecnologico Padano, 26900 Lodi, Italy

*Corresponding author, e-mail: carla.scotti @entecra.it


In the genus Medicago saponins are a complex mixture of glycosidic compounds whose aglycone moieties

are formed by triterpenoid skeletons. In spite of their importance as antimicrobial compounds and their

possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still lagging

behind. In Medicago spp. all these triterpenic compounds are synthetized from the isoprenoid pathway via

the cyclization of 2,3-oxidosqualene to �-amyrin. The �-amyrin skeleton is then transformed into the various

aglycones by means of oxidative modifications mediated by cytochrome P450 (P450s); glycosyl transfer

reactions, mediated by glycosyltransferases (GTs) are responsible for the addition of sugar moieties.

Our aim is to identify and functionally characterize P450 genes involved in the biosynthetic pathway of

saponins in M. truncatula and to transfer this information to the cultivated alfalfa (M. sativa L.). A reverse

genetic TILLING approach on M. truncatula M2 plants of an EMS-mutagenized collection established in

Italy (Porceddu et al., 2008) is used to screen for P450 candidate genes. These genes have been chosen

mainly on the basis of the transcriptome analysis of a mutant (lha-1) we have identified lacking haemolytic

saponins (Carelli et al., 2011). The gene responsible for the mutation is CYP716A12 catalyzing the three-

step oxidation at C-28 position necessary to trasform �-amyrin into oleanolic acid. This oxidative step has

been demonstrated to be a key step in hemolytic saponin pathway as its disruption causes the block of the

synthesis of any aglycones of hemolytic saponins. The project involves the growth and chemical

characterization of the M3 progenies of the mutants for the P450 genes identified by TILLING analysis and

the expression of these genes in a yeast system for functional characterization.

References

Porceddu A, Panara F, Calderini O, Molinari L, Taviani P, Lanfaloni L, Scotti C, Carelli M, Scaramelli L, Bruschi G,

Cosson V, Ratet P, de Larembergue H, Duc G, Piano E, Arcioni S, 2008. BMC Research Notes 1: 129.

Carelli M, Biazzi E, Panara F, Tava A, Scaramelli L, Porceddu A, Graham N, Odoardi M, Piano E, Arcioni S, May S,

Scotti C, Calderini O. 2011. Plant Cell 23: 3070-3081.


��

��

P-5

An omic approach to unravel the metabolism of the highly valuable medicinal alkaloids from

Catharanthus roseus Carqueijeiro, I.1 ,2, Gardner, R.3, Duarte, P.1, Goossens, A.4, *Sottomayor, M.1,2 1IBMC – Instituto de Biologia Molecular Celular, Universidade do Porto, Portugal 2Departamento de Botânica da Faculdade de Ciências da Universidade do Porto, Portugal 3IGC – Instituto Gulbenkian de Ciência, Oeiras Portugal 4Plant Systems Biology, VIB/UGent.



Catharanthus roseus accumulates in low levels the terpenoid indole alkaloids (TIAs) vinblastine and

vincristine, used in cancer quimiotherapy, as well as ajmalicine, used as an antihypertensive, and serpentine,

used as sedative. Although much is known about the biosynthesis and regulation of TIAs, gene/enzyme

characterization is still lacking for many biosynthetic steps, the membrane transport mechanisms of TIAs are

basically uncharacterized despite its importance for TIA accumulation, and no effective master switch of the

TIA pathway has been identified.

In C. roseus leaves, the first steps of TIA biosynthesis occur in the epidermis, while the late steps and TIA

accumulation occur in differentiated mesophyll cells, the idioblasts, characterized by a conspicuous blue

fluorescence credited to the TIA serpentine. Therefore, transport of TIA intermediates is thought to occur

between the two cell types. Here, we implemented a targeted strategy involving the isolation of idioblast

protoplasts from those of common mesophyll cells by FACS (Fluorescence Activation Cell Sorting),

followed by differential transcriptomic analysis in order to discover new candidate genes involved in the

biosynthesis, regulation and transport of C. roseus TIAs. The fraction of idioblast cells obtained by FACS

showed a high purity, and cDNA-AFLP-based transcript profiling was performed for roots, leaves, leaf

protoplasts and sorted idioblast protoplasts. At this moment, we have identified sequence tags for i) six

putative transcription factors significantly up-regulated in idioblasts, which are candidate regulatory genes of

the TIA pathway, and ii) five putative ABC transporters differentially expressed in idioblasts, which are

candidate genes either for cell extrusion, cell uptake or vacuole uptake of TIAs. Isolation and

characterization of these genes is being initiated, further in silico analysis should still lead to the

identification of more candidate genes, and the idioblasts will also be characterized by differential

metabolomics.


��

��

P-6

Different approaches and plant materials to disentangle the control of biosynthesis of

condensed tannins Passeri, V., Panara, F., Calderini, O., Paolocci, F., *Damiani, F.

Institute of Plant Genetics CNR, UOS Perugia. Perugia. Italy



The Condensed tannins (CT), flavonoids that share with anthocyanins the biosynthetic route, have beneficial

effects for humans and animals. Therefore, engineering their biosynthesis in feed and food species is our

long-term goal. Here we report on studies carried out to unveil genetic determinants controlling the synthesis

of CT in model (Medicago truncatula, Tobacco) and legume species (Medicago sativa, Lotus corniculatus).

In L. corniculatus, a model legume for CT analysis, in that it accumulates these metabolites in all organs, the

structural genes of the pathway were cloned and extensively characterized (Paolocci et al., 2007). Recently

RNAi was approached in L. corniculatus with the aim to understand the in vivo function and the contribution

of each gene family to the overall CT accumulation. In M. truncatula mutant lines for anthocyanins and/or

CT have been produced by transposon tagging and their molecular and metabolic profiling is also in

progress.

The role of bHLH and MYB transcription factors in CT accumulation has been acknowledged (Baudry et al.,

2004; Robbins et al. 2003), however their ectopic expression turned out to be insufficient to induce de novo

CT biosynthesis. Nevertheless, we recently observed that in tobacco CT can be produced at expenses of

anthocyanins once a MYB gene (VvMYBPA1) specific to CT is overexpressed. Whether and to what extent

this outcome is specific to tobacco and/or to the transgene used is currently investigated by expressing a

number of CT-specific MYBs in M. truncatula, M. sativa and tobacco.

Overall, by combining transcriptomic, metabolomic and functional genomics analyses we expect to identify

new gene functions and provide a comprehensive picture on key genes and limiting factors controlling the

CT metabolic flux in different organs and plant species.

References

Baudry A, Heim MA, Dubreucq B, Caboche M, Weisshaar B, Lepiniec L. 2004. The Plant Journal 39: 366–380.

Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F. 2007. Plant Physiol 143: 504-516

Robbins MP, Paolocci F, Hughes JW, Turchetti V, Allison G, Arcioni S, Morris P, Damiani F. 2003. J Exp Bot 54:239-

248


��

��

P-7

Hypericum perforatum cells accumulate anthocyanins and flavonoids at the expense of

xanthone biosynthesis during light adaptation

*Franklin, G., Dias, A.C.P.

CITAB – Department of Biology, University of Minho, 4710-057 Braga, Portugal



Hypericum perforatum L. (HP) is well-known for its medicinal uses. Extracts of HP have been used in

traditional medicine worldwide for several ailments (Franklin et al 2009). Pharmacological properties of HP

extracts such as antidepressant, antioxidant, anti-inflammatory, hepatoprotective, and anticancer activities

can be attributed to the phenolic compounds such as naphtodianthrones, xanthones, flavonoids etc., produced

by HP (Conceição et al 2006).

The present study was conducted to analyse the secondary metabolic changes in HP cells under light stress.

HP cell suspension cultures were yellowish green in colour under 20 µmol s–1 m–2 intensity light

illumination. Whereas, when transferred to 60 µmol s–1 m–2 light intensity, these cultures gradually attained a

pink colouration within 15 days. HPLC-DAD analysis revealed that these cells present higher accumulation

of anthocyanins and flavonoids, while decreasing the otherwise predominant xanthone production. Transfer

of these pink cultures back to 20 µmol s–1 m–2 light condition resulted in xanthone accumulation, while

diminishing anthocyanins and flavonoids production. Simultaneous to this metabolic change, the cells also

regained their normal colour within 15 days. Interestingly, maintenance of pink cultures under high light

intensity for about 60 days (6 subcultures) also completely reversed the cultures to their normal colour

indicating that the differential accumulation of phenolics in HP cultures is associated with their

acclimatization to high light condition. It is known that xanthones, flavonoids and anthocyanins are

biosynthetically related compounds, and share a common pool of precursors (Liu et al 2003, Conceição et al

2006). Hence, under light stress, these precursors might have been shifted towards flavonoids and

anthocyanins production by shutting down the xanthone biosynthesis temporarily. We propose that a two-

way molecular switch operates at the key branch point, where benzophenone synthase (BPS) and chalcone

synthase (CHS) operates. This is currently under investigation.

References

Franklin G, Conceição L, Kombrink E, Dias ACP. 2009. Phytochemistry 70: 60-68.

Conceição LFR, Ferreres F, Tavares RM, Dias ACP. 2006. Phytochemistry 67: 149-155.

Liu B, Falkenstein-Paul H, Schmidt W, Beerhues L. 2003. Plant Journal 34:847-855


��

��

P-8

Promoter region analysis of a putative Arabidopsis thaliana peroxidase involved in

lignification *Gómez-Ros, L.1, Ros Barceló, A.1, Herrero, J.2, Esteban Carrasco, A.2, Zapata, J.M.2 1 Department of Plant Biology. University of Murcia. Murcia. Spain. 2Department of Plant Biology, University of Alcalá, E-28871, Alcalá de Henares, Spain



The basic peroxidase isoenzyme from Zinnia elegans (ZePrx), an enzyme involved in lignin biosynthesis, is

sensitive to a plethora of hormones (auxins, cytokinins, brassinosteroids and giberellic acid) which control

xylem lignifiation. In fact, auxins and cytokinins induce ZePrx, similarly to the way in which they induce

xylem differentiation, while brassinosteroids and giberellic acid inhibit ZePrx, similarly to the way in which

they inhibit xylem differentiation. This hormonal response of ZePrx is supported by analysis of the ZePrx

promoter, which contains cis-elements directly responsive to auxins, cytokinins, brassinosteroids and

giberellic acid and cis-elements target of the plethora of transcription factors, such as NAC, MYB, AP2,

MADS and class III HD Zip, which are up-regulated during auxin- and cytokinin-induced xylem

differentiation. In a search for Arabidopsis thaliana homologues to the ZePrx, it was found that a high degree

of homology at 1D, 2D and 3D between certain peroxidases from A. thaliana and ZePrx is necessary but not

sufficient for involving an A. thaliana peroxidase (AtPrx) in lignification, since some promoters of AtPrxs do

not contain cis-elements target of the NAC, MYB, AP2, MADS and class III HD Zip transcription factors,

nor cis-elements responsive to auxins, cytokinins, brassinosteroids and giberellic acid. They therefore lack

xylem-specific regulatory cis-elements in their respective promoters. This communication describes attempts

to establish the minimal structural and regulatory elements contained in the promoter region of an AtPrx, a

putative peroxidase involved in lignification.

The results showed that AtPrx are responsive to the two main hormones that govern vascular differentiation

by the in silico analysis of the AtPrx promoters, which contain (a) cis-elements responsive to auxins, (b)

cytokinins and (c) cis-elements targets of transcription factors (NAC, MYB, AP2, and class III HD Zip)

which are up-regulated by auxins and cytokinins during secondary growth.

References:

Gutierrez J, López Núñez-Flores MJ, Gómez Ros L V, Novo Uzal E, Esteban Carrasco A, Díaz J, Sotomayor M, Cuello

J, Ros Barceló A. 2009. Planta. 230:767-778.

Acknowledgments: This work was supported by a grant from the MEC (BFU2009-08151)-FEDER and Fundación

Séneca (08610/PI/08).


��

��

P-9

A reverse genetics approach to identify cardoon (Cynara cardunculus) genotypes with

improved polyphenolic content Ferro, A.1,2, Oliveira, M.M. 3, *Gonçalves, S.1,2 1Centro de Biotecnologia Agrícola e Agro-Alimentar do Baixo Alentejo e Litoral (CEBAL) / Instituto Politécnico de

Beja (IPBeja), 7801-908 Beja, Portugal. 2Centre for Research in Ceramics & Composite Materials (CICECO),

University of Aveiro, 3810-193 Aveiro, Portugal. 3ITQB/IBET, Av. da República, Estação Agronómica Nacional,

2780-157 Oeiras, Portugal



Cynara cardunculus (Cc) is a Mediterranean species used as multipurpose crop. The potential of cardoon for

the production of pharmaceutic bioactive phenolic compounds has gained recent interest. Plant polyphenols

have low toxicity and antioxidant properties, and have been considered useful in prevention and treatment of

different human diseases, including cancer, cardiovascular and chronic disorders. Among the various

phenolic compounds described to be present in Cc extracts, the most abundant are dicaffeoylquinic acids.

Some of these compounds have been demonstrated to be strong anti-hepatotoxic agents in experimental liver

disease and present a great anti-tumorigenic activity in several types of cancer.

The improvement of cultivated varieties largely depends on the use of new genetic variability in crop species

with narrow genetic basis. This has led to a surge of interest in exploring natural biodiversity as a source of

novel alleles to improve the productivity, adaptation, quality and nutritional value of crops. Mutation

breeding based on the screening of natural biodiversity leading to the identification of improved alleles has

been successfully experimented in various species.

This project applies a reverse genetics approach to identify cardoon plants with allelic variations in genes

involved in the phenylpropanoid pathway. Our strategy is to screen cardoon plants for base mutations in the

gene sequences of hydroxycinamoyltransferase HQT and p-coumaroyl ester 3'-hydrolase C3'H as these

genes are involved in the synthesis of clorogenic acid (CGA), precursor of the dicaffeoylquinic acids. The

natural variation for the selected genes will be assessed by TILLING, using a representative collection of

biological diversity of cardoon from Portugal, Spain and Italy. This cardoon plants identified with sequence

variations in the target genes will be evaluated for improved biological activity. Total phenol content,

antioxidant activity and antitumorigenic capacity will be determined in these plants. Results obtained so far

will be presented.


��

��

P-10

Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes

Juárez, P., Presa, S., Espí, J., Pineda, B., Antón, M.T., Moreno, V., *Buesa, J., Granell, A., Orzaez, D.

Crop Breeding and Biotechnology Department. Instituto de Biología Molecular y Celular de Plantas, CSIC-Universidad

Politécnica de Valencia. Valencia. Spain.



Edible fruits are inexpensive biofactories for human health promoting molecules that can be ingested as

crude extracts or partially purified formulations. We show here the production of a model human antibody

for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits.

Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against

the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ±

0.8% of the total soluble protein (TSP) in the fruit of the transformed plants. Minimally-processed fruit-

derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus

infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily

distinguishable from wild type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a

transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription

factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits

expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits

contain high levels of recombinant anti-rotavirus neutralizing human IgA (33,5 ± 4,2 µg/g of fresh weight) in

combination with increased amounts of health-promoting anthocyanins.


��

��

P-11

Research activities on bioactive compounds from native Norwegian plants: Generation of

yellow-colored poinsettia by engineering of the flavonoid biosynthetic pathway as an example *Liu Clarke, J., Almvik, M., Martinussen, I.

Bioforsk- Norwegian Institute for Agricultural & Environmental Research, Norway



Genetic engineering of a flavonoid biosynthetic pathway in floriculture would provide a powerful method for

obtaining novel flower colors beyond genetic constraints that conventional breeding is not able to overcome.

In Norway, poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is one of the most important pot plants

with a yearly production close to 6 million plants. Its ornamental value and innovation potential has laid the

basis for extensive research in Norway, especially at Bioforsk. Aurone flavonoids confer a bright yellow

color on flowers such as Antirrhinum majus and Dahlia variabilis. A. majus aureusidin synthase (AmAS1)

was identified as the key enzyme in aurone biosynthesis. In addition, chalcone 4’-O-glucosyltransferase

(4’CGT) is essential for aurone biosynthesis and yellow coloration in vivo. We have attempted to produce

pure yellow-coloured poinsettia, a highly desirable colour for the poinsettia industry, using genetic

engineering of the flavonoid biosynthetic pathway. Co-expression of AmAS1 and 4’CGT genes, which has

proven successful for yellow color formation in transgenic Torenia hybrida flowers, was used in the

generation of yellow-coloured poinsettia through the Agrobacterium-mediated genetic transformation

method we have developed for poinsettia.

