Scientific Report 2005-2006 Leibniz Institute of Plant Biochemistry

SCIENTIFIC REPORT 2005-2006 LEIBNIZ INSTITUTE OF PLANT BIOCHEMISTRY

Weinberg 3 06120 Halle (Saale)

Phone: (03 45) 55 82 11 10 Fax: (03 45) 55 82 11 09

www.ipb-halle.de TABLE OF CONTENTS

Presentation of the Institute 4 Departmental Organization 7 Organs of the Institute 8

DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY 10 Head: Professor Toni M. Kutchan until March 2006, afterwards Claus Wasternack (prov.) Alkaloid Biosynthesis 12 Head: Toni M. Kutchan until March 2006

Opium Poppy Biotechnology 14 Head: Susanne Frick until March 2006

Mode of Action of Jasmonates 16 Heads: Claus Wasternack & Otto Miersch

Papaver Gene Expression Analysis 18 Head: Jörg Ziegler

Publications and Other Scientific Activities 2005 and 2006 20

DEPARTMENT OF BIOORGANIC CHEMISTRY 22 Head: Professor Ludger Wessjohann Natural Products 24 Heads: Norbert Arnold & Jürgen Schmidt

Chemoenzymatics 26 Heads: Ludger Wessjohann & Wolfgang Brandt

Synthesis 28 Head: Ludger Wessjohann & Bernhard Westermann

Spectroscopy 30 Heads: Andrea Porzel & Jürgen Schmidt

Screening 32 Heads: Norbert Arnold & Bernhard Westermann

Computational Chemistry 34 Heads: Wolfgang Brandt & Andrea Porzel Publications and Other Scientific Activities 2005 36 Publications and Other Scientific Activities 2006 38

DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY 40 Head: Professor Dierk Scheel Molecular Communication in Plant-Pathogen Interactions 42 Head: Wolfgang Knogge 2 Cellular Signaling 44 Heads: Dierk Scheel & Justin Lee

Induced Pathogen Defense 46 Heads: Dierk Scheel & Sabine Rosahl

Metal Homeostasis 48 SCIENTIFIC REPORT 2005 - 2006 LEIBNIZ INSTITUTE OF PLANT BIOCHEMISTRY

Head: Stephan Clemens until September 2006

Bioinformatics & Mass Spectrometry 50 Head: Steffen Neumann

Metabolite Profiling in Arabidopsis and Crop Plants 52 Heads: Stephan Clemens & Dierk Scheel in cooperation with Ludger Wessjohann & Jürgen Schmidt

Publications and other Scientific Activities 2005 and 2006 54

DEPARTMENT OF SECONDARY METABOLISM 56 Head: Professor Dieter Strack

Metabolism of Phenylpropanoids 58 Heads: Dieter Strack & Carsten Milkowski

Molecular Physiology of Mycorrhiza 60 Head: Michael H. Walter

Cell Biology of Myccorrhiza 62 Head: Bettina Hause

Metabolite Profiling & Biochemistry of Proteins 64 Heads: Willibald Schliemann & Thomas Vogt

Publications and other Scientific Activities 2005 and 2006 66

ADMINISTRATION AND TECHNICAL SERVICES 68 Head: Lothar Franzen Administration Employees 70 Employee Statistics 71 Resources and Investments 72 Funds from External Sources in 2005 and 2006 73 Involvement of the IPB in National and International Scientific Networks 77 Seminars, Workshops and Colloquia 2005 78 Seminars, Workshops and Colloquia 2006 80 Guest Researchers and Fellows 82

PRESS AND PUBLIC RELATIONS 84 Head: Sylvia Pieplow Media Presence of the IPB in 2005 and 2006 86

Map & Impressum 88

3 PRESENTATION OF THE INSTITUTE

MESSAGE FROM THE MANAGING DIRECTOR The Leibniz Institute of Plant Biochemistry (IPB) is a non-university research center of the Leibniz Association. It was originally founded in 1958 by Kurt Mothes as one of the research institutes of the German Academy of Sciences at Berlin. From the beginning, the research focus of the IPB was on plant natural products including hormones. Within four research departments, more than 150 staff scientists, students and guest researchers investigate in a multidisciplinary style structure and function of natural products from plants and fungi, analyze interactions of plants with pathogenic and symbiotic microorganisms and study molecular interactions as part of complex biological processes.

The years 2005 and 2006 were very productive and the new scientific developments are highlighted within the research group reports. Scientists of the IPB have been participa- ting in many regional, national and international research networks. Among the local activities, the Collaborative Research Center, SFB 648 Molecular Mechanisms of Information Processing in Plants and the Network of Excellence Structures and Mechanisms of Biological Information Processing have been established and are coordinated in cooperation with the Martin Luther University Halle-Wittenberg and the Leibniz Institute of Plant Genetics and Crop Plant Research in Gatersleben. At the national level, the Priority Program, SPP 1152 Evolution of Metabolic Diversity of the German Research Foundation has been coordinated by scientists of the IPB. In addition, scientists of the institute have been involved in projects of the German Program GABI (Genome Analysis of the Plant Biological System). Beside many bilateral cooperations between research groups at the IPB and groups from different countries worldwide, one trilateral project in the frame of GABI was carried out in cooperation with groups in France and Spain and the institute was involved in two international projects supported by the European Community.

Toni Kutchan, Head of the Department of Natural Product Biotechnology accepted the offer of an attractive position of the Donald Danforth Plant Science Center in St. Louis, Mis souri, USA, and left the IPB in March 2006.

Stephan Clemens, Head of the Research Group Metal Homeostasis of the Department of Stress and Developmental Biology accepted the offer of the W3 Professorship of Plant Phy siology at the University of Bayreuth and left the institute in fall 2006. We wish both colleagues success at their new positions.

The facilities of the IPB have been further improved by expanding the greenhouse space and adding two new buildings, one of which is accommodating gardeners and craftsmen and the second junior research groups and bioinformaticians.

4 SCIENTIFIC REPORT 2005 - 2006 LEIBNIZ INSTITUTE OF PLANT BIOCHEMISTRY

RESEARCH MISSION STATEMENT The large manifold of plant species is reflected in the enormous diversity of their natural products. This content of natural compounds is made more complex by the change in metabolite patterns during development as well as when a plant is responding to its environment. Knowledge of structure and function of natural products is requisite to understanding plant diversity, developmental and adaptation processes. New resources can then become available for innovative application in plant production, plant protection, biotechnology and in the development of biologically active compounds. Furthermore, together with the growing gain in knowledge available from plant genome research, this information is of fundamental importance to functional genome analysis.

The comprehensive analysis of plant and fungal natural products is a priority in the re - search mission of the Leibniz Institute of Plant Biochemistry. An interdepartmental competence group develops and applies suitable analytical methods for a qualitative and quantitative analysis of natural products in biological materials. This forms the basis for investigation of function and biosynthesis of natural products and for discovering new biologically active compounds. Structure analysis, synthesis and derivatization of natural products contribute to an understanding of their function and to an increase in their structural diversity. Subsequent identification and isolation of biosynthetic enzymes can pro - vide access to the encoding genes, which in turn enables study of the regulation of the biosynthesis and of the cellular and organismic organization of its components.

The genetic determination of plant development and its modulation during environmental adaptation rely on receptor-mediated perception of endogenous signals or biotic and abiotic environmental factors. The different signals are evaluated, compared and conver- ted into physiological responses via altered gene expression patterns that are controlled by cellular and systemic signal transduction networks. The physiological responses are usually based on transiently and locally altered profiles of natural products. Molecular interactions form the basis of the cellular functions responsible for these processes. An interdisciplinary analysis of these interactions is therefore of central importance to the research mission of the Leibniz Institute of Plant Biochemistry. Receptor-ligand, enzyme- substrate and protein-protein interactions form the molecular principle of these processes and have application in the development of new biologically active agents. From this perspective, the mechanisms of communication between plants and their symbionts and pathogens are investigated as are biosynthetic pathways and signal transduction networks. Interdepartmental cooperation includes transcriptomic and proteomic ap - proaches, but supports also the development of novel cell biological methods for the analysis of the dynamics of molecular interactions in the life organism. Chemical structures of the interacting components are modified using gene technological methods, directed evolution and chemical derivatization. The effects of these changes can be monitored in model systems or with activity screens until a molecule with the desired characteristic (e.g. a drug, signal compound or an enzyme) is achieved. The development of new syntheses, screening tests, assays and analytical methods is supported by visualization of mole- 5 cular interactions via computer modeling. PRESENTATION OF THE INSTITUTE & DEPARTMENTAL ORGANIZATION

RESEARCH MISSION STATEMENT The tight combination of natural product chemical, biochemical, molecular and cell biological approaches allows new access to gene function analysis, the third research priority of the Leibniz Institute of Plant Biochemistry. In the frame of a comprehensive research concept of functional genome analysis that is based on transcriptome, pro teome and metabolome data, genes are identified and characterized, which are essential for plant development and environmental adaptation. The use of mutants and transgenic plants makes the direct analysis of gene functions possible and allows the generation of model plants with altered patterns of natural products, with novel health promoting compounds or plants adapted to specific sites or environmental conditions. Such plants will be bene- ficial for sustained production of valuable substances and biocatalysts, for use as biological indicators and for plant breeding.

A nexus of natural product research and the study of molecular interactions is storage and evaluation of the large amount of data that is generated. In particular, high throughput processes used in metabolome and proteome analysis and in the production of combinatorial libraries make necessary the development of new methods in information technology. To this end, a new junior group in information technology has been established at the Leibniz Institute of Plant Biochemistry. Together with the Research Group Computational Chemistry this forms a new research priority that will be extended towards an interdepartmental competence area. This priority aims at the integral linkage and analysis of structurally diverse data sets generated within the other research areas towards a better understanding of the biological system of plants.

6 SCIENTIFIC REPORT 2005 - 2006 LEIBNIZ INSTITUTE OF PLANT BIOCHEMISTRY

FOUNDATION COUNCIL Ministerialrat Thomas Reitmann Senior Superior Counsellor Ministerialrat Dr. Jürgen Roemer-Mähler SCIENTIFIC ADVISORY BOARD Superior Counselor Prof. Alfons Gierl Chairman since September 2006 Prof. Wilhelm Boland Chairman until December 2005 OARD OF IRECTORS B D Prof. Sabine Flitsch PUBLIC RELATIONS Prof. Dierk Scheel Vice-Chairwoman since September 2006 Managing Director since November 2005 Sylvia Pieplow Prof. Toni M. Kutchan Personal Assistent to the Managing Director Managing Director until November 2005 Lothar Franzen Head of Administration Prof. Dieter Strack Prof. Claus Wasternack provisional since April 2006 Prof. Ludger Wessjohann

Department of NATURAL PRODUCT Department of Department of Department of Department of BIOTECHNOLOGY BIOORGANIC CHEMISTRY STRESS AND DEVELOPMENTAL SECONDARY METABOLISM ADMINISTRATION AND Prof. Toni M Kutchan Prof. Ludger Wessjohann BIOLOGY Prof. Dieter Strack TECHNICAL SERVICES until March 2006 Prof. Dierk Scheel Lothar Franzen Prof. Claus Wasternack, provisional since April 2006

Molecular Commu ni- Metabolism of Natural Products Accounting Alkaloid Biosynthesis cation in Plant-Pathogen Phenylpropanoids Norbert Arnold & Human Resources Toni M. Kutchan Interactions Dieter Strack & until March 2006 Jürgen Schmidt General Administration Wolfgang Knogge Carsten Milkowski

Opium Poppy Chemoenzymatics Cellular Signaling Molecular Physiology of Library Services Biotechnology Ludger Wessjohann & Dierk Scheel & Mycorrhiza Stockroom Susanne Frick until March 2006 Wolfgang Brandt Justin Lee Michael H. Walter Graphic Arts

Mode of Action of Induced Synthesis Cell Biology of Facilities Management Jasmonates Pathogen Defense Ludger Wessjohann & Mycorrhiza Computer Support Claus Wasternack & Dierk Scheel & Bernhard Westermann Bettina Hause Horticulture Otto Miersch Sabine Rosahl

Metabolite Profiling & Papaver Gene Expression Spectroscopy Metal Homeostasis Biochemistry of Proteins Analysis Andrea Porzel & Stephan Clemens Willibald Schliemann & Jörg Ziegler Jürgen Schmidt until September 2006 Thomas Vogt

Screening Bioinformatics & Mass Norbert Arnold & Spectrometry Bernhard Westermann Steffen Neumann

Computational Chemistry Wolfgang Brandt & Andrea Porzel 7

Metabolite Profiling in Arabidopsis and Crop Plants (Part of the German Plant Genome Project, GABI) Stephan Clemens, Dierk Scheel, Ludger Wessjohann & Jürgen Schmidt ORGANS OF THE INSTITUTE

BOARD OF DIRECTORS Prof. Dierk Scheel Managing Director since November 2005 Head of the Department of Stress and Developmental Biology

Prof. Toni M. Kutchan Managing Director until November 2005 Head of the Department of Natural Product Biotechnology until March 2006

Lothar Franzen Head of Administration and Technical Services

Prof. Dieter Strack Head of the Department of Secondary Metabolism

Prof. Claus Wasternack Provisional Head of the Department of Natural Product Biotechnology since April 2006

Prof. Ludger Wessjohann Head of the Department of Bioorganic Chemistry

BOARD OF TRUSTEES Ministerialrat Thomas Reitmann Chairman of the Board of Trustees Ministry of Education and Cultural Affairs of the State of Saxony Anhalt

Ministerialrat Dr. Jürgen Roemer-Mähler Vice Chairman of the Board of Trustees Federal Ministry of Education and Research

Prof. Wilhelm Boland Chairman of Scientific Advisory Board until December 2005 Max Planck Institute for Chemical Ecology, Jena

Prof. Wulf Diepenbrock since January 2006 Rector of the University of Halle

Prof. Alfons Gierl Chairman of Scientific Advisory Board since September 2006 Technical University of Munich

Prof. Sabine Flitsch Vice Chairwoman of Scientific Advisory Board since September 2006 Manchester Interdisciplinary Biocentre (MIB)

Prof. Reinhard Neubert Vice Rector for Research and Postgraduate Students of the University of Halle 8 Member of the Board of Trustees until September 2006

Prof. Jörg Stetter Gesellschaft Deutscher Naturforscher und Ärzte e.V. SCIENTIFIC REPORT 2005 - 2006 LEIBNIZ INSTITUTE OF PLANT BIOCHEMISTRY

SCIENTIFIC ADVISORY BOARD Prof. Wilhelm Boland Chairman of Scientific Advisory Board until December 2005 Max Planck Institute for Chemical Ecology, Jena

Prof. Alfons Gierl Chairman of Scientific Advisory Board since September 2006 Technical University Munich, Department of Genetics

Prof. Sabine Flitsch Vice Chairwoman of Scientific Advisory Board since September 2006 Manchester Interdisciplinary Biocentre (MIB)

Prof. Christoph Benning Michigan State University, Department of Biochemistry and Molecular Biology

Prof. Raoul J. Bino University of Wageningen, Laboratory of Plant Physiology

Prof. Thomas Boller University of Basel, Institute of Botany

Prof. Jonathan Gershenzon Max Planck Institute for Chemical Ecology, Jena

Prof. Horst Kunz University of Mainz, Institute of Organic Chemistry

Prof. Rainer Metternich Schering AG, Research Center Europe, Berlin

Prof. Birger Lindberg Møller University of Copenhagen, Department of Plant Biology

Prof. Andreas Schaller University of Hohenheim, Institute of Plant Physiology and Biotechnology

Dr. Günther Strittmatter KWS SAAT AG, Einbeck Member of the Scientific Advisory Board until December 2005

Prof. Lutz F. Tietze University of Göttingen, Institute of Organic Chemistry

Prof. Lothar Willmitzer Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm

Prof. Ulrich Wobus Institute of Plant Genetics and Crop Plant Research, Gatersleben Member of the Scientific Advisory Board until December 2005 9 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY Head: Professor Toni M. Kutchan until March 2006, afterwards Professor Claus Wasternack (prov.) Secretary: Christine Dietel

lants synthesize and accumulate numerous secondary compounds beside their primary metabolism functioning in growth and development. Among these secondary Pcompounds the benzylisoquinoline alkaloids are of great importance, since many of them are of pharmaceutical interest. That is preferentially the case for morphinane compounds, such as the antitussive codeine and the pain-reducing morphine. Three working groups of the Department of Natural Product Biotechnology were studying morphine biosynthesis by molecular genetic techniques in 2005 and 2006. The goal of the work is the establishment of molecular tools to analyze the branched biosynthetic pathways of benzylisoquinolines. Using overexpression and suppression of genes coding for enzymes in morphine biosynthesis, an altered profile of intermediates and products (Meta bolic Engineering) is envisaged (Research Group Alkaloid Biosynthesis, Toni M. Kutchan, Research Group Opium Poppy Biotechnology, Susanne Frick). This could be achieved after establishment of proper transformation protocols for Papaver somniferum with cDNA constructs related to the following enzymes: Berberine bridge enzyme, (S)-N- methylcoclaurin-3’-hydroxy lase, (R,S)-reticuline-7-O-methyl-transferase, salutaridinol-7- O-acetyltransferase and co deinone reductase. Additional interest was given on biosynthesis of isoquinoline terpenoid alkaloids and of naphthoquinones (Research Group Alkaloid Biosynthesis). Ipecac alkaloid biosynthesis of Psychotria ipecacuanha has been studied by cloning enzymes involved. The biosynthesis of the naphthoquinone plumbagin has been analyzed by cloning and characterization of a type III polyketide synthase of Plumbago indica L.

New insights in morphine biosynthesis were achieved by analysis of ESTs (Research Group Papaver Gene Expression Analysis, Jörg Ziegler). Comparison of gene expression and alkaloid profiles between numerous opium poppy varieties and Papaver species led to the identification of new genes functioning in biosynthesis and regulation of morphine. Among them the salutaridine reductase, an O-methyltransferase and an ABC-transporter were studied in detail. Homology modeling of the salutaridine reductase led to the characterization of the substrate binding product.

The research groups of Toni M. Kutchan and Susanne Frick went to the Donald Danforth Plant Science Center in St. Louis (USA) in April 2006, and the research group of Jörg Zieg - ler is working now at the University of Calgary (Canada) since October 2006. Conse - quently, scientific activities of all three working groups are finished at the Department of Natural Product Biotechnology of the IPB.

A fourth Research Group (Mode of Action of Jasmonates, Claus Wasternack & Otto Miersch) of the department is working for many years on functional analysis of jasmonic acid (JA) and its metabolites, commonly named jasmonates. Their compounds act as sig -

10 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

nals in plant stress responses and development. Using reverse genetic approaches, cell biological techniques, and quantitative analyses of these compounds, the role of JA biosynthesis and jasmonates in stress responses and de - velopment of Arabidopsis thaliana and tomato were studied. New aspects in regulation were found by transgenic approaches and analyses of knockout mutants of the JA biosynthesis enzyme allene oxide cyclase (AOC) in Arabidopsis and its cell type-specific and organ-specific occurrence in tomato. Among the metabolites increasing interest is given to 12-hydroxyjasmonic acid due to its JA-independent properties and its specific accumulation in distinct developmental processes. Numerous collaborative works in the field of jasmonates were continued in 2005 and 2006.

11 ALKALOID BIOSYNTHESIS Head: Toni M. Kutchan until March 2006

The main topic of research in the group is the investigation of natural product biosyn thetic pathways. Approximately 25 % of current pharmaceuticals are either isolated directly from medicinal plants or are modified from natural products. Multiple stereocenters present in many of these natural products prohibit commercially feasible total syntheses, thereby making an understanding of the enzymes and genes involved in the biosynthesis of central interest to developing improved sources of these medicinals through either metabolic engineering or biomimetic synthesis. The study of the biosynthesis of isoquinoline terpenoid alkaloids and of naphthoquinones has recently yielded intriguing preliminary results.

GROUP MEMBERS Containing more than 1800 members rich in which cDNA has been isolated from the mono- Domenika Arndt structural diversity, the monoterpenoid indole terpenoid indole alkaloid-producing species Technician until March 2006 alkaloids are a fruitful source of physiologically Rauwolfia serpentina and Catharanthus roseus. Maria Luisa Diaz Chavez PhD Student until March 2006 active molecules. Historically, the use of mono- Root cultures of Psychotria ipecacuanha were terpenoid indole alkaloid-producing plants, developed from rootless in vitro plantlets by Verona Dietl Technician until March 2006 such as Rauwolfia serpentina, in traditional modification of the hormone components of Nadja Grobe medicines can be traced back in India more the tissue culture medium. HPLC-MS analysis PhD Student until March 2006 than 3000 years. Selected examples of pharma- of extracted tissues showed that the root cul- Nils Günnewich PhD Student until March 2006 ceuticals derived from monoterpenoid indole tures accumulate emetine, at approximately alkaloid-producing plants are the antineoplastic 10 % of the level accumulated in in vitro plant- Gabriele Herrmann Postdoctoral Position until March 2006 drugs vinblastine and vincristine and the antihy- let leaves. Cultured roots were chosen for pre- Aphacha Jindaprasert pertensives ajmalicine and ajmaline. The alkaloi- paration of a cDNA library to be randomly se - PhD Student until January 2006 dal glucoside strictosidine has been recognized quenced. Even though less alkaloid is present Robert Kramell Postdoctoral Position until December as a biosynthetic precursor to the monoterpe- than in leaf material, the root tissue cultures 2006 noid indole alkaloids. The enzyme that catalyzes are not photosynthetic and should be, relatively Tobias Kurz the formation of strictosidine, strictosidine syn- speaking, “enriched” for alkaloid biosynthetic PhD Student until December 2006 thase, was one of the first indole alkaloid bio- transcripts. The EST sequences thus obtained Alfonso Lara PhD Student until December 2006 synthetic enzymes isolated and the cDNA en - were compared to sequences present in the Christin Richter coding this enzyme was the first alkaloid bio- GenBank and in this way a cDNA with homo- PhD Student until March 2005 synthetic gene isolated. Biosynthetically related logy to the Rauwolfia serpentina strictosidine Khaled Sabarna to the monoterpenoid indole alkaloids are the synthase gene, STR1, was identified. In addition, PhD Student until November 2006 ipecac alkaloids, such as emetine. Emetine is cDNAs with homology to Rauwolfia serpentina Karin Springob Postdoctoral Position until March 2006 produced in Psychotria ipecacuanha, which glucosidases involved in monoterpenoid indole Marco Steen grows wild in Central and South America, and alkaloid biosynthesis were also identified. The Technician until March 2006 is used in medicine as an emetic and amoebi - ipecac homologs were generated as full-length Annika Wirth cide. Subcutaneous injections of the alkaloid clones using RT-PCR and the conditions for Diploma Student until December 2005 emetine, the chief active principle present in functional heterologous expression are now ipecac, produces a rapid cure of amoebic dys- being developed in Escherichia coli. The entery, while oral administration promotes the strictosi dine synthase-like enzyme will be emetic properties of the drug. tested for ac tivity with secologanin and dopamine as well as with tryptamine and dopamine. Based upon catalytic mechanism, we postulate For the recombinant glucosidase, we have a that the first enzyme of ipecac alkaloid biosyn- series of alkaloidal glucosides that have been thesis is related in primary sequence to stricto- tested as sub strates. The recombinant glucosi- sidine synthase. Strictosidine synthase catalyzes dase appears to hydrolyze the glucose moiety the Pictet-Spengler condensation of the bioge- of both ipecoside and strictosidine. nic amine tryptamine and the iridoid secologa- 12 nin, whereas the first enzyme of emetine bio- Plumbago indica L. is a traditional medicinal synthesis condenses dopamine and secologanin plant from South East Asia. The roots of P. indica to the alkaloidal glucoside deacetylipecoside. accumulate the naphthoquinone plumbagin, We have attempted to identify the ipecac which exhibits antimicrobial, insecticidal, antitu- cDNA by homology to strictosidine synthase, mor, and antifertility activities. Plumbagin origi- SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

chalcone synthase (CHS) that synthe sizes nar- COLLABORATORS ingenin chalcone, the precursor of fla vo noids, Gerhard Bringmann University of Würzburg, Germany by condensing three acetate units derived from Wanchai De-Eknamkul malonyl-CoA with a p-coumaroyl-CoA starter Chulalongkorn University, Bangkok, unit. To characterize the enzyme potentially Thailand catalyzing the first step in the biosynthesis of Anthony Fist Tasmanian Alkaloids, Westburry, Australia the naphthoquinone plumbagin, a cDNA enco- Phil Larkins ding a type III PKS was isolated from roots of P. Scientific and Industrial Research Orga ni - indica. The translated polypeptide shared 47 % za tion Plant Industry, Canberra, Australia to 60 % identical residues with PKSs from other Friedrich Lottspeich Max Planck Institute of Biochemistry, plant species. Re combinant P. indica PKS expres- Martinsried, Germany Plumbago indica sed in Escherichia coli accepted acetyl-CoA as Markus Piotrowski starter and carried out five decarboxylative University of Bochum, Germany Joachim Stöckigt nates from six acetate units. This was demons - condensations with malonyl-CoA. The resulting University of Mainz, Germany trated for the first time by tracer experiments hexaketide was not folded into a naphthalene with Droso phyl lum lusitanicum cultures and derivative. Instead, an a-pyrone, 6-(2',4'-dihy- Plumbago europaea plants. In plants, polyketi- droxy-6'-methylphenyl)-4-hydroxy-2-pyrone, des are synthesized by type III polyketide syn- was produced. In addition, formation of a-pyro- theses (PKS). Type III PKSs are typically homo- nes with li near keto side chains derived from dimeric proteins with two functionally indepen- three to six acetate units was observed. Since dent active site cavities. They use thioesters of phenylpyrones could not be detected in P. indica coenzyme A as substrates and ca talyze up to roots, it is possible that the novel PKS is invol- seven decarboxylative condensa tion reactions ved in the biosynthesis of naphthoquinones, and followed by cyclization of the in termediate additional co-factors are probably required for polyketide. The prototype of type III PKSs is the biosynthesis of these secondary metabolites

Proposed PKS reaction mechanism OPIUM POPPY BIOTECHNOLOGY Head: Susanne Frick until March 2006

Opium poppy (Papaver somniferum L.), which contains more than 80 different alkaloids, remains one of the most important industrial medicinal plants. Poppy serves as a renewable resource of a number of medicinally relevant alkaloids. These include the analgesic and narcotic drug morphine, the cough suppressant codeine, as well as the muscle relaxant papaverine, the antineoplastic agent noscapine and the antimicro bial sanguinarine. We developed a transformation system for opium poppy, that will allow us:

to investigate the regulation and ecological function of these alkaloids in plants,

and to alter the alkaloid metabolism in commercial poppy varieties in order to obtain varieties lacking alkaloids or carrying tailored alkaloid profiles for industrial a n d pharmaceutical use.

GROUP MEMBERS During the last years several genes from the restricted. By completely suppressing morphi- Kathleen Bräuer biosynthetic pathways for reticuline, sanguinari- nane alkaloid biosynthesis, opium poppy could Technician until February 2006 ne and morphine have been cloned (Fig. 1-). Al potentially be grown more widely for the food Stefanie Haase though the biosynthesis is well understood at and oil industries. PhD Student until December 2006 the enzyme level, the molecular and biochemi- Elke Hillert Technician until March 2006 cal mechanisms that regulate these pathways We have used an Agrobacterium-mediated Katja Kempe are not known. approach to introduce different cDNAs enco- PhD Student until December 2006 ding enzymes of reticuline, morphine Heike Riegler and sanguinarine biosynthesis as sense, Diploma Student until December 2005 antisense- or RNAi-constructs into ex - plants to attempt to alter the alkaloid profile. Alkaloid-free plants developed in this manner will be used to test the chemical and ecological function of morphinane and benzophenanthridine alkaloids in planta. After the regenera - tion of plants, the qualitative and quantitative determination of alkaloids was determined by HPLC and LC-MS in latex, leaves and roots of opium poppy. Finally the heredity of the alkaloid pattern was confirmed.

Overexpression of the cDNA encoding (S)-N-methylcoclaurine 3’-hydroxylase (CYP80B3) resulted in an up to 450 % Figure 1: Biosynthetic pathway from L-tyrosine to sanguinarine, lau- increase in the amount of total alkaloid danosine and morphine in P. somniferum. Enzymes are highlighted in red. in latex (Fig. 2). This increase occurred either without changing the ratio of the A major goal of this project was the develop- individual alkaloids, or together with an overall ment of a stable transformation and regenera- increase in the ratio of mor phine. Cor res pon - tion method for opium poppy, which will make dingly, CYP80B3-antisense cDNA expressed in the metabolic engineering of the above mentio- opium poppy caused a re duction of total ned compounds possible. Poppy seed oil finds amount of alkaloids in latex up to 84 %, sugge- 14 use in chemical industry for the production of sting that the observed pheno types were pigments and lacquer, but residual morphine le - dependent on the presence of the transgene. vels prevent a more widespread applications. The increase or decrease of the amount of Because opium is the raw material for the illicit alkaloids in the transgenic cell lines is heritable production of heroin, cultivation of poppy is at least to the T3 generation. The transcript SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

O-methyltransferase gene (7- COLLABORATORS OMT) was deter mined by Nor - Anthony Fist Tasmanian Alkaloids, Westbury, Australia thern blot analysis from the T0 Phil Larkins to the T2 generation. Southern Scientific and Industrial Research Orga ni - blot analysis re vealed the inte- za tion Plant Industry, Canberry, Australia gration of one to five copies Sylvestre Marillonnet Icon Genetics AG, Biozentrum Halle, into the plant genome. The Germany amount of reticuline, laudanine Jürgen Schmidt Leibniz Institute of Plant Biochemistry, and laudanosine was changed in Halle, Germany 40 % of the T1 and 75 % of the T2 generation. Some plants of T1 and T2 generation displayed also changed levels of morphinane alkaloids.

The suppression of the sa lu - taridinol 7-O-acetyltransferase gene (SALAT) with an RNAi construct pro duced transgenic plants, in which pathway in ter - me diates of morphinane biosynthesis accumulated. This al - kaloid pattern was heritable to the T1 generation. The suppres - sion of SALAT was confirmed by nor thern blot analysis and real time PCR. With Southern blot analysis, two to four copies could be detected in transgenic plants.

In parallel, we initiated the first crossing experiments. Crossing Figure 2: Comparative alkaloid analysis of latex from P. somniferum. A-B wild-type (WT), A, T1 plant 1850 and T3 plant 15.7/13, both from transformation with CYP80B3-sense3. of the transgenic plant B, T1 plant 579 and T3 plant 17.6/10, both from transformation with CYP80B3-antisense. CYP80B3-sense with a transge- Alkaloids in latex were calculated as µg alkaloid/100 µg soluble protein, then normalized to 100 % to produce the individual divisions within pie graphs, representing relative ratios nic plant overexpressing a of the latex alkaloids. Total WT alkaloids were set to 100 % to produce the relative dia- NADPH:cytochrome P-450 meters of the pie graphs, representing the relative total alkaloid contents of the transgenic compared to the WT. oxidoreductase (CPR) produced all four possible phenoty- levels of CYP80B3 were determined by a mul- pes: wild-type, CPR overexpressing plants, tiplex RT-PCR with 18S rRNA as an en - CYP80B3 overexpressing plants and plants dogenous standard. In plants overexpressing overexpressing both transgenes. Plants overex- CYP80B3, higher ratios of CYP80B3:18S rRNA pressing both transgenes inherited five to six in comparison to wild-type was observed. In copies of CYP80B3 and one copy of CPR from an tisense-CYP80B3 plants the ratio their parental plants. The transcript levels of CYP80B3:18S rRNA was reduced. Southern CYP80B3 and CPR were determined by multi- blot analysis re vealed patterns that are in plex RT-PCR with 18S rRNA as an endogenous agreement with five to six copies of CYP80B3 standard. The mRNA levels of CYP80B3 and 15 in CYP80B3-sense plants and three to five CPR were increased in the trait-stacked plants copies in CYP80B3-antisense plants. as well as in their parental plants. The new transgenic plants displayed either increased or The overexpression of the (R,S)-reticuline 7- decreased amount of alkaloids. MODE OF ACTION OF JASMONATES Heads: Claus Wasternack & Otto Miersch

Jasmonates and their precursors, the octadecanoids, are signals in plant stress responses and in plant development. A mechanistic analysis of the mode of action of jasmonic acid (JA) and its metabolite 12-hydroxy-JA was performed by a reverse genetic approach using the allene oxide cyclase (AOC)-catalyzed step in jasmonate biosynthesis and the 12-hydroxy-JA-sulfotransferase. Gain of function and Loss of function studies with transgenic tomato and Arabidopsis thaliana plants revealed mo dulation of jasmonates and allowed to inspect the role of jasmonates in response to biotic and abiotic stresses as well as flower and seed development. Functional analysis of AOC using genetic approaches is also performed in Arabidopsis to ask on specific versus redundant functions of the four AOCs in this plant. These data were linked to metabolite profiles on jasmonates and octadecanoids under various stress conditions and in different developmental stages. In addition to this analytical work, chemical synthesis of standards and labeled substrates is an essential part of this work.