The terrestrial adaptogenic plant species of Nordic areas are well adapted to the challenging growing

conditions and represent an interesting resource for bioprospecting. Bioforsk has started a strategic work to

exploit these natural resources and increase the knowledge about commercially interesting compounds from

our northern flora, e.g. for development of new, targeted biopesticides. The chemical analysis is performed

with GC-MS and LC-MS/MS. Through the COST Action FA1006, Bioforsk seeks new partners for

cooperation on these subjects.


��

��

P-12

A modular cloning system for metabolic engineering in eukaryotes or prokaryotes

Engler, C., Weber, E., Gruetzner, R., Ehnert, T.M., Werner, S., *Marillonnet, S.

Icon Genetics GmbH, Weinbergweg 22, D-06120 Halle, Germany



Synthetic biology requires methods that allow streamlined assembly of standard basic genetic elements into

transcription units and into increasingly complex multigene constructs of choice. We have developed a DNA

assembly method, called Golden Gate cloning, that allows seamless assembly of multiple DNA fragments in

a one-pot reaction. Based on this cloning method, we developed a modular cloning system (MoClo) that

allows efficient assembly of multigene constructs from libraries of basic genetic elements that include

promoters, coding sequences and terminators. This cloning system can be used for generation of constructs

for expression in either eukaryotes or prokaryotes. As an example, the Janthinobacterium lividum operon

coding for violacein biosynthesis was reengineered for expression in E.coli and Nicotiana benthamiana. This

cloning system can also be used for construction of artificial biosynthetic pathways that combine genes from

different organisms, and for optimization of production of the desired metabolites in either plant or microbial

hosts.


��

��

P-13

Metabolic engineering of carotenoid synthesis in tomato fruit

*Nogueira, M.N., Enfissi, G., Bramley, P.M., Fraser, P.D.

School of Biological Sciences, Royal Holloway University of London, Egham, UK.



Carotenoids are isoprenoid molecules and represent the most widespread class of natural pigments, found in

animals, plants and microorganisms. However, animals are unable to synthesise carotenoids de-novo, and

rely upon the diet as a source of these compounds. The health benefits and colorant aspects conferred by

numerous carotenoids have led to attempts to elevate their level in foodstuffs.

Tomato fruit and its products are the main source of the potent antioxidant lycopene and �-carotene in

human diet. In contrast to chemical synthesis, which is the present method of choice used to produce most

carotenoids, the tomato crop represents a cheap renewable bioresource with less environmental impact.

Therefore, metabolic engineering of the carotenoid pathway in tomato seems to be a valuable option to

produce useful secondary metabolites such as carotenoids and other isoprenoids.

Stable varieties of tomato have been generated in which individual step in the carotenoid biosynthetic

pathway have been amplified with heterologous enzymes. In order to combine these steps to create a

pathway in which multiple steps have been enhanced, a genetic crossing approach has been used.

The following genes have been used for this purpose, GGPP synthase (CrtE; from Erwinia uredovora),

phytoene synthase (CrtB; from Erwinia uredovora), phytoene desaturase (CrtI; from Erwinia uredovora).

The analysis of the genetic crosses with multiple transgenes suggests that in tissues where the pathway is

under strict regulation, it may not be necessary to utilise several pathway enzymes simultaneously. Instead, a

single optimised enzyme having influence over the pathway flux may suffice.

Analysis of the carotenoids levels in the sub-chromoplast fractions isolated from tomato control Ailsa Craig

and the genotype harbouring the CRTB and CRTI heterologous enzymes has also been performed. The data

suggest that the cellular structures can adapt to facilitate the sequestration of the increased carotenoid

synthesized from the action of CRTB and CRTI.


��

��

P-14

GoldenBraid: an iterative cloning system for standardized multigene assembly in plants

Sarrion-Perdigones, A., Falconi, E.E., Zandalinas, S.I., Juárez, P., Fernández-del-Carmen, A., Granell, A.,

*Orzaez, D.

Crop Breeding and Biotechnology Department. Instituto de Biología Molecular y Celular de Plantas, CSIC-Universidad

Politécnica de Valencia. Valencia. Spain.



Multigene engineering in plants has the potential to create new synthetic products with added value. As an

example, we engineered “identity preserved” purple transgenic tomatoes that produce high levels of a human

antibody against the diarrhea agent rotavirus. However, this was not an easy task: combining traits for Plant

Biotechnology is a difficult “puzzle” due to the lack of DNA assembly standards. Plant Synthetic Biology

requires efficient and versatile DNA assembly systems to facilitate the building of new genetic

modules/pathways from basic DNA parts in a standardized way. To facilitate multigene engineering in

plants, here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction

enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The

GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite

assemblies made of standard DNA parts and to combine them binarily to build increasingly complex

multigene constructs. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic

Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant

transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made

of five reusable devices each, and comprising a total of 19 basic parts will be described.


��

��

P-15

Pathway to polyketide derived 2-pyrones in Gerbera

Pietiäinen, M., *Teeri, T.H.

Department of Agricultural Sciences. University of Helsinki. Helsinki. Finland



The ornamental plant Gerbera hybrida is rich in two glucosidic lactones, gerberin and parasorboside. We

have earlier determined that the first step in their biosynthesis is catalysed by 2-pyrone synthase (2PS, a

polyketide synthase) using acetyl-CoA as starter substrate (Eckermann et al. 1998). Gerbera antisense lines

down regulated for 2PS lack these metabolites and show increased sensitivity to fungal attack (Koskela et al.

2011) and insect herbivory (unpublished). In order to elucidate further the enzymatic steps from

triacetolactone formed by 2PS to the reduced and glucosylated derivatives gerberin and parasorboside, we

have determined the transcriptomes in gerbera inflorescence stem, petal and anther tissues using next

generation sequencing of mRNA (Illumina). Based on correlation with 2PS expression, we have defined

candidates for reductases and glucosyl transferases and are proceeding in determining their involvement in

gerberin and parasorboside biosynthesis.

References

Eckermann S, Schröder G, Schmidt J, Strack D, Edrada RA, Helariutta Y, Elomaa P, Kotilainen M, Kilpeläinen I,

Proksch P, Teeri TH and Schröder J. 1998. Nature 396:387-390

Koskela S, Söderholm P, Ainasoja M, Wennberg T, Klika KD, Ovcharenko VV, Kylänlahti I, Auerma T, Yli-

Kauhaluoma J, Pihlaja K, Vuorela P and Teeri TH. 2011. Planta 233:37-48


��

��

P-16

Is metabolic engineering the best approach to modify the monoterpene profile in spike

lavender (Lavandula latifolia Med) essential oil? *Mendoza, I., Muñoz-Bertomeu, J., Arrillaga, I., Segura, J.

Department of Plant Biology. University of Valencia. Valencia. Spain.



Spike lavender (L. latifolia Med) is an aromatic shrub of economical importance due to its essential oil,

which is composed mainly of monoterpenes. Basic C5 (IPP and DMAPP) units for monoterpene formation

in this species are primarily synthesized in the plastids via the MEP pathway. The enzymes 1-deoxy-D-

xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR),

responsible for the first and second reactions, respectively, of the MEP pathway, are important regulatory

steps for monoterpene production in spike lavender. Thus, we have demonstrated that constitutive

overexpression of any of the above mentioned enzymes (DXS or DXR) increases essential oil yield in leaves

without affecting the oil profile (1, 2). Nevertheless, leaf monoterpene profile can be altered by

overexpressing monoterpene synthases as limonene or linalool synthases. In both cases, developing leaves

from spike lavender transgenic plants showed increased amounts of limonene or linalool, respectively

without altering essential oil yield (3, 4,5).

In a further attempt to modify monoterpene profile in both developing and mature spike lavender leaves, we

obtained, by cross-pollination, transgenic plants containing both the DXS gene and either limonene or

linalool synthase genes. In any event, the plants that inherited the DXS gene produced more essential oil than

control plants, but surprisingly none of those that inherited both genes produced a specific linalool or

limonene increase. This suggests the presence of a fine regulatory mechanism for accumulative transgenes in

spike lavender.

References

1. Muñoz-Bertomeu J, Arrillaga I, Ros R, Segura J. 2006. Plant Physiology 142:890-900.

2. Mendoza I, Muñoz-Bertomeu J, Segura J, Arrillaga I. 2009. VIII Reunión de la Sociedad Española de Cultivo In

Vitro de Tejidos Vegetales. Murcia, Spain. Abstract Book: 68

3. Muñoz-Bertomeu J, Ros R, Arrillaga I, Segura J. 2008. Metabolic Engineering 10:166-177.

4. Mendoza I, Navarro A, Muñoz-Bertomeu J, Segura J, Arrillaga I. 2011. IX reunión de la Sociedad Española de

Cultivo In Vitro de Tejidos Vegetales. Tenerife, Spain. Abstract Book: SIV-P47, page 85

5. Muñoz-Bertomeu J, Ros R, Arrillaga I, Segura J. 2005 Córdoba, Spain. VI Reunión de la Sociedad Española de

Cultivo In Vitro de Tejidos Vegetales. Córdoba, Spain. Abstract Book: 82

Acknowlegments: This work was funded by a FPU Research Fellowship from the Spanish Government (to I.M.) and by

the Generalitat Valenciana (Prometeo 2009/075).


��

��

P-17

Expression and characterization of two isoformes of cis-prenyltransferase AtCPT6 and

AtCPT7 in Arabidopsis thaliana Surmacz, L., *Swiezewska, E.

Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw,

Poland



Polyisoprenoid alcohols (dolichols and polyprenols) are widespread in nature from bacteria to mammalian

cells. They modulate the properties of the biological membranes, participate in glycosyl-phosphoinosytol

membrane (GPI) anchor, protein C- and O-mannosylation, and protein N- and O-glycosylation.

Polyisoprenoids are formed via condensations of isopentenyl diphosphates (IPP) molecules with farnesyl

diphosphate (FPP) performed by cis-prenyltransferase (CPT).

Several genes encoding CPT have been cloned from bacteria, plants and mammals. Interestingly,

Arabidopsis thaliana ten putative genes (AtCPT1 to AtCPT9 and LEW1) with homology to yeast CPTs

revealed the tissue-specific expression. Here we present data on the preliminary characterization of two A.

thaliana CPT isoformes AtCPT6 and AtCPT7.

AtCPT6 is expressed exclusively in roots whereas AtCPT7 in leaves, stems, flowers and roots. The

microsomal and cytosolic fractions were isolated from leaves and roots of A. thaliana Col-0 ecotype,

separated by SDS-PAGE and probed with specific anti-peptide AtCPT6 and AtCPT7 antibodies. AtCPT6

antibody revealed the immunoreactive polypeptide ~35 kDa in both cytosolic and microsomal fractions from

roots only. As expected AtCPT7 antibody gave positive signal in both fractions from both roots and leaves.

The AtCPT6 and AtCPT7 genes were amplified by RT-PCR and cloned into yeast expression vector pYES-

DEST52 and expressed in S. cerevisiae INVSc1 strain using galactose induction for 32h and 28h,

respectively. Affinity purified His tag fusion proteins were analyzed by Western blot. Specific anti-peptide

AtCPT6 antibody showed positive signal only with ~40 kDa in size product of pYD52-AtCPT6 construct

whereas specific anti-peptide AtCPT7 antibody only with ~45 kDa product of pYD52-AtCPT6. These

specific antibodies did not show cross reactivity with AtCPT6 and AtCPT7 recombinant proteins,

respectively. Furthermore, mass spectrometry analysis clearly confirmed the sequences of AtCPT6 and

AtCPT7 overexpressed in S. cerevisiae.

Currently, functional analysis of AtCPT6 and AtCPT7 is in progress.


��

��

P-18

The molecular basis for chemotypes in Thymus caespititius

Lima, S.1, Novak, J.2, Mendes, M.1, Figueiredo, A.C.1, Pedro, L.1, Barroso, J.1, *Trindade, H1 1Universidade de Lisboa, Faculdade de Ciências de Lisboa, DBV, Instituto de Biotecnologia e Bioengenharia, Centro

de Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisboa, Portugal 2Institute for Applied Botany and Pharmacognosy, University of Veterinary Medicine, Veterinärplatz 1, 1210 Wien,

Austria



Thymus caespititius belongs to the Lamiaceae family and possess an essential oil composed mainly of

monoterpenes. Previous studies have shown that this species shows several chemotypes, namely thymol,

carvacrol, sabinene and �-terpineol, with mixed chemotypes existing as well (Trindade et al., 2008). We

have been studying the molecular basis for the different chemotypes in this plant species, aiming at

identifying the different terpene synthase genes.

Terpene synthases (TPS) are the key enzymes in the formation of terpene metabolites that catalyze the

formation of a single or a complex mixture of reaction products from the same prenyl diphosphate substrate.

In order to elucidate the molecular mechanisms of reaction responsible for terpene biosynthesis in Thymus

caespititius, the genomic structure of two putative monoterpene synthases, sabinene synthase (Tctps1) and �-

terpinene synthase (Tctps2) was characterized. Genomic DNA was isolated following the CTAB extraction

method. Primers were designed from previous information available from Origanum vulgare (Crocoll et al.,

2010). Sequencing results were aligned using Geneious software.

A genomic characterization of exon and intron numbers, sizes and placement, showed that both genes were

organized in seven exons and six introns. Tctps1 had an open reading frame of 1812 bp while Tctps2 an open

reading frame of 1794 bp. cDNA synthesis from floral aerial parts was performed and studies on

heterologous expression are underway.

References

Crocoll C, Asbach J, Novak J, Gershenzon J, Degenhardt J (2010) Terpene synthases of oregano (Origanum vulgare L.)

and their roles in the pathway and regulation of terpene biosynthesis. Plant Mol. Biol. 73: 587-603

Trindade H, Costa MM, Lima AS, Pedro LG, Figueiredo AC, Barroso JG (2008) Genetic diversity and chemical

polymorphism of Thymus caespititius from Pico, São Jorge and Terceira islands (Azores). Biochem. Sys. Ecol. 36: 790-

797

Acknowledgments: FCT - PTDC/AGR-GPL/101334/2008.


��

��

P19

Dalmatian sage extracts: aromatic profile and antioxidant capacity

Ortiz, V.1, Carrasco, A.1, Martinez-Ruiz, J.1, Parra, M.1, Martinez, F.J.2, Sanchez, M.3, *Tudela, J.1 1GENZ-Grupo de investigacion Enzimologia (www.um.es/genz), Dept. Bioquimica y Biologia Molecular-A, Facultad

de Biologia, Universidad de Murcia. 2Esencias Martinez-Lozano (www.esenciaslozano.com). 3Alissi Brontë

(www.alissibronte.com). Murcia, Spain.


Topic: Molecular tools and approaches.

Dalmatian sage (Salvia officinalis) from Murcia country, was milled and extracted with water, ethanol and n-

hexane. The extracts were analysed on an Agilent SB C18 5 cm x 4.6 mm x 1.8 microm column, with 0.1%

formic acid water/acetonitrile gradient as mobile phase, using an Agilent 1200 HPLC-RR liquid

chromatograph, DAD-SL detector and ChemStation software. Water extract showed high proportions of

chlorogenic and caffeic acids and other phenolic acids. Hexane extract contains broad and high

concentrations of terpenoids and some flavonoids such as quercetin and luteolin. Ethanol extract consist of

phenolic acids, flavonoids and some terpenoids.

Hexane extract: The main aromatic terpenoids were alpha-thujone (64.52%), camphor (19.14%), eucalyptol

(3.56%), o-cymene (3.40%), borneol (2.38) and caryophyllene oxide (1.94%). Ethanol extract: alpha-thujone

(60.62%), camphor (17.11%), eucalyptol (3.47%), o-cymene (3.37), alpha-humulene (3.37%), caryophyllene

(1.77%) and myrcene (1.69%). These values were determined from calibration straights on a Supelco SLB

5ms 15 m x 0.1 mm x 0.1 microm column, with hydrogen gas carrier, using a Gerstel MPS2 autosampler on

an Agilent 7890 gas chromatograph, the 5975 EI-SQ mass spectrometer detector, the ChemStation software

and the NIST and Wiley mass spectrometry libraries.