GROUP MEMBERS The role of JA biosynthesis (Fig. 1) was analy- single copy gene is Domenika Arndt zed in tomato and Arabidopsis. Preferentially, specifically ex pressed Technician until August 2006 the AOCs, the 12-hydroxy-JA sulfotransferase in ovules of young Christian Böttcher and the profile of jasmonates and other oxyli- flowers and all vascu- Technician since April 2006 pins were studied to elucidate specific func - lar bundles, which led Carolin Delker PhD Student tions in plant stress responses and develop- together with data Verona Dietl ment. from various transge- Technician since April 2006 nic tomato plants to Figure 2: Pollen tube growth in Stefan Götz a proposed model on tomato flowers 24 hours upon Diploma Student until August 2006 amplification in wound pollination of the stigma. Anja Hellwege Photo: S. Götz Diploma Student until November 2006 signaling with prefe- Elke Hillert rential signaling properties of JA. This is suppor- Technician since April 2006 ted by detection of JA biosynthetic enzymes Nils Kirmse including AOC in sieve elements of tomato leaf Diploma Student since October 2006 veins. Robert Peter Lange Postdoctoral Position since March 2005 Jana Neumerkel Abundant accumulation of AOC in ovules of PhD Student since December 2003 tomato flower buds and detection of a specific Birgit Ortel oxylipin signature in flower organs of wild-type Technician and AOC overexpression lines led to a concept Markus Otto PhD Student since October 2006 of specific functions of oxylipins in flower deve- Kathrin Rehagel lopment (Figs. 2-4). Corresponding to the fe - Diploma Student until August 2005 male sterility of the JA-insensitive mutant jai1, a Nadine Schumann role of oxylipins in early stages of embryo de - Diploma Student since October 2006 velopment of tomato was shown. An inte - Marco Steen Technician since April 2006 resting and important new facet in the role of Stefanie Thumm JA is the identification and characterization of Technician since February 2005 metabolites of JA, such as 12-hydroxy-jasmonate, its sulfated derivative and its glucose ester,

Figure 1: Jasmonate biosynthesis takes place in chloroplasts and both in wounded tomato leaves as well as du - peroxisomes. The final reactions occur via fatty acid b-oxidation ring flower development. Their content is dra- steps. In the activation to the corresponding CoA esters 4 CL-like CoA ligase is involved (from Wasternack, C. 2006 Oxy li pins– matically altered upon wounding and during flo- 16 Biosynthesis, Signal Trans duc tion and Action. In Plant Hormone Signaling (Hedden, P. & Thomas, S., eds.) Ann. Plant Reviews, pp. 185- wer, embryo and seedling development. In case 228. Blackwell, Oxford, UK). of 12-hydroxy-JA a sulfotransferase of to mato was cloned and transgenic lines overexpressing The first AOC was cloned from tomato (Zieg - or repressing the corresponding gene revealed ler et al. 2000, patent No. DE 1000 4468.9). This a flowering phenotype of the day-neutral toma- SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

Figure 3: Developing em - bryos in the tor- pedo stage of to - mato flowers (18 hours after pollination). Photo: S. Götz to plant. In contrast, in the photoperiod-depen- lowed by wounding of leaves revealed that mu - COLLABORATORS dent Ara bi dopsis plants the position of the cor- tants were able to perform b-oxidation of the Guillermina Abdala Universidad Nacional de Rio Cuarto, responding sulfotransferase within the various carboxylic acid side chain of JA precursors. Argentina flowering time control pathways is deter mined These data correspond to recently pu blished Ivo Feussner due to hints on flowering time control by 12- results of other labs. University of Göttingen, Germany hydroxy-JA (in cooperation with Luc Varin, Bettina Hause, Sabine Rosahl, Dierk Scheel, Jürgen Schmidt Montreal, Canada). Collaborative work was done with groups in Leibniz Institute of Plant Biochemistry, Germany and abroad and could attribute to the Halle, Germany In Arabidopsis the AOC is encoded by four following published working areas: Gerd Hause Biocenter, University of Halle, Germany genes. Inspection of transgenic Arabidopsis Gregg Howe plants expressing a reporter gene driven by (1) Role of NO in alternative oxidase activity University of Michigan, East Lansing, USA each AOC promoters, revealed redundant and of ozone-treated tobacco plants. Erich Kombrink non-redundant functions of the AOCs. Pre fe - (Ederli et al. 2006) Max Planck Institute for Plant Breeding Research, Cologne, Germany rentially, in root growth and seedling develop- (2) Interaction of salicylic acid and JA signaling. Johan Memelink ment (Fig. 5) as well as in flower development (Mur et al. 2006) University of Leiden, The Netherlands there are tissue and organ specific promoter (3) Hormones and gene expression pattern Thomas Roitsch activities. These data and analyses of knock-out during barley seed development. University of Würzburg, Germany lines indicate that Arabidopsis is able to per- (Sreeni vasulu et al. 2006) Paul Staswick University of Nebraska, Nebraska, USA form a fine-tuning of regulation by spatial and (4) Singlet oxygen and cell death in A. thaliana. Luc Varin temporal differences in the expression of the (Danon et al. 2005) Concordia University, Montreal, Canada four AOCs. In contrast, the four recombinant (5) AOC of hop and its role in organogenesis. AOCs exhibited similar enzymatic properties (Fortes et al. 2005) and substrate specificities. Using Arabidopsis (6) Lipoxygenase-mediated metabolism of sto- mutants affected in the multifunctional protein rage lipids in germinating sunflower cotyle- of fatty acid b-oxidation (aim1) or exhibiting a dons. (Gerhart et al. 2005) defect in protein import during peroxisome (7) 4-CL-like enzymes of Arabidopsis and their biogenesis (pex6) the role of fatty acid b-oxida- role in JA biosynthe sis. tion in final steps of JA biosynthesis was shown. (Schneider et al. 2005). Quantitative analysis of octadecanoids, jasmonate (8) Ethylene and JA in CDPK and MAPK sig- and its intermediate in biosynthesis upon fee- naling of stress responses. ding of deuterated 12-oxophytodienoic acid fol - (Ludwig et al. 2005). (9) Modified oxylipin signature of barley by overexpression of a 13-lipoxygenase. (Sharma et al. 2005).

The know-how of the group on JA and oxylipin analytics is used and re quested by many groups. A minor ac tivity is linked to collaboration with the Pro biodrug GmbH in Hal le. The company is working on role of glutami- nylcyclases (QCs) of hu man, animal and plant sources. QCs at tribute in mamma- lian sys tems to a functionalization of peptide hormones and proteins by py - roglutamate formation at the N-terminus. Our effort is to clone plant homo- Figure 4: The allene oxide cyclase (AOC) establi shing the correct loges of the QC as a tool for de sign of enantiomeric structure of the cyc lopentanone ring of jasmonate is Figure 5: Non-redundant promoter activity of AOC3 17 encoded by a single copy gene in tomato, which is specifically potent QC inhibitors. So far the potato and AOC4 in roots of seven-day-old Arabidopsis thalia- expressed in ovules of young flower buds and in early embryo na plants. Each promoter of the four AOC genes of A. development as revealed by AOC promoter activity tests (blue QC and a QC of Arabi dop sis were clo- thaliana was fused to the uidA gene coding for b-glucu- staining, left bottom) and immunocytological analysis (right) of a ned, overexpressed as re com binant pro- ronidase (GUS). Promoter activity was recorded by cross section. The structure of jasmonic acid (bottom) and its pre- blue staining, which appeared in tissues carrying GUS cursor 12-oxophytodienoic acid (top) is given. tein and characterized in terms of enzy- activity. Photo: B. Hause matic properties (Schilling et al. 2006). Photo: I. Stenzel PAPAVER GENE EXPRESSION ANALYSIS Head: Jörg Ziegler

The benzylisoquinoline alkaloids comprise a group of roughly 2,500 compounds identified so far. Among these are compounds of high pharmaceutical value, such as the analgesic morphine, the antitussive codeine, and the antimicrobial sanguinarine. The benzylisoquinolines mainly occur in the Ranunculales within the Papaver species ac cumulating the most extensive diversity of structures. The biosynthesis of all monomeric benzylisoquinolines is conserved up to the central intermediate (S)-reticuline, and biochemical and molecular data are available for every step. After that, the biosynthetic pathways diverge leading to the extensive structural diversity of this group of alkaloids. However, most of these steps are unknown yet. Similarly, factors regulating the accumulation of benzylisoquinolines, as well as components facilitating the transport of intermediates between cell types are not characterized yet. In order to ad dress these issues, we took advantage of the species- specific accumulation of dis tinct benzylisoquinoline profiles in Papaver species. Correlation of gene expression with the alkaloid profile of each Papaver species was successfully used to detect cDNAs involved in the establishing a distinct benzylisoquinoline profile in individual plants.

Group Members As a first step, an alkaloid profiling method was type. Unexpectedly, a morphinane derivative was Christin Fellenberg developed using FT-ICR/MS as analytical me thod also found at very low levels in P. arenarium. Practical course in September 2005 followed by the development of a program to vi - René Geißler sualize the data. The high mass accuracy of the Based on the profiling results, the gene ex - Diploma Student until September 2006 FT-ICR/MS allows the correct as signment of pression analysis of 2,000 ESTs was performed Andreas Gesell PhD Student until September 2006 empirical formulas to the masses. Although in order to de tect cDNAs whose expression Romy Klausnitzer many benzylisoquinolines possess the same could be related to the accumulation of morphi- Diploma Student until August 2006 mass, the FT-ICR/MS ap proach gave a fast and, nane al kaloids. The analysis of 16 different Pa - Silke Pienkny by inclusion of a standard, quantitative over- paver species yielded 69 cDNAs showing higher PhD Student until September 2006 view over the distribution of alkaloids among ex pression in plants containing morphinanes. In - Susan Voigtländer Diploma Student until September 2005 different Papaver species. The most noticeable te restingly, eight of these cDNAs code for pro- Silvia Wegener masses were further analyzed by LC-MS/MS for teins shown to be involved in benzylisoquino - Technician until September 2006 structure elucidation. As already known from line biosynthesis (Fig. 1). 43 cDNAs sho wed no literature, it was shown that only varieties of homology to any known protein in the data - Papaver somniferum and P. bracteatum accumu - base, whereas nine code for house-keeping late high amounts of alkaloids of the morphinane genes.The remaining cDNAs could be classified

Figure 1: Clustered display of gene expression analysis of Papaver species. One cluster showing increased expression in P. somniferum varieties is shown. Indicated in blue: cDNAs coding for enzymes in benzylisoquinoline biosynthesis; red: cDNAs currently characterized. SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

as ABC transporter (1), reductases (2), and active site, suggests a methylating activity of the Collaborators P450-monooxygenases (6). With the exception 3‘-hydroxy group of simple benzylisoquinolines. Christian Ammer, Wolfgang Brandt, Jürgen Schmidt of the house-keeping genes, these cDNAs Currently, work is in progress to substantiate Leibniz Institute of Plant Biochemistry, could putatively be involved in the accumula - these suggestions experimentally. Halle, Germany tion of alkaloids in P. somniferum and their cha- Milton Stubbs University of Halle, Germany racterization has been initiated. One reductase showing higher expression le - vels in morphine containing plants showed ho - Biochemical evidence showed the involvement mology to the large family of the short chain of ABC transporter in the translocation of in - dehydrogenases/reductases (SDRs), with the termediates of morphine biosynthesis in P. highest similarity to the men thone: neomenthol somniferum. The full-length sequence of an re ductase (MNR) in volved in menthol biosyn- ABC transporter was obtained and its charac- thesis. After overexpression, the recombinant terization is in progress. protein could be identified as salutaridine re - ductase (SalR), catalyzing the stereospecific re - Among the six P450 monooxygenases showing duc tion of salutaridine to 7-(S)-salutaridinol, an higher expression levels in morphinane alkaloid in termediate step in the biosynthesis of mor - containing plants, three full-length sequences phine. This previously un known cDNA showed were isolated and successfully expressed in in - high substrate specificity. Neither other benzyl - sect cells. One of these was able to convert an isoquinolines of similar structure, nor substra- intermediate in morphine biosynthesis, but LC- tes of other SDRs involved in plant secondary MS/MS analysis showed ambiguous spectra, metabolism, such as tropinone or menthone, which could not be assigned to a distinct com- are accepted as substrates. Similarly, the over- pound.This was due to the low quantity of pro- expression of the MNR from Mentha piperita ducts formed in the first experiments, and re - also showed exclusive substrate specificity to - petition should result in the structure elucida- ward the natural substrate, showing the high tion of this compound, so that this P450 mono- selectivity of the SDRs involved in plant secon- oxygenase can finally be characterized with re - dary metabolism. In order to elucidate the mo - spect to its role in benzylisoquinoline metabo- lecular basis for the high substrate specificity of lism. SalR, a model of the tertiary structure was cre - ated and the substrate salutaridine was docked After isolation of their full-length sequence, into the active site (Fig. 2). Careful examination two cDNAs showing tight clustering to the of the model proposed several amino acids to cDNAs involved in benzylisoquinoline biosyn- be involved in the selective binding of salutari- thesis exhibited homology to class 2 O-methyl- dine and in the active site of the enzyme. transferases. One cDNA could easily be identi- Substitution of the active site residues Asn, Ser, fied as 3‘-hydroxy-N-methylcoclaurine 4’-O Tyr and Lys resulted in a strong decrease in the me thyltransferase based on its high homology catalytic efficiency or the complete loss of en - to previously isolated sequences coding for the zyme activity, confirming the catalytic mecha- same enzyme. Biochemical characterization of nism of SDRs. The catalytic efficiency was also the bacterial ex pressed protein, which cataly- decreased by substitutions zes the last step in the basic benzylisoquinoline of amino acids in the pro- pathway to (S)-reticuline, showed to possess posed substrate-binding the highest substrate specificity compared with site, which was mainly due other O-methyltransferases isolated from P. to the increases in the Km- somniferum. The other O-methyltransferase values. With these results, showed homology to several methyltransfera- the SalR model could be ses in benzylisoquinoline biosynthesis, but en - sub stan tiated experimen- zyme assays performed with the overexpressed tally, and the first clues 19 protein suggested a new function. Homology about the mode of binding modeling of the tertiary structure of this me - of benzyl isoquino line sub- Figure 2 left: Model of the tertiary structure of salutaridine reductase. The active site residues are highlighted in blue; the substrate-binding site thyltransferase, and subsequent docking studies strates to enzymes were is indicated by the arrow. right: Amino acid residues forming the binding of many different benzylisoquinolines into the obtained. site for salutaridine (in turquoise). PUBLICATIONS AND OTHER SCIENTIFIC ACTIVITIES 2005 AND 2006

PUBLICATIONS 2005 Kutchan, T. M. Predictive plant metabolic engi- Andrade, A., Vigliocco, A., Alemano, S., Miersch, neering - still full of surprises. Trends Bio tech - Ziegler, J. & Kutchan, T. M. Differential gene ex - O. & Botella, M.A. Endo ge nous jasmonates nol. 23, 381-383. pres sion in Papaver-species in comparison and octadecanoids during germination and with alkaloid profiles. Acta Hort. 675, 173- seedling development: their relation with hy- Kutchan, T. M. A role for intra- and intercellu- 178. persensitive tomato mutants to abiotic stress. lar translocation in natural product biosynthe- Seed Sci. Res. 15, 309-318. sis. Curr. Opin. Plant Biol. 8, 292-300. Ziegler, J., Diaz Chavez, M. L., Kramell, R., Am - mer, C., & Kutchan, T. M. Comparative macro- Cenzano, A. M., Vigliocco, A., Miersch, O. & Ab- Kutchan, T. M. & Dixon, R. A. Physiology and array analysis of morphine containing Papaver dala, G. Octadecanoid levels during stolon to me ta bolism. Secondary metabolism: nature’s somniferum and eight morphine free Papaver tuber transition in potato. Potato Res. 48, 107- chemical re servoir un der deconvolution. species identifies an O-me thyltransferase in - 115. Curr. Opin. Plant Biol. 8, 227-229. volved in benzylisoquinoline biosynthesis. Planta 222, 458-471. Danon,A., Miersch, O., Felix, G., op den Camp, Ludwig,A.A., Saitoh, H., Felix, G., Freymark, G., R. G. L. & Apel, K. Concurrent activation of cell Miersch, O., Wasternack, C., Boller, T., Jones, BOOKS AND BOOK CHAPTERS 2005 death-regulating signaling pathways by singlet J.D.G. & Romeis, T. Ethylene-mediated cross- Frick, S., Kramell, R., Larkin, P.J. & Kutchan, T.M. oxygen in Arabidopsis thaliana. Plant J. 41, 68- talk between calcium-dependent protein ki - Stu dying morphine biosynthesis using transge- 80. nase and MAPK signaling controls stress re - nic opium poppy (Papaver somniferum L.). In: sponses in plants. Proc. Nat. Acad. Sci. USA Proc. WOCMAP III, Vol.6: Traditional Medicine Durgbanshi, A., Arbona, V., Pozo, O., Miersch, 102, 10736-10741. & Nutraceuticals (Pala nis wamy, U.R., Craker, O., San cho, J. V. & Gómez-Ca denas, A. Si mul - L.E., & Gardner, Z.E., eds.), Acta Hort. 680, pp. taneous determination of multiple phytohor- Meixner, C., Ludwig-Müller, J., Miersch, O., 37-43. mones in plant extracts by liquid chromato- Gresshoff, P., Staehlin, C. & Vierheilig, H. Lack graphy-electrospray tandem mass spectrome- of mycorrhizal autoregulation and phytohor- Ounaroon, A., Frick, S. & Kutchan, T.M. Mo- try. J. Agric. Food Chem. 53, 8437-8442. monal changes in the su pernodulating soybe- lecular genetic analysis of an O-methyltransfe- an mutant nts1007. Planta 222, 709-715. rase of the opium poppy Papaver somniferum. Fortes, A.M., Miersch, O., Lange, P.R., Malho, In: Proc. WOCMAP III, Vol.1: Bioprospecting & R., Tes til lano, P.S., del Risueno, M.C., Was ter- Nualkaew, N., De-Eknamkul, W., Kutchan, T. M. Ethnopharmacology (Bernáth, J., Né meth, É., nack, C. & Pais, M.S. Ex pression of allene oxide & Zenk, M.H. Geranylgeraniol formation in Craker L.E. & Gardner, Z.E., eds.), Acta Hort. cyclase and accumulation of jasmonates du - Croton stellatopilosus proceeds via successi- 675, pp. 167-171. ring or ganogenic nodule formation from hop ve mo node phos pho rylations of geranylgera- (Hu mu lus lupulus var. Nugget) internodes. nyl di phos phate. Tetrahedron Lett. 46, 8727- Wasternack, C. Jasmonates – overview on bio- Plant Cell Physiol. 46, 1713-1723. 8731. synthesis and diversity in actions. In: Jasmo - nates, Special Issue of J. Plant Growth Reg. 23 Frick, S., Kramell, R., Larkin, P.J. & Kutchan, T.M. Ounaroon, A., Frick, S. & Kutchan, T. M. Mole- (Wasternack, C., ed.), Sprin ger Verlag, Berlin, Stu dying morphine biosynthesis using transge- cu lar ge netic analysis of an O-methyltransfe - pp. 167-169. nic opium poppy (Papaver somniferum L.). rase of the opium pop py Papaver somniferum. Acta Hort. 680, 37-43. Acta Hort. 675, 167-171. PATENTS 2005 Kutchan, T. M., Frick, S. & Kempe, K. Modula - Frick, S., Kramell, R., Schmidt, J., Fist, A. J. & Rudus, I., Kepczyñska, E., Kepczyñski, J., tion of alkaloid biosynthesis in plants and Kut chan, T.M. Comparative qualitative and Wasternack, C. & Miersch, O. Changes in jas- plants having altered alkaloid biosynthesis. quantitative determination of alkaloids in nar- monates and 12-oxophytodienoic acid con- PCT International Patent Appli cation filed on cotic and condiment Papaver somniferum cul- tents of Medicago sativa L. du ring somatic em - 11 August 2005 tivars. J. Nat. Prod. 68, 666-673. bryogenesis. Acta Physiol. Plant. 27, 497-504. No.PCT/ EP2005/ 009232

Gerhard, B., Fischer, K., Balkenhohl, T.J., Poh - Schneider, K., Kienow, L., Schmelzer, E., Colby, DIPLOMA THESES 2005 nert, G., Kühn, H., Wasternack, C. & Feussner, T., Bartsch, M., Miersch, O., Wasternack, C., Grobe, Nadja: Genexpressionsanalysen in I. Lipoxygenase-mediated metabolism of sto- Kombrink, E. & Stuible, H.- P. A new type of Esch schol zia californica und heterologe rage lipids in germinating sunflower cotyle- peroxisomal acyl-coenzyme A synthetase from Expres sion zweier O-Methyltransferasen. dons and b-oxidation of (9Z,11E,13S)-13-hy - Arabidopsis thaliana has the ca talytic capacity University of Halle-Wittenberg, Department droxy-octadeca-9,11-dienoic acid by the co - to activate biosynthetic precursors of jasmo- of Bio che mistry/Biotechnology 08/09/2005. tyledonary glyoxysomes. Planta 220, 919-930. nic acid. J. Biol. Chem. 280, 13962-13972. Riegler, Heike: Analyse und Kreuzung der Isayenkov, S., Mrosk, C., Stenzel, I., Strack, D. & Stumpe, M., Carsjens, J. G., Stenzel, I., Göbel, transgenen Mohnlinien CYP80B1-sense und Hause, B. Suppression of allene oxide cyclase C., Lang, I., Pawlowski, K., Hause, B. & Feussner, CPR-sense. University of Halle-Wittenberg, in hairy roots of Medicago truncatula reduces I. Lipid metabolism in arbuscular mycorrhizal Department of Bioche mis try/ Biotechnology, jasmonate levels and the degree of mycorrhi- roots of Medicago truncatula. Phytochemistry 26/08/2005. zation with Glomus intraradices. Plant Physiol. 66, 781-791. 139, 1401-1410. Rehagel, Kathrin: Die Bedeutung der Sul fo - 20 Yu, C. K. Y., Springob, K., Schmidt, J., Nicholson, transferase AtST2a für den Blühzeitpunkt von Kramell, R., Schmidt, J., Herrmann, G. & Schlie - R. L., Chu, I. K., Yip, W. K. & Lo, C. A stilbene Arabidopsis thaliana im Zusammenhang mit mann, W. N-(Jasmonoyl)tyrosine-derived com- synthase gene (SbSTS1) is involved in host and Genen des photoperiodischen Signalweges. pounds from flower of broad beans (Vicia fa - nonhost defense re sponses in Sorghum. Plant University of Halle-Wittenberg, Department ba). J. Nat. Prod. 68, 1345-1349. Physiol. 138, 393-401. of Biochemistry/Biotechnology, SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

07/09/2005. del, R. R., Hau se, B. & Schulze, J. Transgenic bar- Wasternack, C., Hause, B., Stenzel, I., Götz, S., ley plants overexpressing a 13-lipoxygenase to Feuss ner, I. & Miersch, O. Jasmonate signaling Voigtländer, Susan: Identifizierung einer Short- modify oxylipin signature. Phyto chemistry 67, in tomato – The input of tissue-specific occur- Chain-Dehydrogenase aus Papaver somnife- 264-276. rence of allene oxide cyclase and JA metabo- rum als Saluta ri din-Reduktase (EC 1.1.1.248), lites. In: Current Advances in the Biochemistry ein Enzym im mor phiumspezifischen Weg der Sreenivasulu, N., Radchuk, V., Strickert, M., and Cell Biology of Plant Lipids, Proceeding of Benzylisochinolin-Syn the se. University of Hal - Miersch, O., Weschke, W. & Wobus, U. Gene the 17th Int. Symp. on Plant Lipids. July 16-21, le-Wittenberg, Department of Bioche mis try/ expression patterns reveal tissue-specific sig - 2006, East Lansing (J. Ohl rogge and C. Biotechnology, 29/09/2005. naling networks controlling programmed cell Benning, eds.), pp.107-111. death and ABA-regulated maturation in deve- DOCTORAL THESES 2005 loping barley seeds. Plant J. 47, 310-327. DIPLOMA THESES 2006 Schäfer, Ursula: Isolierung und funktionelle Geißler, René: Untersuchungen zur Katalyse Cha rak te risierung eines an der Trichom ent - Wasternack, C., Stenzel, I., Hause, B., Hause, und Sub stratbindung der Salutaridin-Reduk- wicklung beteiligten MYB-Transkriptions fak- G., Kutter, C., Maucher, H., Neumerkel, J., tase aus Papaver bracteatum L. University of tors aus Nicotiana benthamiana. University of Feussner, I. & Miersch, O.The wound res ponse Halle-Wittenberg, De partment of Bioche mis - Halle-Wittenberg, De part ment of Biochemis- in tomato – Role of jasmonic acid. J. Plant try/ Biotechnology, 07/09/2006. try/Biotechnology, 12/07/2005. Physiol. 163, 297-306. Götz, Stephan: Die Rolle von Jasmonaten in Nualkaew, Natsajee: The study of enzyme and Wasternack, C. Jasmonates–Biosynthesis, sig - der Em bryoentwicklung der Tomate. Univer - gene involved in plaunotol biosynthesis in nal transduction and action. Annals of Botany sity of Halle-Wittenberg, Department of Bio- Croton stellatopilosus Ohba. Chulalangkorn Lecture. Reg. Plant Growth & Dev. 41, Suppl. chemistry/Biotech nology, 02/08/2006. University, Faculty of Phar maceutical Sciences 2006, p. 11. Payathai Road, Bangkok 10330, Thailand, 2005. Hellwege, Anja: Die Rolle von AtTFL2 und sei- Winkler, A., Kutchan, T. M., Glieder, A. & Ma - nem D. melanogaster Homologen DmHP1 in PUBLICATIONS 2006 cheroux, P. Berberine bridge enzyme posses- der Blühinduk tion von A. thaliana. University Delker, C., Stenzel, I., Hause, B., Miersch, O., ses a novel bi-covalently attached FAD cofac- of Halle-Wittenberg, Department of Bioche - Feussner, I. & Wasternack, C. Jasmonate bio- tor linked to a histidine and cysteine residue. mistry/Biotechnology, 27/11/2006. synthesis in Ara bi dop sis thaliana – Enzymes, J. Biol. Chem. 281, 21276-21285. products, regulation. Plant Biol. 8, 297-306. Klausnitzer, Romy: Untersuchungen zur Be - Ziegler, J., Voigtländer, S., Schmidt, J., Kramell, deutung von ABC-Transportern für die Mor - Ederli, L., Morettini, R., Borgogni, A., Was ter - R., Miersch, O., Ammer, C., Gesell, A. & Kut - phinbiosynthese in Papaver somniferum L. nack, C., Miersch, O., Reale, L., Ferranti, F., chan, T.M. Comparative transcript and alkaloid Uni versity of Halle-Witten berg, Department Tosit, N., & Pas qua lini, S. Interaction between profiling in Papaver species identifies a short of Biochemistry/Biotechnology, 02/08/2006. nitric oxide and ethylene in the induction of chain dehydrogenase /reductase involved in alternative oxidase in ozone-treated tobacco morphine biosynthesis. Plant J. 48, 177-192. Schulz, Melanie: Untersuchungen zur Rolle plants. Plant Physiol. 142, 595-608. von Was serstoffperoxid bei der Expression BOOK CHAPTER 2006 des Allen oxid cyc lase-Gens und anderen wun- Frick, S., Kramell, R. & Kutchan, T.M. Metabolic Wasternack, C. Oxylipins – Biosynthesis, Sig - dinduzierten Genen der Tomate. University of engineering of a morphine biosynthetic P450 nal Transduction and Action. In: Plant Halle-Wittenberg, Department of Chemistry, in opium poppy surpasses breeding. Metab. Hormone Signaling, Ann. Plant Reviews 23/06/2006. Eng. 9, 169-176. (Hedden, P. & Tho mas, S., eds.) Blackwell, Oxford, UK. pp. 185-228. Wirth, Annika: Charakterisierung einer Glu - Mur, L. A. J., Kenton, P., Atzorn, R., Miersch, O. cosyl transferase und einer Methyltransferase & Wasternack, C. The outcomes of concentra- BOOKS AND BOOK CHAPTERS IN PRESS aus Dro sophyllum lusitanicum. University of tion specific interactions between salicylate Frick, S., Kramell, R., Larkin, P.J. & Kutchan, T.M. Halle-Wittenberg, Department of Pharmacy, and jasmonates sig nalling include synergy, an - Stu dying morphine biosynthesis using transge- 27/02/2006. ta gonism and the activation of cell death. Plant nic opium poppy (Papaver somniferum L.). In: Physiol. 40, 249-262. Proc. WOCMAP III, Vol. 1: Bioprospecting & DOCTORAL THESIS 2006 Eth nopharmacology (Bernáth, J., Németh, È., Sabarna, Khaled: Approach of isolating the - Nualkaew, N., De-Eknamkul, W., Kutchan, T. M. Cra ker, L.E. & Gardner, Z.E., eds.). baine synthase from Papaver somniferum. & Zenk, M. H. Membrane-bound geranylgera- Uni versity of Halle-Wittenberg, Department nyl diphosphate phosphatases: Purification and Kutchan, T. M., Frick, S. & Weid, M. Engineering of Biochemistry/Biotech nology, 16/11/2006. characterization from Croton stellatopilosus plant alkaloid biosynthetic pathways – Pro - leaves. Phytochemistry 67, 1613-1630. gress and prospects. In: Advances in Plant Bio - chemistry and Molecular Biology (Lewis, N. & Sharma, V.K., Monostori, T., Hause, B., Maucher, Nes, D. W., eds.) Vol. 1, Bio en gi nee ring and H., Gö bel, C., Hornung, E., Hänsch, R., Bittner, Mo lecular Biology of Plant Pathways F., Was ter nack, C., Feussner, I., Mendel, R.R. & (Bohnert, H.J. & Nguyen, H.T., eds.) Elsevier Schulze, J. Ge ne tic transformation of barley to Science Ltd., Ox ford, UK. mo dify expression of a 13-lipoxygenase. Acta 21 Biol. Sze ge di ensis 49, 33-34. Wasternack, C. & Abel, S. Plant hormones. In: Mo lecular Plant Physiology, chapter 15, (Shar - Sharma, V. K., Monostori, T., Göbel, C., Hänsch, ma, R., ed.) Harvard Press. R., Bittner, F., Wasternack, C., Feussner, I., Men- DEPARTMENT OF BIOORGANIC CHEMISTRY Head: Professor Ludger Wessjohann

Secretary: Elisabeth Kaydamov / Ines Stein

MOLECULES IN (INTER)ACTION Research of the department is based on three main topics: (1.) Learning from nature’s chemistry through both elucidation of natural structures as well as understanding basic principals of natures application of chemistry in a biological context. (2.) Total and diversity oriented synthesis of natural products and derivatives for applications, e.g., in biology or medicine. (3.) Understanding molecular interactions and develop new tools for recognition compounds and their application.

Many biological questions to be answered are developed in cooperation with the other departments at the IPB. Most efforts directed towards the profiling of secondary metabolites (metabolomics) are done in an interdepartmental collaboration, e.g. together with the Department of Stress and Developmental Biology. Common projects also exist with the Department of Secondary Metabolism in the areas of glucosidases and glucosyl and methyl transferases and in the application of our microtubuli-active compounds on plant cells.

The isolation, characterization, and modification of secondary metabolites and enzymes from plants and fungi is the basis of our efforts to understand the properties of these compounds or even to disclose their function in nature, and finally to explore their use in chemistry and biology. Applications are driven by the discovered properties and in - clude such diverse areas as nutriceuticals, lead structures in medicinal chemistry or cos- metics, biological research tools, or the utilization of enzymes as biocatalysts in synthesis or as screening targets. This is backed by an extensive synthesis program to increase compound availability and molecular diversity by combinatorial chemistry, method development, and de novo synthesis. Advanced projects often proceed in collaboration with industrial partners.

22 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

A project in biological chemistry usually will re quire the competences of several re search groups, e.g., re search on an active principle of a plant initially requires expertise in na tural product isolation, then spectroscopic characterization, in parallel screening is required, at some point synthesis, then synthetic derivatization, again analytics and screening, perhaps metabolic studies and modeling. The demands and expertise needed at any given project time point shift with the progress and discoveries made. At the Department of Bioorganic Chemistry we believe that projects as well as the individual groups strongly profit from the excellent horizontal as well as vertical integration that is reflected in the organisational presentation (see figure), in which the three major "wet" chemistry based competence areas (natural products, synthesis and chemoenzymatics) are complemented by the three predominantly analytical ones (spectroscopy, computational chemistry, and screening). The latter also comprise a major service unit for our department and the institute in general.

The department maintained a high publication frequence with some 30 research papers per year, including top chemistry journals such as Angewandte Chemie, Journal of the Ame rican Chemical Society, Organic Letters, or Chemical Communications. It offers research op portunities in a highly international and competitive environment to students wishing to at tend advanced practical courses, visiting scientists and postdoctoral fellows, but primarily to students who wish to pursue a master or Ph.D. in all areas connected to bioorganic chemistry such as synthetic, analytical and physical chemistry, pharmacy, biochemistry, food/ nutritional chemistry, as well as for candidates of related areas like informa- tics inclusive bio- and cheminformatics, mathematics, or biology.

23 NATURAL PRODUCTS Heads: Norbert Arnold & Jürgen Schmidt

The group is engaged in the production, isolation, and structural elucidation of plant and fungal metabolites. The evolutionary selection undergone by these compounds provides a bias for bioac- tivity and thus can be used for the discovery of novel lead structures for drug candidates or other applications. The investigation of the native function of natural products in natural systems can aid the identification of new targets, the understanding of ecological interactions, or the discovery of new biocatalysts or enzymatic transformations.

GROUP MEMBERS CONSTITUENTS OF FUNGI mena. The production of fungitoxic substances Nasser Abdullah Awadh Ali Mushrooms are among the largest groups of can be interpreted as pre formed defense in the Guest Researcher until October 2006 higher organisms on our globe with an estima- "fight" over the nutritional substrate. To clearify Sanela Bacinovic ted 106 species. At present only some 7% this hypothesis, crude extracts of different Diploma Student until October 2005 (75.000) of the estimated total are described. Sepedonium species were tested for their fun- Ivana Correa Ramos Leal PhD Student until December 2006 During their evolution fungi developed a large gicidal and bactericidal properties, e.g., S. Kanchana Dumri variety of strategies to utilize different sub - ampullosporum Damon proved to possess high DAAD-Leibniz Fellow strates for their nutrition. They frequently live antifungal activity. The future aim is the isolation Katrin Franke in close symbiosis with plants (mycorrhiza). and characterization of the re sponsible sub- Postdoctoral Position Some genera use dead organisms or even stances. Gudrun Hahn Technician other toadstools as a source for their nutrition. Myint Myint Khine The species diversity of the latter, so-called my - Constituents of Higher Fungi Daimler-Benz Fellow until March 2006 cophilic fungi is tremendously rich, also in their Fruit bodies of the mycorrizal genus Hygro - Christine Kuhnt chemical arsenal. phorus are rarely attacked by parasitic fungi or Technician insects. In continuation of our research on their Monika Kummer Technician Constituents of Mycophylic Fungi secondary metabolites, the alkaloids harmane Martina Lerbs Species of the genus Sepedo ni um are pa ra si tes Technician on fruit bo dies of ge nera in Bo letales. They re - Tilo Lübken present the asexual synanamorph of ascomyce- Guest Scientist since March 2006 tes of the teleomorph genus Hypomyces, Katharina Michels Diploma Student since April 2006 whose perithezia can only be observed rarely. Yulita Mitei Species of the perthophytic genus Sepedonium PhD Student until September 2006 are first settling on the living host, and in a later Nguyen Hoang Anh stage they kill the host and use the organic Guest Scientist until July 2006 material of the host for their own nutrition. Re - Luay Rashan Guest Scientist until October 2005 mar kably, Boletales fruit bodies, which are Axel Teichert attacked by Sepedonium in their enviroment PhD Student are rarely attacked by other parasitic fungi, Carlo Tiebe whose spores are perva sive. This let us assume (1) and norharmane (2) could be isolated from Diploma Student until November 2006 that secondary metabolites produced by Hygrophorus eburneus (Bull.) Fr. So far, 35 Trinh Thi Thuy Guest Scientist until July 2006 Sepedonium are re sponsible for such pheno- selected Hygro phorus species were investiga- Alexander Voss ted for these alkaloids by LC/ESI-SRM (selected Diploma Student until May 2005 reac tion monitoring), using the reaction of the [M+H]+ ion to the base peak ion at m/z 115. Both alkaloids are widely distributed in the genus Hygrophorus, independent of the geo- graphic origin, and the occurrence also appears to be quite constant within a species. Therefore, the compounds might be useful chemotaxonomic markers for the genus 24 Hygrophorus.

CONSTITUENTS OF PLANTS Sepedonium spec. growing on fruit bodies of Bolets HEANTOS - a traditionalistic SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

vietnamese opiate detoxification remedy human breast cancer cell line and a mouse Collaborators The project HEANTOS was originally coordi- fibroblast cell line. Six cardenolides exhibit Helmut Besl University of Regensburg, Germany nated by the United Nations (UNESCO and noteworthy antiproliferative activity (IC50 valu- Stefan Dötterl UNOPS). Dr. Dan (Peoples Republic of Viet - es < 1mM), and four cardenolides show the University of Bayreuth, Germany nam) developed the original herbal remedy to induction of apoptosis in a hu man leukemic cell Günter Gerlach aid opioid detoxification and called it HEAN- line. The project is finished. Botanical Garden Munich, Germany TOS (“heat of sun”). The formulation was sub- Carola Griehl Hochschule Anhalt (FH), Köthen, sequently improved with the Vietnamese Medicinal plants from Socotra Island Germany Academy of Science and Technology (Institute (Republic of Yemen) Matthias H. Hoffmann of Che mistry, Prof. Sung). HEANTOS consists Socotra Island is the largest and most easterly Botanical Garden Halle, Germany of at least 13 components and was constantly island of a small archipelago in the Indian Wolfgang Steglich University of Munich, Germany improved in composition, also with the aid of Ocean. Access is restricted to few researchers. Peter Spiteller our findings. In cooperation with Vietnamese The island is rich in unique and distinguished Technical University of Munich, Germany partners our research is focused on the isola - species of plants: nearly half of them are consi- Tran Van Sung tion and characterization of the constituents of dered to be endemic. So far, extracts from Academy of Science and Technology these plants. So far, more than 200 compounds Soco traous medicinal plants belonging to twelve have been isolated from twelve plants. A sub- plant families showing, e.g. antifungal and anti - stance based overview and extensive literature oxidant activities, are under investigation. survey was conducted. Simultaneously the very complex coextracted Heantos mixture (capsu- Floral Oils les) as produced for applications was examined Flowers produce a variety of constituents, by means of coupled HPLC and mass spectro- often specific to this part of the plant, e. g., vola- metric methods to determine the lead substan- tiles, floral oils, resins, or waxes. Floral oils are ces for the standardization of the medicine. secreted by specialized glands, so-called elaio - Through this the first standardization protocol phores. The lipid profiles of floral oils and resins could be worked out and was utilized to allow of various species and in other cases of flowers a clinical study to be performed in Germany. It in various stages of development are conduc- was also the first one to be applied to such a ted by GC/MS and ESI-FT-ICR-MS. complex traditional Asian medicine. Thus, several Diascia species Traditional medicinal plants from Myanmar (Scrophu la riaceae) occur- The aim of the project was the isolation, struc- ring in Southern Africa are tural characterization and study of selected pollinated by oil-collecting pharmacological activities of phytoconstituents bees of the genus Rediviva from the Myanmar (Burmese) medicinal plants (Meli t tidae). b-Hydroxy fat ty Streptocaulon tomentosum Ellis & Arthur (As - acids, mo no-, di- and tri acyl - clepiadaceae), Curcuma comosa Roxb. (Zingi - glycerols could be identified be raceae) and Vitis repens Wight & Arm (Vita - as main constituents of their ceae). About 20 constituents belonging to tri- floral oils by GC-MS. While terpenoids, cardenolides, lignanes, and steroidal the monoacylglycerols pos- saponines including three new substances were sess a long-chain b-acet oxy isolated from the roots of S. tomentosum. fatty acid (C14, C16 and C18), About 27 constituents belonging to curcumi- the di- and tri acylglycerols noids and sesquiterpenoids including nine new additionally contain one or sesquiterpenes could be characte rized from two short-chain b-acetoxy the rhizome of C. comosa. Poly phe nols, fatty fatty acids, respectively. acids, lignanes were obtained from the rhizome of V. repens. Extracts of each plant were tested Similarily, the "chemical" for their antifungal properties against relationship of the Eu ropean 25 Cladosporium cucumerinum. Six cardenolides Lysi machia punctata flowers isolated from S. to mentosum were tested for with Macropis fulvipes bees their antiprolifera tive activity in vitro against a was studied. Here the col- CHEMOENZYMATICS Heads: Ludger Wessjohann & Wolfgang Brandt

Enzymes are the molecular machines of organisms catalyzing chemical conversions. As such they can be utilized in two different ways: either as targets to modify biological processes within its producing organism, or as biocatalyst to perform its task either in a different organism or ex vivo in synthetic chemistry. Of special interest to us is the latter point, complemented by the desire to understand the mechanisms of biological catalysis.

GROUP MEMBERS ISOPRENOID CONVERTING ENZYMES experimental results and structural classifica - Cristiano Bohn-Rhoden The synthesis of the complex natural isopre- tion of prenyl enzymes, a new highly relevant PhD Student noids is based on prenyl group transfering en - 3D-model could be developed. This model is Lars Bräuer PhD Student until September 2006 zymes, which include e.g. oligoprenyl synthases, able to explain a wide range of substrate speci- terpene cyclases or aromatic prenyltransfera- ficities and is in complete agreement with the Gudrun Hahn Technician ses. results of site-directed mutagenesis. Josef Skopek Guest Scientist until July 2005 The ubiA-Enzyme of E. coli belongs to the lat- At the same time, ubi A could be used for syn- Roman Weber PhD Student ter group. The enzyme and its relatives are cru- thetic purposes with artificial substrates. It ca - cial for the production of the essential electron talyzed a crucial step in a total synthesis of ku - Heike Wilhelm Postdoctoral Position until April 2006 carrier ubiquinone in pro- and eucaryotic histanols, the natural meroterpenoids. Svetlana Zakharova organisms. In order to study its active site, an Postdoctoral Position until December 2006 intense bio- and cheminformatic analysis was Using comparative homology modeling techni- performed and combined with site-directed ques reasonable 3D-structure models of two mutagenesis studies. Based on previous mode- terpene synthases from Cannabis sativa could ling analyses, amino acids identified as impor- be developed. To prove the correctness of the tant for the catalytic mechanism were selecti- models and to clarify the catalytic mechanism vely replaced to obtain five new mutants. Their of these two enzymes both site-directed muta- involvement in the catalytic mechanism was genesis and quantum mechanical calculations strongly supported but at the same time a re - were performed. Combined ab initio (catalytic modeling of the previously proposed enzyme active site) and force field (the whole enzyme structure appeared necessary to combine two except the active site) calculations, the so-cal- active sites, which had to be placed into close led QM/MM method, is applied to study the proximity in the new model. Based on these catalysis mechanism in realistic surroundings.

Space filling model of the active site of ubiA-prenyltransferase with the prenyldiphosphate buried (in magenta) and the aromatic benzoate on top (in orange). SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

In practice, the applicability of these enzymes was only possible by kinetic resolution and thus Collaborators for chemoenzymatic syntheses was explored stopped below the theoretical value of 50% Martin Biendl Hallertauer Hopfenveredelungs GmbH, with the aim to alter product specificity to - yield. In order to generate a dynamic kinetic Mainburg, Germany wards non-native products through rational resolution scheme, base- or acid-catalyzed ra - Uwe Bornscheuer protein design. cemization in principal can be used. However, University of Greifswald, Germany both substrate and product are prone to this, Lutz Heide University of Tübingen OTHER ENZYMES and additionally can undergo acyloin shift and György Horvath The selective synthesis of plant benzopyranes aldol reaction under such conditions. A coope- University of Antwerp, Belgium (flavonoids, isoflavonoids etc.) could be im - rative study elucidated the best conditions for Udo Kragl proved considerably by utilizing plant O-methyl a differential racemization of either species, University of Rostock, Germany transferases. which proved to run best under acid catalysis Toni M. Kutchan Donald Danfort Plant Science Center, St. in a compartimentalized reactor, separating ra - Louis, USA The enantioselective O-acylation and deacylati- cemization and resolution spatially. Shuming Li on of acyloins with lipases or esterases so far University of Düsseldorf Markus Pietzsch University of Halle, Germany Orgentis Chemicals GmbH Gatersleben, Germany

Rational protein design for biocatalysis: geranylpyrophosphate in the pocket of a terpenoid cyclase. SYNTHESIS Head: Ludger Wessjohann & Bernhard Westermann

GROUP MEMBERS Chemical genomics aims to use small molecules to probe phenotypic and genetic variations. Muhammad Abbas Specifically, experimental design in chemical genomic screening takes into account genetic and Postdoctoral Position genomic differences in identifying cellular reactions to small mo lecules. Furthermore, this Muhammad Ayaz approach provides a temporary control over cellular states not achieved normally by molecular PhD Student since April 2006 biology methods. Very recently we have started and expanded successfully programs to evaluate Carlos Boluda target- and diversity-driven synthetic approaches to provide small molecules. This focus furnishes Postdoctoral Position until February 2006 vehicles to study biological interactions, which we envision as one of the main goals of our scientific work. Kristin Brand PhD Student since October 2006 Andriy Buchynskyy In the context of target-oriented total synthe- extension of b-amyloid plaques. The total syn- Postdoctoral Position since September sis we could realize the synthesis of Hygropho - thesis has been achieved as well as the synthe- 2006 ron (1), Galanthamin (2) and Tubulysin (3). sis of dimeric structures employing the “click” Marco Dessoy Postdoctoral Position approach. Victor Dick Hygrophorones have PhD Student until July 2006 been isolated in our Alexander Dömling de partment and have Guest Scientist until June 2006 been shown to exhibit Simon Dörner PhD Student a broad spectrum of 1 Tobias Dräger very in teresting bio - PhD Student logical and pharmaceutical profiles, e. g. activity Uwe Eichelberger against Van co mycin-resistant strains. The synthe- Tubulysin, which at this moment is the most Postdoctoral Position until May 2006 sis providing the enantiomerically pure natural active natural product for Tubulin stabilization, Otilie Eichler-Vercillo PhD Student since September 2005 product is star ted with a Sharpless dihydroxyla- has been the synthetic goal of a couple of Lars Gabriel tion reaction, which is providing the chiral, non- groups worldwide. We have been able to achive Trainee since September 2006 racemic in termediates. Fur ther reactions are the total synthesis first and reported it in Daniel Garcia-Rivera highly diastereoselective and afford the hygro- September 2006. Up to the end of 2006, two PhD Student phorones in eight steps. Variations in order to additional total syntheses have been reported. Matthäus Getlik Master Student since July 2006 commit these products in chemical genomics The key reaction of the synthetic route at our Gergely Gulyas studies can be carried out easily by using diffe- de partment is a multicomponent reaction, PhD Student rent starting materials employing the same which is most atom efficient and provides effi- Alexander Gutsche strategy. ciently the central heterocycle in one step. Diploma Student until April 2006 Michael Henze PhD Student since October 2006 Galanthamin, iso- The aspects of atom- and eco-efficiency are 2 Nicole Hünecke lated some 50 one of the key factors in our synthetic plans Trainee years ago from and have been considered largely for the syn- Oliver Kreye Cau ca sian snow thesis of 1 and 2, too. This topic is also one of PhD Student drops, is used for the main aspects in our diversity driven multi- Fredy Leon-Reyes PhD Student since May 2005 the treatment of component reaction approach towards macro- Christiane Neuhaus Alz hei mer disea- cycles. PhD Student until February 2006 se. In addition, di me ric derivatives of this natu- Angela Schaks ral product are intensively discussed to exhibit For the evaluation of molecular interactions, Technician inhibitory activities within the context of fibrill macrocycles are well suited due to conforma- Gisela Schmidt Technician Alex Schneider PhD Student Jasquer Alonso Sehnem Guest Scientist until April 2005 Tran Thi Phuong Thao PhD Student until August 2006 Marcio Weber Paixao 28 PhD Student until March 2006 Katharina Wolf Trainee Mingzhao Zhu SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

tional preorganization and the incorporation of tural peptidic counterparts. The high degree of Collaborators binding units as well as monitoring units. In our diversity can be generated by a single MiB reac- Bionorica AG Neumarkt, Germany approach towards ma crocycles of type 4, we tion (5), consecutive MiB reaction led to even Antonio Luiz Braga have been able to tune these products by inte- higher diversified products (6, 7). Federal University of Santa Maria, Brazil grating a sensing unit, which is monitoring bin- Alexander Dömling, ding events through changes in the absorption Concomitantly, the synthetic progress to these Wolfgang Richter R & D Biopharmaceuticals GmbH bands. This facilitates the determination of pro- highly complex natural and natural-like pro- Martinsried, Germany ductive binding through molecular interactions. ducts, respectively, is enabled by the develop- Gregor Fels, Nikolaus Risch Parallel to these attempts, optical switches have ment of new synthetic methodologies. In this University of Paderborn, Germany been incorporated in or der to alter the confor- context, the main focus has been directed to - Sabine Flitsch University of Edinburgh, UK mation of the macrocycle and the interacting wards organocatalysis. Organocatalysis has gai- Carlos Kleber Zago de Andrade unit itself. This may regulate the binding from an ned its impact due to the non-utilization of ha - University of Brasilia, Brazil on to off status. zardous metals and resource-saving proce - Francisco Coll Manchado dures. In our group the main emphasis has been University of Havana, Cuba The inherent complexity of natural products drawn towards Mannich reactions and hydro- Romano Orru Free University of Amsterdam, could be generated by expanding the MiB (mul- xylations. The arylation of aromatic aldehydes, Netherlands tiple multicomponent macrocyclizations inclu- which inevitable needs Zinc as a metal, could be PharmaZell GmbH ding bifunctional building blocks) approach, extended into a catalytic version reducing the Raubling, Germany which has been established by us recently. By amount of metals to only a minimal amount. In Polymer Laboratories Ltd. Shropshire, UK variation of the starting compounds products addition, the chiral, non-racemic ligands for the Priaton GmbH exhibiting a high degree of functional, topologi- asymmetric version has been developed fur - Tutzing, Germany cal and skeletal diversity could be provided. The ther, they now rely mostly on renewable re - Laszlo Somsak peptoidic framework, which is due to the Ugi sources such as amino acids. Moreover, the ap - University of Debrecen, Hungary multicomponent reaction, offers the advantage plicability is strengthened by the short synthe- Symrise AG Holzminden, Germany of high metabolic lability compared to their na - sis to obtain these ligands. WeylChem GmbH Mannheim, Germany

29 SPECTROSCOPY Heads: Andrea Porzel & Jürgen Schmidt

The spectroscopy group is engaged in the identification and structural elucidation of small molecules with modern analytical techniques, including questions in the area of metabolomics and physicochemical screening. Modern mass spectrometric methods, NMR-spectroscopic experiments, and methods of the optical spectroscopy (IR, UV, CD) are used and developed for solving structural problems and for the study of noncovalent interactions of molecules. Other departments of the IPB and external research groups are supported in solving structural and analytical problems.

GROUP MEMBERS MASS SPECTROMETRY sents a powerful tool for evaluating biochemi- Christine Kuhnt The mass spectrometry group has carried out cal pathways and facilitates the study of the flux Technician a series of mass spectrometric investigations, of di stant precursors into these natural pro- Martina Lerbs both with respect to structural investigations ducts. Technician of plant and fungal metabolites and the mass Annett Siebenhüner Diploma Student until April 2005 spectral service for all groups of the IPB and NMR AND OPTICAL SPECTROSCOPY Maritta Süße external cooperation. The electrospray Modern NMR spectroscopy experiments were Technician Fourier-transform-ion cyclotron resonance used for the structural elucidation of bioactive (ESI-FT-ICR) mass spectrometer was success- natural products isolated from plants and hig- fully applied to a series of analytical problems her fungi and for synthetic compounds. Routine including profiling experiments of alkaloids, flo- spectra (1H/ 13C/ 19F/ 31P NMR, IR, CD, ORD, UV) ral oils, and triacylglycerols. Furthermore, a were recorded as a service for the Department series of dendrimers and macrocycles as well of Bioorganic Chemistry as well as other re - as other synthetic compounds were investiga- search groups of the IPB. The NMR laboratory ted. took successfully part in a Round Robin test on the quantitative analysis of an active pharma- Phenolic compounds from lichens belonging to ceutical ingredient (rutin) by 1H NMR spectros- the family Physciaceae were investigated in col- copy. laboration with Siegfried Huneck. In that case the depsides atranorin and chloroatranorin In collaboration with the Institute of Organic could be identified as main components. Chemistry, University of Darmstadt, we started testing of residual dipolar couplings (RDCs) as In collaboration with the Department of Na- a tool for determination of the relative stereo- tural Product Biotechnology (Susanne Frick, chemistry of small organic molecules. For these Robert Kramell, Jörg Ziegler, Toni Kutchan) and purpose, 1J(1H,13C) coupling constants of a the Biocenter Halle a series of LC-ESI-MS/MS hygrophorone derivative were investigated in investigations of Papaver alkaloids and benzyl - the chiral liquid crystal system poly-g-benzyl-L-

iso qui noline alkaloids were performed. Thus, glutamate/CDCl3. the alkaloid pattern after feeding with [ring- 13 1 15 C6]-tyramine as a biogenetic precursor of H, N heteronuclear 2D NMR correlation ex - these alkaloids to Papaver somniferum seed- periments were established in order to charac- lings was elucidated both by direct infusion terize peptoids with repetitive structural ele- high-resolution ESI-FT-ICR mass spectrometry ments. The method was used to determine the and liquid chromatography/electrospray tan- number of N-CO cis/trans isomers. dem mass spectrometry. Based on this procedure, the struc ture of about 20 alkaloids dis- Gentianae radix (dried rhizome and roots of playing an incorporation of the labeled tyrami- Gentiana lutea L.) extracts were investigated as ne could be elucidated. These alkaloids belong example for the usability of evaporative light to different classes (morphinane, benzylisoqui- scattering (ELS) and online-1H-NMR as HPLC noline, protoberberine, benzo[c]phenanthridi- detection methods for investigations of crude 30 ne, phthalideisoquinoline and protopine type). plant extracts. ELSD was proven to be a valu- The valuable in formation gained from the alka- able tool for detection of UV active as well loid profile de monstrates that the combination inactive constituents in a virtually quantitative of these two spectrometric methods repre- manner. Online 1H-NMR detection suffered SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

from the low sensitivity (regarding the instru- alkaloids isolated from the fungus Cortiarius Collaborators mentation of the department), however, direct brunneus were elucidated. From the same Ivo Feussner University of Göttingen, Germany investigation of the crude extracts in proton- source, N-glucosidic indole alkaloids were iso- Christina Thiele ated solvents with sophisticated (WET) solvent lated. Also for these compounds, available in Technical University of Darmstadt, suppression technique yielded in promising sub-milligram amounts only, structural elucida- Germany results. tion by means of NMR spectroscopy was suc- Meinhart Zenk Biocenter, University of Halle, Germany cessful. Using one- and two-dimensional NMR experiments the structures of a series of carboline The attenuated total reflection (ATR) FT-IR

Expansion of the 1H-15N HMBC NMR spectrums of cis and trans peptoid isomers

31 SCREENING Heads: Norbert Arnold & Bernhard Westermann

The new competence group was established in January 2006, opening the door for the investigation of properties of small molecules by biological, chemical, and virtual screening methods. Thereby it is not only limited to the Department of Bioorganic Chemistry, but is open to all other departments of the IPB and cooperation partners wishing to engage in chemical biology driven by the impact of small molecule on.

GROUP MEMBERS BIOLOGICAL SCREENING CHEMICAL SCREENING Claudia Bobach Secondary metabolites isolated from fungal The chemical screening projects are embedded PhD Student fruit bodies or plants as well as synthetic com- in the research topics related to molecular in - Monika Kummer Technician pounds are tested in simple cell or organism- teractions. Chemical reactions or interactions based assays for their biocidal activity profile, can be screened by physicochemical methods Ernst Roemer Postdoctoral Position until February e.g. for fungicidal, bactericidal, herbicidal, or such as MS, NMR, or optical spectroscopy. E.g., 2006 algaecidal properties. These bioassays are com- macrocycles, cryptands, and cages were made Fredy Leon-Reyes PhD Student since May 2005 plemented by cytotoxicity and more detailed accessible by the MiB-strategy in unprecedented enzymatic and receptor assays in cases of veri- diversity (see Research Group Synthesis). The fied positive results or of special interest. Thus high de gree of complexity and the prospect of in depth tests of steroid-like activities have sui table binding properties should render them been established during the first year of this amenable for the selective binding of me tal ions. group. This fact has been proven by the template synthesis of macrocycles (Scheme 1). Addition of va rious earth alkali ions clearly changed the product spectrum as shown by mass spectrometry. In continuation of these studies we will deter - mine whether cationic and anionic guests can be selectively bound and how macrocycles se lec tive for certain species are best generated and selected. Structure-activity driven screen ing will help to identify those func tional groups and conformational preferences most suited for selective host-guest in teractions.

VIRTUAL SCREENING Many compounds are too scarce or valuable to Figure left: Algaecidal assay (Spirulina laxissima) of crude fungal extracts. right: Assay on developed TCL plates. be tested randomly on various targets. In other cases, e.g. if a compound acting on a defined target is to be sought, random screening will re - quire high throughput set-ups not available at the IPB or too costly.

In cases of a (bio-)chemically defined target, a solution can provided by the fast pre-selection of candidates out of databases with millions of compounds by pharmacophore search tech - niques. Subsequently, docking of identified po - tential ligands to the specific hosts (receptors, enzymes etc.) leads to the prediction of compounds worthwhile for experimental tes ting or/ and synthesis, offering a higher probability of 32 success compared to a random selec tion. Also

Results of an antibacterial assay of a fungal fatty acid on the Gram- other parameters such as calculated cell perme - negative bacterium Vibrio fischeri. ability can be used for the preselection of useful candidates. SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

Especially for effective screening for host-guest Collaborators interactions of conformationally flexible mole- Dagmar Riemann, Barbara Seliger cules, new software tools have been developed University of Halle, Germany with the Research Group Computational Che - Probiodrug AG mistry to automate the entire process of phar- Halle, Germany macophore screening, docking, energy optimi- Wella AG zation of the host-guest complexes and identi- Darmstadt, Germany fication of most favoured guests for the specific host.

Pharmacophor model of a receptor with a ligand fragment (turquoise polyeder).

Scheme 1: Left: generation of the dynamic and frozen imine macrocycle library with selected structures shown. Right: MS-spectra of the libraries under various challanges: a) without any template, b) presence of Mg2+, shift to smaller rings like 4, c) presence of Ba2+, shift to larger rings like 5-7. COMPUTATIONAL CHEMISTRY Heads: Wolfgang Brandt & Andrea Porzel

The computational chemistry activities comprise cheminformatics, molecular modeling, and to some extent theoretical chemistry and quantum mechanical calculations. With these methods, the research group investigates molecular structures (natural products; 3D protein structures), their interactions and reaction mechanisms (e.g. of enzyme catalysis), and physicochemical properties. New bio- and cheminformatics tools for the evaluation of spectroscopic, structural and biological data are developed in cooperation with other departments and external partners.

GROUP MEMBERS PROTEIN HOMOLOGY MODELING of the pH- and neighboring group dependency Susanne Aust Thioredoxin reductase is a selenocysteine con- of selenocysteine redox properties studied by Guest Scientist taining homodimeric NADPH-dependent flavo- electrochemical analysis of peptide fragments Lars Bräuer PhD Student until September 2006 enzyme and a key player of the cellular redox of thioredoxin reductases of various or - milieu. Based on our recently developed model ganisms. Andrea Brock Guest Scientist until December 2005 we suggested that a highly conserved glu ta - Frank Broda mate477 in the neighborhood of His472 could es - As part of our isoprenoid research, in coopera- System Administrator sentially participate in the catalytic mechanism tion with the Department of Natural Product Stephanie Gulde PhD Student since August 2005 of this enzyme, acting as proton sink in a pro- Biotechnology, two homology models of ter - ton charge relay mechanism (Fig. 1). It thus can pene cyclases were created and extensively in - Tobias Heintz PhD Student since October 2006 favor the formation of the selenolate required vestigated by site-directed mutagenesis (for Martin Jess for substrate reduction. Based on our theoreti- more see report of the Research Group Che - Diploma Student since September 2006 cal calculations we postulated a new, swapping moenzymatics). Robert Klein 477 472 59 Diploma Student since May 2006 catalytic triad between Glu , His , and Cys followed by Glu477, His472, and Sec498 which reacts Recently, the NADPH-dependent short chain Anna-Carolin Meier PhD Student since July 2005 with Cys32’ (or Cys35') of the substrate thiored - dehydrogenase/reductase "salutaridine reduc - Diana Schulze oxin. Site-directed mutagenesis of Glu477 to glu- tase" involved in morphine biosynthesis was clo- PhD Student since May 2006 tamine, alanine, or lysine led to a significant ned from Papaver somniferum by Ziegler et al. Bianca Wachsmuth Diploma Student until May 2005 drop in enzymatic activity as predicted. This (IPB). A model of the Papaver bracteatum ho - provided not only a quantitative limit for the mologe was created based on the x-ray struc - energetic contribution of glutamate477 in the ca - ture of human carbonyl reductase 1. The homo- talytic mechanism of thioredoxin reductase and logy model shows the typical a/b folding pattern related enzymes but furthermore validates in of such enzymes. Docking of salutaridine into si lico DFT-calculations as a useful tool for quan- the active site revealed the first data on the mo - titative considerations in enzymology. The lecular interaction of benzylisoquinoline alkaloid results aided the design of experimental studies with enzymes. Site-directed mutagenesis clari- fied the involvement of residues relevant in substrate binding and for the proton transfer sys - tem.