The antioxidant capacities of the extracts have been evaluated with the ABTS-TEAC and ORAC-TEAC

assays. The disappearance of the ABTS radical has been monitored at 734 nm with a Perkin Elmer dual

beam Lambda 35 spectrophotometer and UV-WinLab software. The fluorescein disappearance was recorded

at 485 nm and 530 nm as excitation and emission wavelengths, respectively, after 3D optimization with a

Perkin Elmer LS55 luminescence spectrometer and FL-WinLab software.

This work has been partially supported by grants from several Spanish organizations. Projects BIO2009-12956

(MICINN, Madrid) and 08856/PI/08 (Fundacion Seneca, CARM, Murcia). Predoctoral fellowships JMR BES-2007-

16208 (FPI, MICINN, Madrid), MP 09378/FPI/08 (Fundacion Seneca, CARM, Murcia), AC (Esencias Martínez

Lozano, Murcia), VO (Alissi Brontë, Murcia).


��

��

P-20

In vitro cultures for production of Plantago major and P. lanceolata secondary metabolites

Dziadczyk, P., Sochacka-Pi�tal, M., *Tyrka, M., Bocian, A., Ruman, T., Semik, M., Buczkowicz, J.

Department of Biochemistry and Biotechnology. Rzeszow University of Technology. Rzeszów. Poland



Plantago major L. has been widely used as a medicinal plant for centuries. The plant extracts have wound

healing, anti-inflammatory, immunomodulatory, antiviral, antioxidant, and free radical scavenger biological

properties. Active compounds of P. major are grouped into pectins, alkaloids, caffeic acid derivatives,

flavonoids, and iridoid glycosides (Samuelsen 2000). Similarly, medicinal value of extracts from Plantago

lanceolata L comes from the presence of aucubin, catalpol, tannins, and sterols.

In our newly started research program, direct and indirect organogenesis was applied to establish root

cultures in liquid media for preliminary testing of the extracts chemical composition. In a frame of long term

optimization of biotechnological process devoted to production of selected secondary metabolites, in vitro

derived plants will be characterized for the presence of endobiotic microorganisms with molecular tools. To

better explore biosynthetic capacity of the two diploid Plantago species an efforts will be undertaken to

obtain polyploids. Transcriptome and proteome analyses will be applied to reveal genetic factors responsible

for different levels of target metabolites in selected clones and interaction between genotypes and active

compounds’ elicitors.

Genetic variation in key and bottleneck genes including upstream and downstream regions will be analyzed.

Transformation procedures will be applied for up-regulation of secondary metabolites synthesis.

References

Samuelsen AB. 2000. The traditional uses, chemical constituents and biological activities of Plantago major L. A

review. Journal of Ethnopharmacology 71: 1-21.


��

��

P-21

Boosting the biosynthesis of bioactive abietane diterpenoids by overexpressing the AtDXS or

AtDXR genes in Salvia sclarea hairy roots *Vaccaro, M.C., Ocampo, V.E., Malafronte, N., De Tommasi, N., Leone, A.

Department of Pharmaceutical and Biomedical Sciences, Faculty of Pharmacy, University of Salerno, Fisciano


Topics: Molecular tools and approaches

Diterpenoids are the most important compounds responsible of the biological activities found in numerous

Salvia species. Salvia sclarea is a cash crop, cultivated mainly for production of essential oil, sclareol and

sclareol derivatives, but roots of this species accumulate several other bioactive diterpenoids. These abietane

diterpenes (e.g. aethipione, 1-oxoaethiopinone, salvipisone and ferruginol) have known pharmacological

value, including citotoxicity and antitumoral activity against different human tumoral cell lines (Rozalski et

al. 2006). However, as for most secondary metabolites, the content of these interesting molecules is too low

(less than 1%) for their economical exploitation. To boost their synthesis the A. thaliana heterologous DXS

(D-xylulose 5-phosphate synthase) and DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) genes,

known to be rate-limiting enzymes of the plastidial pathway of plant terpenoids, were overexpressed

constitutively in S. sclarea hairy roots.

Several independent transgenic hairy root lines for each exogenous gene were generated and three root lines

expressing different level of the exogenous DXS or DXR protein were analysed further. Quantitative targeted

metabolic analysis (HPLC-DAD) revealed that the total diterpenoid content enhanced significantly in AtDXS

and AtDXR overexpressing hairy roots, compared to transgenic roots transformed with the empty vector.

However, overexpression of AtDXR was more effective than AtDXS in boosting the synthesis of

aethiopinone, the most interesting molecule, with of up to 5 fold-increase over the empty vector control,

probably due to the detrimental effect of AtDXS overexpression exerted on hairy root growth. Altogether

these data suggest that this strategy might be promising towards a large-scale production of S. sclarea hairy

roots as a source for the extraction of this interesting class of plant secondary metabolites.

References

Rozalski M, Kuzma L, Krajewska U, Wysokinska H. 2006. Z Naturforsch 61(7-8):483-8.


��

��

P-22

Production of aspartic proteases in arthichoke (Cynara scolymus L.) suspension cell cultures

Celikkol Akcay, U.

Department of Agricultural Biotechnology. Suleyman Demirel University. Isparta. Turkey.



Various animal, plant and microorganism-originated enzymes are used for their milk precipitation activity in

cheese technology. Rennin which is prepared from calf rennet is the most common enzyme preparation used

for this purpose. Various plant-based proteinases also have milk clotting ability. Wild artichoke (Cynara

cardunculus L.) flower extracts are being used traditionally for cheese production in several Mediterranian

countries. Four different aspartic proteases, which are encoded by a multigene family and designated as

cardosin A, B, C and D, were identified from the plant. In recent studies, three different cardosins (A, B and

C) were also izolated from artichoke (Cynara scolymus L.) flowers and their milk precipitating potential

which are similar to wild artichoke was identified. Among cardosin A, B, C and D proteins, which are

characterized and purified through various studies, only the cDNA sequences encoding A and B proteases

and their cristal structures were determined.

In this present study, cardosin B gene was cloned under the control of ribulase biphosphate carboxylase

promoter following the preparation of cDNA library from artichoke (Cynara scolymus L.) flowers.

Artichoke leaves are being transformed by Agrobacterium and the milk precipitation/protein proteolysis

efficiencies of cell suspensions, which are being developed from leaf callus cultures, are currently under

investigation. In addition to the genetic transformation approach, the total aspartic protease activity of cell

suspension culture extracts originated from artichoke pistils is being determined by examining milk

precipitating efficiency and proteolytic effectiveness on casein. Several compounds like jasmonic acid,

carboxymethyl celullose and galactose are known to enhance secondary metabolite production in plant cell

cultures. In this study, the effect of these particular compounds on the production of aspartic proteases in

artichoke suspension cultures are also being determined in comparison to commercial calf rennet,

recombinant microbial rennet and extracts from intact artichoke flowers.

References

Chazarra et al. (2007) International Dairy Journal, 17:1393–1400.

Sıdrach et al. (2005) .Phytochemistry, 66 41– 49.

Sılva et al. (2003) International Dairy Journal 13:559-564.

Gala et al. (2005) Journal of Plant Physiology 162:782-784

Nagamori et al. (2001) Biochemical Engineering Journal, 9: 59–65.

Saito & Mizukami (2002). Plant Biotechnology and Transgenic Plants (Plant Cell Cultures as Producers of Secondary Compounds)

Marcel Dekkel Inc, New York, ABD.


��

��

P-23

Terpene transport issues

*van der Krol, S., Ting, H-M., Bouwmeester, H.

Wageningen University, Lab. of Plant Physiology, Droevendaalsesteeg 1, Wageningen, The Netherlands



Terpenoid producing cells usually also contain high levels of specific lipid transfer proteins (LTP’s) and we

are investigating this suggestive link between terpenes and LTP’s. For this purpose we identified three LTP

genes from Artemisia annua which are highly expressed in glandular trichomes, where also the

sesquiterpene artemisinin is produced. Two LTP homologs were subsequently identified in Arabidopsis

which produces the sesquiterpene caryophyllene. Homozygous single mutants of these two LTP genes were

obtained and analysis of floral extracts by GCMS showed reduced caryophyllene levels in both mutants.

Currently we are trying to obtain the homozygous double LTP mutant plants. RNAi constructs of the

A.annua LTP genes have been transformed into Artemisia plants to test the effect on artemisinin production.

In another set of experiments we tested whether vesicle transport is involved in the accumulation of terpenes.

For this purpose we employed RNAi constructs of specific SNARE genes which are involved in vesicle

fusions. Inhibition of SNAREs by RNAi, in combination with transient expression in N. benthamiana of

different (mono- and sesqui-) Terpene Synthases resulted in higher production of the terpene products. We

are currently trying to elucidate the mechanism behind this effect of SNARE RNAi on terpene

accumulation.


��

��

P-24

Isoflavone synthase in legumes and non-leguminous plants

Pi�manová, M., R�ži�ka, P., *Honys, D.

Institute of Experimental Botany AS CR, Rozvojová 263, 165 02, Praha 6, Czech Republic



Isoflavones belong among important food supplements due to their numerous biological activities. Their

health-protecting and health-prolonging effects have been repeatedly demonstrated. The vast majority of

over 1600 known isoflavone structures were found in legumes. However, isoflavonoids have been identified

in many other species from over 60 families. Most enzymes catalyzing individual steps of the isoflavone

metabolic pathway belong among common methyltransferases and glycosyltransferases. However, the first

step of isoflavone biosynthesis is catalysed by a specific enzyme, isoflavone synthase (IFS). Known IFS

genes belong to the CYP93C subfamily of cytochrome P450. Due to very specific nature of the catalysed

reaction, we hypothesised that IFS genes in other species share high degree of homology with already cloned

genes. We aimed to identify new IFS orthologues in isoflavone-producing species, both legumes and non-

leguminous plants. IFS orthologous sequences were amplified from selected species by touchdown PCR of

genomic DNA with specific set of nested and degenerate primers. Flanking sequences were amplified by

TAIL-PCR. Sequences were analysed by Vector NTI9 and ClustalW. IFS orthologues were identified from

the following species: Pisum sativum (Fabaceae, positive control with yet non-cloned IFS gene), Cannabis

sativa and Humulus lupulus (Cannabaceae), Ruta montana and Ruta graveolens (Rutaceae) and Nicotiana

tabacum (Solanaceae). IFS sequences showed significant degree of similarity, over 70% at the nucleotide

level. The phylogenetic map demonstrates the evolutionary relation of IFS genes from congenial species and

their relationship to other taxa. Non-leguminous isoflavone producers contain close IFS orthologues that are

likely to catalyse the first step of isoflavone biosynthesis. This finding has the potential to be exploited for

the control of isoflavone production in crop plants.

Authors acknowledge the support from GACR (525/09/0994) and MSMT CR (OC10054).


��

��

P-25

Different conversion routes of L-methionine into aroma volatiles in melon fruit

Gonda, I.1,2, Bar, E.1, Sikron, N.2, Burger, J.1, Schaffer, A.A.1, Tadmor, Y.1, Katzir, N.1, Fait, A.2,

*Lewinsohn, E.1 1Institute of Plant Sciences, Newe Ya’ar Research Center, Agricultural Research Organization, PO Box 1021, Ramat

Yishay 30095, Israel 2Ben-Gurion University of the Negev, Jacob Blaustein Institutes for Desert Research, Institute of Drylands

Biotechnology and Agriculture, Midreshet Ben-Gurion, 84990



Melons fruits produce and emit many volatiles that contain sulfur. Previous studies showed that thioether

esters have a significant contribution to the overall aroma perception of ripe melons. We show here that

sulfur-containing aroma volatiles in melon fruits are derived from the amino acid L-methionine. Exogenous

supplied 13C5-L-mehionine was incorporated into melon sulfur aroma volatiles in 3 different patterns: +4

labeled compounds, +1 labeled compounds and mixed +1 and +2 labeled compounds (mainly

dimethyldisulfide and dimethyltrisulfide). These patterns indicate that at least two different metabolic

pathways lead from methionine into sulfur aroma volatiles in melon fruits. In the first pathway the carbon

skeleton of L-methionine is preserved except for the alpha-C, suggesting that the enzyme initiating this

pathway is a methionine aminotransferase. In the second and third pathways only the methylthio group is

preserved from the original L-methionine molecule, suggesting that in this pathway the first catabolic

enzyme is a methionine-�-lyase (MGL) that generates methanethiol. To further support this hypothesis,

MGL activity was detected in cell-free extracts of ripe fruits. In addition, the Melon EST Database

(http://www.icugi.org/) contains a candidate unigene annotated for MGL (CmMGL) composed of 9 clones, 4

of them from ripe fruit. Functional characterization of this gene is under progress. This work is one step

forward towards the understanding of the formation of sulfur aroma volatiles in melon fruits.


��

��

P-26

A role for protein acetylation in modulating the carbon flux towards isoprenoids and other

metabolic pathways? Xing, S., *Poirier, Y.

Department of Plant Molecular Biology, University of Lausanne. CH-1015 Lausanne, Switzerland

*Correspoding author, e-mail: [email protected]


Controlling and increasing the carbon flux towards valuable products in plants is a target for many projects.

Unfortunately, overexpression of genes of a pathway often leads to only marginal improvement in carbon

flux, indicating a large contribution for post-translational regulation of metabolic pathways. In recent years,

acetylation of lysine residues in a large number of metabolic enzymes has been discovered in both animal (1)

and bacteria (2). Lysine acetylation have been shown to up- or down-regulate the activity of a number of

key enzymes involved, for example, in glycolysis, TCA cycle and ß-oxidation (1, 2). The first studies in

plants also showed that numerous proteins are lysine acetylated and that their activity was affected by it (3,

4). These studies raise the question as to whether protein acetylation could be exploited to control and

increase the carbon flux to certain metabolic pathways. In that aspect, we are interested in analyzing whether

pathways utilizing acetyl-CoA as the building block, such as the isoprenoid pathway and fatty acid

biosynthesis, would themselves be regulated by lysine acetylation, and whether modulating the level of

cytosolic level of acetyl-CoA could affect such process.

References:

1- Science 327 : 1000-1004 (2010)

2- Science 327 : 1004-1007 (2010)

3- Plant Physiol. 155 : 1769-1778 (2011)

4- Plant Physiol. 155 : 1779-1790 (2011)


��

��

P-27

Pregnane-modifying enzymes in plants: occurrence, catalysis and function

*Müller-Uri, F., Kreis, W.

Department Biology, FAU Erlangen-Nürnberg, Germany



When investigating the biosynthesis of 5�-cardenolides two enzymes were isolated from Digitalis (foxglove)

plants that were capable of converting pregnenolone to isoprogesterone (3�HSD) and progesterone to 5�-

pregnane-3,20-dione (P5�R), respectively. Meanwhile, the respective plant genes have been isolated and it

was demonstrated that these genes code for functional enzymes not only in plants producing 5�-cardenolides

but also in cardenolide-free plant species. This finding challenges their specific role in cardenolide

metabolism and motivated us to investigate these two enzymes in some depth.

In analogy to the formation of steroids in animals, pregnenolone is assumed to be produced from a sterol

precursor. This initial step is thought to be catalyzed by P450scc, which, however, has never been

characterised in detail in plants. As yet, pregnenolone has not be demonstrated unambiguously to occur in

plants. 3�-Hydroxysteroid dehydrogenases (3�HSD; EC 1.1.1.145) act on pregenenolone to form

progesterone. Several 3�HSDs have been isolated from animals, bacteria and higher plants. In animals the

enzyme is essential for biosynthesis of all classes of steroid hormones, whereas in bacteria 3�HSDs are

involved in steroid degradation. In higher plants they are supposed to be involved in the formation of

secondary plant products, including 5�-cardenolides. 3�HSDs are members of the short-chain

dehydrogenase/reductase (SDR) superfamily of proteins. Bifunctional 3�HSDs are known that also catalyze

the reaction of the �5-3-ketosteroid isomerase (Simard et al., 2005). Even trifunctional 3�HSDs exist such as

guinea pig enzyme which in addition to its 3�HSD and �5-3-ketosteroid isomerase activities also shows 17�-

HSD type II-like activity (Durocher et al., 2005). The human type 1 and type 2 3�HSD isoforms are known

to be encoded by two distinct genes that are expressed in a tissue-specific pattern (Thomas et al., 2002). The

molecular biology and phylogeny of the animal 3�-hydroxysteroid dehydrogenase/�5-3-ketosteroid

isomerase gene family has been studied in some detail. On the other hand, not too much is known about 3�-

HSDs from higher plants. Arabidopsis thaliana, a plant not capable of producing cardenolides, contains

several putative HSDs. Erysimum is a cardenolide-containing genus closely related to Arabidopsis. Two E.

crepidifolium cDNAs have been cloned and expressed in Escherichia coli. The cDNAs both code for

enzymes that catalyse the oxidation of pregnenolone to isoprogesterone. Isoprogesterone is readily converted

to progesterone either by chemical isomerisation or by a 3-ketosteroid isomerase.