In close cooperation with the Department of Secondary Metabolism, structures of the serine carboxypeptidase-like (SCPL) enzymes 1-O-si - na poyl-b-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-b-glucose:choline sinapoyltransferase (SCT) were modeled to gain in - sight into determinants of specificity and substrate recognition. The structures reveal the a/b-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The binding sites of the sub- 34 strates could be characterized and explain the substrate specificity and aspects of the catalytic Fig. 1: Active site of human thioredoxin reductase showing the suggest and now proven catalytic mechanism. Several mutations in the active site triad Sec498 – His472 and Glu477. of these enzymes confirmed the principal cor- SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

rectness of the models. lity. An automatic procedure has been deve - Collaborators In cooperation with Birgit Dräger (University loped to identify the most likely stable confor- Apogepha GmbH Dresden, Germany of Halle) a protein model of putrescine N-me - mers. A subsequent in silico screening including Birgit Dräger thyltransferase was developed. In comparison large compounds libraries can be used to find University of Halle, Germany to related enzymes, the substrate putrescine matching interactions. In silico screening is per - Monika Frey and the cosubstrates S-adenosylmethionine formed by calculation of so-called pharmaco- Technical University of Munich, Germany and de carboxylated S-adenosylmethionine ap - phore queries (Fig. 2), e.g. hydrophobic re gions, Stephan Gromer University of Heidelberg, Germany pear to bind differently. The modeling studies hydrogen bonding donor or acceptor po sitions, Alexander Hinneburg helped to identify amino acid residues in the and searching fast in databases to find good University of Halle, Germany active site, which will be subject to mutation to matching guests for the host enzyme or macro- Volker Lipka evaluate both the model and possibility to alter cycle, or vice versa. John Innes Centre,Norwich, UK the substrate specificity of the enzyme. OntoChem GmbH Halle, Germany CHEMINFORMATICS Probiodrug AG In cooperation with Volker Lipka (University of AND SOFTWARE DEVELOPMENTS Halle, Germany Tübingen) a model of an Arabidopsis PEN2 gly- New concepts Ernst Reiss cosyl hydrolase has been developed. Current methods of structural comparison Institute of Resistance Research and Pathogendiagnostics, Aschersleben, allow substructure searches, but they are not Germany Furthermore, in cooperation with Ernst Reiss very effective for similarity searches. Especially Ute Wittstock (Aschersleben) eight different barley PR-5 pro- in huge databases with billons of chemical Technical University of Braunschweig, teins (TLPs 1 to 8, TLP for thaumatin-like pro- structures severe limitations appear, and new tein) were modeled. The models highlighted the search concepts and algorithms have to be de - effects of sequence differences between the veloped. In cooperation with external partners, TLP isoforms in terms of their secondary a first set of new methods and the correspon- structures and their molecular electrostatic ding software applications were developed and potentials. We did propose that these sequence offer the chance for much faster and more ef - differences have implications for the target pre- fective tools. Within IPB, these new tools will ferences of the different isomers. be applied for the analysis of natural products or bioactive compounds (vs. other compounds PROTEIN-LIGAND INTERACTION, SMALL MO LECULE vs. medicinal compounds), in order to expand MODELING AND QSAR* the currently used cheminformatic analysis As a contribution to the investigation of the tools. evolution of isoprenoids, the binding sites of di - phosphates in 771 proteins with known 3D- 2D NMR data base system structure have been analyzed, characterized, In continuation of our work on an 2D NMR and correlated to data of prenyltransferases. spectra data base system, we could show This analysis led not only to classifications into (diploma thesis of Karina Wolfram under super- metal cation mediated and non-cation media- vision of Alexander Hinneburg, University of ted recognition sites of diphosphates but also Halle) that probabilistic semantic indexing to characteristic binding motives with different (PLSI), which is types of amino acid residues. designed for text data crea- Another important group of plant isoprenoids ted by humans, are steroids and steroid mimics, e.g. isoflavo - can effectively noids. The QSAR of their binding to common handle mapped steroid receptors and steroid converting en - NMR data origi- zymes was studied by theoretical methods. nating from HSQC 2D NMR Artificial ligands and hosts are created by syn- spectra. Ad di tio - thesis, e.g. macrocyclic compounds by very effi- nally, PLSI com- 35 cient Ugi four-components reactions. The lat- bined with the ter, despite of the formation of a cyclic struc - new mappings is Fig. 2: Example structure of a macrocycle with pharmacophore queries (red hydrogen bonding acceptor and yellow hydrophobic sites). ture, exhibit rather high conformational flexibi- ab le to find

*QSAR = Quantitative Structure Activity Relationship PUBLICATIONS AND OTHER SCIENTIFIC ACTIVITIES 2005

PUBLICATIONS 2005 Hause, G., Lischweski, S., Wessjohann, L.A. & Hause, B. Abbas, M., Neuhaus, C., Krebs, B. & Westermann, B. Epothilone D affects cell cycle and microtubular pat- Catalytic addition of homoenolates to imines- The tern in plant cells. J. Exp. Bot. 56, 2131-2137. Homo-Mannich reaction. Synlett 16, 473-476. Kramell, R., Schmidt, J., Herrmann, G. & Schliemann, Barth, A., Thondorf, I., Gebauer, S., Brandt, W., Neu - W. N-(Jasmonoyl)tyrosine-derived compounds from bert, K., Stano, J., Psenak, M. & Kovacs, P. Ge dan ken flowers of broad beans (Vicia faba). J. Nat. Prod. 68, zum Mechanismus der Serinproteasen. Teil 2. Dipepti - 1345-1349. dyl-Peptidase IV (DP IV/CD 26). Ac ta Facultatis Phar maceuticae Universitatis Come nianae , LII, 7-20. Lipka, V., Dittgen, J., Bednarek, P., Bhat, R., Wierner, M., Stein, M., Landtag, J., Brandt, W., Rosahl, S., Scheel, D., Basso, A., Ebert, C., Gardossi, L., Linda, P., Phuong, T. T., Llorente, F., Molina, A., Parker, J., So merville, S. & Schul - Zhu, M. & Wessjohann, L. A. Penicillin G amidase-cata- ze-Lefert, P. Pre- and postinva sion defenses both lysed hydrolysis of phenylacetic hydrazides on a solid contribute to nonhost resistance in Arabidopsis. phase: A new route to enzyme-cleavable linkers. Ad v. Science 310, 1180-1183. Synth. Catal. 347, 963-966. Nguyen, T.H.V., Nguyen, T.H.A., Sung, T.V., Franke, K. & Baumert, A., Milkowski, C., Schmidt, J., Nimtz, M., Wessjohann, L.A. Four phthalides from the roots of Wray, V. & Strack, D. Formation of a complex pattern Angelica sinensis. Tap Chi Hoa Hoc 43, 228-231. of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyl- Nguyen, T.H.V., Nguyen, T.H.A., Sung, T.V., Franke, K. & transferase family? Phytochemistry 66, 1334-1345. Wessjohann, L.A. Three phthalide derivatives from Angelica sinensis rhizomes. Tap Chi Hoa Hoc 43, 119- Braga, A.L., Alves, E.F. , Sivera, C.C., Appelt, H.R. & 122. Wessjohann, L.A. A new cystein derived ligand as ca - talyst in the addition of diethylzinc to aldehydes: The Odman, P., Wessjohann, L.A. & Bornscheuer, U.T. importance of a "free" sulfide site for enantioselecti- Chemoenzymatic dynamic kinetic resolution of acy- vity. Synthesis 558-594. loins. J. Org. Chem. 70, 9551-9555.

Braga, A.L., Lüdtke, D.S., Wessjohann, L.A., Pai xao, Ruijter, E., Schültingkemper, H. & Wessjohann, L.A. M.W. & Schneider, P. H. A chiral disulfide from (R)- Highly substituted tetrahydropyrones from hetero- cys teine in the enantioselective addition of diethyl- Diels-Alder reactions of 2-alkenals with stereochemi- zinc to aldehydes: Loading effect and asymmetric cal induction from chiral dienes. J. Org. Chem. 70, amplifica tion. J. Mol. Catal. A 229, 47-50. 2820-2823.

Braga, A.L., Lüdtke, D.S., Schneider, P.H., Vargas, F., Schmidt, J., Raith, K., Boettcher, C. & Zenk, M.H. Schneider, A., Wessjohann, L.A. & Paixao, M.W. Cata - Analysis of benzylisoquinoline-type alkaloids by elec- lytic enantioselective aryl transfer: Asymmetric addi- trospray tandem mass spectrometry and atmos phe - tion of boronic acids to aldehydes using pyrrolidinyl- ric pressure photoionization. Eur. J. Mass Spectrom. methanols as ligands. Tetra he dron Lett. 46, 7827- 11, 325-333. 7830. Teichert, A., Lübken, T., Schmidt, J., Porzel, A., Ar nold, Brandt, W. & Wessjohann, L.A. The functional role of N. & Wessjohann, L.A. Unusual bioactive 4-oxo-2-al - selenocysteine (Sec) in the catalysis mechanism of kenoic fatty acids from Hygrophorus eburneus. Z. large thioredoxin reductases: Proposition of a swap- Na turforsch. B 60, 25-32. ping catalytic triad including a Sec-His-Glu state. Chembiochem 6, 386-394. Wessjohann, L.A. & Ruijter, E. Macrocycles rapidly produced by multiple multicomponent reactions in - Dörner, S. & Westermann, B. A short route for the cluding bifunctional building blocks (MiBs). Mol. synthesis of "sweet" macrocycles via a click-dimeriza- Divers. 9, 159-169. tion-ring-closing metathesis approach. Chem. Com - mun. 2852-2854. Wessjohann, L.A. & Ruijter, E. Strategies for total and diversity-oriented synthesis of natural product(-like) Frick, S., Kramell, R., Schmidt, J., Fist,A.J. & Kut chan, macrocycles. Top. Curr. Chem. 243, 137- 184. T.M. Comparative qualitative and quantitative determination of alkaloids in narcotic and condiment Wessjohann, L. A., Ruijter, E., Garcia-Rivera, D. & Papaver somniferum cultivars. J. Nat. Prod. 68, 666- Brandt, W. What can a chemist learn from na ture's 673. macrocycles? A brief, conceptual view. Mol. Divers. 9, 171-186. Greiner, D., Bonaldi, T., Eskeland, R., Roemer, E. & Im - 36 hof, A. Identification of a specific inhibitor of the his - Wessjohann, L.A., Voigt, B. & Garcia-Rivera, D. Diver - tone methyltransferase SU(VAR)3-9. Na ture Chem. sity oriented one-pot synthesis of complex macrocy- Biol. 1, 143-145. cles: Very large steroid-peptoid hybrids from multiple multicomponent reactions including bifunctional buil- SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

ding blocks. Angew. Chem. 117, 4863-4868; Angew. tivität von Naturstoffen. University of Halle-Witten - Chem. Int. Ed. 44, 4785-4790. berg, 04/04/2005. Westermann, B. & Dörner, S. Synthesis of multivalent Annett Siebenhüner: Untersuchung von nicht UV-ak - aminoglycoside mimics via the Ugi multicomponent tiven Substanzen in Gentianae radix mittels HPLC reaction. Chem. Commun. 2116-2118. und NMR. University of Halle-Wittenberg, 30/04/2005. Westermann, B. & Neuhaus, C. Dihydroxyacetone in amino acid catalyzed Mannich-type reactions. Angew. Bianca Wachsmuth: Anwendung und Grenzen des Chem. 117, 4145-4147; Angew. Chem. Int. Ed. 44, Protein-Homologie-Modellings am Beispiel von zwei 4077-4079. Prenyltransferasen und einer Terpensynthase. Uni ver - sity of Halle-Wittenberg, 31/01/2005. Yu, C.K.Y., Springob, K., Schmidt, J., Nicholson, R.L., Chu, I.K.,Yip, W.K. & Yip, L.C. A stilbene synthase DOCTORAL THESES gene (SbSTS1) is involved in host and nonhost defen- Jens Hasenjäger: Chirale Phosphorliganden in der ho - se responses in Sorghum. Plant Physiol. 138, 393-401. mogenen, asymmetrisch katalysierten Hydrierung. University of Paderborn, 2005. PATENTS Lübken, T., Arnold, N., Wessjohann, L., Locher, H. Lars Müller: Zur homogenen, katalysierten, stereose- Hy grophorone und deren Derivate. Patent lektiven Hydrierung von Sacchariden. University of PCT/EP2005/001957. Pa derborn, 2005.

Wessjohann, L., Wilhelm, H., Biendl, M. Verfahren zur Eelco Ruijter: Design and Synthesis of Ratjadone Herstellung von Naringeninderivaten aus Xan tho - Analogues. Vrije Universiteit Amsterdam, 15/12/2005. humol. Patent 10 2005 013 258.8-44.

DIPLOMA THESES Stephanie Gulde: Virtuelles Screening auf Hormonak -

37 PUBLICATIONS AND OTHER SCIENTIFIC ACTIVITIES 2006

PUBLICATIONS 2006 Gromer, S., Wessjohann, L.A., Eubel, J. & and an indole alkaloid skeleton bearing identi- Abbas, M., Bethke, J. & Wessjohann, L. A. One Brandt, W. Mutational studies confirm the ca - cal acylated glycosidic groups from yellow pe - pot syn thesis of selenocysteine containing talytic triad in the human selenoenzyme thio- tals of Papaver nudicaule. Phy tochemistry 67, peptoid libraries by Ugi multicomponent re - redoxin reductase predic ted by molecular 191-201. actions in water. Chem. Commun. 541-543. modeling. ChemBioChem 7, 1649-1652. Schneider, A., Rodrigues, O. E. D., Paixao, M. W., Augustin, T., Schlosser, D., Schmidt, J., Baum- Hornung, E., Krüger, C., Pernstich, C., Gip - Appelt, H. R., Braga, A. L. & Wessjohann, L. A. bach, R., Grancharov, K., Krauss, G. & Krauss, mans, M., Porzel, A. & Feussner, I. Production of Stereoselective synthesis of Boc-protected L- G. J. Biotrans formation of 1-naphthol by a (10E,12Z)-conjugated linoleic acid in yeast seleno- and tellurolanthionine, L-seleno- and strictly aquatic fungus. Curr. Microbiol. 52, and tobacco seeds. Bio chim. Biophys. Acta, tellurocystine and derivatives. Tetrahedron 216-220. Mol. Cell Biol. Lipids 1739, 105-114. Lett. 47, 1019-1021.

Braga, A.L., Vargas, F., Sehnem, J. A. & Wess- Horvath, G., Guissez, Y., Wessjohann, L. A., Selent, J., Brandt, W., Pamperin, D. & Gober, B. johann, L.A. Microwave-mediated palladium- Caubergs & R. J., Horemans, N. Differential Enan tiomeric N-methyl-4-piperidyl benzilates catalyzed asymmetric allylic alkylation using distribution of tocopherols and tocotrienols as muscarinic receptor ligands: Radioligand chiral ß-seleno amides. Eur. J. Org. Chem. over photosynthetic and non photosynthetic binding studies and docking studies to models 4993-4997. tissues in higher plants. Phy to che mistry 67, of the three muscarinic re ceptors M1, M2 and 1185-1195. M3. Bioorg. Med. Chem. 14, 1729-1736. Brandt, W. Thaumatin-like protein 1. - 8. Pro- tein data bank, entries: 2DOV, 2DOW, 2DOZ, Huang, X., Roemer, E., Sattler, I., Moellmann, U., Sontag, B., Rüth, M., Spiteller, P., Arnold, N., 2DOX, 2DOY, 2DPO1, 2DP2 & 2DP0. Christner, A. & Grabley, S. Lydiamycins A-D: A Steglich, W., Reichert, M. & Bringmann, G. new class of small cyclodepsipeptides with an- Chromogenic meroterpenoids from the Brandt, W. & Stehle, F. 1-O-sinapoyl-b-glu cose: ti-mycobac terial properties. Angew. Chem. mushrooms Russula ochroleuca and R. viscida. choline sinapoyltransferase from Arabi dopsis 118, 3138-3143; Angew. Chem. Int. Ed. 45, Eur. J. Org. Chem. 1023-1033. thaliana. Protein Data Bank, Entry: 2DRG. 3067-3072. Stehle, F., Brandt, W., Milkowski, C. & Strack, D. Brandt, W. & Stehle, F. 1-O-sinapoyl-b-glu cose: Huneck, S. & Schmidt, J. Phenolische Ver bin - Struc ture determinants and substrate recog - choline sinapoyltransferase from Brassica na - dungen einiger Flechten aus der Familie Phy - nition of se rine carboxypeptidase-like acyl- pus. Protein Data Bank, entry: 2DTP. siaceae. Herzogia 19, 199-203. transferases from plant secondary metabolism. FEBS Lett. 580, 6366-6374. Brandt, W. & Stehle, F. 1-O-sinapoyl-b-glucose: Kreye, O., Westermann, B., Rivera, D. G., L-malate sinapoyltransferase from Johnson, D. V., Orru, R. V. A. & Wessjohann, L. A. Tchinda, A. T., Teshome, A., Dagne, E., Arnold, Arabidopsis thaliana. Pro tein Data Bank, Dye-modified and photoswitchable macrocy- N. & Wessjohann, L. A. Squalene and amento- entry: 2DRF. cles by multiple multicomponent macrocycli- flavone from Anti desma laciniatum. Bull. zations including bifunctional building blocks Chem. Soc. Ethiopia 20, 325-328. Czifrak, K., Hadady, Z., Docsa, T., Gergely, P., (MiBs). QSAR & Comb. Sci. 25, 461-464. Schmidt, J., Wessjohann, L.A. & Somsak, L. Syn- Thuy, T. T., Franke, K., Porzel, A. Wessjohnn, L.A. thesis of N-(beta-D-glucopyranosyl) mannosi- Lübken, T., Arnold, N., Wessjohann, L.A. Bött - & Sung, T. V. Quaternary protoberberine alka- des of dicarboxylic acids as potential inhibi- cher, C. & Schmidt, J. Analysis of fungal cyclo- loids from Ste pha nia rotunda. J. Chem. tors of glycogen phosphorylase. Carbohydr. pentenone deriva tives from Hygrophorus Vietnam VAST (Tap Chi Hoa Hoc) 44, 259- Res. 341, 947-956. spp. by liquid chromatography/electrospray- 264. tandem mass spectrometry. J. Mass Spectrom. de Greef, M., Abeln, S., Belkasmi, K., Dömling, 41, 361-371. Thuy, T. T., Sung, T. V., Franke, K. & Wessjohann, A., Orru, R.V. & Wessjohann, L.A. Rapid com- L.A. Ben zylisoquinoline and tetrahydroproto- binatorial ac cess to macrocyclic ansapeptoids Paixao, M.W., de Godoi, M., Bohn Rhoden, berberine alkaloids from Stephania rotunda and ansapeptides with na tural-product-like C.R., Wes termann, B., Wessjohann, L.A. Lüdt - Lour. J. Chem. Vietnam VAST (Tap Chi Hoa core structures. Synthesis 3997-4004. ke, D.S. & Braga, A.L. The application of chiral, Hoc) 44, 372-376. non-racemic N-alkylephedrine and N,N,-dial- Dömling, A., Beck, B., Eichelberger, U., Saka mu - kylnorephedrine as ligands for the enantiose- Thuy, T. T., Sung, T. V., Franke, K. & Wessjohann, ri, S., Menon, S., Chen, Q.-Z., Lu, Y. & Wessjo - lective aryl transfer reaction to aldehydes. J. L.A. Morphinane and oxoaporphine alkaloids hann, L.A. Total synthesis of tubulysin U and V. Mol. Catal. A 261, 120-124. from Ste pha nia rotunda Lour. J. Chem. Angew. Chem. 118, 7393-7397; Angew. Chem. Vietnam VAST (Tap Chi Hoa Hoc) 44, 110- Int. Ed. 45, 7235-7239. Reiss, E., Schlesier, B. & Brandt, W. cDNA se - 114. quences, MALDI-TOF analyses, and molecular Franke, K., Hoffmann, M., Schmidt, J., Porzel, A., modelling of barley PR-5 proteins. Phyto che - Vetter, C., Wagner, C., Schmidt, J. & Steinborn, Ar nold, N. & Wessjohann, L.A. 2"-O-Gluco syl - mistry 67, 1856-1864. D. Syn thesis and characterisation of platinum vi te xin, a chemotaxonomic marker for the (IV) complexes with N, S and S,S heterocyclic genus Crypto coryne (Araceae). Biochem. Rivera, D. G. & Wessjohann, L. A. Supramole cu - ligands. Inorg. Chim. Acta 359, 4326-4334. Syst. Ecol. 34, 546-548. lar compounds from multiple Ugi multicomponent macrocyclizations: Peptoid-based Wessjohann, L. A., Fulhorst, M. & Zakharova, S. 38 Gray, I.J., Ren, R., Westermann, B. & Kluger, R. cryptands, cages, and cryptophanes. J. Am. Synthe sis of mechanistic probes and inhibitors Lan tha num-catalyzed aqueous acylation of Chem. Soc. 128, 7122-7123. for preny la ting enzymes. Polish J. Chem. 80, monosaccharides by benzoyl methyl phospha- 673-678. te. Can. J. Chem. 84, 620-624. Schliemann, W., Schneider, B., Vray, V., Schmidt, J., Nimtz, M., Porzel, A. & Böhm, H. Flavanols Wessjohann, L. A., Rivera, D. G. & Coll, F. Syn - SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF BIOORGANIC CHEMISTRY

thesis of steroid-biaryl ether hybrid macrocycles with high skeletal and side chain variability by multiple Carlo Tiebe: Isolierung von Naturstoffen aus Sepedo - multicomponent macrocyclization including nium spp. und Testung der biologischen Aktivität. Mas - bifunctional building blocks. J. Org. Chem. 71, 7521- terarbeit University of Halle-Wittenberg, 28/08/2006. 7526. DOCTORAL THESES Lars Bräuer: Modelling- und Mutationsstudien an aus- Wilhelm, H. & Wessjohann, L. A. An efficient synthesis gewählten prenylierenden Enzymen. University of of the phytoestrogen 8-prenylnaringenin from xan - Halle-Wittenberg, 24/08/2006. thohumol by a novel demethylation process. Tetra he - dron 62, 6961-6966. Viktor Dick: Totalsynthese von Galantamin und Deri- vaten. University of Halle-Witten berg, 29/09/2006. Wolfram, K. & Hinneburg, A. Similarity search for mul - ti-dimensional NMR spectra of natural products. Simon Dörner: Synthesen zu Aminoglycosid-Mime - Knowlegde Discovery in Databases: PKDD 2006. tika. University of Halle-Wittenberg, 29/09/2006. Pro cee ding Lecture Notes in Computer Science 4213, 650-658. Myint Myint Khine: Isolation and charac te riza tion of phytoconstituents from Myanmar me dicinal plant. Yu, C. K. Y., Lam, C. N. W., Springob, K., Schmidt, J., University of Halle-Wittenberg, 03/03/2006. Chu, I. K. & Lo, C. Constitutive accumulation of cis-pi - ceid in transgenic Ara bidopsis overexpressing a Sor - Tilo Lübken: Hygrophorone – Neue antifungische Cy- ghum stilbene synthase gene. Plant Cell Physiol. 47, clopentenonderivate aus Hygrophorus-Arten (Basi - 1017-1021. dio myceten). University of Halle-Wittenberg, 16/03/2006. Zanatta, N., Schneider, J. M. F. M., Schneider, P. H., Wou - ters, A. D., Bonacorso, H. G., Martins, M. A. P. & Wess - Tran Thi Phuong Thao: Peptoid synthesis by sequential johann, L. A. Regiospecific synthesis of 4-alkoxy and 4- multicomponent reactions coupled with enzyme- amino substituted 2-trifluoromethyl pyrroles. J. Org. induced macrocyclization. Uni versity of Halle-Wit- Chem. 71, 6996-6998. tenberg, 24/08/2006.

BOOK Mingzhao Zhu: Development of acylation patterns as Nuhn, P. & Wessjohann, L. A. Natur stoff chemie- Mi - site selective probes for macro lide pharmacology krobielle, pflanzliche und tierische Naturstoffe. S. Hir- profiles. University of Halle-Wittenberg, 30/11/2006. zel Verlag, Stuttgart, 4. Auflage. ISBN-10: 3-7776-1363- 0 und ISBN-13: 978-3-7776-1363-5.

PATENT Wessjohann, L., Tran Thi Phuong, T., Wes termann, B. Estereleongationsverfahren zum se quenzgesteuerten Aufbau alternierender Pep tid-Peptoid-Polymere. 10 2006 039 615.4

DIPLOMA THESES Alexander Gutsche: Chrom(II)-vermittelte Homoal - dol-Reaktionen. University of Halle-Wittenberg, 09/01/2006.

Tobias Heintz: Cheminformatische Unter su chungen von Naturstoffen. University of Halle-Wittenberg, 22/03/2006.

Fadime Mert: Herstellung von Bausteinen zur Syn these chiraler verbrückter Terpyridine. University of Pa - derborn, 2006.

Katharina Michels: Isolierung und Charakteri sierung von biologisch aktiven Substanzen aus der Mikroalge Eustigmatos magnus. Hoch schu le Anhalt (FH) Köthen, 15/12/2006.

Diana Schulze: Charakterisierung von Diphos phatbin - dungsstellen in Proteinen und Homo logie model lie - 39 rung eines Proteins als Beitrag zur Untersuchung der Evolution prenylierender Enzyme. University of Hal- le-Wittenberg, 22/03/2006. DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY Head: Professor Dierk Scheel

Secretary: Susanne Berlin

lant development, although genetically determined, is largely modulated by biotic and abiotic environmental factors. In this way, developmental programs are adapted Pto specific local conditions and protective as well as defense reactions are initiated during stress situations – an advantageous situation for sedentary living plants.

The basis for those processes is the ability of plants to perceive environmental factors and initiate signal transduction networks that modify gene expression patterns. In addi - tion, post-transcriptional reactions play an important role. The investigation of the molecular mechanisms underlying this course of events is the main topic of the Department of Stress and Developmental Biology.

Plant pathogens play a major role in biotic stress. Research in this area is focusing on interactions of pathogens with plants that are not their hosts. In these cases plants display a durable resistance (basal or non-host resistance) based on an efficient multi-component defense response. Host plants react with similar if not identical defense responses against infection with avirulent pathogen races expressing avirulence genes complementary to the host’s resistance gene. In the absence of such complementary pairs of genes, pathogens suppress the basal defense response and cause disease. The work of several research groups of the department focuses on the analysis of recognition, signal transduction and gene activation processes in plant-pathogen interactions.

The work on abiotic environmental factors centers around studies of the effects of metals on plant development. Using hyper-accumulating model organisms, the Research Group Metal Homeostasis analyzed structure and function of genes responsible for the tolerance of plants towards exposure to normally toxic metal concentrations. This work will be continued at the University of Bayreuth, where the head of this research group, Dr. Stephan Clemens, accepted a Professorship for Plant Physiology.

40 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

Plant responses to biotic and abiotic environmental factors finally result in altered patterns of proteins and metabolites. In order to be able to directly monitor such altera - tions, mass spectrometry-based methods have been established allowing the comprehensive profiling of proteins and secondary metabolites. Both methods are also being employed for biochemical phenotyping of mutants. Comprehensive metabolite profiling required the establishment of metabolomics and bioinformatics platforms including the development of appropriate databases and tools for analysis and annotation of primarily mass spectrometry data.

41 MOLECULAR COMMUNICATION IN PLANT-PATHOGEN INTERACTIONS Head: Wolfgang Knogge

Rhynchosporium secalis, the causal agent of a leaf spot disease on several grasses, is of particular economic importance as a pathogen of barley. The fungus belongs to the group of phytopathogenic microorganisms colonizing the intercellular spaces of their host plants. This region is relatively poor in nutrients and in order to optimize their life style pathogens developed strategies to improve the nutrient supply by the host cells. To prevent just this, plants on their part evolved mechanisms enabling the efficient recognition of invaders as the prerequisite for their rejection. At the molecular level, the interaction between a pathogen and a plant requires therefore exchange and perception of signals. On the pathogen side, receptors may be involved that sense stimuli of plant origin and, hence, allow the adaptation of the pathogen physiology to the specific environment of the host. In the plant, the communication processes are mediated by membrane- localized or intracellular receptors, which are the targets of pathogen-secreted molecules, frequently of proteinaceous nature. The final outcome of an interaction is therefore determined by protein-protein interactions, for which two scenarios can be envisaged. The plant defense is suppressed and the plant metabolism is redirected in favor of the pathogen with disease ensuing. Alternatively, upon recognition of the pathogen through secreted compounds the plant defense is in duced leading to the expression of plant resistance.

GROUP MEMBERS Our research ultimately aims at identifying the play a role during pathogenesis, random muta- Jolly Basak processes on the plant side that are involved in, genesis strategies are being applied. Loss-of- Postdoctoral Position since August 2006 if not pivotal for the development of disease or function mutations in genes, whose products Barbara Degner resistance in barley.To this end, the response of contribute to optimal pathogenesis (virulence Technician until June 2006 host and non-host plants, respectively, to inocu- genes) or that are even essential for microbial Andreas Kirsten Diploma Student since September 2006 lation with fungal wild-type strains is compared penetration and development in planta (patho- Susanne Kirsten to that upon inoculation with mutant strains, in genicity genes) can be presumed to generate a Technician since August 2006 which single candidate genes were knocked phenotype that deviates more or less clearly Claudia Mönchmeier out. Deviations from the wild-type are being from the wild-type situation. Isolation and de - PhD Student since November 2005 followed at the phenotypic level, as well as at tailed molecular analysis of the involved genes Sylvia Siersleben PhD Student the level of gene expression (transcriptome) and their protein products will lead to detec- Ronny Völz and protein patterns (proteome). R. secalis is ting those pathogen processes that affect the Diploma Student until March 2006 characterized by its subcuticular growth in host host physiology. Targeted disruption of the Stefanie Wetzel leaves. After penetration the fungus grows in genes will generate the appropriate fungal PhD Student until September 2006 the extracellular region between the leaf cuti- mutants that are capable of identifying the cor- Marina Wibe Diploma Student until March 2005 cle and the epidermal cells of susceptible responding plant processes. An alternative mo - plants. During early stages of pathogenesis epi- lecular genetic approach (signal sequence trap dermal cells collapse, whereas necrotic lesions strategy, SST) aims at identifying and isolating do not become visible until later stages when fungal genes that encode secreted proteins (se - the mesophyll cells are affected as well.The epi- cretome). These proteins can be regarded to dermis is therefore the only tissue in close pro- either be required for the synthesis of fungal ximity to fungal hyphae. Consequently, this tis- extracellular structures or to be candidates for sue represents the prime target of investiga - factors involved in the interaction with the tions aiming at identifying the mechanisms of host. plant susceptibility or resistance to R. secalis and other pathogens such as Blumeria graminis Several molecular tools have been established (powdery mildew). or improved for the use with R. secalis. Impor - tantly, targeted disruption of fungal genes can Key point of our strategy is the availability of be achieved by homologous recombination fol- informative pathogen mutants, suitable for pro- lowing Agrobacterium tumefaciens-mediated 42 bing the plant response. To identify fungal can- transformation with specific vectors. These vec- didate genes, whose products are involved in tors are optimized to facilitate the discrimina - the communication with the host and, hence, tion between homologous and heterologous SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

recombinants. In addition, an SST system has mutagenesis approaches, insertion mutagenesis COLLABORATORS been established and shown to function pro- and promoter trapping. The deduced func tions Patrick Schweizer, Hans-PeterMock perly with fungal control genes known to en - of the gene products, although biochemically Institute of Plant Genetics and Crop code intracellular or secreted proteins. This not yet confirmed, suggest their role in various Plant Research, Gatersleben, Germany system can now be used to screen a fungal pathogenetic processes such as fungal uptake Barbara Howlett University of Melbourne, Australia cDNA library for genes that encode proteins of specific plant nutrients (GABA/amino acid Bruce McDonald carrying a secretor signal sequence. permease), oxidative reactions of as yet un - Swiss Federal Institute of Technology, known nature (P450 protein) and intracellular Zurich, Switzerland A function in disease development has long information processing (histidine protein kina- been suspected for several small proteins se, MAP ki nase). In addition, a protein with se - secreted by R. secalis. Phenotypic analysis of quence similarity to a class of transcription fac- fungal knockout mutants has now revealed that tors may control fungal genes that are only ex - these proteins are indeed virulence factors pressed during fungal growth in the plant. required for full expression of the disease symp - Finally, a gene was identified that encodes an toms. Furthermore, one of these virulence fac- intramembrane protease. A subgroup of these tors is turned into a specific recognition sig nal enzymes catalyzes the release of extracellular in host plants carrying resistance gene Rrs1, signals from transmembrane precursor pro- i.e., an offensive fungal tool now serves as the teins. However, neither the identity of the signal trigger of plant resistance reactions. A specific ge nerated by the R. secalis enzyme nor its tar- binding site for this fungal protein was detected get or the role of regulated intramembrane on membranes from resistant and susceptible proteolysis in fungus-plant interactions in gene- barley cultivars as well as from other cereals. ral are known to date. Fungal knockout This suggests that the receptor is not encoded mutants are now being generated to allow a by the Rrs1 gene and that (an) additional com- compara tive analysis using a barley cDNA ponent(s) are required for resistance-related macroarray to unravel the impact of these fun- sig nal transduction. gal genes on plant gene expression.

Several fungal pathogenicity and virulence genes have resulted from two different random

Pathogenesis of R. secalis in barley leaf tissues. (Top) Schematic presentation (not in scale) of the early stage with two-cel- led spore (S), germ tube (G) and subcuticular hypha (H); cuticle (C), epidermis (E), mesophyll (M). (Bottom) Disease sym- 43 ptoms on a barley primary leaf; at this late stage necrotic lesions originate from the collapse of epidermis and mesophyll cells beneath fungal hyphae. CELLULAR SIGNALING Heads: Dierk Scheel & Justin Lee

Plants are constantly under the attack of potential pathogenic microbes, inverte brate pests and even parasitic plants. In contrast to vertebrate immune systems where specialized cells are mobi- lized to sites of infection, plants possess non-circulatory de fense mechanisms that are nevertheless equally effective in delimiting the invading orga nism. Research in our group concentrates on plant defense signal transduction pathways in non-host plant-pathogen interaction and the so-called gene-for-gene type of disease resistance. We also investigate the infection strategies of the parasitic plant Cuscuta reflexa.