Session 3: System engineering approach

Session 3: System engineering approach 54

��

��

O-12

Elucidating regulatory aspects of the response of plant gene expression, metabolism and cell

biology to stressful environments Galili, G.

Department of Plant Science, The Weizmann Institute of Science, Rehovot, Israel


Topic: System engineering approach

Plants, as sessile organisms, which continuously face environmental stresses and other external cues and

adapt to them through synchronized changes in gene expression, metabolism and cellular remodeling. The

concerted operation of these processes is translated into specific phenotypic outputs that help plants survive

these harmful stress conditions or even tolerate them. Elucidating such harmonized responses, particularly

the changes in gene expression and metabolism, is not simple because gene expression and metabolic

programs may be extensively modified in response to different stresses as well as along the progression of a

given stress. I will present a dedicated bioinformatics approach developed in our laboratory, which focuses

on the elucidation of compound changes in the expression of genes encoding metabolism-associated proteins

and transcription factors in response to stress1. I will also show how it was effectively used to identify gene

networks in the model plant Arabidopsis, regulating metabolism in response to various biotic and abiotic

stresses as well as some hormonal and nutritional cues. Finally, I will also present some additional results

from our laboratory, exposing novel insights into the association of selective autophagy processes in plant

stress interactions.

Reference: 1Less H, Angelovici R, Tzin V, Galili G (2011) Coordinated gene networks regulating Arabidopsis plant metabolism in

response to various stresses and nutritional cues. Plant Cell 23: 1264-1271.


��

��

O-13

Computational modeling of intracellular networks beyond flux balance analysis

Hamacher, K.

AG Bioinformatics and theo. Biology. Technische Universität Darmstadt. Darmstadt, Germany



The computational modeling of intracellular networks is a fundamental concept of bioengineering and

synthetic biology. Intracellular networks - and metabolic networks in particular - are extremely versatile

biological entities and need to be understood in several dimensions: from optimized productivity, over

selectivity, to robustness. The traditional approach to investigate solely one aspect (productivity) is flux

balance analysis as a mathematical framework for a quasi-static picture of metabolic networks. As we have

recently shown, stochasticity and fluctuations are of tremendous importance for the production

characteristics of metabolic networks (Phys. Rev. E 84:021913, 2011). In this talk, I will describe modeling

approaches to include such effects, the underlying technological challenges, and the impact the progress in

computational biology can have for the COST action "Plant Metabolic Engineering for High Value

Products” when it comes to engineering synthetic circuits and networks.


��

��

O-14

An in silico compartmentalized model of Brassica napus central metabolism

*Pilalis, E.1,2, Chatziioannou, A.2, Kolisis, F.N.1 1School of Chemical Engineering, National Technical University of Athens 2Institute of Biological Research and Biotechnology, National Hellenic Research Foundation



Biochemical network reconstructions represent valuable tools for the computational metabolic modeling of

organisms that present a great biotechnological interest. An in silico multi-compartmental model of the

central metabolism of the plant Brassica napus (Rapeseed) was constructed, aiming to investigate the

metabolic properties of the Brassicaceae family. In order to study network properties of seed metabolism,

various methods were employed, like Flux Balance Analysis and reaction deletion studies, which simulate

the effect of gene knock-out experiments. Additionally, due to the fact that the large-scale metabolic

reconstructions are typically underdetermined systems with a large space of possible solutions, a systemic

regulatory analysis was performed in order to explore the whole steady-state flux space. The aforementioned

analysis comprised the uniform, random sampling of the solution space, followed by Singular Value

Decomposition of the obtained sample matrix. By this approach we obtained small reaction sets, which

variability accounts for a certain important level of the total flux variability in the entire biochemical

network. Furthermore, the attribution of a global regulatory score to every reaction enabled the identification

of important enzymes as targets of network regulation. As an illustration, we successfully investigated the

regulatory role of the WRI1 transcription factor, which has been shown to control lipid biosynthesis in

Arabidopsis thaliana seeds, by adapting the model to a lipid biosynthesis maximization problem.


��

��

O-15

Regulation of the production of trans-resveratrol in elicited grapevine cell cultures by

signalling compounds. *Belchi-Navarro, S.1, Almagro, L. 1, Miras-Moreno, B. 1, Fernández-Pérez, F. 1, Bru, R.2, Pedreño, M.A. 1 1Department of Plant Biology. University of Murcia. Murcia. Spain. 2Departamento de Agroquímica y Bioquímica. University of Alicante. Alicante. Spain



Elicitors have been widely employed to increase secondary metabolite production in plant cell cultures. Cell

cultures typically respond effectively to oligosaccharide elicitors and to methyl jasmonate (MJ) by inducing

different biosynthetic pathways of secondary metabolites. The use of cyclic oligosaccharides like

cyclodextrins (CDs), alone or combined with MJ, as elicitors has been proved very effective in stimulating

the production of trans-resveratrol (t-R) and its secretion to the medium in V. vinifera cell cultures,

producing more than 3000 mg/L (1). As the use of elicitors is one strategy to increase secondary metabolite

production in cell cultures, understanding the molecular mechanism would improve the management of plant

cells as factories of these compounds. Such molecular mechanism include the early signalling pathways

(protein phosphorylation/dephosphorylation events, cytosolic calcium flux, mitogen-activated protein kinase

activity and H2O2 and NO production (2)) and its relationship with late events, particularly the biosynthesis

of t-R. The results obtained in this study provide evidence for a role of calcium in t-R production triggered

by CDs separately or in combination whit MJ in grapevine cell cultures. In fact, the biosynthesis of t-R is

provoked by an increase in cytosolic free calcium concentration which, in turn, is promoted by an uptake of

Ca2+ from the extracellular medium, and by Ca2+ mobilization from organelles. Protein

phosphorylation/dephosphorylation events appear also to be involved in the signal transduction pathways

triggered by these elicitors. However, while tyrosine protein dephosphorylation events and mitogen-activated

protein kinases seem to be involved in the production of t-R, the involvement of serine/threonine protein

kinases and phosphatases in the signal transduction pathway leading to the production of t-R was not

observed. In the same way, our results indicated that the production of t-R is dependent on both H2O2 and

NO, which are involved in the signal transduction pathways triggered by CDs in grapevine cells.

References

1. Belchí-Navarro S, Almagro L, Lijavetzky D, Bru R and Pedreño MA. 2011. Plant Cell Reports. DOI 10.1007/s00299-

011-1141-8

2. Lecourieux, D; Ranjeva, R; Pugin, A. 2006. New Phytologist. 171:249.


��

��

O-16

1H-NMR based metabolomics as a new tool for variety selection of flaxseed

Ramsay, A.1,2,3, Molinie, R.1, Fliniaux, O.1, Jousse, C.1, Guillot, X.4, Roscher, A.2, Grand, E.3, Kovensky, J.3,

Gontier, E.1, *Mesnard, F.1 1EA 3900 Biologie des plantes et contrôle des insectes ravageurs, Faculté de Pharmacie, 1, rue des Louvels et Faculté

des Sciences, 33, rue Saint Leu, 80000 Amiens, France, 2Génie Enzymatique et Cellulaire, UMR CNRS 6022,

Université de Picardie, 33 rue St. Leu, 80039 Amiens Cedex, France 3Laboratoire des Glucides CNRS FRE 2779,

Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens, France 4Laboulet Semences

S.A., BP5, 80270 Airaines



Since the last decade, there is an increasing interest in the use of flaxseed (Linum usitatissimum) in relation

to human health. The seedcoat of flaxseed is rich in lignans and the embryo is rich in oil with a high omega-3

fatty acid content. The beneficial effects of these compounds on human health are now well recognised.

Lignans - and other phenylpropanoid derivatives - appear to be anticarcinogenic compounds, whereas

omega-3 fatty acids are known to reduce heart desease and would be helpful in the case of inflammatory

deseases such as rheumatoid arthritis. Besides applications of flaxseed components reported in

pharmaceutical, food and cosmetic products, it can be used to feed animals and poultry, once processed. It is

therefore of interest to have a variety selection tool of flaxseed based on its metabolite content.

Here we report the development of such a method by using NMR-based metabolomics after optimisation of

the extraction process. It was indeed necessary to adapt the analytical constraints to those of the plant

material. This step was achieved using a design of experiments. 1H NMR spectra of flaxseed extracts

coupled with multivariate data analysis were applied to investigate the metabolite variations of the polar

fraction - containing the phenylpropanoid derivatives - in varieties of flaxseed different in fatty acid content.


��

��

O-17

Carotenogenesis in the potato tuber

Taylor, M.A.

Cell and Molecular Sciences, James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK

*Corresponding author, email: [email protected]


Despite extensive studies characterising the biosynthetic genes involved in the carotenoid pathway little is

known about the mechanisms regulating carotenoid accumulation in non-green tissues. Early transgenic

studies have demonstrated that tubers have the capacity to accumulate nutritionally significant levels of a

range of carotenoids, either by manipulation of biosynthesis using single or combinations of transgenes, or

by altering the sink capacity for storage of carotenoids. We have used a range of techniques in an attempt to

discover the genes that underpin natural variation in potato tuber carotenoid content. A conventional

genome-wide QTL approach has been used to develop our understanding of the genetic architecture of the

tuber carotenoid trait. Two major and numerous minor QTL were discovered. A genetical genomics

approach was used to identify candidate genes for QTL.

This work was funded by EU-FP7 METAPRO 244348


��

��

O-18

Temporary immersion systems for contained plant propagation and biomass production

Wawrosch, C.

Department of Pharmacognosy. University of Vienna. Vienna. Austria.



Standard in vitro-systems for shoot or whole plant culture are mostly based on semi-solid nutrient medium.

This involves large numbers of relatively small containers, large culture areas, and extensive manual

handling., thus resulting in certain costs as well as limited scale-up options. Micropropagation systems

basing on liquid nutrient media offer various advantages like e.g. uniform culture conditions, easy renewal of

medium, scale up to bioreactor size, and possibilities of automation. However, in most plant species

continuous contact of the explants to liquid medium leads to hyperhydration and subsequent loss of the

propagules.

This problem can be overcome with temporary immersion systems (TIS) where the plant material is only

periodically wetted with liquid medium. After proper, species dependent adjustment of immersion frequency

and duration, TIS can yield high biomass, and can be scaled up to fermenter size. As an advantage over cell

suspension cultures, usually no problems occur with somaclonal variation. Selected applications will be

presented and compared to „standard“ cultivation on semisolid medium. TIS should have a good potential as

contained production systems for otherwise not ex vitro-cultivable GM plants


��

��

O-19

Proteomic analysis of cyclodextrin- and methyl jasmonate-mediated resveratrol accumulation

in grapevine cell cultures

Martinez-Esteso, M.J.1 , Vilella-Antón, M.T.1, Sellés-Marchart, S.2, Morante-Carriel, J.1, Vera-Urbina, J.C.1,

Pedreño-García, M.A.3, *Bru-Martínez, R.1 1Plant Proteomics and Functional Genomics Group, Department of Agrochemistry and Biochemistry, Faculty of Science, University

of Alicante, Alicante, Spain. 2Research Technical Facility, Proteomics and Genomics Division, University of Alicante, Alicante,

Spain. 3Plant Peroxidases Group. Department of Plant Physiology, Faculty of Biology, University of Murcia, Murcia, Spain.



Grapevine (Vitis vinifera cv. Gamay) cell suspension cultures accumulate large extracellular amounts of the

stilbenoid resveratrol along several days, in response to elicitation with methylated cyclodextrins (MBCD)

and methyl jasmonate (MeJA) [1,2]. The aim of this study was to investigate changes in the cellular

proteome potentially related to resveratrol production and accumulation in response to the above elicitors.

The quantitative gel-based DIGE technique was used to detect statistically significant changes in the cell’s

proteome during an incubation time of up to 120 hours. In a pilot experiment [3], 67 unique spots were found

de-regulated after 96 hours of incubation with elicitors. In a larger proteomic kinetic experiment sampling

four data points (6, 24, 72 and 120 hours) from each experimental condition (control, MeJA, MBCD and

MBCD+MeJA treatments), the number of de-regulated unique spots detected increased to 212. Protein

identification based in nLC-MS/MS analysis and NCBInr database search revealed a large complexity in the

abundance pattern of several proteins relevant to resveratrol synthesis and accumulation. Stilbene synthases

(STS), found in 29 unique spots, encoded by up to 40 paralogs, were classified into 6 phylogenetic groups.

Such groups showed specific abundance patterns in response to the different elicitor treatments. The

comparison of STS protein and stilbene metabolite profiles lead to detect potential STS groups specifically

involved in the response to MBCD alone or combined with MeJA elicitors that lead to resveratrol

accumulation. Likewise, glutathione-S-transferases (GST) were found in a high number of spots. These

belonged to two tau phylogenetic subgroups, one phi and one lambda class. Interestingly, the five GST

whose abundance profiles correlated with profiles of STS and metabolites composed one of the two tau class

clusters, thus pointing to a potential role in resveratrol trafficking within the cell and across plasma

membrane. Finally, the profile of alpha-SNAP, a protein required for vesicle trafficking suggest that this

process may be involved in metabolite accumulation. Other findings will be presented and discussed in

relation to the effect of MBCD and MeJA elicitation.

References: (1) Bru et al. (2006) J. Agric. Food Chem. 54, 65-71. (2) Lijavetzky et al. (2008) BMC Res. Notes 1: 132. (3) Martinez-

Esteso et al. (2011) Journal of Proteomics doi:10.1016/j.jprot.2011.02.035

Acknowledgements

This work has been supported by research grants from MICINN-FEDER (BIO2008-2941). Protein identification was carried out at

the Alicante University Proteomics Facility, a laboratory member of PROTEORED (http://www.proteored.org). MJME holds a

research grant from CajaMurcia; JCVU holds a grant from the International Cooperation Unit of the Alicante University for Latin

American students.


��

��

O-20

The relationship between taxane production and the expression of several genes involved in

taxol biosynthesis in different in vitro Taxus systems *Onrubia, M.2, Bonfill, M.1, Moyano, E.2, Cusidó, R.M.1, Goossens, A.3,4, Palazon, J.1

1Sección de Fisiologia Vegetal, Fac.Farmacia, Universidad de Barcelona, E-08028 Barcelona, Spain 2Departament de Ciències Experimentals i de Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain 3Department of Plant Systems Biology, VIB, B-9052 Gent, Belgium 4Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Gent, Belgium



Some steps and the regulation of taxol biosynthesis remain incompletely understood. For this reason it is

essential to explore how different factors that improve taxane biosynthesis and accumulation also affect the

metabolic profiles and genetic expression in different in vitro Taxus spp.systems.

We have studied the relationship between the pattern of taxane production and the expression of several

genes involved in taxol biosynthesis in cell cultures of T. baccata, of T. media cell cultures overexpressing

the TXS gene from T. baccata as well as of T. baccata plantlets growing for one year in vitro.

Results obtained from both transgenic and non-transgenic cell cultures showed that taxane production

depends completely on the presence of elicitors in the culture medium. While elicitation with 100 µM methyl

jasmonate considerably increased the production of taxol and baccatin III and the expression of TXS and

BAPT genes, 50 µM vanadyl sulphate mainly increased taxol production and BAPT expression. When cells

cultures were elicited with 1 µM Coronatine, total taxane production was 4 times higher than with MeJ and

almost 8 times higher than in the unelicited cell line. Although cultures grown with MeJ and Cor had a

higher gene expression compared to control, the clearest difference between them was the earlier expression

of taxol biosynthesis genes under Coronatine elicitation. In their first year, the most abundant taxane in the

aerial parts of plantlets was 10-deacetylbaccatin III and low transcript accumulation of the DBAT gene was

observed.

TXS expression is required for all taxanes synthesis, low expression of DBAT restricts convertion from 10-

deacetylbaccatin III into baccatin III and BAPT expression is correlated with highest production of taxanes

with lateral chain attached. All these results allow us to conclude that the expression of genes involved in

taxol biosynthesis is related with the pattern of taxane production.


��

��

P-28

Early signalling network in tobacco cells elicited with methyl jasmonate and cyclodextrins

*Almagro, L.1, Pugin, A.2, Pedreño, M.A.1 1Dpto de Biología Vegetal. Facultad Biología. Universidad de Murcia. Spain. 2UMR Plante-Microbe-Environnement. INRA. Université de Bourgogne, Dijon. France.



The events occurring in elicitor-induced defence responses include reversible phosphorylation of plasma

membrane and cytosolic proteins, cytosolic calcium flux, plasma membrane depolarization, defence gene

expression, MAPK and NADPH oxidase activation, H2O2 and NO and secondary metabolite production (1).

Methyl jasmonate (MJ) has been proposed as a key compound of the signal transduction pathway involved in

the production of secondary metabolites which take part in plant defence reactions (2). Likewise, special

attention has been paid to the use of cyclodextrins (CDs) as elicitors that induce defense responses, including

phytoalexin synthesis (3). However, the intracellular signalling pathway induced by CDs alone or in

combination with MJ in cell cultures is completely unknown. Our results showed that the application of MJ

and CDs to tobacco cells provoked an increase of cytosolic free Ca2+ concentration ([Ca2+]cyt), which was

promoted by Ca2+ influx from the extracellular medium. The Ca2+ signatures in response to elicitors differed

in peak time, intensity and duration. Likewise, the [Ca2+]cyt rise triggered by MJ was independent of both

staurosporine-sensitive protein phosphorylation and H2O2, whereas CDs activated a protein kinase that

would act upstream of the [Ca2+]cyt rise, or Ca2+ influx which in turn, is dependent on H2O2-sensitive

channels. These results suggest the existence of different intracellular signalling pathways for both elicitors.

Likewise, CDs might act by regulating the signalling pathway triggered by MJ since, in the presence of both

compounds, CDs neutralized the strong oxidative and nitrosative bursts triggered by MJ and therefore, they

regulate both H2O2 and NO levels. Moreover, an alkalinization of the extracellular medium and the induction

of capsidiol are observed as a result of elicitation.

References

1. Lecourieux, D; Ranjeva, R; Pugin, A. 2006. New Phytologist. 171:249.

2. Gundlach H, Müller MJ, Kutchan TM and Zenk MH (1992) PNAS 89: 2389.

3. Bru, R; Sellés, S; Casado, J; Belchí-Navarro, S; and Pedreño, MA. 2006. J. Agric.Food Chem. 54: 65


��

��

P-29

Metabolic profiling of citrus peel tissues reveals an important role of phenylpropanoids in

induced resistance

Ballester, A.R1,2,3, Lafuente, M.T1, de Vos, R2,3, Bovy, A2,3, *González-Candelas, L1. 1Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC). Paterna, Valencia. Spain. 2Plant Research

International. Wageningen. The Netherlands. 3Centre for Biosystems Genomics. Wageningen. The Netherlands.



Penicillium spp. are among the major postharvest pathogens of citrus fruit in Mediterranean climate regions.

The application of fungicides constitutes the most common method used to control postharvest diseases.

However, due to the development of resistance to fungicides among fungal pathogens and the growing

public concern on their effects on human health and environment, there is a trend to develop alternative

methods to control postharvest diseases. Induction of natural resistance in fruits constitutes one of the most

promising alternatives to chemical fungicides. We have recently used an infection+curing treatment that

induces resistance in citrus fruit. Resistance coincided with the induction of PAL, soluble peroxidase, basic

�-1,3-glucanase and chitinase at both gene expression and enzyme activity levels (Ballester et al., 2010).

Moreover, using a transcriptomic approach we have shown that secondary metabolism, mainly

phenylpropanoid biosynthesis, and ethylene play important roles in the induction of resistance in citrus fruit

(Ballester et al., 2011) and in the defence against pathogens (Gonzalez-Candelas et al., 2010).

In order to better understand the biological basis of induced resistance in citrus fruit, we have used a

metabolomics approach in elicited oranges using HPLC coupled to a PDA and a fluorescence detector.

Further analyses by HPLC coupled to an accurate mass spectrometer allowed the identification of several

compounds that may be relevant for induced resistance. In elicited fruits, a greater diversity of phenolic

compounds was observed in the flavedo (outer coloured part of the peel) as compared to the albedo (inner

white part of the peel). Only small changes were detected in the most abundant flavonoids, such as flavones,

flavanones and polymethoxylated flavones. The coumarin scoparone was among the compounds with

highest induction upon elicitation. Two other induced compounds were identified as citrusnin A and

drupanin aldehyde. All three compounds are known to exert antimicrobial activity. Our results indicate that

phenylpropanoids and flavonoids play an important role in the induction of resistance in citrus fruit.

References

Ballester AR, Izquierdo A, Lafuente MT, González-Candelas L. 2010. Postharvest Biology and Technology 56: 31-38.

Ballester AR, Lafuente MT, Forment J, Gadea J, De Vos CHR, Bovy AG, González-Candelas L. 2011. Molecular Plant

Pathology. doi: 10.1111/j.1364-3703.2011.00721.x.

Gonzalez-Candelas L, Alamar S, Sanchez-Torres P, Zacarias L, Marcos J. 2010. BMC Plant Biology 10, 194-211.


��

��

P-30

Resveratrol production in grapevine suspension cultured cells and evaluation of its

antitumoral activity

Belchi-Navarro, S.1, *Fernández-Pérez, F. 1, Bru, R.2, Pedreño, M.A. 1 1Department of Plant Biology. University of Murcia. Murcia. Spain. 2Departamento de Agroquímica y Bioquímica. University of Alicante. Alicante. Spain



In this work, we used elicitation with cyclodextrins and methyl jasmonate (Belchí-Navarro et al. 2011) as a

strategy to increase the production of trans-resveratrol (trans-R) in suspension cultured cells (SCC) of Vitis

vinifera cv Monastrell as a previous step to scaling-up in a bioreactor . We studied how both elicitors and

different factors might enhance trans-R production since this could be particularly valuable in large-scale

systems. Thus, when SCC of Monastrell were elicited for eight continuous elicitation cycles, trans-R

production increased until the third elicitation cycle when it reached 0.82±0.05 mg/L.day, using

cyclodextrins and methyl jasmonate as elicitors. When grapevine SCC was scaled-up using a cell biomass

ranging from 0.1 to 1.5 L in flasks that ranged in size from 0.25 to 5 L, maximum trans-R productivity was

obtained in both the 0.5 and 5 L flasks, containing 0.2 or 1.5 L, respectively, of cells suspension elicited with

a combination of cyclodextrins and methyl jasmonate. In these conditions, the final productivity was

9.89±0.10 mg/g fresh weight (FW) in 0.5 L flask and 8.65±0.10 mg/g FW in 5 L flask.

In addition, in vitro trials were carried out to better characterize trans-R role in protection against cancer. For

this, the antitumoral activity of trans-R obtained from our grapevine SCC was tested in JURKAT E.6

(Human acute T cells leukaemia), THP-1 (Human acute monocytic leukaemia) and MCF-7 (Human breast

adenocarcinoma) cell lines. After 72 hours of incubation we determined the IC50 values with different

concentrations of trans-R. The values obtained were 29.3±2.5 �M for JURKAT E.6, 21.73±3.2 �M for THP-

1 and 60.47±2.9 �M for MCF-7. Cell cycle distribution was determined by flow cytometry analysis. A

concentration of 30 µM trans-R led to the accumulation of cells in their S phase, accompanied by a

significant decrease in the G0/G1 and G2/M states in all cell lines after 48 hours of incubation. Increasing the

concentration to 90 µM trans-R restored the G0/G1 cell population to close of their initial control levels,

while the total number of cells and cells in the S phase were reduced.

References

Belchí-Navarro S, Almagro L, Lijavetzky D, Bru R and Pedreño MA. 2011. Plant Cell Reports. DOI 10.1007/s00299-

011-1141-8


��

��

P-31

Phospholipid signalling in Silybum marianum elicited cell cultures

Corchete, P.

Department of Plant Physiology. University of Salamanca. Salamanca. Spain.



Several evidences show that phospholipids have function in the signalling transduction pathway leading to

the extracellular accumulation of the flavonolignan silymarin in elicited cultures of Silybum marianum.

Activation of PLA and PLD was produced by methyljasmonate in cell cultures. Using fatty acid fluorescent

labelled phosphatidylcholine, both fluorescent fatty acid and phosphatidic acid were detected in “in vivo”

labelled cultures. The magnitude of the activation by the elicitor was both time- and dose- dependent.

Inhibitors of PLA and PLD inhibited the activation of both enzymes by MeJA and also blocked silymarin

release into the medium.

The powerful secretagogue mastoparan strongly activated PLA and PLD and also elicited silymarin

accumulation and release.

The three indicators of PLA and PLD activities, namely lysophosphatidylcholine, free fatty acids and

phosphatidic acid, when added alone to control cultures emulated the action of MeJA, inducing the synthesis

and extracellular accumulation of silymarin.

The presence of the lysophospholipids LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and

the high levels of PA, represented by the species 34:3, 34:2 and 36:5 and 36:4, indicates high basal levels of

phospholipase activity and a high phospholipid turnover. Elicitation did not quantitatively alter total lipid

membrane content nor overall fatty acid composition, but induced changes in some lipid molecular species.

A detailed ESI-MS/MS-based analysis showed that mole percentages of 36:6 PC and 36:5 PC and PE species

were lower in the MeJA –treated cells than in the control, suggesting that the decrease of these species, with

a probably acyl composition of di-18:3 and 18:3-18:2, might have resulted from a preference or partial

accessibility to hydrolytic enzyme(s), like PLD, for more unsaturated species. These results suggest that

elicitation causes remodelling of membranes. The connection of these events with the extracellular release of

secondary metabolites needs further research.


��

��

P-32

Psoralen: bioproduction and regulation in Bituminaria bituminosa

*Del Río, J.A.1, Díaz, L.1, Pérez, I.1,Correal, E.2, Dabauza, M.2, Walker, D.2, Ortuño, A.1 1 Department of Plant Biology. University of Murcia. Murcia. Spain. 2 IMIDA. La Alberca. Murcia, Spain.

* Corresponding author, e-mail [email protected]


Psoralen is a linear furanocoumarin, a subgroup of phenolic compounds included in the coumarins group,

whose presence in Bituminaira bituminosa has ben demonstrated in previous reports (Martínez et al. 2010;

Del Río et al. 2010). This compound is used for the treatment of psoriasis, vitiligo and other type of

dermatitis due to its effect on skin pigmentation/depigmentation. This furanocoumarin makes the skin more

sensitive to radiation, so that it can be activated when combined with ultraviolet light in the therapy called

PUVA. This is one possible treatment for skin diseases.

This work quantifies by HPLC-MS the furanocoumarin content in different varieties of B. bituminosa plants

and cell cultures. The objective is to identify and select lines that have a high psoralen content that can be

used as a source for the extraction of this compound for pharmaceutical purposes.

The results obtained reveal that the high concentration of sporalen recorded in B. bituminosa var. bituminosa,

accession Calnegre, make it an ideal source for the extraction of this secondary metabolite. The use of

different cell cultures obtained from plants of B. bituminosa var. bituminosa, organogenic calli and root

cultures submitted to different elicitation protocols (UV, heavy metals) is receiving attention as an

alternative production system of this furanocumarin.

References

Martínez S, Correal E, Real D, Ortuño A, Del Río JA. 2010. Recent Progress in Medicinal Plants, Vol. 27, Drug Plants

I (Awaad A.S., Govil J.N., Singh V.K., Eds.) Studium Press LLC, USA pp. 307-322. ISBN 1-933699-17-5.

Del Río JA, Ortuño A, Pérez I, Bennett RG, Real D, Correal E. 2010. The contribution of grasslands to the conservation

of Mediterranean biodiversity (C. Porqueddu, S. Ríos, Eds.), pp. 67-70, ISBN 2 85352-435-3.

Acknowledgements: This work was supported by a grant (BFU2010-19599) from Ministerio de Ciencia e Innovación,

Spain.


��

��

P-33

Ethylene signalling drives the accumulation of antioxidants in tomato fruit

*Di Matteo, A.1, Ruggieri, V.1, Sacco, A.1, Carriero, F.2, Rigano, M.M1, Frusciante, L.1, Barone, A.1 1DiSSPAPA, University of Naples Federico II, Via Università 100, 80055 Portici (Italy) 2Metapontum Agrobios, S.S. Jonica 106, km 448.2, 75010 Metaponto (Italy)



Tomatoes are valuable sources of antioxidants such as phenolic compounds and ascorbate. In the last years

increasing evidences suggest the possible role of antioxidants in the prevention of cardiovascular disease and

cancer. Antioxidants are plant secondary metabolites that are important for both sensory and nutritional

quality of fruits and vegetables. Therefore, increasing antioxidants in tomato fruit is a major claim of

breeding in order to meet consumer requirements and create new market opportunities. So far, many key

genes have been reported that control the accumulation in planta of antioxidants. However, additional

insights are required in order to highlight genetic and physiological mechanisms controlling the

accumulation of antioxidants in tomato fruit and to support breeding programs for increased fruit quality.

The aim of the work was the identification of major genes and gene networks that regulate the level of

antioxidant compounds in tomato fruit.

The screening of Solanum pennellii x S. lycopersicum introgression lines (ILs) over three year trials allowed

the identification of a stable QTL for increased fruit content of total phenolics in the IL7-3. In particular,

chlorogenic acid mainly accounted for the higher performance of this line. In addition, IL7-3 also expressed

higher ascorbate and soluble solid content in red-ripe fruit. To investigate candidate genes controlling

nutritional quality parameters in IL7-3 fruit, we performed a comparative transcriptomic analysis in tomato

pericarp between this line and the control cv. M82. The transcriptomic approach allowed identifying 149 up-

regulated and 142 down-regulated probes. Based on functional annotation, clustering and networking

outputs, subsets of differentially expressed transcripts were used to develop model networks that describe

mechanisms controlling accumulation of antioxidants in tomato fruit. The network explains the variation in

phenols levels in terms of interactions between ethylene signalling, plant responses to stress and biosynthesis

of phenolics. Upon validation of key transcripts of our model by RT-qPCR we undertook a functional

characterization of candidate genes by the TILLING approach. In particular, tomato plants homozygous for

mutations in the coding sequence of an ethylene-responsive factor ERF1 revealed to drive ripening-

associated accumulation of antioxidants in fruit. These results suggested us to design new strategies of

precision breeding for increased fruit nutritional quality in tomato.


��

��

P-34

Rosmarinic acid production in salicylic acid-treated Thymus membranaceus shoots

López-Orenes, A., Martínez-Pérez, A., Calderón, A.A., *Ferrer, M.A.

Department of Agricultural Science and Technology. Universidad Politécnica de Cartagena, Spain



Thymus membranaceus Boiss. subsp. membranaceus is an endemic species in Southeast Spain, belonging to

the Lamiaceae family. As occurs with many other members of this plant family T. membranaceus has been

used for ethnopharmacological purposes since ancient times. However, data on phytochemistry or biological

activity of the species are scarcely documented. An environmental friendly approach for studying the

biological activity of endemic and rare species is the use of in vitro culture techniques since only a small

amount of plant material is needed to initiate the culture. One additional advantage of using in vitro culture is

that the accumulation of phytochemicals can be enhanced by elicitation with stress related compounds such

as salicylic acid (SA). Taking into account the above-mentioned statements the aims of this work were (a) to

characterise the antioxidant activity of Thymus membranaceus shoots grown in vitro using different

analytical methods, (b) to evaluate the correlation between the antioxidant properties and the phenolic

content of the shoots, and (c) to investigate the effect of SA doses on antioxidant properties, total phenolic

content and rosmarinic acid (RA) accumulation in T. membranaceus shoots. The results obtained showed

that a single application of SA (10 µM) on culture media resulted in an increase in RA and phenolic levels,

which in turn improved extracts’ antioxidant properties. These current findings open new opportunities for

obtaining valuable natural antioxidants for commercial exploitation by using tissue culture systems


��

��

P-35

Volatile phytochemicals production by hairy root cultures

*Figueiredo, A.C., Pedro, L.G., Barroso, J.G.