GROUP MEMBERS Non-host resistance mechanism was initially Gerit Bethke studied using the Pep-13-parsley cell suspen - PhD Student sion culture model system. Pep-13, an oligo- Mandy Birschwilks peptide derived from a Phytophthora sojae cell Postdoctoral Position wall transglutaminase, serves as a pathogen- Stefan Bornschein Diploma Student since June 2005 associated molecular pattern (PAMP) to trigger Jutta Elster defense reactions in parsley cells. The concept Technician of PAMP recognition and activation of innate Anja Halbauer immunity was first characterized in animal sys - Diploma Student until May 2005 Fig. 2: DIGE 2D-PAGE image of two protein samples labe- tems but is similarly conserved in plants and led with different fluorescent dyes. The abundance of Franziska Handmann Rubisco in green tissues of plants hinders the analysis of PhD Student has been documented in many other plant low abundant proteins in proteomics studies. A simple Jan Heise defense models e.g. the bacterial flagellin-derived separation procedure to remove Rubisco was developed. Postdoctoral Position until February Here a Rubisco-depleted protein extract (green) from 2006 flg22 peptide elicitation in Arabidopsis. The first Arabidopsis is compared to a fraction that contains pre- step to defense response activation is the per- dominantly Rubisco (pink) to show the efficiency of this Sylvia Krüger Technician ception of pathogen-derived signals. Peptide method. Marlen Mrotzek and proteinaceous elicitors studied in our labo- Diploma Student since February 2006 ratory include the above-mentioned flg22 and tion. The signaling events include ion fluxes at Kai Naumann Postdoctoral Position Pep-13, as well as the elongator factor(EF-Tu)- the plasma membrane, mitogen-ac tivated pro- derived elf18 peptide, harpin from tein kinases (MAPK) ac tivation and the accu- Stefanie Ranf PhD Student Pseudomonas syringae or a P. sojae necrosis- mulation of signaling molecules, such as reac - Christel Rülke tive oxygen species, phospholipids and phytohor- Technician mones. Dirk Schenke Postdoctoral Position since April 2006 Studies involving the calcium-sensitive repor- Rita Schlichting PhD Student ter, aqueorin, as well as patch-clamp techniques Claudia Spielau revealed that changes in cytosolic calcium le - PhD Student vels represent an early key step that is essen tial Nicole Staroske PhD Student since April 2006 for all other downstream events studied. The calcium ions may arise from extracellular, as Esther van der Zalm Postdoctoral Position since January 2006 well as intracellular pools. Recent work from Tino Unthan Fig. 1: Imaging of cytosolic calcium levels with the lumines- other groups suggested that the two-pore- PhD Student since June 2005 cent calcium-sensitive protein aequorin. Leaf disks from channel, TPC1, might be the elusive channel Ivy Widjaja aequorin transgenic plants were placed in a multi-well plate and time series images taken with a luminescence- mediating calcium fluxes during defense res - recording camera after treatment with flg22 pep tide (F) ponses. A parsley TPC1 homolog was cloned, or with water (W) as a control. For eventual calibration but studies, especially those performed using of calcium levels, the total luminescence of the re maining aequorin reporter was discharged at the end of the expe- Arabidopsis tpc1 mutants and TPC1-overex- riment. pressing lines, failed to support this concept.

inducing protein. MAPK activation was previously shown in the A network of cellular signaling events is trig - parsley system as essential signaling event for 44 gered upon perception of PAMPs by plasma- controling defense gene expression. Yet, the membrane-localized receptors, and this coordi- same subset of MAPKs appears to be activated nates the subsequent defense response activa- by a plethora of diverse stress signals. The une- SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

Fig. 3: A protein array consisting of 1,700 unique Arabi- dopsis proteins was used for in vitro ki - nase screening of MAPK substrates. The left image (green spots) is an antibody-ba sed la - and a transcription factor of the ethylene- COLLABORATORS beling of all proteins on the ar ray response factor (ERF) family. This interaction Jeff Dangl University of North Carolina, Chapel Hill, and the image on was disrupted upon flg22 elicitation, suggesting USA the right is an en- larged section of some biological relevance of this dynamic inter- Petra Dietrich the chip showing action during the flg22-signaling. The transcrip- University of Erlangen, Germany radioactively-la be - tion factor was phosphorylated by active Minna Haapalainen, Suvi Taira, led spots of phos- Martin Romantschuk phorylated sub - AtMPK6 and may potentially regulate flg22- University of Helsinki, Finland strates. induced defense gene expression. Bettina Hause Leibniz Institute of Plant Biochemistry, Halle, Germany Approximately 1700 unique recombinant pro- Gerd Hause qual number of genes in the Arabidopsis ge - teins of Arabidopsis were purified and spotted Biocenter, University of Halle, Germany nome coding for the different MAPK cascade onto a protein array chip. In vitro kinase assays Rainer Hedrich components, namely the MAPKs, MAPK-kina- on this chip identified a series of potential sub- University of Würzburg, Germany ses (MKK) and MKK-kinases (M3K), also indi- strates of AtMPK3 and/or AtMPK6. One of the Heribert Hirt University of Vienna, Austria cate that the same protein must, in certain ca - AtMPK6 substrates was ACS6 (ACC syn thase-6, Sophien Kamoun ses, cater to more than one signaling pathway. the rate-limiting enzyme for ethylene biosyn- Ohio State University, Wooster, USA These observations raise the question of how thesis), which was previously identified as one Birgit Kersten, signal specificity is maintained for MAPK path- of the first plant MAPK substrates. Hence, the RZPD German Resource Centre for Genome Research, Berlin, Germany ways. The specificity may reside at the level of in vitro screen with this protein array may lead John Mundy the MAPK substrate diversity as well as the to other authentic MAPK substrates. University of Copenhagen, Denmark presence of pathway-specific protein com - Teun Munnik plexes that precludes erroneous crosstalk in The biochemical functioning in a cell is ultima- University of Amsterdam, The Netherlands signaling. We are currently pursuing different tely executed by the diverse regulatory roles of Thorsten Nürnberger, strategies to isolate MAPK substrates and scaf- proteins (e.g. as en zymes or structural ele- Andrea Gust (Ludwig) fold proteins. These include yeast-two-hybrid ments). Hence, proteome studies are increasing University of Tübingen, Germany screens, biochemical isolation of MAPK-inter- in im portance in the post-genomic research Ralf Oelmüller University of Jena, Germany acting proteins by tandem-affinity purification era. High resolution two-dimensional polyacryl - Jane Parker, Imre Somssich (TAP) and a protein array-based screen for amide gelelectrophoresis (2D-PAGE) is com- Max Planck Institute for Plant Breeding MAPK substrates. monly used to study changes in protein levels Research, Cologne, Germany and post-translational modifications. We have Gunter Reuter, Raffael Schaffrath The existence of MAPK protein complexes improved current 2D-PAGE methods through a University of Halle, Germany could be shown by size-exclusion chromato- number of technical advances such as Rubisco Joachim Uhrig graphy. Transgenic plants with tandem-affinity depletion, economical alternative dyes for University of Cologne, Germany tagged versions of the elicitor-activated MAPKs DIGE (Fluorescence 2D Difference Gel Elec - have been generated to facilitate the isolation trophoresis) and phosphoprotein analysis. of such complexes. The functionality of the Currently, 2D-PAGE is used to study changes in TAP-tagged MAPKs was verified either by phe- protein patterns in systemic acquired resi- notypic complementation of mutant plants or stance and the AvrRPM1 gene-for-gene model by the ability of elicitors to activate these tag- system in Arabidopsis. ged proteins (as an indication that they have incorporated into the signaling protein com- Not unlike other phytopathological systems, plex). Purification of associated proteins is cur- successful invasion of the holoparasitic plant, rently in progress. The isolated proteins will be Cuscuta reflexa, requires the directed transfer identified through mass-spectrometry-based of nu trients and water from the host into the peptide fingerprinting and sequencing methods. parasite, in this case via a multicellular hausto- rial structure. Xylem- and phloem-mo bile dyes, Fig. 4: Post-translational modificati- To verify the putative MAPK-interacting candi- respectively, de monstrate functional con nec - on of proteins can be vi su alized in 2D-gels. Here a puta tive lipid raft dates identified in the yeast-two-hybrid screen, tion of the invading haustorium to the host vas- component protein (black we set up fluorescence resonance energy cular system. This parasitism can eventually re - arrowhead) is found to exist in various forms, including a phos- transfer (FRET) technique in Arabidopsis pro- sult in death of the host. A screen in various phorylated state (red arrowhead), 45 toplasts. As an example, we could confirm a ecotypes and defense-related mu tants has been in the AvrRpm1 gene-for-gene specific in vivo interaction between AtMPK6 initiated to elucidate the components that may mo del sys tem. INDUCED PATHOGEN DEFENSE Heads: Dierk Scheel & Sabine Rosahl

Late blight is one of the most devastating diseases of potato and is caused by the oomycete Phytophthora infestans. Interestingly, the model plant Arabidopsis thaliana is able to successfully defend itself against infections by this pathogen. We are interested in studying the interaction of P. infestans with both its host plant potato and the non-host plant Arabidopsis. The major focus of our work is the elucidation of the role of the signaling compounds, salicylic acid and jasmonic acid, for pathogen defense in potato and the genetic dissection of non-host resistance of Arabidopsis against P. infestans.

GROUP MEMBERS Basal defense of potato in response to infec tion 9-lipoxygenase pathway, such as hydroxylino- Simone Altmann with P. infestans involves recognition of the pa - le(n)ic acid and the divinyl ether containing po - PhD Student since August 2005 thogen, signal transduction and the activation lyunsaturated fatty acids colnele(n)ic acid, were Lennart Eschen-Lippold PhD Student of defense gene expression. Both salicylic acid shown to act as antimicrobial compounds. The and jasmonic acid have been identified as signa- role of these oxylipins for defense against P. in - Vincentius A. Halim PhD Student until January 2006 ling molecules in plant pathogen interactions. festans is being analyzed by generating potato Anja Kurth Our studies aim at the elucidation of the role plants expressing RNA interference constructs Diploma Student since April 2006 of these compounds for pathogen defense res - targeted against the pathogen-induced 9-lip- Jörn Landtag PhD Student until April 2005 ponses in potato. To address this issue, transge- oxygenase and 9-divinyl ether synthase. Basal nic plants were generated that express the bac- resistance of potato against virulent isolates of Gabi Reichenbach Diploma Student until October 2006 terial NahG gene encoding salicylate hydroxy - P. infestans is not affected by the reduction of Grit Rothe lase. As a consequence, these plants are unable 9-lipoxygenase-derived compounds. Presently, Postdoctoral Position until June 2005 to accumulate salicylic acid. On the other hand, potato plants carrying the resistance gene R1, Jenny Teutschbein Diploma Student until April 2006 RNA interference constructs against jasmonic against P. infestans are transformed with the acid biosynthetic enzymes were expressed in RNAi constructs in order to analyze the role of Annett Weichert Diploma Student until August 2006 transgenic potato plants resulting in significant- oxylipins for race/cultivar-specific resistance. Ralf Weigel ly decreased levels of jasmonic acid. In a com- Postdoctoral Position since July 2005 patible interaction of the NahG plants with a Angelika Weinel Technician vi rulent isolate of P. infestans, significantly higher growth of the oomycete was observed, Lore Westphal Senior Scientist sugges ting that salicylic acid is required for Dorothea Wolf basal de fense in potato. Diploma Student until February 2005 The oligopeptide elicitor, Pep-13, a pathogen- associated molecular pattern (PAMP) from oomycetes, induces an oxidative burst, accumulation of salicylic acid and jasmonic acid, de - fense gene expression and hypersensitive-like cell death in potato. None of these defense re - actions are observed in transgenic plants un - able to accumulate salicylic acid. In plants with reduced levels of jasmonic acid, salicylic acid still accumulates while the oxidative burst, hy - persensitive cell death and defense gene ex - pression are reduced. These data suggest that Pep-13 activates defense responses via a signal transduction chain which requires both salicylic acid and jasmonic acid.

In addition to jasmonic acid, which is synthe - 46 sized via the lipoxygenase pathway, other oxyli- Fig. 1: Infection of the host plant potato leads to disease pins are assumed to play a role in pathogen de - (upper panel). In contrast, Arabidopsis can successfully prevent pathogen entry and stays healthy (lower panel). fense responses. In particular, products of the SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

resistance (SAR) in Arabidopsis, which confers COLLABORATORS immunity against secondary infections. NPR Ivo Feussner 1 University of Göttingen, Germany serves as a mediator between the signaling Markus Frank compound salicylic acid and the salicylic acid- BASF Plant Science, Ludwigshafen, induced expression of pathogenesis-related Germany (PR) genes by interacting with a group of basic Joëlle Fournier, Marie-Thérèse Esquerré-Tugayé leucine zipper transcription (TGA) factors. Université Paul Sabatier, Toulouse, France These in turn bind to promoter elements of PR Christiane Gebhardt, Paul genes in a salicylate-dependent manner. PR Schulze-Lefert Fig. 2: Defense reactions in Arabidopsis leaves in re - Max Planck Institute for Plant Breeding gene expression generally correlates with the Research, Cologne, Germany sponse to drop inoculation with P. infestans can be visua- establishment of SAR. lized under UV light. Left: wild-type, right: pen2, a mutant Volker Lipka impaired in penetration resistance. University of Tübingen, Germany Additional interaction partners of NPR , the William Albert Rensink 1 The Institute for Genomic Research, Since Arabidopsis is not a host plant for P. infes - NIMIN proteins, were identified. In a yeast-one- Rockville, USA tans, we are interested in elucidating the me - hybrid system, NIMIN1 can establish a ternary Jose Sanchez-Serrano Universidad Autonoma de Madrid, Spain chanisms responsible for successful defense complex with NPR1 and TGA factors, which against this pathogen. For this purpose, genetic bind to a cis element of the PR1-promoter via Otto Miersch approaches are applied in order to identify genes the TGA factors. Analyses of nimin1 overex- involved in non-host resistance. P. infestans spo- pressing and nimin1 knock-out plants revealed res germinate on Arabidopsis leaves and attempt that NIMIN1 is a negative regulator of NPR1- to penetrate cells, which react with the deposi- mediated gene expression. These data suggest tion of callose, accumulation of autofluorescent that activation of the PR1 promoter coincides material and hypersensitive cell death. The with the binding of the negative regulator Arabidopsis mutant, pen2, which was isolated by NIMIN1, thereby ensuring a fine tuning of PR Volker Lipka and Paul Schulze-Lefert (Max gene expression. Planck Institute for Plant Breeding Research, Co - logne) as allowing enhanced penetration of the Activation of the salicylic acid pathway in Ara - non-host pathogen, barley powdery mildew bidopsis leads to inhibition of jasmonic acid-me - (Blu meria graminis f. sp. hordei), shows a higher diated defense responses. Nimin1 knock-out number of penetrated epidermal cells and in - plants exhibit increased PR gene ex pression, creased cell death in response to infection by P. however, possibly due to redundance, no en - infestans. Seeds of the pen2 mutant were muta- hanced resistance to biotrophic pathogens is genized and approximately 70,000 M2-plants observed. Therefore, multiple knock-out mu - were screened for alterations in their response tants were generated and analyzed. The nimin1- to infection by P. infestans. Fourteen independent 1;nimin2-1 double mutant and the ni min1-1; mu tants were isolated, which show increased nimin2-1;nimin1b-1 triple mutant show en - hy persensitive cell death and in some cases hanced PR gene expression as well and are hy - enhanced growth of P. infestans. Mapping of the persensitive to chemical inducers of the salicylic position of the affected genes is in progress. acid signal transduction pathway. This hy per - sensitivity leads to chlorosis and eventually to The protein, NPR1, is a central regulator in the necrosis of the treated plants, rendering them signal transduction leading to systemic acquired more susceptible to necrotrophic pathogens.

47 METAL HOMEOSTASIS Head: Stephan Clemens until September 2006

Plants – like all other organisms – are able to tightly regulate the intracellular concentration and the distribution of essential metals such as zinc and copper. Also, the cytosolic concentrations of non-essential toxic metals (e.g. cadmium, lead) have to be minimized. Some plant species (so-called metallophytes) can tolerate otherwise toxic concentrations and grow on metal contaminated soil. The projects of our group are aiming at elucidating the molecular mechanisms underlying plant metal homeostasis, metal tolerance and metal hyperaccumulation. Plants under investigation are Arabidopsis thaliana and its close relative Arabidopsis halleri, which grows, for instance, on medie- val mining sites in the Harz mountains. In addition, we are working with Schizosaccharomyces pombe as a cellular model for metal homeostasis. Our main em phasis in the past two years has been on (i) comparative root transcriptome studies of A. thaliana and A. halleri; (ii) screening of genetic diversity in A. thaliana for metal homeostasis phenotypes; (iii) functional analysis of two low-molecular weight metal cation ligands: phytochelatins and nicotianamine.

GROUP MEMBERS Comparative transcript profiling of A. thaliana and Roland Eils (Heidelberg) trying to develop Annegret Bährecke and A. halleri via cross-species hybridization of respective bioinformatic tools. The data sets PhD Student since June 2004 Affymetrix GeneChips led to potentially im - this work is based upon were obtained through Thomas Fritsche portant insights into the molecular mechanisms genomic hybridizations of three different Ara - PhD Student until September 2006 of metal hyperaccumulation. Previously, we had bidopsis species. Marina Häußler Technician seen that several metal homeostasis genes Beate Schulz show strong overexpression in A. halleri roots The constitutive strong expression of several Diploma Student until April 2006 relative to A. thaliana roots. Nicotianamine syn- metal homeostasis genes in A. halleri relative to Pierre Tennstedt thases (NAS) and transporters of the ZIP fami- A. thaliana raised the question as to what the PhD Student until August 2005 ly were identified as hyperaccumulation/hyper- molecular mechanisms behind this altered re - Alexandra Trampczynska Postdoctoral Position until September tolerance candidate genes. More recently, we gulation are. We have therefore embarked on 2006 have been studying transcriptome responses to comparative promoter studies for various NAS Michael Weber Postdoctoral Position until September Fe deficiency, because it is not clear how A. hal- and ZIP genes using reporter lines. For NAS2 leri manages to acquire enough Fe even in the and ZIP4 we found evidence that the A. halleri presence of vast excess of Zn (at several A. hal- promoter is sufficient to drive strong expres - leri sites in the Harz mountains, for instance, sion in A. thaliana. Therefore we initiated the we measured soil Zn content exceeding 1.5 ge neration of various transgenic lines carrying mg/g). Zn and Fe are often competing for the truncated versions of the A. halleri promoters. same uptake and translocation pathways. These will hopefully lead to the identification of cis elements required for the activity. Microarray data on excess metal responses in A. halleri and A. thaliana roots provided clues as Our comparative transcriptome studies of A. to the modes of toxicity and helped identifying thaliana and A. halleri had indicated a potential common as well as species-specific transcrip- role of nicotianamine synthases (NAS) for Zn tional changes that will guide functional studies. hypertolerance/hyperaccumulation of A. halle- In particular, “core” response genes, genes ri. Loss-of-function studies for A. halleri NAS responsive specifically to excess of one particu- genes and the other candidate genes we identi- lar metal and putative signal transduction com- fied require RNAi lines of A. halleri. The suc- ponents were identified. Several of the respon- cessful transformation of A. halleri has been sive genes appear to be specific for Cd2+ stress. achieved owing to a collaboration with the group of Ute Krämer in Golm. RNAi lines for To date, the mining of microarray data for A. some of the candidate genes are currently halleri is still limited by the fact that the probe being characte rized. sets are designed for the A. thaliana genome. In 48 order to identify reliable probes and to esta- A second hypothesis concerning nicotianamine blish a way of better normalizing the data, we (NA) that emerged from the experiments on are in a collaboration with Ute Krämer (Golm) A. halleri postulates NA function not only as a SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

li gand for long-distance transport in xylem and transpeptidation activities can be separated by COLLABORATORS phloem, but also as an intracellular ligand for analyzing this protein. Our collaborators in Ca - Roland Eils German Cancer Research Center, Zn(II). In order to obtain additional evidence darache, France, have been able to crystallize Heidelberg, Germany supporting this hypothesis we started a series the bacterial protein (Nostoc PCS) using our E. Rüdiger Hell, Andreas Meyer of experiments on Neurospora crassa. coli clones. They obtained high resolution University of Heidelberg, Germany Genome sequencing had revealed that several structure which now serves as our template Ute Krämer Max Planck Insitute of Molecular Plant filamentous fungi carry NAS homologous for the targeted mutagenesis of both the ance- Physiology, Golm, Germany sequences. We therefore cloned a putative stral proteins and plant PC synthases. We are Youngsook Lee nicotianamine synthase from Neurospora cras- trying to find determinants for the two activi- POSTECH, Pohang, South Korea sa and showed by functional expression in a ties and to understand the evolution of this Dietrich H. Nies University of Halle, Germany Zn2+-hypersensitive fission yeast mutant that family of proteins. Also, we generated trunca- Kuo-Chen Yeh this protein indeed possesses NAS activity. ted ver sions of bona fide PC synthases that Institute of BioAgricultural Sciences, Using capillary liquid chromatography coupled show GSH degrading activity. One of these is Academia Sinica, Taipeh, Taiwan to electrospray ionization quadrupole time-of- currently being expressed in E. coli in order to flight mass spectrometry (Cap-LC-ESI-QTOF- enable our collaborators to crystallize this pro- MS) we unequivocally proved the formation of NA in N. crassa mycelium. Expression of the N. IZS 72 Col-0 IZS 72 Col-0 crassa NAS gene is strongly up-regulated under conditions of Zn deficiency as shown by quantitative real-time PCR. These findings demon- strate that NA is more widespread in nature than previously an ticipated. Also, they show occurrence of NA in organisms that do not require extracellular long-distance transport. A hallmark of filamentous fungi is cytoplasmic continuity. The cellular compartments of hyphae are delineated by in complete septa that control 7.0 µM Ni2+ allow passage of organelles and cytoplasmic components. The izs72 mutant of A. thaliana is Ni2+ hypersensitive.

The other low molecular weight ligands we are interested in, phytochelatins (PC), are still rai- tein. sing biologically important questions. PCs are non-translationally derived from glutathione by A major em phasis of our work in the past two the enzyme phytochelatin synthase (PCS). It years has been on using genetic diversity in A. appears unlikely that the proven contribution thaliana to identify new components of the of PCS genes in Cd and As detoxification alone plant metal homeostasis network. Firstly, we can explain the almost ubiquitous presence in established a screening for metal hypersensitive the plant kingdom, the widespread distribution mutants. More than 25 mutants have now been in other domains of life and their constitutive confirmed that differ in their metal specificity. expression. Thus, we are searching for additio- Complementation analysis and mapping experi- nal functions of PC synthases and PC synthesis. ments are ongoing. Secondly, within the frame Two lines of evidence have been obtained by of a GABI-Genoplante program we started to our group and collaborators in Heidelberg for explore natural diversity in Arabidopsis for Zn (i) a role of PC synthesis in essential metal ho - tolerance/accumulation, Cd tolerance/accu- meostasis and (ii) potential other roles of PCS mulation, Cu tolerance and micronutrient defi- proteins in GSH metabolism: PC-deficient Ara - ciency tolerance. Most of the ecotypes in the bidopsis mutants are less Zn2+ tolerant and ac - Versailles Core24 collection of Arabidopsis ac - cumulate less zinc than wildtype.A bacterial an - cessions have been screened. We identified cestor of PC synthases catalyzes only the first ecotypes that clearly differ with respect to ei - 49 step of PC synthesis, the degradation of GSH ther Zn tolerance, Cd tolerance or Cu tole - to g-glutamylcysteine. Thus, peptidase and rance. Using recombinant inbred lines we are in BIOINFORMATICS & MASS SPECTROMETRY Head: Steffen Neumann

The research group is the most recently established group in the Bioinformatics Center Gatersleben-Halle, operational since January 1, 2005. Goal of the group is to establish a bioinformatics and metabolomics platform at the IPB by developing databases and tools to support data- driven analysis and annotation, especially for mass spectrometry measurements using advanced pattern recognition methods and database technology.

GROUP MEMBERS MASS SPECTROMETRY DATABASES For the liquid chromatography-coupled mass Björn Egert The development of databases for mass spec- spectrometry (LC-MS) setup at the IPB the PhD Student since October 2005 trometry data is the basis of further high- signal processing parameters, such as peak Yvonne Pöschl throughput analysis. The modern software de - width, S/N and others have been systematically PhD Student since March 2005 velopment paradigm Model Driven Archi tec - optimized to obtain good results. Different Ralf Tautenhahn PhD Student since March 2005 ture (MDA) allows to create large portions of peak models (e.g. the empirically transformed the required software and databases from a Gaussian model) have been used to fit peaks graphical UML model. from actually measured data, and several classes of peaks have been obtained through clu- To facilitate data exchange stering in the model parameter space. with other laboratories and analysis tools, the A set of annotation methods has been imple- mzData and ArMet stan- mented, which aim to group together mass sig - dards developed in the nals measured from a single metabolite based context of the Pro teomics on rules for mass differences and peak shape Standards Initiative (PSI) comparison. These annotations greatly reduce have been chosen as data the time necessary to interpret mass spectro- model. The MetArchive metry data. data base has been created as repository for mass DATA WAREHOUSE spectrometry data genera- FOR MASS SPECTROMETRY DATA ted at the IPB. Several mass A data warehouse is a database optimized for spectrometry instruments online analytical processing (OLAP), and allows from different vendors can to retrieve subsets of data based on arbitrary already submit to this ar - filter criteria.The BioMart framework has been chive. The MetArchive used to establish the MetHouse for preproces- Fig. 1: A rich set of clustering and visualization algorithms collec- ted in the metabolomics and datamining toolboxes. tools include an editor, sed peaks obtained e.g. by XCMS. which al lows experimenta- lists to edit and annotate their mass spectro- BioMart provides several frontends, including metry data. stand-alone and web interfaces, and a powerful command line and scripting client. The current SIGNAL PROCESSING content of the MetHouse includes ca. 240 mass OF RAW MASS SPECTROMETRY DATA spectrometry runs with ca. 1.2 million peaks. The first steps of an analysis pipeline processing Possible filters against the warehouse are e.g. mass spectrometry data are signal processing plant genotype, growth treatment, model of the and peak picking tasks. The XCMS software mass spectrometry machine, ionization mode developed at the Scripps Research Institute has or institute. been adapted to the specific settings at the IPB. It has an Open-Source license, and in coopera- CLUSTERING OF METABOLITE DATA tion with the upstream authors, the software Metabolite data can be interpreted as pheno- has been improved regarding the set of input typic feature and used to distinguish different formats and signal processing. Data import is metabolic states of a system or to identify e.g. 50 also possible through a direct database link to ecotypes. Different hierarchical clustering algo- the MetArchive. rithms implemented in the R statistics software have been used with several distance measure- SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

441.73 414.21 432.24 533.07 349.13 1542 442 2858 652 376

0.875 / 0.855

430.02 440.73 386.18 313.15 406.13 495 1542 380 449 1739

756.24 1608

504.19 416.19 318.17 385.20 609.18 571 382 437 1213 1549

442.21 444

240.07 303.16 308.15 450.22 638.28 1391 433 437 540 514

224.13 0.855 / 0.843 476

336.06 668.28 472.22 532.22 620.27 458.22 476.23 COLLABORATORS 410 781 449 1550 1262 1596 1069 selected a subset of 19 mass signals, which are known or believed to be metabolites involved in Masanori Arita, Kazuki Saito 446.20 209.13 516.67 391.13 506.24 381 390 368 371 1338 Riken Plant Science Center, Yokohama the phenylpropanoid pathway as core data set. 0.985 / 0.985 City, Japan

296.15 284.15 208.14 345.11 We randomly selected an additional set of 21 450 511 696 361 Sebastian Böcker mass signals resulting in the extended dataset. University of Jena, Germany 254.14 495 Ivo Große, Falk Schreiber Leibniz Institute of Plant Genetics and Fig. 2: Connections between metabolites (nodes labelled with mass The structure of the Bayesian network learned Crop Plant Research, Gatersleben, Germany and retention time) for the core (grey) and extended (white) dataset. from the core dataset remains stable even if the Roeland van Ham 21 randomly chosen mass signals were ad ded to Plant Research International, Wageningen, ments to create a hierarchical clustering. In the core dataset. The Netherlands several datasets, such as Arabidopsis ecotypes, Nigel Hardy University of Wales, Aberystwyth, UK single gene mutants or stress responses to It is possible to represent extra information, Henning Hermjakob copper treatment, a clear separation of the such as the type of the tissue or experiment European Bioinformatics Institute, groups could be achieved. treatments in additional nodes. Bayesian net- Hinxton, Cambrigde, UK works provide a valuable annotation of metabo- Oliver Kohlbacher University of Tübingen Density based clustering algorithms, such as lites and connect influences, such as knock- out Joachim Selbig DBScan and Optics, fractal clustering like the or stress conditions to the metabolic state of Max Planck Instiute for Molecular Plant dimension induced clustering (DIC) algorithm the system. Physiology, Golm, Germany have been implemented as described in the literature and/or integrated into the pipeline and WEB APPLICATION AND INTEGRATION applied to metabolite data. Advanced visualiza- INTO THE PLANT BIOINFORMATICS PORTAL tion methods (see Fig. 1 for examples), such as To achieve an easy integration of R and biocon- parallel coordinate plots or MA plots known ductor-based applications into web applications from expression analysis have also been inte- Rserve is employed as a communication grated. Differential abundance of metabolites sys tem between Java clients and an R ser- between experiments offers insights into the ver.A tag library has been created to cou- metabolic difference between the samples. ple the JSF pages with the backend com- pute server, an overview of the architec- NETWORK RECONSTRUCTION ture is shown in Figure 3. WITH BAYESIAN NETWORKS To uncover the causal relationships between Applications implemented this way inclu- metabolites can be considered the ultimate (and de: ambitious) goal of metabolomics. Training of Bay - - XCMS mass spectrometry signal pro esian Networks might be one of the tools on cessing and alignment, the way. - several normalization and clustering algo Fig 3:Distributed architecture of the MetWare rithms, software. Each component could run on a dif- Bayesian Networks are graphical models used to - SiteSeer motif search. ferent machine. model and visualize biological processes. A Bay - esian Network consists of a directed acyclic Applications developed by external co o pe- graph representing metabolites with edges for rations have been web-enabled and in tegrated their relationships and a conditional probability into the portal, including: distribution. The Sparse Candidate Algorithm - DISOP Elemental Composition (developed by (SCA) has been implemented in Java to efficient- A. Peruvkin, Uni versity of Jena), ly find a network that models the measured da - - Macroarray overshining correction (developed ta. Interventions in metabolic pathways cause a by D. Müller-Lukas, Univer sity of Halle). disturbance of the distribution and can help to identify regulation. The applications are available to in-house users and to the public on the IPB web site. The inte- The tt4 mutant harbors a non-functional chal - gration into the distributed BioMoby infrastruc- cone synthase gene causing the disruption of fla- ture has already started and will enable to access 51 vonoid, tannin, and anthocyanin biosyntheses re - metabolomics resources such as the Met Ar - sulting in a different colour of the seeds. We chive, MetHouse and MetWare remo tely. METABOLITE PROFILING IN ARABIDOPSIS AND CROP PLANTS Heads:Stephan Clemens & Dierk Scheel in cooperation with Ludger Wessjohann & Jürgen Schmidt

In order to get a better understanding of how plants are using their arsenal of natural products in the daily battle of survival in an ever-changing environment, methods are required which allow sensitive detection, quantification and identification of se condary compounds. Metabolomics approaches are one possible answer to these requirements. Our group has established a reliable liquid chromatography-coupled mass spectrometry (LC-MS) platform for the analysis of various developmental and stress-induced changes, as well as for the biochemical phenotyping of mutants in Arabidopsis thaliana and relevant crop plants.

GROUP MEMBERS Because of the chemical diversity of the meta- MetaboliteID and MetAlign was shifted to an Christoph Boettcher bolome, which for any given multi-cellular spe- open source software called XCMS and tai - Postdoctoral Position cies comprises a mixture of thousands of com- lored to the needs of the LC-MS profiling plat- Frank Brettschneider PhD Student pounds differing in size, polarity and abundance, form. In order to facilitate the identification the need for multi-parallel analytical techniques and structural elucidation of metabolites, the Mathias Franz Student Helper is obvious and well accepted. LC-MS is a versa- commercial software package ACD was ac - Tobias Heintz tile analytical tool. It covers a wide mass range quired.The realms of this software were exten- Student Helper since October 2006 and allows to target many compound classes ded to allow - next to managing LC-MS and Edda von Roepenack-Lahaye Postdoctoral Position not detectable by gas chromatography-coupled MS-MS spectra - sophisticated search algo- mass spectrometry (GC-MS). In addition mo - rithms within the steadily increasing database. Conny Schmotz Student Helper dern LC-MS set-ups offer superior options to In addition, an LC-MS-Profile database was set structurally elucidate unknown metabolites up in the institute providing additional data- Edith Willscher Student Helper until June 2006 using accurate mass determination and tandem mining tools. The developed data mining tools MS. The main emphasis of this group has been for the ACD and XCMS software, as well as the Michaela Winkler on further developing the already established ACD/ESI-MS database will be publicly available Technician profiling platform, constantly reevaluating and at http://msbi.ipb-halle.de/MetWare. Michael Zerjeski extending it towards the analysis of crop plants Student Helper or more specific various rapeseed lines. Doubts In the frame of the Gabi1 project, the metabo- about the feasibility and reliability of LC-MS-ba - lomic profiling was originally based on using sed metabolite profiling have been raised re - Arabidopsis thaliana leaves and roots as the peatedly, because LC-MS - especially with elec- source of plant material. Within Gabi2 the fo - trospray ionization - can be subject to matrix cus shifted to crop plants, with a special inte- effects. The term matrix effect refers to altera- rest in rapeseed. Extraction and profiling proto- tions of ionization efficiency of analytes by the cols were optimized using as a first step Ara - presence of co-eluting substances, which can bidopsis seed material. Seeds apparently con- severely compromise quantification. We evalua- tain in general lower amounts of those classes ted matrix effects for our metabolomics plat- of secondary metabolites that can be examined form with several methodological approaches. with our profiling platform. This was indicated Our data demonstrated that there are indeed by a reduced number of mass signals (roughly significant absolute matrix effects when compa- about 800 signals in contrast to 1,500 signals in ring highly divergent samples. However, relative leaves) reliably detected by LC-MS. In addition, matrix effects are negligible - unless extremely in contrast to roots and leaves, seeds contain divergent matrices are compared - and do not high levels of choline esters. To structurally cha- compromise the relative quantification that is racterize this important compound class, ex - aimed for in non-targeted metabolomics stu- tensive MS-MS analyses were performed resul- dies. ting in the identification of 25 of these nitrogen containing esters.As initial optimization experi- Data analysis was and is still a major bottleneck ments, seed material from Arabidopsis lines of metabolomics. In cooperation with the Re - harboring mutations in the well known flavo - search Group Bioinformatics and Mass Spec - noid pathway were analyzed. The transparent 52 tro metry, methods for raw data deconvolution testa mutants, tt4 and tt5, harbor non-functio- have been developed and improved. The former nal chalcone synthase and chalcone isomerase use of the data deconvolution softwares enzymes, respectively (Fig. 1). The metabolic SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

COLLABORATORS Ulf-Ingo Flügge University of Cologne, Germany Gunhild Leckband Norddeutsche Pflanzenzucht, Hans Georg Lembke KG, Hohenlieth, Germany Enrico Martinoia University of Zurich, Switzerland Shauna Sommerville Carnegie Institute of Washington, Stanford, USA Paul Schulze-Lefert Max Plank Institute for Plant Breeding Research, Cologne, Germany

Fig.1: In the tt4 and tt5 mutants, the flavonoid pathway leading from 4-coumaroyl-CoA to kaempferol is blocked at the stages of chalcone synthase and chalcone isomerase, respectively. The Hierarchical Cluster Analysis showed a clear dis tinc - tion in the degree of relationship between the two mutant and the wild-type seed materials, as well as between the two wild-type seed batches (due to their different age). phenotype of these plants results in a more or was up to 300) were either increased or re - less complete lack of kaempferol and its de - duced in the transformants (Fig.2). As the trans- rived compounds, hence the light yellow colo- formant lines showed a ring of the seed testa. The non-targeted profi- reduced long chain fat ty ling ap proach showed not only the expected acid content (for ex - ab sence of specific members of the flavonoid ample oleic acid) and pathway. In addition, several flavonoids, which increased shorter chain have not been described before, accumulated fatty acid deriva tives to high le vels in the mutant lines indicating a re - (for example palmitin) direc tion of metabolic fluxes. Hierarchical clu- it was not unexpected ster analysis (HCA) of this experiment showed that quite a number of not only a clear separation of the different mu - the variable metabo lites tant and wild-type materials, but also a clear ap pear to be in volved in dis tinction between seed batches of different the fatty acid pathway. age (seed batch WT-3/4 was one year older than WT-5/6, Fig. 1). Additional examinations To increase the range of pointed to a changing metabolome during seed metabolites that can be storage, em phasizing the need for truly compa- analyzed with the profi- rable starting materials for profiling experi- ling platform, a GC-MS ments. station was established within the last year. It Fig.2: LC-MS Profiling of rape- Having optimized the metabolomics approach has undergone a tho- seed. The heatmap shows the comparison of the rape wildtype for Arabidopsis thaliana the system was applied rough evaluation pro- line, Drakkar, and the FatB3 to crop analysis. The comparison of the wild - cess com parable to the transformants. The co lour red is type rape line, Drakkar, with transformants car- LC-MS system and will indicating areas of higher concen tra tion, white is indicating rying the thioesterase CIFatB3 gene revealed soon start broa den the lo wer concentrations of the 53 that about 10 % of the detected mass signals scope of our profiling roughly 300 reliably detected mass sig nals. (the total number of reliably integrated m/z abilities. PUBLICATIONS AND OTHER SCIENTIFIC ACTIVITIES 2005 AND 2006

PUBLICATIONS 2005 tein docking. Bioinformatics 21, 683-684. Feilner, T., Hultschig, C., Lee, J., Meyer, S., Im mink, DIPLOMA THESES 2005 R.G.H., Koenig, A., Possling, A., Seitz, H., Beve rid ge, A., Altmann, Simone: Die Rolle des oxidative burst für Scheel, D., Cahill, D. J., Lehr bach, H., Kreutz berger, J. & die Pathogenantwort in Solanum tuberosum L.. Kersten, B. High-throughput identification of poten - University of Halle-Wittenberg, Depart ment of tial Arabi dopsis mitogen-activated protein kinases Biochemistry/ Biotechnology, 28/7/2005. substrates. Mol. Cell. Pro teomics 4, 1558-1568. Halbauer, Anja: Genexpressionsanalyse von Arabi dop- Lanquar, V., Lelièvre, F., Bolte, S., Hamès, C., Al con, C., sis-Pflanzen mit erhöhter mitogen-aktivierter Pro - Neumann, D., Vansuyt, G., Curie, C., Schroder, A., Krä - tein-Kinase-Aktivität. Fach hochschule Jena, 20/6/2005. mer, U., Barbier-Brygoo, H. & Thomine, S. Mo bi lization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is es - Mönchmeier, Claudia: Charakterisierung ei nes Histi - sential for seed germination on low iron. EMBO J. 24, din-Kinase-Gens aus Rhynchosporium se calis. 4041-4051. Univer si ty of Halle-Wittenberg, De part ment of Biochemis try/Biotechnology, 23/9/2005. Li, C.-M., Haapalainen, M., Lee, J., Nürnberger, T., Ro - mantschuk, M. & Taira, S. Harpin of Pseu domo nas Peisker, Daniel: Funktionelle Charak terisie rung von syringae pv. phaseolicola harbors a protein binding Phytochelatinsynthase-Mutationen. University of Hal - site. Mol. Plant Microbe Inter act.18, 60-66. le-Wittenberg, Department of Biochemistry/Bio - technology, 3/11/2005. Lipka, V., Dittgen, J., Bednarek, P., Bhat, R., Wiermer, M., Stein, M., Landtag J., Brandt, W., Rosahl, S., Scheel, D., Wibe, Marina: Charakterisierung der Mutante Llorente, F., Molina, A., Parker, J., Som merville, S. & UK7Dnip2 des phytopathogenen Pilzes Rhyn cho spo - Schulze-Lefert, P. Pre- and postinvasion defenses rium secalis. Fachhochschule Jena, 22/6/2005. both contribute to nonhost resistance in Arabidopsis. 310 Science , 1180-1183. Wolf, Dorothea: Die Rolle des F-Box-Pro teins COI1 in S. tuberosum. University of Halle-Wit tenberg, De - Miersch, J., Neumann, D., Menge, S., Bärlocher, F., partment of Bioche mis try/Bio technology, 2/3/2005. Baumbach, R. & Lichtenberger, O. Heavy metals and thiol pool in three strains of Tetra cladium marchalia- DOCTORAL THESES 2005 num. Mycol. Prog. 4, 185-194. Nickstadt, Anja: Charakterisierung der jasmonatin- sensitiven Arabidopsis thaliana Mutanten jin1 und Miroshnichenko, S., Tripp, J., zur Nieden, U., Neumann, jin4. University of Halle-Wittenberg, Department of D., Conrad, U. & Manteuffel, R. Immuno mo dulation of Bio logy, 17/10/2005. function of small heat shock proteins prevents their assembly into heat stress granules and results in cell Wassersleben, Susan: Metall-Detoxifikation durch death at sublethal temperatures. Plant J. 41, 269-281. Silizium in Silene und Arabidopsis. Uni versity of Halle- Wittenberg, Department of Biochemistry / Biotech - Overmyer, K., Brosché, M., Pellinen, R., Kuittinen, T., nology, 2/11/2005. Tu ominen, H., Ahlfors, R., Keinäen, M., Saar ma, M., Scheel, D. & Kangasjärvi, J. Ozone-induced pro - Weber, Michael: Identifizierung und Charak te ri sie - grammed cell death in the Ara bi dopsis radical-indu- rung von Hyperakkumulationsfaktoren bzw. schwer- ced cell death1 mu tant. Plant Physiol. 137, 1-13. metallregulierten Genen in Ara bi dopsis halleri und Ara bidopsis thaliana. Univer sity of Halle-Wittenberg, Prost, I., Dhondt, S., Rothe, G., Vicente, J., Rodri guez, De partment of Bio logy, 14/3/2005. M.J., Kift, N., Carbonne, F., Griffith, G., Es querré-Tu - gayé, M.T., Rosahl, S., Castresana, C., Hamberg, M. & PUBLICATIONS 2006 Fournier, J. Evaluation of the anti-microbial activities Birschwilks, M., Haupt, S., Hofius, D. & Neu mann, S. of plant oxylipins supports their involvement in de - Transfer of phloem-mobile substances from the host fense against pathogens. Plant Physiol. 139, 1902- plants to the holoparasite Cus cuta sp. J. Exp. Bot. 57, 1913. 911-921.

Racape, J., Belbahri, L., Engelhardt, S., Lacombe, B., Lee, Clemens, S. Evolution and function of phytochelatin J., Lochmann, J., Marais, A., Michel, N., Nürn berger, T., synthases. J. Plant Physiol. 163, 319-332. Parlange, F., Puverel, S. & Keller, H. Ca2+-dependent li - pid binding and membrane integration of PopA, a har- Clemens, S. Toxic metal accumulation, responses to pin-like elicitor of the hypersensitive response in to - exposure and mechanisms of tolerance in plants. bacco. Mole cul. Micro bio l. 58, 1406-1420. Biochimie 88, 1707-1719.

Weigel, R.R., Pfitzner, U.M. & Gatz, C. Interac tion of Consonni, C., Humphry, M.E., Hartmann, H.A., Livaja, NIMIN1 with NPR1 modulates PR gene ex pression M., Durner, J., Westphal, L., Vogel, J., Lipka, V., Kem mer - in Arabidopsis. Plant Cell 17, 1279-1291. ling, B., Schulze-Lefert, P., Somerville, S.C. & Pan struga, 54 R. Conserved re quirement for a plant host cell pro- Zöllner, F., Neumann, S., Kummert, S. & Sage rer, G. Da - tein in powdery mildew pathogenesis. Nat. Genet. 38, tabase driven test case generation for protein-pro- 716-720. SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF STRESS AND DEVELOPMENTAL BIOLOGY

filamentous fungi. FEBS Lett. 580, 3173-3178. Grzam, A., Tennstedt, P., Clemens, S., Hell, R. & Meyer, A.J. Vacuolar sequestration of gluta thione S-conju - Weber, M., Trampczynska, A. & Clemens, S. Com pa - gates outcompetes a possible degradation of the glu- rative transcriptome analysis of toxic metal responses tathione moiety by phytochelatin synthase. FEBS Lett. in Arabidopsis thaliana and the Cd2+-hypertolerant 580, 6384-6390. facultative metallophyte Arabidospsis halleri. Plant, Cell Environ. 29, 950-963. Halim, V. A., Vess, A., Scheel, D. & Rosahl, S. The role of salicylic acid and jasmonic acid in pa thogen defence. BOOKS AND BOOK CHAPTERS 2006 Plant Biology 8, 307-313. Clemens, S., Böttcher, C., Franz, M., Willscher, E., v. Roepenack-Lahaye, E. & Scheel, D. Ca pil lary HPLC Hamel, L.P., Nicole, M.C., Sritubtim, S., Moren cy, M.J., coupled to electrospray ionization quadrupole time- Ellis, M., Ehlting, J., Beaudoin, N., Bar bazuk, B., Klessig, of-flight mass spectrometry. In: Biotechnology in Agri - D., Lee, J., Martin, G., Mundy, J., Ohashi, Y., Scheel, D., culture and Forestry, Vol. 57, Plant Metabolomics Sheen, J., Xing, T., Zhang, S., Seguin, A. & Ellis, B.E. An - (Saito, K., Dixon, R.A. & Willmitzer, L. eds.), Springer- cient signals: comparative genomics of plant MAPK Verlag, Berlin-Heidelberg, pp. 65-79. and MAPKK gene families. Trends Plant Sci. 11, 192- 198. Krämer, U. & Clemens, S. Functions and home os tasis of zinc, copper and nickel in plants. In: Molecular Bio - Klie, S. & Neumann, S. Storage and processing of lo gy of Metal Homeostasis and De toxification. From mass spectrometry data. In: Proc. of 17th Int. Con - Mi crobes to Man. Topics in Cur rent Genetics, Vol.14 ference on Databases and Expert Sys tems (Ta más, M. J. & Mar ti noia, E. eds.), Springer Verlag Applications (DEXA`06), Krakow, Poland. IEEE 211 – Berlin-Heidel berg, pp. 215-271. 215. PUBLICATION IN PRESS Knogge, W. & Scheel, D. LysM receptors re cognize Tautenhahn, R., Böttcher, C. & Neumann, S. Anno - friend and foe. Proc. Nat. Acad. Sci. USA 103, 10829- tation of LC/ESI-MS Mass Signals. BIRD 2007 Proc. of 10830. BIRD 2007-1st International Con ference on Bio in for - ma tics Research and De velopment.(Hochreiter S. & Pöschl, Y., Böttcher, C., Clemens, S., Scheel, D., Posch, Wag ner R. eds.) Springer Lecture Notes in Bio in for- S. & Neumann, S. Analysis of Meta bolite relations in matics 4414 Berlin. LCMS data using Bayesian Net works. In: German Con ference on Bioinformatics, Short Papers and DIPLOMA THESES 2006 Poster Ab stracts (Huson, D., Kohlbacher, O., Lupas, A., Teutschbein, Jenny: Kartierung und Cha rak terisierung Nieselt, K. & Zell. A. eds.), ZBIT, 17-18. von Nichtwirtsresistenz-Genen in Arabidopsis thaliana. University of Halle-Wit tenberg, Department of Qutob, D., Kemmerling, B., Brunner, F., Küfner, I., Biology, 24/4/2006. Engelhardt, S., Gust, A.A., Luberacki, B., Seitz, H.U., Stahl, D., Rauhut, T., Glawischnig, E., Schween, G., Schulz, Beate: Untersuchungen zur Rolle von Ni - Lacombe, B., Watanabe, N., Lam, E., Schlichting, R., cotianamin für die Zink-Toleranz und -Akku mula - Scheel, D., Nau, K., Dodt, G., Hubert, D., Gijzen, M. & tion.University of Halle-Witten berg, Department of Nürnberger, T. Phyto toxicity and innate immune res - Biology, 24/4/2006. ponses induced by NEP1-like proteins. Plant Cell. 18, 3721-3744. Völz, Ronny: Funktionelle Analyse des MAP-Kinase- Gens RsPMK1 aus Rhynchosporium secalis . Roth, U., v Roepenack-Lahaye & Clemens, S. Pro teome University of Halle-Wittenberg, De partment of changes in Arabidopsis thaliana roots upon exposure to Biology, 23/5/2006. Cd2+. J. Exp. Bot. 57, 4003-4013. Weichert, Annett: Untersuchung von Phy toph thora Sarret, G., Harada, E., Choi, Y.-E., Isaure, M.-P., in festans-aktivierten Genen aus Arabi dopsis thaliana. Geoffroy, N., Fakra, S., Marcus, M. A., Birschwilks, M., University of Halle-Witten berg, Department of Bio - Clemens, S. & Manceau, A. Trichomes of tobacco ex - logy, 28/9/2006. crete zinc as zinc-substituted calcium carbonate and other zinc-contai ning compound. Plant Phy siol.141, DOCTORAL THESES 2006 1021-1034. Halim, Vincentius Andrianto: The role of salicylic acid and octadecanoids for pathogen de fense in potato, Tautenhahn, R., Ihlow, A. & Seiffert, U. Adaptive fea ture University of Halle-Witten berg, Department of Bio - selection for classification of micros cope images, In: logy, 23/5/2006. Fuzzy Logic and Applications, 6th International Workshop, WILF 2005, Crema, Italy, Lecture Notes in Landtag, Jörn: Untersuchung des Pathosys tems Ara - Computer Science 3849, 215-222. bidopsis thaliana (L.) – Phytophthora infestans und Cha rakterisierung von Mutanten mit einem veränder- 55 Trampczynska, A., Böttcher, C. & Clemens, S. The tran- ten Nichtwirtsresistenz-Phänotyp, University of sition metal chelator nicotianamine is synthesized by Halle-Wittenberg, Department of Biology, 12/7/2006. DEPARTMENT OF SECONDARY METABOLISM Head: Professor Dieter Strack

Secretary: Ildikó Birkás

ork of the department is concerned with the molecular regulation of plant secondary metabolism, evolution of the enzymes involved and the role of Wsecondary products in interactions of plants with their environment. The work on metabolic regulation includes isolation and characterization of the en zymes and the encoding genes, focusing on transferases. We investigated malate and choline hydroxycinnamoyltransferases as well as several hydroxycinnamate glucosyltransferases from Arabidopsis thaliana and oilseed rape (Brassica napus) as well as flavonoid and beta- nidin glucosyltransferases from betacyanin-accumulating plants or flavonoid methyltransferases from the ice plant (Mesembryanthemum crystallinum) and Arabidopsis.

The aim of the work on glucosyl- and hydroxycinnamoyltransferases is to elucidate their evolutionary origin and structure-function relations to predict substrate specificity. Glucosyltransferases involved in betacyanin biosynthesis are considered to be oligophy- letic and originate from different clusters of flavonoid glucosyltransferases. A new class of methyltransferases was identified, whose substrate specificities can be modula ted by metal cations and N-terminal deletions. Hydroxycinnamoyltransferases, which are dependent on b-acetal esters as acyldonors, are vacuolar serine carboxypeptidase-like (SCPL) proteins as found for the enzymes involved in the formation of sinapoylmalate in Arabidopsis and sinapoylcholine (sinapine) in oilseed rape. Homology modeling of these acyltransferases identified structure determinants and amino acids involved in substrate recognition. The existence of these enzymes proves a new concept in the regulation and evolution of plant secondary metabolism. Additional work on a sinapine esterase from rape and a chlorogenate-specific caffeoyltransferase from tomato (Solanum lycopersicum) indicates functional shifts from lipases to a phenylpropanoid-specific esterase and acyltransferase, respectively.

56 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF SECONDARY METABOLISM

Special emphasis is also placed on programs focusing on the molecular symbiotic interactions of plant roots with soil-borne fungi, i.e. arbuscular mycorrhizal fungi.The work of two groups is concerned with fungus-induced alterations in plant isoprenoid metabolism, in particular carotenoid biosynthesis and degradation leading to massive accumulation of apocarotenoids. This is accompanied by a dramatic reorganization of plastid population in arbuscule-harboring root cells. Another main objective is the analysis of the role of phytohormones, in particular jasmonates, in development and functional maintenance of mycorrhizal symbiosis. It was found that jasmonates obviously play a crucial role in the establishment of arbuscular mycorrhizas. These studies are supported by comprehensive analysis of primary and secondary metabolites (metabolite profiling) in wild-type and transgenic mycorrhizal plants. In another project on adventitious root formations of Petunia hybrida the possible function of wound-related jasmonates is studied.

57 METABOLISM OF PHENYLPROPANOIDS Heads: Dieter Strack & Carsten Milkowski

The work of our group is focused mainly on metabolism of sinapate esters in oilseed rape (Brassica napus) and the model plant Arabidopsis thaliana with the aim to im prove rapeseed qua- lity by metabolic engineering. Target genes, i.e. UDPglucose:sinapate glucosyltransferase (SGT) and sinapoylglucose:choline sinapoyltransferase (SCT), to lower the content of the antinutritive seed constituent sinapine (sinapoylcholine) were identified. This is a key requirement for establishing rape as a protein crop. With Arabidopsis the basic role of SGT in phenylpropanoid metabolism and some aspects of plant physiology were studied. To study the evolutionary recruitment of enzymes from primary metabolism to produce highly diverse patterns of metabolites for novel functions in plant secondary metabolism we are interested to eluci date the structural changes necessary for conversion of hydrolases (peptidases, lipases) to acyltransferases by analysis of structure-function relationships of these enzymes. This work includes the SCT and the related acyltransferase sinapoylglucose:malate sinapoyltransferase (SMT) as well as the chlorogenate-dependent biosynthesis of caf feoylglucarate (CGT) from tomato.

GROUP MEMBERS The enzyme UDP-glucose:sinapate glucosyl- BnSGT/BnSCT double suppression construct Alfred Baumert transferase (SGT) catalyzes the formation of three homozygous single copy insertion lines Research Scientist 1-O-sinapoylglucose, the principle acyldonor in reduced in seed sina pate ester content were Kathleen Clauß transacylation reactions leading to a wide array developed. Screening PAL suppressing plants PhD Student since February 2005 of sinapate esters in species of the Brassicaceae (T seeds) several low sinapine lines were iso- Claudia Horn 2 Technician family. Seed-specific suppression of the gene in lated. In order to identify the sinapine hydroly- Dirk Meißner oilseed rape (Brassica napus), BnSGT1 zing enzyme, sinapine es terase, the activity was PhD Student (UGT84A9), led to a significant decrease in purified from germinating seeds. Based on pep- Juliane Mittasch total sinapate ester content, establishing tide sequences the full-length cDNA was isola- PhD Student UGT84A9 as key target gene for breeding of ted and shown to en code sinapine esterase by Ingrid Otschik Technician low sinapine cultivars. To support TILLING functional expression in Nicotiana benthamia- Diana Schmidt approaches for isolation of low sinapine na. Sequence analysis revealed that sinapine PhD Student until April 2006 mutants a genomic BAC library was screened esterase shares homology with lipases. Related Felix Stehle to assess the number of UGT84A9 alleles and cDNAs from Arabidopsis were cloned and PhD Student genomic loci in the allotetraploid genome of B. shown to be active towards sinapine. Jenny Teutschbein PhD Student since May 2006 napus. Sequence polymorphisms and Southern Sylvia Vetter Blot patterns of isolated BAC clones reveal Technician since February 2006 two alleles and four genomic loci of UGT84A9 in winter oilseed rape cultivar “Express”. For the gene encoding the final enzyme in sinapine biosynthesis, sinapoylglucose:choline sinapoyltransferase (SCT), two alleles were identified in the B. napus ge nome. Since BnSCT expression is restricted to developing seeds the promotor was isolated as a potential tool for future transgenic strategies aimed at modification of seed- specific traits. The DNA fragment covering 729 bp upstream of BnSCT translation start was sufficient to drive GUS expression specifically to the em bryo and the aleurone layer of Arabidopsis seeds (Fig1). Approaches to sup- Fig.1: The BnSCT1 promoter drives seed-specific GUS expression press sinapine accumulation were put forward in Arabi dopsis. The BnSCT1 promoter-GUS fusion cassette was by seed-specific dsRNAi knock-down of inserted into the vector pGREEN (pBnSCT1GUSpGREEN) to transform Arabidopsis. The empty plasmid was used as negative BnSGT/BnSCT and PAL (phenylalanine-ammo- control (pGREEN). The upper images show GUS activities in tis - sues of the testa and aleurone layer, the middle ones of the hypo- 58 nia lyase), the enzyme catalyzing the entry step cotyls and the lower of the cotyledons. Bars represent 20 µm. into phenylpropanoid metabolism. With the SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF SECONDARY METABOLISM

Since soluble hy droxycinnamate es ters have glucarate caffeoyltransferase) were chosen. The COLLABORATORS been as cribed for UV screen function in Ara bi - CGT was purified from tomato leaves. Peptide Peter Dörmann Max Planck Institute of Molecular dopsis, ex pression of ester forming HCA-GTs sequences indicate homology of this acyltransfe- Physiology, Potsdam-Golm, Germany (UGT84A1-4) was rase with lipases. A Martin Frauen, measured under full-length cDNA en - Gunhild Leckband Norddeutsche Pflanzenzucht, Hans Georg UV-B stress condi - coding CGT was iso- Lembke KG, Hohenlieth, Germany tions. Treatment of lated. For Ara bi dop - Manfred Nimtz, Victor Wray Arabidopsis plants sis SMT a yeast ex - Helmholtz Centre for Infection Research, Braunschweig, Germany for ten hours under pression system was José Orsini conditions of eleva - optimized. By codon Saaten-Union Resistenzlabor GmbH, ted UV-B radiation usage adaptation of Leopoldshöhe, Germany Wolfgang Brandt, resulted in a four- Fig. 2: Proposed fold of the ternary structure of SMT (left) and quaternary the SMT-cDNA to structure of SCT (right), each with the docked ligand of sinapoylglucose. Jürgen Schmidt fold increase in The catalytic triad (Ser, Asp, His) is colored in red. the requirements of Leibniz Institute of Plant Biochemistry, UGT84A2 tran- Sa ccha romyces and Halle, Germany Gopalan Selvaraj script abundance in rosette leaves accompanied translational fusion with the signal peptide of a Plant Biotechnology Institute, Saskatoon, by corresponding increases in enzyme activities yeast vacuolar protease enzymatically active Canada towards sinapate. Promoter-GUS fusion expe- SMT was expressed from a multicopy vector Milton T. Stubbs University of Halle, Germany riments corroborated the UV-B stress induc - with a yield of about five milligram per liter. Thus tion of UGT84A2 transcription. Under stan- kinetic measurements to study the reaction me - dard conditions, UGT84A2 expression was hig- chanism of SMT catalysis could be started. In a hest in cotyledons of three-day-old seedlings parallel approach, structure models of the SMT whereas UV-B stress clearly induced transcrip- and SCT proteins were generated that reveals tion in ro sette leaves. functional elements like the hydrogen bond network for substrate recognition, the “oxyanion To investigate the evolutionary recruitment of hole” stabilizing the transition states and the ca - enzymes from primary metabolism for novel talytic triad (Ser, His, Asp) for the nucleophilic at - functions in plant secondary metabolism the hy - tack onto the carbonyl carbon of the acyldonor drolase-related acyltransferases sinapoylgluco- sinapoylglucose. Site-di rected mutagenesis of the se:malate sinapoyltransferase (SMT) from Ara - recombinant SMT approved the functional signi- bidopsis and the tomato-CGT (chloro ge na te: ficance of several amino acid residues.

Fig. 3: Stereo picture of the structure model of the SMT active side (above). Amino acid residues forming the catalytic triad (Ser-173, Asp- 358, His-411) and some of those forming the hydrogen bond network for sinapoylglu cose recognition (Asn-73, Asp-172, Lys-268, His-272, Arg- 322) were substituted in site-directed mutagenesis of the recombinant en zymes, giving the relative specific activities as shown below.

59 MOLECULAR PHYSIOLOGY OF MYCORRHIZA Head: Michael H. Walter

The arbuscular mycorrhizal symbiosis of plants with specialized fungi in the soil provides roots with improved access to mineral nutrients, particularly phosphate, while plant carbohydrates are directed to the fungus in return. A main focus of the group is the elucidation of biosynthesis and function of certain apocarotenoids, which accumulate in later stages of this interaction. An early biosynthetic step in the methyl erythritol phosphate (MEP) pathway catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), is used as a target for an RNAi approach. A specific isogene (DXS2) dedicated to apocarotenoid and other secondary isoprenoid biosynthesis allows for specific manipulation of this step without interfering with primary metabolism. Additional functions of DXS2 independent of mycorrhization are being studied in tomato leaves and their trichomes. Furthermore, the ancestry of diverged DXS genes of angiosperms in land plant evolution is investigated by working with the gymnosperm Picea abies and the moss Physcomitrella patens.

GROUP MEMBERS Arbuscules are tiny intracellular organs of the the general isoprenoid pathway leading to Daniela Floß fungus with a tree-like morphology, which are isopentenyl diphosphate / dimethylallyl di - PhD Student the symbiotic transfer sites of phosphate from phos phate in plastids (MEP pathway), Jessica Leuchte the fungi into plant cells. Arbuscules are ephe- the carotenoid biosynthetic pathway, and PhD Student since June 2006 meral structures with only a few days of func - the carotenoid cleavage and subsequent Kerstin Manke Technician tionality in nutrient transfer. There is thus a modification steps. Heike Paetzold need for constant renewal of arbuscules but PhD Student since March 2005 the mechanisms of their turnover are un - A major recent focus of the group has been the known. During colonization by arbuscular my - first step of the MEP pathway catalyzed by corrhizal (AM) fungi plant roots frequently ac - 1-deoxy-D-xylulose 5-phosphate synthase cumulate two types of apocarotenoids (carote- (DXS), since there are at least two distantly re - noid cleavage products). These compounds lated isogenes, which are differentially regulated localize preferentially to cells with degrading and appear to have dedicated roles in primary

arbuscules. Both compounds, yellow linear C14 metabolism (DXS1) or secondary metabolism

mycorradicin and colorless C13 cyclohexenone (DXS2). derivatives, are predicted to originate from a

common C40 carotenoid precursor. Apoca ro - The MtDXS2-1 gene of the model legume Me - tenoid biosynthesis can be subdivided into dicago truncatula, one of two closely related three pathways: MtDXS2 genes organized in a tandem repeat, exhibits strongly elevated transcript levels in

Fig. 1: Scheme of arbuscule development, maturation and breakdown in root cells. Arbuscule degradation is accompanied by local apocarotenoid deposition shown as small yellow vesicles (center panel); Con fo cal images of arbuscule development featuring young, ma ture and degrading stages from left to right (below); Simplified biosynthetic pathway of apoca- 60 rotenoids and position of DXS2 (right panel). SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF SECONDARY METABOLISM

Fig. 2: Tomato trichomes isolated from leaves. The head of a glandular trichome is visible at the upper left. mycorrhizal roots, whereas the on ly known exception is COLLABORATORS MtDXS1 is unaffected. RNAi con- Arabidopsis thaliana, which has two Harro Bouwmeester, Plant Research International, Wageningen, structs to knock down expres sion DXS1 co pies, but no DXS2 copy. The Netherlands of both MtDXS2 genes, but not Orthologs of both angiosperm DXS Helge Küster, MtDXS1, were introduced into classes have been identified in the University of Bielefeld, Germany transgenic hai ry roots of M. trunca- gymnosperm Picea abies indicating Mike Philips, Max Planck Institute for Chemical tula by transformation using Agro - that an ancient gene duplication has Ecology, Jena, Germany bacterium rhizogenes and the evolved into the present-day diver- Ralph Reski, pRNAi/pRed Root-vector system. The current gence of DXS genes. The P. abies DXS genes are University of Freiburg, Germany results indi cate a correlation of reduced levels also differentially regulated and appear to have Klaus von Schwartzenberg, University of Hamburg, Germany of MtDXS2 transcripts and a significant reduc- a similar division of labor between primary and tion in apocarotenoid levels in transgenic roots se condary metabolism as in the angiosperm after nine weeks of root colonization by case. This is confirmed by a recent report on Glomus intraradices. This coincides with fewer ginkgolide formation in Ginkgo biloba roots lin- stainable mycor rhizal structures, particularly king its biosynthesis to expression of a DXS arbuscules, and an even more pronounced drop gene belonging to class 2. However, in several in transcript levels of MtPT4, a mycorrhiza-spe- gymnosperms the DXS2 class is further split cific plant phosphate transporter gene. MtPT4 into two more distantly related DXS2 genes. is used as a molecular marker for the functionality of ar buscules. Additional experiments To trace back the separation of DXS genes indicated that this effect is dependent on the even further in evolution work with the moss growth stage of the plants or growth conditi- Physcomitrella patens has been started. This ons. However, under the conditions of reduced moss produces a tetracyclic diterpene (16-a- apocarotenoid levels in transgenic roots there hydroxykaurane), which is excreted and can be appears to be a general accumulation of older observed as crystals on leaf tips or on agar degenerate stages of arbuscules at the expense medium next to the plants. The function of this of young and mature arbuscules as compared compound is unknown, but it has been implica- to empty vector control plants. ted in microbial defense mechanisms. Since this diterpene can be classified as a secondary com- Involvement of DXS2 genes in non-mycorrhizal pound, it constitutes a suitable target to find isoprenoid pathways is being investigated in out, whether particular subfunctionalized DXS tomato (Solanum lycopersicum). Glandular tri- isogenes of P. patens are involved in its biosyn- chomes of tomato are known to produce a thesis. Starting with EST data four different number of different mono- and diterpenes DXS isogenes of the moss have been identified involved in insect resistance and these com- and full-length cDNAs obtained from „Phys - pounds are derived from precursors provided cobase“ were sequenced. The deduced pro- by the MEP pathway. The SlDXS2 gene of to - teins are all closely related and belong to the mato is expressed in leaves, roots and flowers, DXS1 class of angiosperms and gymnosperms. but not in the ripening fruit. In isolated leaf tri- Several conditions of diterpene biosynthesis chomes SlDXS2 transcript levels are strongly and a number of other stimuli have been stu- elevated indicating a preferential expression in died to find out about potential differential re - leaf trichomes. Peptide antibodies specific for gulation of the four PpDXS genes. There are SlDXS2 were generated and are currently be - differences in expression levels between the ing used in immunolocalization experiments. four genes, but at this point a condition, where Suppression of the biosynthesis of MEP path- they are differentially regulated, was not found. way-derived secondary isoprenoids by a knock- In future work individual PpDXS genes of P. down of SlDXS2 expression in transgenic to - patens will be knocked out and the outcome mato similar to the apocarotenoid suppression on general viability and diterpene biosynthesis Fig. 3: Crystal needles of 16-a- in M. truncatula will be performed. will be determined. Additional experimental hydroxykaurane appearing at the systems located between the mosses and the leaf tip of the moss Physcomitrella patens (top) or on the moss culture In angiosperms the diversification of DXS gymnosperms in land plant evolution will be ap - agar medium (below). 61 genes into DXS1 and DXS2 is widespread and proached to clarify their DXS gene divergence. CELL BIOLOGY OF MYCCORRHIZA Head: Bettina Hause

The mutualistic interaction between plants and arbuscular mycorrhizal (AM) fungi is believed to be regulated in part by the action of phytohormones. Jasmonates, known as regulators in plant response to biotic or abiotic stresses, are good candidates for such a role. Therefore, the functional analysis of jasmonates during the interaction between Glomus intraradices and barrel medic (Medicago truncatula) is the main ob jective of our group. Moreover, we are interested in the regulatory role of apoplastic invertases in the supply of the AM fungus with carbohydrates by the plant. In a third project, the massive proliferation of plastids in AM-colonized root cortical cells is studied. Metabolic and cytological changes as well as respective molecular mechanisms are investigated. In a new project the role of jasmonates in the formation of adventitious roots in Petunia hybrida will be analyzed.