Universidade de Lisboa, Faculdade de Ciências de Lisboa, Departamento de Biologia Vegetal, Instituto de

Biotecnologia e Bioengenharia, Centro Biotecnologia Vegetal, C2, Campo Grande, 1749-016 Lisboa, Portugal.



Hairy root cultures are a flexible system, grown in hormone-free media, which can be controlled in terms of

nutrition and environmental conditions, constituting an alternative technology for large-scale production of

valuable products.

Our work has focused mainly on the volatiles production by hairy roots from members of Asteraceae

(Achillea millefolium) and Apiaceae (Anethum graveolens, Levisticum officinale and Pimpinella anisum)

(Figueiredo et al. 2006 and references therein) and more recently from Convolvulaceae (Ipomoea batatas),

Plantaginaceae (Digitalis purpurea) and Solanaceae (Solanum lycopersicum and S. tuberosum). Hairy root

cultures are kept in batch culture in darkness, or different photoperiod conditions, at 24ºC, 80 r.p.m., and

subcultured every month.

In order to assess how physiological and nutritional factors affect volatiles production, the culture medium

composition, namely nitrogen sources and ratios, illumination conditions, addition of precursors or elicitors,

induction of regenerants, culturing in bioreactor and in two-phase systems and biotransformation, have been

evaluated (Figueiredo et al. 2006 and references therein, Costa et al. 2008, Nunes et al. 2009, Faria et al.

2009). Presently the root morphology and growth, the biotransformation capability and the root-organism’s

relationships, is being evaluated using foxglove (Digitalis purpurea), sweet potato (Ipomoea batatas), potato

(Solanum tuberosum) and tomato (Solanum lycopersicum) hairy roots as model systems.

References

1. Figueiredo AC, Barroso JG, Pedro LG, Scheffer JJC. 2006. Potentialities of hairy root cultures for in vitro essential

oil production. In: Teixeira da Silva JA (Ed.), Floriculture, Ornamental and Plant Biotechnology: Advances and

Topical Issues, Vol. II, pp. 478-486. Global Science Books, UK.

2. Costa MM, Figueiredo AC, Barroso JG, Pedro LG, Deans SG, Scheffer JJC. 2008. Biotechnol. Letters, 30: 1265-

1270.

3. Nunes IS, Faria JMS, Figueiredo AC, Pedro LG, Trindade H, Barroso JG. 2009. Planta Medica, 75: 387-391.

4. Faria JMS, Nunes IS, Figueiredo AC, Pedro LG, Trindade H, Barroso JG. 2009. Biotechnol. Letters, 31: 897-903.


��

��

P-36

Towards reduced acrylamide potato products: Analysis of precursor accumulation pathways

using 13C-labelling in conjunction with GC/MS

Pont, S.D.A.1, Ponte, A.1,2, Shepherd, L.V.T.1, Davies, H.V.1, *Hancock, R.D.1 1 The James Hutton Institute, Invergowrie, Dundee DD2 5DA. United Kingdom. 2 Ecole Nationale Supérieure d’Agronomie et des Industries Alimentaires, 2 avenue de la Forêt de Haye - BP 172,

54505 Vandoeuvre les Nancy Cedex. France.

* Corresponding author, e-mail: [email protected]


Acrylamide is a neurotoxin and potential carcinogen formed at elevated temperatures by reaction between

asparagine and Maillard reaction intermediates. Acrylamide formation is recognised as a potential problem

in foods containing high levels of free sugars and amino acids that are prepared at high temperatures (frying,

roasting, baking). Food processors are seeking to address these issues through a variety of approaches

including agronomic and storage practices designed to reduce the formation of acrylamide precursors, and

the choice of parameters to reduce acrylamide formation during processing. In the longer term there is scope

for the breeding or metabolic engineering of novel cultivars with reduced accumulation of acrylamide

precursors.

To aid the identification of targets for conventional breeding or metabolic engineering, we undertook an

examination of amino acid metabolism in potato tubers from genotypes with high and low acrylamide

forming potential (AFP). Tuber discs were labelled with [U-13C]Glu or [U-13C]Asp and their metabolic

products monitored by GC/MS. In this system formation of [13C]Asn from [U-13C]Asp was barely detectable

and instead we observed a strong flux from Asp into TCA cycle intermediates. Similarly, both genotypes

diverted significant quantities of Glu into the TCA cycle via the GABA shunt with the low AFP genotype

exhibiting a stronger flux from Glu to Gln (an amino donor for Asn formation) than the high AFP genotype.

The approach taken suggests that synthesis of free Asn in tubers is not a strong contributor to its

accumulation and alternative mechanisms such as import via the phloem or release of Asn via proteolysis

may contribute. Interestingly, we found evidence for the presence of an Asp-4-decarboxylase activity,

previously only described in prokaryotes. Transcript-metabolite correlations suggested a potential gene

encoding this activity which may represent a target for the breeding or metabolic engineering of cultivars in

which levels of the Asn precursor Asp are reduced.


��

��

P-37

A multidisciplinary approach to understand the control of flavonol synthesis in grape berry

*Malacarne, G.1, Coller, E.1, Heppel, S.2, Vrhovsek, U.1, Czemmel, S.2, Bogs, J.2,3, Moser, C.1 1Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all’Adige (Trento -

Italy). 2Centre for Organismal Studies Heidelberg (COS), University of Heidelberg, Im Neuenheimer Feld 360, 69120

Heidelberg-Germany 3Fachhochschule Bingen, Berlinstr. 109 55411 Bingen am Rhein, Germany



Flavonols are flavonoids found in most higher plants, usually in glycosidic forms. They are products of the

flavonoid biosynthetic pathway, which also give rise to anthocyanins and to condensed tannins in grapes. In

grapevine, they are predominantly synthesized in inflorescence and berry skin, while no detectable levels are

found in pulp or seeds (Downey et al., 2003). They act as UV-protectants and as anthocyanin co-pigments in

flowers and fruits. For this reason they play a role in defence and pollinators attraction and in conferring

stability to the colour of red wines.

Although the general flavonol pathway has been genetically and biochemically elucidated in many plant

species and most recently also in grapevine (Matus et al., 2009), (Czemmel et al., 2009), its regulation still

remains not completely characterized.

A population from the cross Syrah x Pinot Noir, segregating for the flavonol content, is being studied to

identify the genetic determinants of flavonol accumulation in the berry cells by a biochemical and

transcriptional characterization. Individuals of the progeny showing high- and low- flavonol content were

selected and characterized at different stages of berry development by a microarray approach. Among the

differentially expressed identified genes, we focused our attention on a transcription factor not yet

characterized in grapevine. To prove its involvement in the control of flavonoid pathway we identified its

targets by a promoter assay approach and we analyzed the phenotype of transgenic tobacco lines

overexpressing this gene. Preliminary results will be presented.

References

Downey et al. .2003. Synthesis of flavonols and expression of flavonol synthase genes in the developing grape berries

of Shiraz and Chardonnay (Vitis vinifera L.). Aust J Grape and Wine Res, 9: 110-121

Matus et al. (2009). Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin

and flavonol synthesis in berry skins of Vitis vinifera. J Exp Bot;60(3):853-67

Czemmel et al. (2009). The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in

developing grape berries. Plant Physiol, 151:1513-30.


��

��

P-38

Activity of plant quinones against oxylipin biosynthesis in-vitro – lipoxygenase and

cyclooxygenase inhibition *Marsik, P., Landa, P.

Laboratory of Plant Biotechnologies, Joint Laboratory of Institute of Experimental Botany Acad. Sci. CR, v.v.i. and

Research Institute of Crop Production, v.v.i., Prague, Czech Republic



Oxylipins are widely spread group of compounds in eukaryotes including plant and animals. Some of them

are important signalling molecules which take part in regulation of essential biological processes

(development, defence, reproduction). In organisms, the oxilipins are products mainly of enzymatic

oxidation of unsaturated fatty acids. Lipoxygenases (LOXs) and cyclooxygenases (COXs) belong to most

important enzymes involved in oxilipin metabolism in higher organisms. In plants, LOXs play crucial role in

biosynthetic pathway of jasmonic acid and related compounds with signal function in plant response to

stress. Human and animal LOXs are key enzymes in biosynthesis of leukotrienes, potent mediators of

chronic inflammatory diseases such as asthma, allergic reactions, and atherosclerosis. COXs are key

enzymes on route leading to the inflammation in animals due to the production of prostaglandins.

Quinone compounds, which are found in various medicinal herbs, had exerted various biological effects

including anti-inflammatory activity. Experimental data indicate that mechanism of anti-inflamatory effect of

some of quinones could be due to inhibition of both of COX and LOX enzymes (Marsik et al. 2005; Werz

2007).

In the present work we try to find potential bioactive quinones of plant origin, which could affect function of

key enzymes of oxylipin pathways. The ability of natural benzo-, naphtho- and anthraquinones to affect

LOX and COXs activity in-vitro was tested. Knowledge of properties of plant secondary metabolites may

help to identify pharmacological targets and modulate their production in plant systems.

References

Marsik P, Kokoska L, Landa P, Nepovim A, Soudek P, Vanek T. 2005. In vitro inhibitory effects of thymol and

quinones of Nigella sativa seeds on cyclooxygenase-1 and -2 catalyzed prostaglandin E2 biosyntheses. Planta Medica

71: 739-742

Werz O. 2007. Inhibition of 5-lipoxygenase product synthesis by natural compounds of plant origin. Planta Medica 73:

1331–1357.


��

��

P-39

Flavonoids levels and mycotoxins expression as part of the defence mechanisms of Citrus sp.

fruits against Alternaria alternate

*Ortuño, A.1, Díaz, L.1, González, J.1, García-Lidón, A.2, Porras, I.2, Lacasa, A.2, Del Río, J.A.1 1 Department of Plant Biology. University of Murcia. Murcia. Spain. 2 IMIDA. La Alberca. Murcia, Spain.

* Corresponding author, e-mail [email protected]


Citrus genus accumulates a series of species-specific flavanones, flavones and polymethoxyflavones, which,

beside shaving important applications in the food and pharmaceutical industries (Benavente et al. 2007;

Ortuño et al. 2006), are also involved in the natural resistance of Citrus against pathogens acting as

phytoanticipins (Ortuño and Del Río 2009).

Alternaria fungi cause several diseases in Citrus, resulting in a substantial loss of production and a lower

value product. The susceptibility to Alternaria alternata depends on the Citrus species, the most susceptible

being the cultivars of Citrus reticulata and its hybrids. Associated with the development of this fungus,

necrosis (known as brown spot) is produced in the infected zone. Studies on the localisation of phenolic

compounds by fluorescent optical microscope and subsequent analysis by HPLC-MS reveal that flavonoids

accumulate around the necrotic zone, which suggests that they might act as phytoanticipins, while in the

necrotic zone itself there is an increase in the activity of oxidising enzymes that cause flavonic catabolism.

The presence of two mycotoxins involved in the evolution of brown spot in these fruits was observed. These

were identified as alternariol (AOH) and alternariol monomethylether (AME). We discuss the possible

participation of these isolated mycotoxins and the flavonoids present in these plant tissues, in the

development of cell death and, therefore, in the evolution of the necrotic zone caused by A. alternata.

References

Benavente-García O, Castillo J, Alcaraz M, Vicente V, Del Río JA, Ortuño A. 2007. Current Cancer Drug Targets 7:

795-809.

Ortuño A, Gómez P, Báidez A, Frías V, Del Río JA. 2006. Potential Health Benefits of Citrus. Oxford University Press,

New York (Patil, B.S., Turner, N.D., Miller, E.G., Brodbelt, J.S., Eds.) pp. 175-185, I.S.B.N. 0-8412-3957-6.

Ortuño A, Del Río JA. 2009. Citrus III. Tree and Forestry Science and Biotechnology 3 (Special Issue 2) (Tennant P.,

Benkeblia N. ,Eds.) pp. 49-53. ISBN 978-4-903313-25-2.

Acknowledgements: This work was funded by the Projects 08676/PI/08 of the Consejería de Educación, Ciencia e

Investigación de la Región de Murcia-Fundación Séneca, Spain, and AGR/11621/08 of the Consejería de Agricultura,

Agua y Medio Ambiente de la Región de Murcia, Spain.


��

��

P-40

New method for the identification of flavonoids, ginkgolides and bilobalide in Ginkgo biloba

extracts by HPLC-ESI-MS-TOF

Hernández-Ruiz, J.1, Sánchez-Godínez, A. 1, Youssef, S.M.2, Sabater-Jara, A.B. 1, Cruz, R.M. 1, Novo-Uzal,

E. 1, *Pedreño, M.A. 1 1Department of Plant Biology, University of Murcia, Murcia, Spain. 2Department of Horticulture, University of Ain

Shams, Egypt. *Corresponding author: e-mail: [email protected]


Ginkgo biloba is one of the oldest living tree species and its extracts or powdered leaves are one of the best

selling herbal preparations. The main bioactive constituents are flavonoids, and terpene trilactones;

ginkgolides and bilobalide; which are considered responsible for their pharmacological activity. In fact,

standardized leaf extracts have been clinically effective in the treatment of Alzheimer’s disease, depression,

diabetic neuropathy, impotency, memory impairment, peripheral vascular disease, intermittent claudication,

vertigo and tinnitus [1]. The positive effects of Ginkgo biloba extracts are thought to result from the

synergistic action of distinct groups of compounds, flavonoids and terpene trilactones [2]. The flavonoids are

responsible for the free radical scavenging effects of G. biloba [3], while the ginkgolides are potent anti-

platelet factor antagonists [4].

Flavonoids and ginkgolides have previously been determined and quantified by gas chromatography–mass

spectrometry (GC–MS) [5], or by high-performance liquid chromatography (HPLC) with UV detector [6].

However, these methods were not validated and required different extractions and separations for the

flavonoid and terpenoid markers, making those methods time consuming and inefficient. In the last few

years, the high performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) has been

shown to be a powerful tool for the identification of natural products in plant extracts owing to their soft

ionization and its high sensitivity and specificity [7]. In this work, a new, fast and sensitive high-

performance liquid chromatography coupled to negative electrospray-ionization-time-of-flight mass

spectrometry (HPLC-ESI-TOF MS) was developed and validated for identification of some G. biloba

compounds. Using this technology, we have well identified six bioactive compounds: ginkgolide A and B,

bilobalide, kaempferol, quercetin and epicatechin. We will expect applying this method to identify and

quantify bioactive compounds in extracts from elicited suspension cultured cells of G. biloba.

References

1. Chavez, M.L, Chavez, P.I. 1998. Hosp. Pharm. 33: 1076–1095.

2. Ndjoko, K. Wolfender, J-L. Hostettmann, K. 2000. J. Chromatogr. B. 744: 249–255.

3. Goh, L.M. Barlow, P.J. Yong, C.S. 2003. Food Chem. 82: 275–282.

4. Chavez, M.L, Chavez, P.I. 1998. Hosp. Pharm. 33: 658-672.

5. Deng, F., Zito, S.W. 2003. J. Chromatogr. A, 986: 121–127

6. Kanga, S.M. et al.2009. J. Biotechnology 139: 84–88

7. Mauri, P. Pietta, P. 2000. J. Pharm. Biomed. Anal. 23: 61–68.


��

��

P-41

Identification and the developmental formation of carotenoid pigments in the yellow/orange

Bacillus spore-formers Perez-Fons, L.1, Steiger, S.2, Khaneja, R. 1, Cutting, S.M. 1, Sandmann, G.2, Fraser, P.D.1 1School of Biological Sciences, Royal Holloway University of London, Egham, U.K. 2Goethe University Frankfurt, Department Biological Sciences, Frankfurt. Germany.



Spore-forming Bacillus species capable of synthesising carotenoid pigments have recently been isolated. To

date the detailed characterisation of these carotenoids and their formation has not been described. In the

present work biochemical analysis on the carotenoids responsible for the yellow/orange pigmentation present

in Bacilli has been carried out and the identity of the carotenoids present elucidated. Chromatographic,

UV/Vis and Mass Spectral data have revealed the exclusive presence of a C30 carotenoid biosynthetic

pathway. The most abundant carotenoids were oxygenated derivatives of diapolycopene, which have

undergone glycosylation and esterification. The presence of fatty acid moieties (C9 to C15) attached to the

hexose sugar residue via an ester linkage was revealed by saponification and MS2 analysis. These bacterial

diapocarotenoids were identified as 5-glycosyl-diapolycopene and 5-glycosyl-4'-methyl-diapolycopenoate

esters. Antioxidant capacity of these C30 carotenoids assessed by TEAC assays showed a >10-fold increase

in antioxidant capacity when compared to plant’s C40 lycopene.

Analysis of these carotenoids over the developmental formation of spores revealed that 5-glycosyl-4'-

methyl-diapolycopenoate was related to sporulation. Sequence of HU36 genome allowed the identification

of six genes responsible for carotenoid biosynthesis and potential biosynthetic pathways was reconstructed

from gene expression during cell cycle. A metabolomic evaluation of the cell cycle of the pigmented strain

HU36 was carried out and correlations established between carotenoid biosynthesis and primary metabolism.

The apocarotenoid biosynthetic genes isolated from these Bacilli are potentially useful tools for the

production of apocarotenoids, particularly apolycopene derivatives, in plants. These gene products could

generate new carotenoid pigments with improved solubility and antioxidant properties capable of functioning

in both lipophilic and hydrophilic cellular compartment.

References:

Perez-Fons, L., S. Steiger, R. Khaneja, P.M. Bramley, S.M. Cutting, G. Sandmann, and P.D. Fraser. Biochim Biophys

Acta. 1811(2011): 177-85.

Acknowlegments: L.P., S.M.C and PDF are grateful for the EU FP7 programme funding for the COLORSPORE project


��

��

P-42

Carotenoid diversity in the genus Citrus: regulation of the biosynthesis and accumulation

*Rodrigo, M.J., Alquézar, B., Alós, E., Zacarías, L.

Postharvest Physiology and Biotechnology. Department of Food Science. Instituto de Agroquímica y tecnología de

Alimentos (IATA-CSIC). Valencia. Spain.



Carotenoids are responsible for the attractive colour of the peel and pulp of Citrus fruits. Moreover, they also

render important health benefits and nutritional properties, mainly due to their antioxidant capacity and

provitamin A activity. Because of the massive world consumption of Citrus fruits both as fresh and juice,

this crop provides an important source of carotenoids for human diet. In last years we have exploited the

high colour diversity and carotenoid composition among the genus Citrus to elucidate key regulatory steps of

the pathway and potential candidate genes for future biotechnological improvement. It is particularly

noteworthy the unusual accumulation of lycopene in fruit of some Citrus species and mutants because its

health promoting properties and attractive colour. We have identified a novel chromoplastic �-lycopene

cyclase gene induced during fruit maturation that, interestingly, displayed a reduced expression and activity

in red grapefruit, suggesting its involvement in the accumulation of lycopene in these fruits. However, the

red-fleshed phenotype of the orange mutant CaraCara appears to be linked to an enhanced expression of

upstream genes of the pathway. Moreover, the use of a yellow orange mutant, named Pinalate, with a 10-fold

increase in colourless carotenes respect to the wild type, suggests an important role for ζ-carotene isomerase.

Other remarkable feature in fruits of some Citrus species (as mandarins) is the high accumulation of �-

cryptoxanthin, a xanthophyll with provitamin A activity. Gene expression analysis revealed that changes in

the relative expression of genes upstream in the pathway respect to downstream genes may be related to the

differential accumulation of �-cryptoxanthin. Overall, these results indicate that multiple steps in the

carotenoid biosynthetic pathway may originate alterations in the carotenoid content and composition in both

peel and pulp of Citrus fruits and potential candidate genes likely associated with a particular carotenoid

composition will be discussed.


��

��

P-43

Enhancement of the production of phytoesterols in Daucus carota cell cultures

*Sabater Jara, A.B. Almagro, L., Belchí-Navarro, S., Pedreño, M.A. Department of Plant Biology. University of Murcia. Murcia. Spain.



Phytosterols are isoprenoid-derived lipids that play essential roles in plant growth and development since

they are integral components of the membrane, and important for its permeability and fluidity. Moreover,

they have important pharmacological effects, including cholesterol-lowering and anti-tumor activities (1).

The beneficial health effects of phytosterols have led to search strategies for enhancement these compounds

from other natural sources. In this sense, the use of plant cell cultures has been developed as a promising

alternative especially when the production of bioactive compounds is difficult or unfeasible, or when it

involves serious damage to the environment. Thus, we have developed a new innovative procedure (2) based

on the use of cyclodextrins (CDs) to increase phytosterol production by using Daucus carota cell cultures.

This new strategy combines the addition of these cyclic oligosaccharides at their optimal concentration, and

both optimal elicitation time and cell growth stage. Other elicitors such as methyl jasmonate (MJ) and UV

irradiation were also considered.

For this, D. carota cell cultures of 10 day-old were elicited with CDs at different concentrations (10, 20, 30,

50, 60, 70 mM) for 96 hours. After elicitation, phytosterols were extracted and analyzed by GC/MS. The

most abundant compounds identified in the elicited culture medium were �-sitosterol and stigmasterol

followed by campesterol and fucosterol. The maximum level of phytosterols produced by cells and secreted

to the media was reached when cell suspensions were incubated in the presence of 50 mM CDs (80.55 ±

12.30 mg/L). In addition, we analyzed the effect of both UV exposure (15 min) and MJ treatment (100µM)

on phytosterol production in the presence of 50 mM �-CDs, separately or in combination. The results

showed that neither UV-light (1.35 ± 0,19 mg/L) nor MJ (2.32 ± 0,49 mg/L) increased the production of

phytosterols compared to the levels obtained in CD treatments (80.55 ± 12.30 mg/L). In the light of these

results, treatment of D. carota cell cultures with CDs, open a new strategy for producing phytochemicals

with nutraceutical and medical applications.

References

1.- Posé D, Castanedo I, Borsani O, Nieto B, Rosado A, Taconnat L, Ferrer A, Dolan L, Valpuesta V, Botella MA.

2009. Plant J. 59: 63–76.

2.- Sabater-Jara, A.B. Almagro, L. Bru, R and Pedreño, MA. 2008. P200803107


��

��

P-44

Stability and degradation of anthocyanins in fruit and ornamentals

*Oren-Shamir, M.1, Bar-Akiva, A.1, Sinilal, B.1, Ovaida, R.1, Weiss, D.2, Perl, A.3 1Department of Ornamental Horticulture, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250,

PO Box 6, Israel; 2 Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food, and Environmental

Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel; and 3Department of Fruit Tree Sciences,

Agricultural Research Organization, Volcani Center, Bet-Dagan 50250, PO Box 6, Israel



Anthocyanins are degraded in plants either due to changes in environmental conditions or to developmental

changes. Here, two studies on anthocyanin degradation in plants are presented: One in which the process is

dependant on a specific developmental stage of the Brunfelsia flowers, resulting in a dramatic change of

flower color from dark purple to white, and the second study is on the slow and constant degradation

occurring in the grape cell suspension of V. vinifera cv. Gamay Red.

Anthocyanin degradation in Brunfelsia flowers is dependant on the induction of genes and synthesis of novel

protein (1). Concomitantly with the degradation of anthocyanins, flowers undergo changes in additional

metabolic pathways related to enhanced flower growth and increased fragrance. An extensive molecular,

biochemical and metabolomic characterization of the events taking place in Brunfelsia flowers during

anthocyanin degradation suggests possible interactions between the pathways preceding and evolving from

the phenylpropanoid pathway (2).

Anthocyanin degradation in the grape cell suspension of V. vinifera cv. Gamay Red is a slow and constant

process, revealed when anthocyanin synthesis in the cell was inhibited. Addition of magnesium to the

inhibited cells prevented the decrease in anthocyanins, suggesting that magnesium increases the stability of

the pigments and prevents the catabolic process occurring in the cells. When magnesium was added to

normal growing grape cells, anthocyanin concentration increased four fold, with no significant induction of

synthesis. Magnesium caused a change in the ratio between the anthocyanins, with a relative increase in less

stable pigments (3). This suggests that the main role of the metals is stabilizing the pigments and increase

their half life time.

Preventing anthocyanin catabolism in fruit and flowers may result in increased accumulation of the pigments

even under conditions in which synthesis rate is low.

References

(1) Vaknin et al. (2005) Planta, 222, 19-26.

(2) Bar-Akiva et al. (2010) Journal of Experimental Botany 61, 1393-1403

(3) Sinilal et al. (2011) Planta 243, 61-71.


��

��

P-45

Transcriptome profiling in Catharanthus roseus

Pomahacova, B., Körbes, A.P., Mustafa, N.R., van Verk, M.C., *Schulte, A.E.

ExPlant Technologies B.V., Einsteinweg 55, 2333 CC Leiden, The Netherlands



Catharanthus roseus is an intensively studied medicinal plant due to the presence of terpenoid indole

alkaloids (TIAs) which possess important medicinal value in treating cancer and hypertension. As the natural

production of those valuable compounds in the plant is limited, there is major interest worldwide to gain

further understanding of the TIA biosynthesis and its regulatory mechanisms with the aim to realize

increased productivity in plants and/or in cell- and hairy root cultures. Nevertheless, the problems to control

TIA biosynthesis and to achieve high production have not been solved yet.

Explant Technologies B.V. has a research program on Catharanthus roseus with the aim to realize cell-

culture based production systems for the bio-active TIAs. Next to advanced chemical assessment of our cell-

lines through targeted analytical methods and NMR-based metabolomics, we explored methods for

transcriptome profiling to identify competitive or limiting steps in the TIA pathway and its regulatory

elements.

Here we present the development of a qPCR micro-array, that currently allows efficient screening for gene

expression of 18 precursor & TIA pathway genes and eight regulators, including some five biosynthesis

genes leading to or involved in competing routes. Most importantly, we developed and validated the qPCR

approach for five Catharanthus house-keeping genes, as these are essential for reliable comparison of target

gene expression within and among different types of biological samples. In this context, the expression of the

candidate house-keeping genes was compared to some TIA related genes in untreated and jasmonic acid

treated cell samples and in different samples from intact, TIA producing plants.

In addition, we initiated a transcriptome sequencing project for Catharanthus gene discovery. Based on these

two transcriptome profiling technologies, we aim to find the essential genes for TIA biosynthesis, and

subsequently, we expect to gain control on TIA production in the cell cultures by our propriety molecular

techniques.


��

��

P-46

Pyrrolizidine alkaloids in Senecio sp. – development of standard references for food safety

assessment Mustafa, N.R.1, Pomahacova, B. 1, *Schulte, A.E. 1, Verhagen, L.C.2, Luijendijk, T.J.C. 2, Lund, K. 2 1ExPlant Technologies B.V., Einsteinweg 55, 2333 CC Leiden, The Netherlands 2PRISNA B.V., Einsteinweg 55, 2333 CC Leiden, The Netherlands



Pyrrolizidine alkaloids (PAs) are a group of plant metabolites that are highly toxic for human and animals.

More than 660 PAs and PA N-oxides have been identified among Leguminosae, Boraginaceae and

Asteraceae families. About half of these PAs cause acute poisoning by severe liver damage and are

associated with DNA damage and cancer development when exposed for long-term at low-doses.

PAs reach the feed and food chain through contamination of staple foods, and indirectly through honey, eggs

and milk; in addition, some PA containing plants are used in herbal preparations and food additives.

Consequently, there is a demand for different PA Standard References to allow Food Safety Assessment.

Here we present our work for the development of such PA Standard References from Senecio sp. through a)

development of isolation procedures from natural resources and b) by realizing optimized production

systems through cell and organ cultures.

Late submitted abstracts 82

��

��

P-47

Phenotypic space of floral volatiles in the Antirrhinaceae

*Weiss, J.1, Mühlemann, J.2, Orlova, I.2, Dudareva, N.2, Egea-Cortines, M.1 1Genetics, ETSIA, Universidad Politécnica de Cartagena, Spain. 2Department of Horticulture and Landscape, Purdue University, USA.



Volatiles can be considered the interface between insects and plants playing a dual role in attraction of

pollinators and repulsion of herbivores. Although important steps have been made towards an understanding

of scent in terms of biochemical pathways and identification of genes involved in discrete steps, little is

know about scent as a trait from an evolutionary perspective. The Antirrhinaceae comprises 25 species that

have the Iberian Peninsula as centre of diversification and origin. We have performed a large-scale analysis

of volatile production in nine wild species and two laboratory lines. As pollination has a strong influence on

scent production, bringing petal life to an abrupt end, we studied floral scent production from day 0 till day 5

after flower opening. Whilst percentages of the different volatiles found for a give species changed along the

stages from full flower opening and onwards, it tended to display a defined set of volatiles, as species

clustered together irrespective of developmental stage before pollination. A total of over 120 different

compounds was identified in the genus but every species analyzed showed a distinct profile of volatiles that

allowed their separation from other species. Although some volatiles like beta-myrcene or linalool are

typical of some A.majus commercial varieties like Mariland pink, it was completely absent in

A.meonanthum. In contrast, large amounts of methyl-cinnamate were found in A. linkianum and

acetophenone was found as a major compound in the laboratory lines of A.majus. A principal component

analysis of the volatiles allowed the separation of the different species that coincided with the classic

phylogenetic and recent molecular reconstruction (Wilson and Hudson 2011), suggesting that scent could be

the major trait under selection as it is a multilocus set of genes, as suggested by the molecular data.

Reference

Wilson, Y. and Hudson, A. (2011) The evolutionary history of Antirrhinum suggests that ancestral phenotype

combinations survived repeated hybridizations. The Plant Journal, 66, 1032-1043.

Acknowledgments

This work was performed under Salvador de Madariaga project PR2010-0592 and MCINN BFU2010-15843

Late submitted abstracts 83

��

��

P-48

Metabolic profiling of Catharanthus roseus cell lines

*Saiman, M.Z.1, Mustafa, N.R.2, Schulte, A.E.2, Choi, Y.H.1, Verpoorte, R.1.

1Institute of Biology, Leiden University, The Netherlands. 2ExPlant Technologies B.V., Leiden, The Netherlands.



Catharanthus roseus is a medicinal plant which produces pharmaceutically valuable terpenoid indole

alkaloids (TIAs) such as the anti-cancer agents vincristine and vinblastine as well as anti-hypertensive

constituents ajmalicine and serpentine. Due to the low level production of TIAs in plant, C. roseus cell

cultures have been studied as an alternative source of important alkaloids. However, the production of TIAs

in C. roseus cell suspension culture is relatively low despite several attempts to enhance the alkaloids by

feeding precursors and elicitation strategies.

Engineering the plant metabolism is a promising strategy, therefore we want to engineer the terpenoid

metabolic pathway of C. roseus which is a limiting factor in the production of TIAs. The first approach is to

select a suitable cell line that fit the experimental purpose. In this work, we have characterized several cell

lines and profiled their metabolite content to search for the best producing cell line accumulating TIAs,

sterols, and carotenoids. Analysis was done with optimized HPLC and GC methods to measure the targeted

metabolites, while 1H-NMR was used for analysis of non-targeted metabolites (Tikhomiroff and Jolicoeur,

2002; Ben-Amotz and Fisher, 1998; Kim et al, 2010). Apparently, different cell lines accumulate different

metabolites and the CRPP cell suspension culture was identified as a suitable cell line for further metabolic

engineering study.

References

Tikhomiroff C, Jolicoeur M. 2002. Journal of Chromatography A 955: 87-93.

Ben-Amotz A, Fisher R. 1998. Food Chemistry 62: 515-520.

Kim HK, Choi YH, Verpoorte R. 2010. Nature. Protocol 5 (3): 536-549.