GROUP MEMBERS Conclusions for a possible involvement of of differentially regulated genes depending on Thomas Fester Jasmonic acid (JA) in mycorrhizal interaction endogenous JA levels (cooperation with Helge Junior Group Leader until July 2006 were drawn first from application experiments, Küster, University of Bielefeld). These changes Susann Gürtler which suggested a concentration-dependent ef - were also reflected by alterations in the pat- Diploma Student since March 2006 fect of JA on mycorrhizal plants. Further results terns of secondary metabolites (cooperation Zakir Hossain G.-Forster-Fellow since April 2006 revealed that JA levels are increased in roots of with Willibald Schliemann, IPB). Ulrike Hintsche/Huth mycorrhizal plants compared to roots of non- Technician mycorrhizal controls. To analyze a possible func - Paul Knick tion of JA during the development of the my - Diploma Student until September 2006 corrhizal symbiosis, roots of M. truncatula were Dagmar Knöfel Technician until July 2006 transformed with sense- and RNAi-constructs Sandra Lischewski for the allene oxide cyclase (AOC). AOC is ca - PhD Student since March 2006 talyzing the crucial step in JA biosynthesis. All Swanhild Lohse constructs were under the control of the PhD Student until November 2005 CaMV 35S promoter. In comparison to samples Fig. 2: Fungal structures in a wild-type tobacco root (a) and Cornelia Mrosk in a root of an invertase-overexpressing plant with mode- PhD Student transformed with a control vector (DsRED), rate invertase activity in leaves (b). Cross sections of G. Anja Nickstadt roots with suppressed ex pression of MtAOC1 intraradices-colonized roots were stained with fluorescent Postdoctoral Position until February exhibited lo wer JA levels and a remarkable de - labelled wheat germ agglutinin (green) and analyzed with a 2006 confocal laser scanning microscope. Bars represent 50 µm. lay in the process of colonization with G. intra- Sara Schaarschmidt PhD Student radices. Both the mycorrhization degree, quan- Rostand Tonleu Tonfack tified by fungal rRNA, and the formation of ar - The mutualistic interaction in AM is characte - Diploma Student until September 2005 buscules, analyzed by the expression level of rized by an exchange of mineral nutrients and Carola Tretner Research scientist the AM-specific phosphate transporter gene carbon. Data about the regulatory function of MtPT4, were af fected. Concluding from this, it carbon availability on AM formation, however, ap pears that jasmonates affect are limited. Therefore, the effect of enhanced mycorrhization, possibly in mul - root hexose levels, the main form of carbohy- tiple ways. drate used by the fungus, on AM formation was analyzed. Modulation of the root carbohydrate Analyses of transcript and meta- status in tobacco was achieved by root-specific bolite patterns by microarrays expression of a gene encoding a yeast-derived and metabolite profiling, both in invertase, targeted to the apoplast (plants kind- wild-type and transgenic roots, ly provided by U. Sonnewald, University of Er - can help to identify processes langen). Despite increased root hexose levels mediated by JA during my - we did not detect any effect on the coloniza - corrhization. First analyses of the tion with G. intraradices analyzing fungal struc- Fig. 1: Immunolabelling of the allene oxide cyclase transcript pattern of transgenic tures as well as the levels of fungus-specific (AOC) in cross section of stem of Medicago truncatula. M. truncatula roots overexpres- fatty acids or the plant phosphate contents. The AOC (green) is located in plastids and occurs mainly in the cortex and the pith as well as in phloem sing or suppressing MtAOC1, Car bohydrate supply for AM fungi, accordingly, cells of the vascular bundles. Cell nuclei are visualized both non-mycorrhizal and my - cannot be improved by increasing specifically 62 by DAPI. corrhizal, revealed a high number root hexose levels, implying that under normal SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF SECONDARY METABOLISM

conditions carbon supply is not limiting for the FtsZ, the plastid enzyme DXR (1-deoxy-D-xylu- COLLABORATORS AM symbiosis. In contrast, plants with decrea- lose 5-phosphate reductoisomerase, involved in Ivo Feussner University of Göttingen, Germany sed acid invertase activity in roots obtained by the formation of apocarotenoids) and a plastid- Philipp Franken, Uwe Drüge root-specific overexpression of an invertase in - targeted green fluorescent protein together Institute of Vegetable and Ornamental hibitor (plants kindly provided by Thomas with fungal structures were colocalized. This Crops, Großbeeren/Erfurt, Germany Roitsch, University of Würzburg) exhibited a analysis allowed a clear dis tinc tion of two phases Gerd Hause University of Halle, Germany diminished mycorrhization. Surprisingly, plants of plastid proliferation in root cortical cells colo- Joachim Kopka with moderately increased invertase activity nized by AM fungi. In the first phase, during the Max Planck Institute of Molecular Plant Physiology, Golm, within the leaves showed a stimulation of my - formation of arbuscules, plastids are intensively Germany corrhization. Compared to wild-type, a higher dividing leading to a large number of lens-shaped Helge Küster degree of my corrhization accompanied by a organelles between the arbuscular branches. The University of Bielefeld, Germany higher density of fungal structures and a higher second phase of proliferation, during arbuscule Jutta Ludwig-Müller Technical University of Dresden, amount of G. intraradices-specific rRNA was degradation, is characterized by a smaller num- Germany de tected. The roots of those plants exhibited ber of largely elongated organelles surrounding Axel Mithöfer lower levels of some metabolites (phenolic the decomposing arbuscule. Some rings of the Max Planck Institute for Chemical Ecology, Jena, Germany compounds, amines and amino acids) as well as division protein FtsZ are located along these Katharina Pawlowski an increased ab scisic acid content compared to tubular plastids indicating either a fusion/fission University of Stockholm, Sweden wild-type (co operation with Joachim Kopka, me chanism or the beginning of the final degrada - Thomas Roitsch MPI Potsdam/Golm and Jutta Ludwig-Müller, tion of these plastids. The observation of tu bular University of Würzburg, Germany Technical University of Dresden). This points to plastids in the second phase corresponds to the Uwe Sonnewald University of Erlangen, Germany changes in metabolism and defense status of formation of apocarotenoids and of re active Claus Wasternack, Otto the plant root due to the increased invertase oxygen species during arbuscule degradation. Miersch, Willibald Schliemann activity within the shoot. One possible functional role of plastids during Leibniz Institute of Plant Biochemistry, Halle, Germany this phase might be the recycling of metabolites (e.g. ammonia) liberated from decaying arbuscules. Further approaches regarded the examina - tion of arbuscule development in tobacco plants severely disturbed in plastid division and the establishment of an inducible system for the analysis of arbuscule degrada tion.

The generation and subsequent growth of ad - ventitious roots in stem tissues of excised leafy cuttings is a crucial physiological process in propagation of many ornamental plant species, among them Petunia hybrida. Despite intensive Fig. 3: Immunolocalization of the plastid-located protein 1- control of environmental factors in the modern deoxy-D-xylulose 5-phosphate reductoisomerase (green) in root cortical cells containing an intact and a de - propagation industry, high economic losses still composing arbuscule (red). Tubular plastids containing the occur due to insufficient rooting. The formation enzyme are predominantly observed close to the decomposing arbuscule. Bar represents 5 µm. of adventitious roots is wound-in duced and re - presents a multistage developmental process in - Previous work had revealed significant reorgani- volving specific gene expres sion events.To inve- zation of plastid shape and metabolism in root stigate the possible function of the wound-rela- cortical cells colonized by AM fungi. Plas tids are ted hormone JA during this process, a cDNA producing a number of primary metabolites (like coding for AOC in P. hybrida (PhAOC) was iso- fatty acids and amino acids) essential for the es - lated.Analyses of transcript and protein levels in tablishment of symbiotic structures, in ad dition the time-course of adventitious root formations they are producing carotenoid cleavage pro- were done. In future, the PhAOC-cDNA will be ducts (apocarotenoids) specific for the AM sym- used for modulating the endogenous JA content biosis. To obtain a clearer correla tion of the in P. hybrida by its constitutive overexpression development of symbiotic struc tures and of pla- (“gain-of-func tion”) or its suppression via RNAi Fig. 4: Flowers of Petunia hybrida cv. 63 Mitchell. stid reorganization the plastid division protein (“loss-of-function”). METABOLITE PROFILING & BIOCHEMISTRY OF PROTEINS Heads: Willibald Schliemann & Thomas Vogt

In the framework of the DFG focus program 1084 Molecular Basics of Mycorrhizal Symbioses metabolite profiling was used to analyze the pattern of primary and secondary metabolites of developing arbuscular mycorrhizal roots of Medicago truncatula colonized with Glomus intraradices with the aim to characterize metabolic processes relevant for mycorrhiza development and symbiotic functioning. The generation and application of different biostatistical tools enabled comprehensive data evaluation and presentation. The established metabolite profiling platform will be applied in a running BMBF project with Canadian partners in the development of low sinapine rape. Recent developments in plant natural product transferase research have lar gely focused on two aspects: first, a detailed molecular study to correlate the observed differences in the structural domains of two distinct clusters of cation-dependent O-me thyltransferases with observed in vitro substrate and position specificities; second, an investigation focused on the natural diversity of the transferase superfamilies to identify and target developmentally and tissue-specific genes and proteins for their specific roles and specificities in planta, one of the primary adaptive mechanisms of plants to meet the changing environmental conditions in a timely and developmentally controlled manner.

GROUP MEMBERS METABOLITE PROFILING of a glucosyltransferase (GT) gene in a similar Christian Ammer In a time course study, the changes of primary system. Sa ponin le vels showed only a growth- Research scientist until August 2006 and secondary metabolites in roots of barrel related in crease. Mycor rhiza-specific apocaro- René Geißler medic (Medicago truncatula) during the sym- tenoids (cy clohexenone and mycorradicin deri- PhD Student since November 2006 biotic interaction with the arbuscular mycor- vatives) ac cumulated parallel to root mycorrhi- Barbara Kolbe Technician rhizal (AM) fungus Glomus intraradices were zation. Struc tures of the cyclohexenone deriva- Jakub Grzegorz Kopycki analyzed by GC-MS, HPLC and LC-MS and tives were elucidated as glucosides of blumenol PhD Student compared to nonmycorrhizal controls recei- C and 13-hydroxyblumenol C and their corre- Dagmar Knöfel ving different phosphate nutrition to discrimi- sponding malonyl conjugates. Typical cell wall- Technician since August 2006 nate between nutrition- and symbiosis-related bound phenolics were not altered, while tyro- Karina Wolfram PhD Student since July 2006 effects. The metabolite identification was based sol with re ported antifungal properties was on authentic standards as well as the plant- found as a major phenolic component only in specific Golm metabolome database. Fungus- mycorrhizal roots. Prin cipal component analy- specific fatty acids from spores and hyphae of G. ses of nonpolar primary metabolites and se - intraradices were used as biomarkers for estima - condary metabolites clearly separate AM roots tion of the mycorrhization degree. Ele va ted levels from those of controls, an additional grouping of certain amino acids (Glu, Asp, Asn) and fatty in harvest days is given by secondary metabo - acids indicate an activation of the plastidial lites. These results are confirmed by hierarchi- metabolism. These results refer to an general ac - cal cluster analysis. Pearson correlation bet- tivation of mitochondrial (tricar- ween primary nonpolar metabolite pairs show- boxylic acid cycle) as well as pla- ed stronger and more frequent relation in my - stid metabolism (lipid biosynthe- corrhizal roots and those supplied with high le - sis, N-assimilation) during vels of phosphate than in the nonmycorrhizal mycorrhiza tion, which correla- controls indicating a tigh ter control of the acti- tes with corresponding increa- vated root primary metabolism. Network cor- sed transcript levels (in si lico relation analyses revealed distinct clusters of and RealTime RT-PCR analyses amino acids and su gars/aliphatic acids with – T. Fester, IPB Halle, Germany). strong metabolic correlations among one anot- Additionally, the biosynthesis of her. Mycorrhizal symbiosis re duces the cluster some constitutive isoflavonoids, separation and enlarges the sugar cluster size. Fig. 1: Retention time – m/z plot of GC/TOF-MS of particularly at later develop- The amino acid clusters re present cliques selected fatty acids from Glomus intraradices. mental stages, was stimulated, (groups of metabolites with strong correlations 9c 11c 11t From left to right: C18:1D , C18:1 D , C18:1 D , which may be related to the among one another) differently composed in 64 C18:0. mycorrhiza-induced expression nonmycorrhizal and mycorrhizal roots. The SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF SECONDARY METABOLISM

results from metabolite profiling provide clear while maintaining the required efficiency may COLLABORATORS distinction between de velopment-, nutrition require further changes on the amino acid level. Wolfgang Brandt, Andrea Porzel, Jürgen Schmidt, (phosphate)- and symbiosis (mycorrhiza)-me - The drop in specificity by changing the bivalent Leibniz Institute of diated alterations of root me tabolism. cat ions within the active site may add additional Plant Biochemistry, Halle, Germany complexity to in vivo roles and specificities. Hermann Bothe University of Cologne, Germany BIOCHEMISTRY OF PROTEINS Martin Hagemann Promiscuous substrate specificities and low The differences in the position specificity of indi- University of Rostock, Germany amino acid sequence identities to the highly con- vidual enzymes re flect the diversity of the corre- Ulrich Hildebrandt served caffeoyl-coenzyme A O-methyltransfera- sponding CCoAOMT-like members on the ge - University of Würzburg, Germany ses (CCoAOMTs) characterize members of a nomic and proteomic level. Apparently, Ara - Joachim Kopka Max Planck Institute of new subcluster of cation-dependent OMTs from bidopsis requires different CCoAOMT-like pro- Molecular Plant Physiology, Golm, various plant species. The conserved position teins of seven transcriptionally active genes, Germany specificity towards aromatic vicinal di- or tri hy - which are developmentally and tissue specifi cally Pål Axel Olsson University of Lund, Sweden dro xy systems is in contrast to their broad sub- regulated. The product of at least one of those Gopalan Selvaraj strate specificity, including aromatics, (iso)flavo - genes shows highest identity in terms of amino Plant Biotechnology Institute, Saskatoon, noids, and phenylpropanoid conjugates, like caf- acid sequence as well as substrate specificity to Canada Milton T. Stubbs feoylglucose. Structural in formation of one the PFOMT, and can clearly be distinguished from University of Halle, Germany newly discovered OMTs from Mes embryan the - the caffeoyl-CoA-specific isoenzyme likely invol- Lloyd W. Sumner mum crystallinum (PFOMT), indi cates that there ved in lignin formation. The expres sion of the Samuel Roberts Noble Foundation, Ardmore, USA are only two domains, which distinguish the 3D- promiscuous variety in reproduc tive organs (flo- Victor Wray, Manfred Nimtz structure of this protein from the 3D-structure wers, seeds) at the end of plant de ve lop ment Helmholtz Centre for Infection Research, of a caffeoyl-CoA-specific enzyme from a re - and its preference for caffeoylglu cose suggests Braunschweig, Germany cently crystallized CCoAOMT from Medicago an interesting in vivo role of this protein other sativa: the first 15 amino acids of the N-terminus than lignin for- and a C-terminal loop. As in case of the GTs, the mation. A pro - N-terminal part of the enzyme is important for posed combina - the posi tion specificity but not for the substrate tion of af finity specificity of the promiscuous type of en zymes. purifica tion, 2-D Va rious N-terminally truncated versions and PAGE, and mass some PFOMT/CCoAOMT chimeric proteins spectrometry were designed to analyze the influence of the should com bine corresponding domains on the specificity and and resolve tis- efficiency. The lack of several amino acids (as sue- and develop- observed also for the native plant enzyme) re - mentally specific sults in a more position-specific, but also less ef - arrays of transfe- ficient enzyme. Chimeric PFOMT/CCoAOMT rases like gluco- constructs, where the N-terminal parts were syl-, acyl- or the derived from the specific CCoAOMT from M. (cation-de pen - sativa resulted in more specific enzymes, but also dent) O-me thyl - had a negative impact on efficiency. The turnover transferases rat- rates dropped with all substrates, but the most her than focusing Fig. 2: Distribution of the seven cation-dependent enzymes from serious drop was observed with caffeic acid, on single en - Arabidopsis (marked in red) within several clusters of promiscuous or which is not accepted at all by the chimeric con- zymes. The ini tial specific plant and microbial CCoAOMT-like proteins. structs. A remarkable change is ob served if both, focus aims at the loop and the N-terminus of PFOMT, are characterizing the pattern and abundance of exchanged simultaneously with the respective these en zymes in seeds of Ara bidopsis and CCoAOMT-domains. In this case a further, dras - Brassica na pus. Both tissues deliver the required tic drop in catalytic efficiency can be detected structurally and developmentally limited sys tems for all substrates, except for caffeoyl-CoA. The to correlate transferase patterns with the se - transformation from a promiscuous towards a condary metabo lite patterns and will also enable caffeoyl-CoA-specific enzyme was accom - to identify new transferases with un known new 65 plished. Modification of substrate specificity specificities. PUBLICATIONS AND OTHER SCIENTIFIC ACTIVITIES 2005 AND 2006

PUBLICATIONS 2005 Stumpe, M., Carsjens, J.-G., Stenzel, I., Göbel, C., Lang, I., Baumert, A., Milkowski, C., Schmidt, J., Nimtz, M., Wray, Pawlowski, K., Hause, B. & Feussner, I. Lipid metabolism V. & Strack, D. Formation of a complex pattern of sina- in arbuscular mycorrhizal roots of Medicago truncatu- pate esters in Brassica napus seeds, catalyzed by en - la. Phytochemistry 66, 781-791. zymes of a serine carboxypeptidase-like acyltransfe - rase family? Phytochemistry 66, 1334-1345. Ziegler, J., Diaz-Chávez, M. L., Kramell, R., Ammer, C. & Kutchan, T. M. Comparative macroarray analysis of Fester, T. & Hause, G. Accumulation of reactive oxygen morphine containing Papaver somniferum and eight species in arbuscular mycorrhizal roots. Mycorrhiza morphine free Papaver species identifies an O-me thyl - 15, 373-379. transferase in volved in benzylisoquinoline biosynthesis. Planta 222, 458-471. Fester, T., Wray, V., Nimtz, M. & Strack, D. Is stimulation of carotenoid biosynthesis in arbuscular mycorrhizal DIPLOMA THESIS 2005 roots a general phenomenon? Phytochemistry 66, Tonleu Tonfack, Rostand: Einfluss von Epo thilonen und 1781-1786. verwandten Substanzen auf das Cytoskelett von Ara - bidopsis thaliana Wurzel spitzen. University of Hal le- Hause, B. & Fester, T. Molecular and cell biology of ar - Wittenberg, De partment of Biochemis try/Bio techno - buscular mycorrhizal symbiosis. Planta 221, 184-196. logy, 20/09/2005.

Hause, G., Lischewski, S., Wessjohann, L. A. & Hause, B. PUBLICATIONS 2006 Epothilone D affects cell cycle and microtubular pat- Delker, C., Stenzel, I., Hause, B., Miersch, O., Feussner, I. tern in plant cells. J. Exp. Bot. 56, 2131-2137. & Wasternack, C. Jasmonate biosynthesis in Ara bi - dopsis thaliana – Enzymes, products, regulation. Plant Hüsken, A., Baumert, A., Milkowski, C., Becker, H. C., Biol. 8, 297-306. Strack, D. & Möllers, C. Resveratrol glucoside (piceid) synthesis in seeds of transgenic oilseed rape (Brassica Frenzel, A., Tiller, N., Hause, B. & Krajinski, F. The con- napus L.). Theor. Appl. Genet. 111, 1553-1562. served AM-specific transcription of the secretory lec- tin MtLec5 is mediated by a short upstream se quence Hüsken, A., Baumert, A., Strack, D., Becker, H. C., Möl - containing specific protein binding sites. Planta 224, lers, C. & Milkowski, C. Reduction of sinapate es ter 792-800. content in transgenic oilseed rape (Brassica napus) by dsRNAi-based suppression of BnSGT1 gene ex pres - Hinneburg, A., Porzel, A. & Wolfram, K. An evaluation of sion. Mol. Breeding 16, 127-138. text retrieval methods for similarity search of mul ti- dimensional NMR-spectra. LWA 2006, 282-289. Isayenkov, S., Mrosk, C., Stenzel, I., Strack, D. & Hause, B. Suppression of allene oxide cyclase in hairy roots of Isayenkova, J., Wray, V., Nimtz, M., Strack, D. & Vogt, T. Medicago truncatula reduces jasmonate levels and the Cloning and functional characterisa tion of two regio- degree of mycorrhization with Glomus intraradices. selective flavonoid glucosyltransferases from Beta vul- Plant Physiol. 139, 1401-1410. garis. Phytochemistry 67, 1598-1612.

Kramell, R., Schmidt, J., Herrmann, G. & Schliemann, W. Kaiser, H., Richter, U., Keiner, R., Brabant, A., Hause, B. N-(Jasmonoyl)tyrosine-derived compounds from flo- & Dräger, B. Immunolocalisation of two tropinone re - wers of broad beans (Vicia faba). J. Nat. Prod. 68, 1345- ductases of potato (Solanum tuberosum L.) in root, 1349. stolon, and tuber sprouts. Planta 225, 127-137.

Liu, S., Chen, K., Schliemann, W. & Strack, D. Isolation Lohse, S., Hause, B., Hause, G. & Fester, T. FtsZ charac- and identification of arctiin and arcti-genin in leaves of terization and immunolocalization in the two phases of burdock (Arctium lappa L.) by polyamide column plastid reorganization in arbuscular mycorrhizal roots chromatography in combination with HPLC-ESI/MS. of Medicago truncatula. Plant Cell Physiol. 47, 1124- Phy tochem. Anal. 16, 86-89. 1134.

Lohse, S., Schliemann, W., Ammer, C., Kopka, J., Strack, Schaarschmidt, S., Roitsch, T. & Hause, B. Arbuscular D. & Fester, T. Organization and metabolism of plastids mycorrhiza induces gene expression of the apoplastic and mitochondria in arbuscular mycorrhizal roots of invertase LIN6 in tomato (Lycopersicon esculentum) Medicago truncatula. Plant Physiol. 139, 329-340. roots. J. Exp. Bot. 57, 4015-4023.

Sharma, V. K., Monostori, T., Hause, B., Maucher, H., Schliemann, W., Schmidt, J., Nimtz, M., Wray, V., Fester, T. Göbel, C., Hornung, E., Hänsch, R., Bittner, F., Waster - & Strack, D. Accumulation of apocarotenoids in mycor - nack, C., Feussner, I., Mendel, R. R. & Schulze, J. Ge netic rhizal roots of Ornitho ga lum umbellatum. Phy to che - transformation of barley to modify expression of a 13- mistry 67, 1196-1205. lipoxygenase. Acta Biol. Szegediensis 49, 33-34. Schliemann, W., Schneider, B., Wray, V., Schmidt, J., Strack, D., Hartmann, T. & Kutchan, T. M. Evolution of Nimtz, M., Porzel, A. & Böhm, H. Flavonols and an indo- 66 metabolic diversity. Phytochemistry 66, 1198-1199. le alkaloid skeleton bearing identical acylated glycosidic groups from yellow petals of Papaver nudicaule. Phyto - SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF SECONDARY METABOLISM

chemistry 67, 191-201. pflanze Medicago truncatula. University of Halle-Wit - Sharma, V. K., Monostori, T., Göbel, C., Hänsch, R., tenberg, Department of Pharmacy, 25/01/2006. Bittner, F., Wasternack, C., Feussner, I., Mendel, R. R., Schmidt, Diana: Die Sinapoylglucose:Cholin-Sinapoyl - Hause, B. & Schulze, J. Transgenic barley plants over- transferase aus Brassica napus. University of Halle- expressing a 13-lipoxygenase to modify oxylipin sig- Wittenberg, Department of Biology, 02/11/2006. nature. Phytochemistry 67, 264-276. HABILITATIONS 2006 Stehle, F., Brandt, W., Milkowski, C. & Strack, D. Struc - Vogt, Thomas: Plant Natural Product Glycosyl- and ture determinants and substrate recognition of se - Methyltransferases, University of Halle-Wittenberg, rine carboxypeptidase-like acyltransferases from Department of Biochemistry/Biotechnology, plant secondary metabolism. FEBS Lett. 580, 6366- 08/03/2006. 6374. Fester, Thomas: Struktur und Metabolismus der Plas - Strack, D. & Fester, T. Isoprenoid metabolism and pla- tiden kolonisierter Wurzelzellen bei der arbuskulären stid reorganization in arbuscular mycorrhizal roots. Mykorrhiza, University of Halle-Wittenberg, De part - New Phytol. (Tansley review) 172, 22-34. ment of Agricultural and Nutritional Science, 18/12/2006. Wasternack, C., Stenzel, I., Hause, B., Hause, G., Kut- ter, C., Maucher, H., Neumerkel, J., Feussner, I. & Miersch, O. The wound response in tomato – Role of jasmonic acid. J. Plant Physiol. 163, 297-306.

Weiss, D., Baumert, A., Vogel, M. & Roos, W. San gui - narine reductase, a key enzyme of benzophenanthridine detoxification. Plant Cell En viron. 29, 291-302.

Wolfram, K. & Hinneburg, A. Similarity search for mul - ti-dimensional NMR spectra of natural products. Knowlegde Discovery in Databases: PKDD 2006. Pro cee ding Lecture Notes in Computer Science 4213, 650-658.

Ziegler, J., Voigtländer, S., Schmidt, J., Kramell, R., Miersch, O., Ammer, C., Gesell, A. & Kutchan, T. M. Comparative transcript and alkaloid profiling in Pa pa - ver species identifies a short chain dehydro ge nase/ reductase involved in morphine biosynthesis. Plant J. 48, 177-192.

PUBLICATION IN PRESS: Cenzano, A., Abdala, G. & Hause, B. Cyto che mical immuno localization of allene oxide cyclase, a jasmonic acid biosynthetic enzyme, in developing potato sto- lons. J. Plant Physiol.

BOOK CHAPTER 2006 Hause, B., Frugier, F. & Crespi, M. Immunocyto che mis - try. In: The Medicago truncatula handbook. http://www.noble.org/MedicagoHandbook/

DIPLOMA THESES 2006 Götz, Stephan: (supervision together with Prof. C. Wasternack, NBT): Die Rolle von Jasmonaten in der Embryonalentwicklung der Tomate, University of Hal - le-Wittenberg, Department of Biochemis try/Bio - technology, 02/08/2006.

Knick, Paul: Die systemische Wundantwort zwischen Wurzel und Spross der Tomate (Lycopersicon esculentum), University of Halle-Wittenberg, Department of Biochemistry/Biotechnology, 07/09/2006.

DOCTORAL THESES 2006 Lohse, Swanhild: Untersuchung der Plastiden entwick - 67 lung bei der arbuskulären Mykorrhiza an der Modell- ADMINISTRATION AND TECHNICAL SERVICES Head: Lothar Franzen

Secretary: Cindy Maksimo

uring the past two years, two construction projects have dominated the activities of the IPB administration. The construction of additional fully climate-con- Dtrolled greenhouses was completed in April 2005. In August 2005, construction of a new building (House E) for the employees of facilities, construction management, graphic arts, horticulture and members of the bioinformatic group was initiated. The project was finished in December 2006. The total construction cost of each building was 2.4 million Euros and was provided by the Federal Ministry of Education and Research and by the Ministry of Education and Cultural Affairs of the State of Saxony-Anhalt.

The new greenhouses, with an additional 350 square meters of floor space, met the growing demand of the departmental research programs involving transgenic plants. The nine air-conditioned chambers were opened for cultivation of tomato, poppy, tobacco, rape, and plants from the mycorrhization projects. In addition, there are two tropical chambers and incubators for Arabidopsis. The technical equipment for the fine control of climatic conditions, as light, shadow, temperature and humidity is state-of-the-art and costs about 1.1 million Euros.

Two new buildings replaced the building that accomodated employees of facilities and construction management, graphic arts and horticulture. Within this new complex, an additional 200 square meters of laboratorys space for 15 new working places for biological and chemical research of junior research groups were created, as well as new offices for the bioinformaticians of the Department of Stress and Developmental Biology.

68 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

Construction of the new green houses was completed in April 2005.

The new building House E.