84

LIST OF AUTHORS

A Almagro, L. ………………………57, 63, 78

Almvik, M. ………………………………36

Alós, E. …………………………………..77

Alquézar, B. ……………………………...77

Antón, M.T. ……………………………...35

Arrillaga, I. ………………………………41

B Bahcevandziev, K. ……………………….28

Ballester, A.R ……………………………64

Bar, E. ……………………………………50

Bar-Akiva, A. ............................................79

Barone, A. ................................................ 68

Barroso, J.G. ........................................43, 70

Bassard, J.E. ...............................................18

Beekwilder, J. ........................................... 23

Belchi-Navarro, S. ….................…57, 65, 78

Beyer, P. ....................................................16

Biazzi, E. ...................................................29

Bocian, A. ..................................................45

Bock, R. .....................................................16

Bogs, J. …………………………………..72

Bonfill, M. ……………………………….62

Bouwmeester, H. .................................23, 48

Bovy, A. ………………………………….64

Bramley, P.M. ............................................38

Brodelius, P.E. …………………………...24

Bru, R. ……………………………57, 61, 65

Buczkowicz, J. ...........................................45

Buesa, J. .....................................................35

Burger, J. ....................................................50

C Calderini, O. ....................................... 29, 31

Calderón, A.A. ...........................................69

Calikowski, T. ............................................13

Cankar, K. ................................................. 23

Carelli, M. ..................................................29

Carqueijeiro, I. ...........................................30

Carrasco, A. ...............................................44

Carriero, F. .................................................68

Celikkol Akcay, U. ………………………47

Chatziioannou, A. ......................................56

Choi, Y.H. ................................................. 83

Coller, E. ....................................................72

Compagnon, V. ..........................................18

Corchete, P. ................................................66

Correal, E. ..................................................67

Cruz, R.M. .................................................75

Cusidó, R.M. ..............................................62

Cutting, S.M. …………………………….76

Czemmel, S. ……………………………...72

D Dabauza, M. ...............................................67

Damiani, F. ................................................31

Davies, H.V. ..............................................71

De Tommasi, N. …………………………46

de Vos, R. ………………………………..64

Del Río, J.A. ....................................... 67, 74

Di Matteo, A. .............................................68

Dias, A.C.P. ...............................................32

Díaz, L. ...............................................67, 74

Dong, L. .................................................... 23

Duarte, P. ...................................................30

Dudareva, N. ............................................. 82

Dziadczyk, P. .............................................45

E Egea-Cortines, M. ..................................... 82

Ehlting, J. ...................................................18

Ehnert, T.M. ...............................................37

Enfissi, G. ..................................................38

85

Engler, C. ...................................................37

Espí, J. ……………………………….…...35

Esteban Carrasco, A. …………………….33

F Fait, A. …………………………………...50

Falconi, E.E. ……………………………..39

Feller, A. …………………………………27

Fernández-del-Carmen, A. ………………39

Fernández-Pérez, F. .............................57, 65

Ferrer, M.A. ……………………………...69

Ferro, A. ………………………………….34

Figueiredo, A.C. ................................. 43, 70

Fischedick, J. .............................................19

Fliniaux, O. ................................................58

Franklin, G. ................................................32

Fraser, P.D. ................................... 16, 38, 76

Frusciante, L. .............................................68

G Galili, G. ....................................................54

García-Lidón, A. ........................................74

Gardner, R. ................................................30

Ginglinger, J.F. ..........................................18

Giorio, G. ………………………………...16

Giuliano, G. ……………………………...16

Goedbloed, M. .......................................... 23

Gómez-Ros, L. …………………………...33

Gonçalves, S. …………………………….34

Gonda, I. …………………………………50

Gontier, E. ……………………………26, 58

González, J. ………………………………74

González-Candelas, L. ……...……………64

Goossens, A. …………….…….…22, 30, 62

Grand, E. …………………………………58

Granell, A. …………...………………35, 39

Gruetzner, R. ……………………………..37

Guillot, X. ………………………………..58

H Hamacher, K. .............................................55

Han, J. ........................................................24

Hancock, R.D. …………………………...71

Heppel, S. ………………………………..72

Hernández-Ruiz, J. ....................................75

Herrero, J. ..................................................33

Hirschberg, J. …………………………….16

Höfer, R. ....................................................18

Honys, D. ...................................................49

J Jousse, C. ...................................................58

Juárez, P. ………………...…………...35, 39

K Katzir, N. ………………………………...50

Khaneja, R. ………………………………76

Kolisis, F.N. ……………………………...56

Körbes, A.P. ……………………………...80

Kovensky, J. ……………………………..58

Kreis, W. ………………………………....52

L Lacasa, A. ………………………………..74

Lafuente, M.T. …………………………...64

Landa, P. …………………………………73

Leone, A. ………………………………...46

Lewinsohn, E. ……………………………50

Lima, A.S. ……………………………28, 43

Liu, Q. ....................................................... 23

Liu Clarke, J. …………………………….36

López-Orenes, A. ………………………...69

Losini, I. ………………………………….29

Luijendijk, T.J.C. ………………………...81

Lund, K. .....................................................81

M Majdi, M. .................................................. 23

Malacarne, G. ……………………………72

86

Malafronte, N. …………………………...46

Marillonnet, S. …………………………...37

Marsik, P. ………………………………..73

Martens, S. ……………………………….27

Martinez, F.J. …………………………….44

Martinez-Esteso, M.J. ……………………61

Martínez-Pérez, A. ……………………….69

Martinez-Ruiz, J. ………………………...44

Martinussen, I. ...........................................36

Matsuno, M. ...............................................18

Mendes, M. ………………………………43

Mendoza, I. ................................................41

Mesnard, F. ................................................58

Miras-Moreno, B. ......................................57

Molinie, R. .................................................58

Morante-Carriel, J. .....................................61

Moreno, V. .................................................35

Moser, C. ...................................................72

Moyano, E. ................................................62

Mühlemann, J. .......................................... 82

Müller-Uri, F. .............................................52

Muñoz-Bertomeu, J. ..................................41

Mustafa, N.R. .................................80, 81, 83

N Nlandu Mputu, M. .....................................26

Nogueira, M.N. ..........................................38

Novak, J. ....................................................43

Novo-Uzal, E. ............................................75

O Ocampo, V.E. ............................................46

Oksman-Caldentey, K.-M. .........................15

Oliveira, M.M. ...........................................34

Olry, A. ......................................................18

Onrubia, M. ................................................62

Oren-Shamir, M. …………………………79

Orlova, I. ................................................... 82

Ortiz, V. ………………………………….44

Ortuño, A. ........................................... 67, 74

Orzaez, D. ............................................35, 39

Ovaida, R. ………………………………..79

P Palazon, J. ..................................................62

Panara, F. ............................................ 29, 31

Paolocci, F. ................................................31

Parra, M. ....................................................44

Passeri, V. ..................................................31

Pedreño, M.A. ..............57, 61, 63, 65, 75, 78

Pedro, L. G. ..........................................43, 70

Pérez, I. …………………………………..67

Perez-Fons, L. ............................................76

Perl, A. .......................................................79

Pi�manová, M. ...........................................49

Pietiäinen, M. .............................................40

Piffanelli, P. ……………………………...29

Pilalis, E. ....................................................56

Pineda, B. ...................................................35

Pinot, F. ......................................................18

Poirier, Y. ..................................................51

Pomahacova, B. ...................................80, 81

Pont, S.D.A. ...............................................71

Ponte, A. ....................................................71

Porras, I. .....................................................74

Presa, S. .....................................................35

Pugin, A. ....................................................63

R Ramsay, A. ................................................58

Rhazi, L. ....................................................26

Rigano, M.M. ............................................68

Rodrigo, M.J. ............................................77

Ros Barceló, A. ..........................................33

Roscher, A. ................................................58

Ruggieri, V. ...............................................68

Ruman, T. ..................................................45

R�ži�ka, P. .................................................49

87

S Sabater-Jara, A.B. ............................... 75, 78

Sacco, A. ....................................................68

Saiman, M.Z. ............................................ 83

Sanchez, M. ...............................................44

Sánchez-Godínez, A. .................................75

Sandmann, G. ……………………………76

Sarrion-Perdigones, A. ..............................39

Sauveplane, V. …………………………...18

Schaffer, A.A. ............................................50

Schulte, A.E. ............................19, 80, 81, 83

Scotti, C. ....................................................29

Segura, J. …………………………………41

Sellés-Marchart, S. ………………………61

Semik, M. ..................................................45

Shepherd, L.V.T. .......................................71

Sikron, N. ...................................................50

Sinilal, B. ...................................................79

Socaciu, C. .................................................16

Sochacka-Pi�tal, M. ...................................45

Sottomayor, M. ..........................................30

Steiger, S. ...................................................76

Surmacz, L. ………………………………42

Swiezewska, E. …………………………..42

T Tadmor, Y. .................................................50

Tava, A. .....................................................29

Taylor, M.A. …………………………16, 59

Teeri, T.H. .................................................40

Thomasset, B. ............................................26

Ting, H-M. .......................................... 23, 48

Tissier, A. ..................................................17

Trindade, H. ........................................ 28, 43

Tudela, J. ....................................................44

Tyrka, M. ...................................................45

U Ullmann, P. ................................................18

Unver, T. …………………………………25

V Vaccaro, M.C. …………………………... 46

van der Krol, S. ................................... 23, 48

Van Herpen, T. ......................................... 23

van Verk, M.C. ..........................................80

Vasseur, G. ................................................26

Vera-Urbina, J.C. .......................................61

Verhagen, L.C. ...........................................81

Verpoorte, R. ............................................ 83

Vilella-Antón, M.T. ...................................61

Vrhovsek, U. ……………………………..72

Vu, T-D. ………………………………….26

W Walker, D. ………………………………..67

Wang, H. …………………………………24

Wawrosch, C. ……………………………60

Weber, E. ………………………………...37

Weiss, D. …………………………………79

Weiss, J. ………………………………… 82

Welters, P. ..................................................21

Werck-Reichhart, D. ……………………..18

Werner, S. ………………………………..37

X Xing, S. ......................................................51

Y Youssef, S.M. ............................................75

Z Zacarías, L. ………………………………77

Zandalinas, S.I. …………………………..39

Zapata, J.M. ……………………………...33

Name Institution Country e-mailAlmagro, Lorena University of Murcia Spain [email protected], Marit Bioforsk Plant Health and Plant Protection Norway [email protected]ós, Enriqueta Instituto de Agroquímica y Tecnología de Alimentos-CSIC Spain [email protected], Kiril Instituto Politecnico de Coimbra - Escola Superior Agraria Portugal [email protected] Frutos, Ana Rosa Instituto de Agroquímica y Tecnología de Alimentos-CSIC Spain [email protected] Navarro, Sarai University of Murcia Spain [email protected], Harro Wageningen University The Netherlands [email protected], Peter E School of Natural Sciences, Linnaeus University Sweden [email protected], Roque University of Alicante Spain [email protected], Tomasz European Commission Belgium [email protected], Raymond James Hutton Institute United Kingdom [email protected], Maria Consiglio per la Ricerca e la sperimentazione in Agricoltura (CRA) Italy [email protected], Ines Instituto de Biologia Molecular e Celular - IBMC Portugal [email protected] Akcay, Ufuk Suleyman Demirel University Turkey [email protected], Constanza Instituto de Biología Agrícola de Mendoza Argentine [email protected], Purificación University of Salamanca Spain [email protected] Ruíz, Rosa Maria University of Murcia Spain [email protected], Rosa María University of Barcelona Spain [email protected], Francesco Consiglio nazionale delle Ricerche Istituto Genetica Vegetale Italy [email protected] Rio Conesa, Jose Antonio University of Murcia Spain [email protected] Matteo, Antonio Department of Soil, Plant, Environmental and Animal Production Sciences Italy [email protected], Alberto Universidade do Minho Portugal [email protected], Patricia Instituto de Biologia Molecular e Celular - IBMC Portugal [email protected], Marcos Universidad Politécnica de Cartagena Spain [email protected], Jack University of Copenhagen Denmark [email protected], Antje Instituto Agrario Di San Michele all'Adige/ Fondazione Edmund Mach Italy [email protected] Perez, Francisco University of Murcia Spain [email protected] Ayala, Maria Angeles Universidad Politécnica de Cartagena Spain [email protected], Ana Cristina Instituto de Biotecnologia e Bioengenharia, Universidade de Lisboa Portugal [email protected], Paul David Royal Holloway University of London United Kingdom [email protected], Gad The Weizmann Institute of Science Israel [email protected], Jean-François Institut de Biologie Moléculaire des Plantes - IBMP France [email protected]ómez Ros, Laura V. University of Murcia Spain [email protected]çalves, Sónia Centro de Biotecnologia Agricola e Agro-alimentar do Baixo Alentejo e Litoral - CEBAL Portugal [email protected], Alain VIB-UGent Belgium [email protected]

Gregory, Franklin Universidade do Minho Portugal [email protected], Kay Technische Universitaet Darmstadt Germany [email protected], Robert James Hutton Institute United Kingdom [email protected] Ruiz, Josefa Universidad de Murcia Spain [email protected], Joseph The Hebrew University of Jerusalem Israel [email protected], David Institute of Experimental Botany ASCR Czech Republic [email protected] Ortega, Paloma Instituto de Biología Molecular y Celular de Plantas Spain [email protected], Fragiskos National Technical University of Athens - NTUA Greece [email protected], Antonella University of Salerno Italy [email protected], Efraim Newe Yaar Research Center, Agricultural Research Organization Israel [email protected],Francisco Instituto de Biologia Molecular e Celular - IBMC Portugal [email protected] Clarke, Jihong Bioforsk- Norwegian Institute for Agricultural & Environmental Research Norway [email protected], Giulia Edmund Mach Foundation Italy [email protected], Sylvestre Icon Genetics GmbH Germany [email protected], Petr Institute of Experimental Botany ASCR Czech Republic [email protected], Stefan Edmund Mach Foundation Italy [email protected]ínez Esteso, María José University of Alicante Spain [email protected] Martinussen, Inger Bioforsk, Norwegian Institute for Agricultural and Environmental Research Norway [email protected], Isabel University of Valencia Spain [email protected], François Université de Picardie Jules Verne France [email protected], Franck Alkion Biopharma United Kingdom [email protected], Claudio Edmund Mach Foundation Italy [email protected], Frieder LS Pharm.Biology, University Erlangen Germany [email protected], Marilise Royal Holloway University of London United Kingdom [email protected] Uzal, Esther University of A Coruña Spain [email protected], Kirsi-Marja VTT Technical Research Centre of Finland Finland [email protected], Juliana Instituto de Biologia Molecular e Celular - IBMC Portugal [email protected], Miriam Universitat Pompeu Fabra Spain [email protected], Michal Volcani Center Israel [email protected]ño Tomas, Ana Maria University of Murcia Spain [email protected] Calatayud, Diego Instituto de Biología Molecular y Celular de Plantas Spain [email protected] Barandela, Javier Universitat de Barcelona Spain [email protected]ño Garcia, Maria Angeles University of Murcia Spain [email protected], Laura Royal Holloway University of London United Kingdom [email protected], Milla University of Helsinki Finland [email protected], Eleftherios School of Chemical Engineering, National Technical University of Athens Greece [email protected]

Piquera Castillo, Abel CEBAS - CSIC Spain [email protected], Yves University of Lausanne Switzerland [email protected], Barbora ExPlant Technologies BV The Netherlands [email protected] Barbeito, Federico University of A Coruña Spain [email protected], Beatriz Universidad San Pablo CEU Spain [email protected] Esteve, Maria Jesús Instituto de Agroquímica y Tecnología de Alimentos-CSIC Spain [email protected] Jara, Ana Belen University of Murcia Spain [email protected], Mohd Zuwairi Leiden University The Netherlands [email protected], Annelies ExPlant Technologies BV The Netherlands [email protected], Juan Universidad de Valencia Spain [email protected], Carmen Proplanta SRL Romania [email protected], Mariana Insituto de Biologia Molecular e Celular - IBMC Portugal [email protected], Agata Technische Universitaet Darmstadt Germany [email protected], Ewa Institute of Biochemistry and Biophysics Polish Academy of Sciences Poland [email protected], Mark James Hutton Institute United Kingdom [email protected], Teeri University of Helsinki Finland [email protected], Brigitte Université de Technologie de Compiègne France [email protected], Alain Leibniz Institute of Plant Biochemistry Germany [email protected], Helena Instituto de Biotecnologia e Bioengenharia, Universidade de Lisboa Portugal [email protected] Sánchez, Libertad Raquel Viveros Bermejo Biotech Division Spain [email protected] Serrano, Jose Universidad de Murcia Spain [email protected], Miroslaw Technical University of Rzeszow Poland [email protected], Turgay Cankiri Karatekin University Turkey [email protected], Mariacarmela University of Salerno Italy [email protected] der Krol, Sander Wageningen University The Netherlands [email protected], Heribert Technische Universitaet Darmstadt Germany [email protected], Christoph University of Vienna Austria [email protected], Julia Universidad Politécnica de Cartagena Spain [email protected], Peter Phytowelt GreenTechnologies GmbH Germany [email protected], Ludger Leibniz Institute of Plant Biochemistry Germany [email protected], Sabry Mousa Soliman University of Murcia Spain [email protected]

Documents

Scientific programme Book of abstracts of Abstracts Murcia... · O9 Manipulation of high value alkaloid products of opium poppy via metabolic engineering ... P7 Hypericum perforatum