69 ADMINISTRATION EMPLOYEES & EMPLOYEE STATISTICS

ACCOUNTING GRAPHIC ARTS Head: Barbara Wolf Head: Christine Kaufmann Maike Hildebrandt Annett Kohlberg Gudrun Schildberg Kerstin Wittenberg HORTICULTURE Head: Iris Rudish CHAUFFEUR Martina Allstädt Jürgen Gaul until January 2006 Annett Grün, Trainee Matthias Barth since February 2006 Christian Müller Philipp Plato, Trainee COMPUTER SUPPORT Kristina Rejall, Head: Hans-Günter König Steffen Rudisch Holger Bartz Katja Scheming Matthias Franke until December 2006 Andrea Voigt Ronald Scheller Sabine Vogt Kevin Begrow, Trainee until September 2006 Marcel Volkmann, Trainee HUMAN RESOURCES Tibor Sari, Trainee since September 2006 Head: Kerstin Balkenhohl Alexandra Burwig CONSTRUCTION MANAGEMENT Claudia Haferung-Bornmann since Head: Heike Böhm September 2006 Catrin Timpel Kathleen Weckerle until August 2006

FACILITIES MANAGEMENT LIBRARY SERVICES Head: Michael Kräge Head: Andrea Piskol Carsten Koth Anja Gärtner, Trainee Jörg Lemnitzer Klaus-Peter Schneider PRESS AND PUBLIC RELATIONS Eberhard Warkus Head: Sylvia Pieplow Susanne Kubenz since June 2005 GENERAL ADMINISTRATION Head: Rosemarie Straßner STOCKROOM Heide Pietsch until April 2006 Hans-Jürgen Steudte Clemens Schinke Elviera Schotte Trainees Ines Deák since December 2006 Caroline Stolzenbach since September 2006 Kristin Weinert since December 2006

70 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

EMPLOYEE STATISTICS 2005 2006

AVERAGE NUMBER OF MMPLOYEES 188 180

Full-time employees in % 61 65

Part-time employees in % 39 35

Permanent positions 92 92

Temporary positions 26 23

Employees externally funded 43 38 Employees remunerated by “University Science Funds Program” (Hochschulwissenschaftsprogramm / HWP) 53

Proportion of female employees in % 58 60

Personnel turnover in % 9 29

Averade age of employees in years 37 37

Scholarship / fellowship holders 27 34

VOCATIONAL TRAINING general administration 3 3 horticulture 2 2 library services 1 1 computer support 2 2 research technical assistant 2 3

Succesfully completed vocational training 24

Average number of apprentices 10 11

71 RESOURCES AND INVESTMENTS & FUNDS FROM EXTERNAL SOURCES IN 2005 AND 2006

Research grants listed on this and the following in Mio. Euro in % pages were given by BASIC FINANCING FUNDS (2005 + 2006) Bionorica Bionorica AG Personnel 9.9 41.9 BMBF Bundesministerium für Bildung und Forschung - Federal Ministry Consumables 4.6 19.6 for Education and Research Grants / Subsidies 0.2 0.8 BPS BASF Plant Science Investments 5.2 22.0 DAAD Deutscher Akademischer Austauschdienst - German Academic Exchange Service University Science Funds Program (HWP) 0.3 1.3

DBU Deutsche Bundesstiftung Subtotal 20.2 Umwelt - German Environmental Foundation FUNDS FROM EXTERNAL SOURCES (2005 + 2006)

DFG Deutsche Forschungsgemeinschaft BMBF 0.9 3.9 German Research Foundation MK-LSA 0.1 0.4 Elsevier Elsevier Science Publisher DFG 1.8 7.6 EU European Union Industry 0.3 1.3 Firmenich Firmenich Company

Hopsteiner Hopsteiner Company EU 0.2 0.8

HWP Hochschulwissenschafts - other 0.1 0.4 programm - University Science Funds Program Subtotal 3.4

Icon Genetics Icon Genetics AG TOTAL 23.6 100.0

MK-LSA Kultusministerium des Landes Sachsen-Anhalt - Ministry of Education and Cultural Affairs of in Million Euro the State of Saxony-Anhalt INVESTMENTS MLU Martin-Luther-Universität Halle- Wittenberg - University of Halle Equipment 2.4

Probiodrug Probiodrug AG Construction 2.8

TOTAL 5.2

Funds from HWP Investments external sources 1.3 % 22.0 % 14.4 %

Consumables 20.4 % Personnel 41.9 %

72 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2005-2006) Funded

DEPARTMENT OF NATURAL PRODUCT BIOTECHNOLOGY

Jasmonate biosynthesis regulation 04/05 DFG 13,000 1 C. Wasternack & O. Miersch

12-Hydroxyjasmonic acid 03/06 DFG 26,100 1 C. Wasternack & O. Miersch

Glutamatcyclase 01/05 Probiodrug 2,900 0 C. Wasternack

Allene oxide cyclase 01/05 Firmenich 7,600 0 C. Wasternack

Analysis of genes 00/05 Icon Genetics 14,200 0 T. Kutchan

Molecular genetics of isoquinoline alk. biosynth. 04/05 DFG 31,200 2 T. Kutchan

Molecular genetics in Liana Triphyoph. Pellatum 03/05 DFG 36,000 1 T. Kutchan 05/06 DFG 42,500

Metabolic engineering 04/06 DFG 78,500 2 S. Frick

12-Hydroxyjasmonat-Arabidopsis 05/08 DFG / SFB 648 176,000 2 C. Wasternack & O. Miersch

HUM-NEU 03/05 Hopsteiner 19,500 1 J. Page & T. Kutchan

Genexpressionsanalyse in Papaver-Spezies 05/07 DFG 32,200 1 J. Ziegler Heterodimerbildung als Regulationsmecha nis mus der Allenoxidcyclase in Arabidopsis 2006 MK-LSA / MLU 4,500 0 C. Wasternack & B. Hause

Subtotal 484,200 11

DEPARTMENT OF BIOORGANIC CHEMISTRY

HEANTOS 05/06 BMBF 96,300 2 L. Wessjohann

MCR ligand synthesis 04/06 DAAD/Probral 11,400 0 L. Wessjohann

Reactivity of selenopeptides 03/05 DFG 7,500 1 L. Wessjohann & W. Brandt 04/06 DFG 49,600 1

CERC-3 04/06 DFG 45,700 1 L. Wessjohann 06/07 DFG 27,800

HUMULUS 03/05 Hopsteiner 9,100 0 L. Wessjohann

Fungi Metabolites 04/06 DFG 33,400 1 N. Arnold & J. Schmidt

Virtual Screening 04/07 Wella 108,200 1 L. Wessjohann

Benzopyrane 05/07 Bionorica 69,600 1 L. Wessjohann 73 FUNDS FROM EXTERNAL SOURCES IN 2005 AND 2006

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2005-2006) Funded

DEPARTMENT OF BIOORGANIC CHEMISTRY

Mannich Diversity 05/07 DFG 82,200 1 B. Westermann

Chalcogen catalysts 06/07 DFG 61,700 1 L. Wessjohann

Prenylierende Enzyme 05/07 DFG 29,700 1 W. Brandt & L. Wessjohann

DBU-Acyloine DBU / 06/08 4,700 0 L. Wessjohann Uni Greifswald

Subtotal 636,900 11

DEPARTMENT OF STRESS- AND DEVELOPMENTAL BIOLOGY

Metalhome 03/06 EU 130,200 1 S. Clemens

NODO 02/05 EU 63,300 1 S. Rosahl

Bioinformatics and mass spectrometry 05/07 BMBF 272,200 2 D. Scheel

Resistence in potatoes 04/06 DFG 28,500 1 D. Scheel & S. Rosahl

GABI-NONHOST 02/05 BPS / BMBF 78,700 2 D. Scheel

Metal homeostasis 04/06 DFG 125,900 1 S. Clemens

GABI-GENOPLANTE 04/06 BMBF 64,100 1 S. Clemens

SARA 04/07 BMBF 116,700 1 D. Scheel

Molekulare Kommunikation von R. secalis 05/08 DFG / SFB 648 68,900 1 W. Knogge

Oxylipine bei Pathogenabwehr 05/08 DFG / SFB 648 103,000 2 S. Rosahl

MAPK-Kaskaden in A. thaliana 05/08 DFG / SFB 648 170,000 2 D. Scheel Transcriptome and proteome analysis MK-LSA of pathogen attacked barley epidermis 05/06 16,900 0 MLU W. Knogge Einfluss der Chromatinstruktur auf die Wechsel- MK-LSA wirkung von A. thaliana mit versch. Pathogenen 2006 9,000 0 MLU D. Scheel Role of MAPKs in female gametophyte/embryo MK-LSA development 2006 9,000 0 MLU J. Lee

Subtotal 1,256,400 15 74 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2005-2006) Funded

DEPARTMENT OF SECONDARY METABOLISM

Metabolism of isoprenoids 04/06 DFG 48,400 1 M. H. Walter The role of jasmonates during the establishment of mycorrhiza 04/06 DFG 59,600 1 B. Hause & D. Strack Carotenoid biosynthesis in arbuscular mycorrhizal roots 04/06 DFG 50,700 1 T. Fester Metabolite Profiling 04/06 DFG 128,800 1 W. Schliemann

Phytochemistry 02/07 Elsevier 44,700 1 D. Strack

HCA-Glucosyltransferases 03/05 DFG 2,000 1 C. Milkowski & A. Baumert 05/06 DFG 31,800

HCA-Glucosyltransferases 06/08 DFG 17,900 1 C. Milkowski

SCPL-Acyltransferases 03/05 DFG 15,300 1 C. Milkowski & D. Strack 05/07 DFG 49,300

DXS-Isoenzyme 05/07 DFG 59,900 1 M. H. Walter

Struktur und Funktion der Sinapinesterase 05/07 DFG 65,500 1 D. Strack

PFOMT 04/06 DFG 9,800 0 T. Vogt

DXS-Evolution 06/07 DFG 20,100 1 M. H. Walter

Caffeoylglucarat-Synthase 06/09 DFG 22,100 1 D. Strack & B. Hause

YellowSin Rape Seed 06/09 BMBF 72,000 3 D. Strack Heterodimerbildung als Regulationsmechanismus MK-LSA der Allenoxidcyclase in Arabidopsis 2006 4,500 0 MLU C. Wasternack & B. Hause Jasmonat, arb. Mykorrhiza, Cytoskelett 2006 DFG 9,800 0 B. Hause

Subtotal 712,200 15

75 FUNDS FROM EXTERNAL SOURCES IN 2005 AND 2006

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2005-2006) Funded

JOINT PROJECTS

GABI 2 04/07 BMBF 296,500 3 D. Scheel, S. Clemens, L. Wessjohann & J.

Subtotal 296,500 3

Projects granted total 3,386,200 55

GENERAL OVERVIEW

BMBF 917,800 12

MK-LSA 43,900 0

DFG 1,860,400 35

Industry 302,200 5

EU 193,500 2

Other Sources 68,400 1

Total 3,386,200 55

HWP 283,100 2

Total 3,669,300 57

76 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

INVOLVEMENT OF THE IPB IN NATIONAL AND INTERNATIONAL SCIENTIFIC NETWORKS

BIOSCIENTIFIC NETWORK OF SAXONY-ANHALT many, France Structures and Mechanisms of Biological In for - HEANTOS mation Processing. A Vietnamese opiate detoxification symp tom medication CERC 3 German Federal Ministry for Education, Re - Chairmen of the European Research Coun cils search and Technology (BMBF) Chemistry Committees German Research Foundation MOLECULAR ANALYSIS OF PHYTOHORMONE ACTION EVOMET Priority Program SPP 1067 of the German Re - Evolution of Metabolic Diversity search Foundation Priority Program 1152 of the German Re search MOLECULAR MECHANISMS OF INFORMATION Foundation PROCESSING IN PLANTS Collaborative Research Center SFB 648 of the GABI German Research Foundation Genome Analysis of the Plant Biological Sys tem MOLMYK German Federal Ministry for Education, Re - Molecular Basics of Mycorrhizal Sym bio ses search and Technology (BMBF) and Business Priority Program 1084 of the German Re search Companies Foundation

GABI-NONHOST ORGANOCATALYSIS A consortium-based functional geno mics ini - Priority Programm 1179 of the German Re - tiative on plant non-host disease resistance search Foundation GABI 1b - Mannich Diversity (Prof. Westermann) - Chalcogen Catalysts (Prof. Wessjohann) METABOLOMICS PLATFORM Metabolite Profiling SELF-ORGANIZATION THROUGH COORDINA TION in Arabidopsis and Crop Plants AND NON- COVALENT INTERACTIONS GABI 2 Graduate Program 894 of the German Re - search Foundation SARA Functional genomics of local and systematic SELENOPROTEINS acquired resistance in Arabidopsis Priority Programm SPP 1087 of the German GABI - trilateral Cooperation, France, Ger many, Research Foundation Spain YELLOWSIN COMPARATIVE GENOMICS Functional genomic approaches for the deve- Comparative genomics between Arabi dopsis lopment of yellow-seeded, low sinapine oilseed and Brassica for genes directing seed-specific rape (Canola; Brassica napus) flavonoid biosynthesis GABI-Kanada, bilateral cooperation GABI - Génoplante - bilateral Cooperation, Ger -

77 SEMINARS, WORKSHOPS AND COLLOQUIA 2005

JANUARY 13 MAY 17 JULY 12 Prof. Udo Johanningmeier, Dr. Charl FJ Faul Dr. Jochen Balbach University of Halle, Ger many University of Bristol, UK University of Halle, Germany Genetic engineering of photosystem II: Functional nanostructures from ionic in- Untersuchung der Proteinfaltung mit Po tential role of methionine residues as teractions - some concepts and general NMR-Spek troskopie. antioxidants. principles. JULY 13 FEBRUARY 10 MAY 25 Prof. Martin Quack Dr. Frantisek Baluska Prof. Claus E. Schäfer University of Copenhagen, Danmark University of Bonn, Germany University of Copenhagen, Denmark Molekülspektroskopie und kinetische Actin cytoskeleton, myosin VIII and polar The present day importance of ligand- Primärpro zesse zwischen Yoctosekun - transport of auxin: Lessons from maize field theory in a historical perspective. den und Jahrmilliar den. root apices. MAY 31 AUGUST 4 FEBRUARY 17 PD Alexander Dömling Dr. Siegbert Melzer Prof. Michael Bölker R&D Biophar maceu ti cals GmbH, Mar- University of Ghent, Belgium University of Marburg, Germany tinsried, Germany Impacts of flowering time genes on plant The small GTPases Cdc42 and Rac1 re- Recent advancement in multicompo- growth. gulate cy tokinesis and dimorphism in nent reaction chemistry. the phytopathogenic fungus Ustilago SEPTEMBER 8 maydis. JUNE 2 Prof. Paola Bonfante Prof. Kurt Eger University of Turin, Italy MARCH 23 University of Leipzig, Germany Roots, mycorrhizal fungi and endobacte- Prof. Karsten Krohn Beiträge zur Arzneimittel(weiter)ent- ria: The development of an underground University of Paderborn, Germany wicklung an der Hochschule. web. Computerunterstützte Synthese von chiralen Bausteinen für Makrolide aus JUNE 7 SEPTEMBER 15 Anhydrozuckern. Dr. Günter Hempel Prof. Cathie Martin, University of Halle, Germany John Innes Centre, Norwich, UK APRIL 6 Verfolgung der Kristallisation von Sei - The molecular genetics underpinning Prof. Karlheinz Drauz tenketten in Polymeren mittels 13C- deve lopment of specialised pollination Degussa AG Düsseldorf, Germany CPMAS. syndromes in So lanaceous plants. Bedeutung der Katalyse in der industri - ellen Fein chemie. JUNE 14 SEPTEMBER 20 Dr. Christoph A. Schalley Stefan Gröger APRIL 13 University of Bonn, Germany University of Halle, Germany Prof. Uwe Conrath Templateffekte und Selbstorganisation NMR-Anwendungsbeispiele aus der University of Aachen, Germany zum Auf bau komplexerer funktionaler pharmazeutischen Industrie. Priming in plants: It is all the world to in - Strukturen. duced di sease resistance SEPTEMBER 22 JUNE 15 Prof. Peter Meyer APRIL 14 Prof. Karl Jug University of Leeds, UK Prof. Petra Dietrich University of Hannover, Germany Natural antisense transcripts in plants. University of Erlangen, Germany Clustermodelle und Molekulardynamik: Redox regulated calcium channels in Neue Me thoden für Festkörper, Ober - SEPTEMBER 29 Arabidopsis mesophyll cells. flächen und Katalyse. Prof. Klaus Harter University of Tübingen, Germany APRIL 21 JUNE 23 Plant two-component systems: Prin ci - Prof. Jerzy Paszkowski Prof. Andrzej Guranowski ples, functions, complexity and cross University of Genf, Switzerland University of Poznan, Poland talk. Genetics of epigenetic regulation of Uncommon dinucleotides-putative sig - transcription in Arabidopsis. naling compounds and enzymes involved OCTOBER 5 in their metabolism. Dr. Erich Glawischnig MAY 11 Technical University of Munich, Ger many Prof. Tim Clark JULY 6 Biosynthesis of camalexin. University of Erlangen, Germany Prof. Christoph Schneider New concepts for theoretical studies of University of Leipzig, Germany intermolecular interactions. Stereoselektive Synthese mit chiralen Auxiliaren und Katalysatoren.

78 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

OCTOBER 6 tors: Iron or not iron? Cycloamidine-wertvolle Bausteine in Pascal Arnoux, NOVEMBER 3 der Syn thesechemie Laboratoire de Bioénergétique Cellu - Prof. Georg G. Gross DECEMBER 5 laire, CEA, Cadarache, France University of Ulm, Germany Prof. Frank Glorius A papain-like enzyme at work: Native Biosynthesis of hydrolyzable plant tan- University of Marburg, Germany and acyl-enzyme intermediate struc - nins - unraveled by enzyme studies. Katalysator- und Reaktionsdesign für tures in phytochelatin synthesis. eine effiziente Synthese. NOVEMBER 10 OCTOBER 12 Prof. Maarten DeWaard DECEMBER 7 Dr. Deepti Dwivedi University of Wageningen, The Nether- Prof. Bernhard Eikmanns University of Basel, Switzerland lands University of Ulm, Germany Interactions between arbuscular mycor- Functions of ABC transporters in plant Die Bedeutung der PEP-Pyruvat-Oxal - rhizal fungi and antagonistic bacteria pathogenic fungi. acetat-Ver zweigung im Stoffwechsel des (Pseudomonas fluorescens and Aminosäure produ zenten Coryne bac te - Alcaligenes faecalis) and their effects on NOVEMBER 16 ri um glutamicum. Vigna mun go. Dr. Joachim Kopka Max Planck Institute of Molecular Plant DECEMBER 8 OCTOBER 13 Physiology, Golm, Germany PD Peter Spiteller Prof. Sabeeha Merchant, Current challenges and developments in University of Munich, Germany University of California, Los Angeles, metabolite profiling. Interaktion zwischen mykoparasitischen USA Pilzen und ihren Wirtspilzen-neue An - Between a rock and a hard place: Cop - NOVEMBER 21 sätze zur Iso lie rung aktiver Naturstoffe. per homeostasis in Chlamydo mo nas. Manfred Knörgen University of Halle, Germany DECEMBER 15 OCTOBER 17 In-vivo Beobachtung von Arzneiträger - Dr. Christian Eckmann, Prof. Zeev Luz, systemen (Tabletten/Pellets) im Magen Max Planck Institute of Molecular Cell Weizmann Institute of Science, Rehovot, mittels Suszepti bili tätskontrast-MRT. Biology and Genetics, Dresden, Ger many Israel Regulating germ cell fate switches at the NMR of methyl groups in the thermal NOVEMBER 23 translational level - a theme of GoLD. and tunneling regimes. Prof. Janos Szabad University of Szeged, Hungary DECEMBER 19 OCTOBER 20 The tubulin-centrosome interplay. Dr. Thomas Bräuniger Dr. Christine Oesterhelt University of Halle, Germany University of Potsdam, Germany NOVEMBER 24 Exotische Anwendungen der NMR- Living on the edge - metabolic versatility Dr. Claude Parsot Spektros kopie. of the unicellular red alga Galdieria sul - Institut Pasteur, Paris, France phuraria. Control for transcription by the activity of the type III secretion apparatus in Shi - OCTOBER 26 gella flexneri. Dr. Matthieu Arlat, Institut National de la Recherche Agro - NOVEMBER 29 no mique/INRA Paris, France Prof. Rainer Beckert Xanthomonas TonB-dependent recep- University of Jena, Germany

79 SEMINARS, WORKSHOPS AND COLLOQUIA 2006

JANUARY 11 form for mass spectrometry based proteo- PD Anke Becker mics. MAY 19 University of Bielefeld, Germany Prof. Sarah Grant Postgenome approaches to Sinorhizobium APRIL 12 University of North Carolina, Chapel Hill, USA meliloti: a model for nitrogen-fixing endo- Olaf Schröder The bacterial virulence factor AvrPpiB ma - symbiotic soil bacteria. Pattern Expert, Borsdorf, Germany kes plants more sensitive to ABA. Detecting biomarkers in mass spectra with JANUARY 17 pattern recognition methods. MAY 24 Dr. Ulf Mazurek Prof. Edward Farmer Hebrew University of Jerusalem, Israel APRIL 25 University of Lausanne, Switzerland Das protonierte Serin-Oktamer in der Gas - Dr. Hanne Schönig Genetic approaches to study lipoxygenase phase. University of Halle, Germany re gulation and oxylipin biogenesis. Ethnobotanisches aus dem Jemen. JANUARY 20 Prof. Martin Feigel Prof. Ing. Miroslav Strnad APRIL 27 University of Bochum,Germany University of Olomouc, Czech Republic Prof. Harry Klee Rings and knots of amino acids in peptide Biotechnological and medicinal applications University of Florida, Gainesville, USA cavitands and steroid peptides. of cytokinin analogues. Synthesis and functions of plant volatile compounds. Some unexpected functions. MAY 29 JANUARY 26 Prof. Paul Staswick Dr. Marcel Bucher MAY 5 University of Nebraska, USA ETH Zürich, Switzerland Prof. Gregory P. Copenhaver Plant hormone regulation by amino acid Mechanisms and interactions in plant phos- University of North Carolina, Chapel Hill, conjugating enzymes; jasmonate, auxins and phate uptake. USA more! The power of four: exploring meiotic re - FEBRUARY 7 combination in Arabidopsis. JUNE 1 Hans Wolf Sünnemann Prof. Gregg A. Howe University of Göttingen, Germany MAY 9 Michigan State University, USA Palladium-catalyzed coupling-sequences as Prof. Peter Imming Regulation of plant antiherbivore defense diversity oriented efficient access to ste - University of Halle, Germany by the jasmonate signaling pathway. roid-skeletons. You can cure by appearances. Similiarity, biomimesis, and the connection between JUNE 7 FEBRUARY 23 molecular and clinical effects. Prof. Francois Buscot Prof. Michael Göttfert Helmholtz Centre for Environmental Re- University of Dresden, Germany MAY 10 search Leipzig-Halle, Germany Regulation of nodulation and type III secre- Prof. Hans-Günter Schmalz Exploring structural and functional diversity tion genes in Bradyrhizobium japonicum. University of Cologne, Germany of soil and mycorrhizal fungi: why and how? Übergangsmetalle in der Synthese bioakti- MARCH 2 ver Verbindungen. Prof. Ute Wittstock Prof. Kazufumi Yazaki University of Braunschweig, Germany University of Kyoto, Japan MAY 11 Metabolic diversity in an activated plant de - Plant ABC transporter, PGP4, catalyzes Prof. John Mansfield fense system. auxin transport in Arabidopsis roots. Imperial College London, UK Evolution of pathogenicity and virulence in JUNE 14 MARCH 16 Pseudomonas syringae: a tale of populations Prof. Lukas Gooßen Prof. Vincent Colot and papillae. University of Kaiserslautern, Germany Unité de Recherche en Génomique Végé - Neue katalytische Methoden von der inno- tale, INRA, CNRS, Evry, France MAY 15 vativen Synthesechemie zur “dream reac - Trans-generational inheritance of epigenetic Dr. Hendrik Metz tion”. variation in Arabidopsis: an epigenomic per- University of Halle, Germany spective. Hallesches NMR-Seminar: EPR in der Phar - JULY 13 mazie. Dr. Antonio Molina MARCH 23 Centro Biotecnología Genómica de Plantas, Dr. Thomas Müller-Reichert MAY 16 Madrid, Spain Max Planck Institute of Cell Biology and Prof. Sabine Flitsch Plant innate immunity and resistance to Ge netics, Dresden, Germany University of Manchester, UK necrotrophic fungi: sense and defense. Electron tomography of centriole assembly Biocatalysis on solid phase. in C. elegans. JULY 20 MAY 18 PD Peter Dörmann Dr. Clemens Groepel Prof. Jeff Dangl Max Planck Institute of Molecular Plant Phy - University of Berlin, Germany University of North Carolina, Chapel Hill, USA siology Potsdam-Golm, Germany OpenMS - An open source software plat- Molecular logic of the plant immune system. Biosynthesis and function of galactolipids

80 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

and vitamins in chloroplasts. Prof. Christiane Gatz Dr. Mark Helm OCTOBER 26 University of Göttingen, Germany University of Heidelberg, Germany Prof. Paul Tudzynski Analysis of transcriptional control mecha- Posttranscriptional modification and struc- University of Münster, Germany nisms in plant defense responses. tural dynamics in tRNA. ROS in biotrophic vs. necrotrophic host-pa - thogen interactions. Prof. Klaus D. Grasser SEPTEMBER 14 Aalborg University, Denmark Prof. Steffen Abel NOVEMBER 2 Architectural chromosomal proteins and University of California, Davis, USA PD Ralph Hückelhoven transcript elongation factors. Phosphate sensing in the Arabidopsis root University of Gießen, Germany meristem. Regulation of actin remodelling and cell Prof. Bernd Weisshaar death in the interaction of barley with pow - University of Bielefeld, Germany SEPTEMBER 15 dery mildew fungi. Regulation of flavonol biosynthesis by MYB Prof. Brian Ellis transcription factors. University of British Columbia, Vancouver, NOVEMBER 9 Ca nada Dr. Giles Oldroyd JULY 21 Map kinases and stress hormone signaling: John Innes Centre Norwich, UK Dr. Jürgen Kroymann old and new connections. Signalling in symbiosis. Max Planck Institute for Chemical Ecology Je na, Germany SEPTEMBER 25 NOVEMBER 14 Functional & evolutionary genomics of Ara - Prof. Birger L. Møller Dr. Marcel Quint bidopsis & friends. Royal Veterinary & Agricultural University, University of Minnesota, USA Copenhagen, Denmark Molecular dissection of auxin response: a Dr. Arp Schnittger Metabolic engineering of secondary meta- ‘complex’ story of plant development. University of Cologne, Germany bolites for crop improvement: lessons lear- Cell cycle control at the interface between ned from cyanogenic glucosides. NOVEMBER 23 development and environment. Prof. Franz Narberhaus SEPTEMBER 28 University of Bochum, Germany PD Joachim Uhrig Dr. Melisa Lim Temperature sensing and membrane lipids University of Cologne, Germany Carnegie Institution, Stanford, USA in plant-interacting bacteria. Protein interactions networks: functional A role for phytochelatin synthase in resi- plant proteomics and host-virus interac - stance to fungal pathogens in A. thaliana. NOVEMBER 28 tions. Dr. Thomas Ott SEPTEMBER 29 INRA-CNRS Toulouse, France Dr. Ute Krämer Prof. Sebastian Böcker Understanding active networks and new Max Planck Institute of Molecular Plant Phy - University of Jena, Germany systems of cell-to-cell communications in siology Potsdam-Golm, Germany Decomposing metabolites: Identifying meta- hi gher plants mediated by proteins har - Metal homeostasis: Extreme natural varia - bolites using high precision mass spectro- bored on lipid rafts. tion among closely related model plants in metry. a complex functional network that is indis- Dr. Verónica Albrecht pensable for plant biology. OCTOBER 4 ETH Zurich, Switzerland Prof. Maria Harrison Snowy cotyledon (sco): a mutant group af - JULY 27 Boyce Thompson Institute for Plant Re - fected in chloroplast development in seed- Prof. Axel Brakhage search, Ithaca, USA lings. University of Jena, Germany The arbuscule mycorrhizal symbiosis: Deve - Secondary metabolites as virulence deter- lopment and function of the arbuscule/cor- NOVEMBER 29 minants of the human-pathogenic fungus tical cell interface. Ulrich Weininger As per gillus fumigatus. University of Halle, Germany OCTOBER 19 Structure and function of a protein folding AUGUST 30 Dr. Christoph Peterhänsel catalyzing protein. Ronald Peltsch, Andreas Lenz University of Aachen, Germany tekom-Gesellschaft für Technische Kom - Signal integration on the chromatin level - a DECEMBER 14 mu ni kation e.V. Halle, Germany case study in maize. Prof. Johan Memelink Übersichtliche und verständliche Präsenta - University of Leiden, The Netherlands tionen und Vorträge. OCTOBER 24 Transcription factor ORA47 - a regulator of Prof. Christian Stark jasmonate biosynthesis in Arabidopsis. SEPTEMBER 7 University of Berlin, Germany Dr. Ali Masoudi-Nejad Katalytische Oxidationsreaktionen zur ste- DECEMBER 21 University of Teheran, Iran reoselektiven Synthese von Sauerstoff hete - Prof. John A. Pickett EGENES & EGassembler: new bioinforma - rocyclen. Rothamsted Research, Harpenden, UK tics tools for the post-genome era. Potential for exploiting biotic stress signal-

81 GUEST RESEARCHERS AND FELLOWS

DEPARTMENT OF NATURAL PRODUCT Cristiano Rodrigo Bohn Rhoden, BIOTECHNOLOGY Bra zil Prof. Guillermina Abdala, Argentina DAAD Fellow 2005-8-30 – 2005-9-30 2004-10-1 - 2006-12-31

Dr. Chotima Böttcher, Thailand Dr. Carlos Boluda, Spain 2005-3-14 – 2006-4-1 Manuel Morales Fellow 2005-2-1 - 2006-2-28 Maria Luisa Diaz Chávez, Mexico Promep Fellow Dr. Andriy Buchynskyy, Ukraina 2003-10-1 – 2007-2-28 2006-9-1 - 2006-12-31

Prof. Andrzej Guranowski, Poland Ivana Correa Ramos Leal, Brazil 2005-6-13 – 2005-7-15 CAPES Fellow 2006-6-2 – 2006-12-22 Aphacha Jindaprasert, Thailand The Thailand Research Foundation Victor Dick, Germany 2004-10-18 – 2006-1-31 Fellow of the Dr. Arnold Hueck Foundation 2004-4-1 - 2006-7-31 Jana Kufová, Czech Republic 2006-2-6 – 2006-4-6 Dr. Alexander Dömling, Germany 2006-4-25 – 2006-6-23 Dr. Mariko Oka, Japan 2005-10-4 – 2006-9-30 Simon Dörner, Germany Fellow Studienstiftung des Deutschen Volkes Alfonso Lara Quesada, Costa Rica 2004-4-1 - 2006-5-30 DAAD Fellow 2003-4-1 – 2006-12-31 Kanchana Dumri, Thailand DAAD Leibniz Fellow Khaled Sabarna, Palestine 2004-3-1 - 2006-12-31 2006-9-1 – 2006-12-1 Otilie Eichler Vercillo, Brazil Anne Schwedt, Germany DAAD Fellow / CNPq 2006-1-2 – 2006-2-28 2005-9-12 - 2006-12-31

Prof. Meinhart Zenk, Germany Daniel Garcia Rivera, Cuba 2000-1-1 – 2006-3-31 Graduiertenkolleg 2003-10-1 - 2006-12-31 DEPARTMENT OF Gergely Gulyas, Hungary BIOORGANIC CHEMISTRY 2005-4-1 - 2006-12-31 Dr. Nasser Abdullah Ali, Yemen DAAD Fellow Alexander Gutsche, Germany 2006-7-15 – 2006-10-1 2006-2-1 – 2006-4-30 Muhammad Ayaz, Pakistan Dr. Nguyen Hoang Anh, Vietnam 2006-4-21 – 2006-12-31 2005-6-1 – 2006-7-31 Dr. Susanne Aust, Germany Myint Myint Khine, Myanmar Probiodrug AG Daimler Benz Fellow 2003-3-1 - 2006-12-31 2002-9-4 - 2006-3-1

82 SCIENTIFIC REPORT 2005 - 2006 DEPARTMENT OF ADMINISTRATION AND TECHNICAL SERVICES

Dr. Tilo Lübken, Germany Dr. Stephan Clemens, Germany 2006-7-1 – 2006-9-1 2006-9-1 - 2006-11-30

Fredy Leon Reyes, Cuba Dr. Emiko Harada, Japan Graduiertenkolleg 2002-2-22 - 2005-7-22 2005-5-1 – 2006-12-31 Dr. Nicholas Harpham, Germany Yulita Mitei, Kenya 2006-6-23 - 2007-6-30 DAAD Fellow 2006-4-1 – 2006-9-30 Dr. Ingo Hofmann, Germany 2004-1-11 - 2008-12-31 Tran Thi Phuong Thao, Vietnam 2001-11-1 – 2006-8-31 Dr. Jens Katzek, Deutschland 2004-11-1 - 2007-5-31 Prof. Luay Rashan, Jordan Humboldt Fellow Dr. Dirk Schenke, Germany 2005-7-5 - 2005-10-5 2006-2-16 - 2007-12-31

Jasqer Alonso Sehnem, Brazil David Sondermann, Germany DAAD Fellow 2006-11-1 - 2007-4-30 2004-10-3 - 2005-4-2 Nicole Staroske, Germany Josef Skopek, Czech Republic Graduiertenprogramm EMBL Fellow 2006-4-1 - 2007-12-31 2005-4-4 - 2005-7-1 Dr. Aleksandra Trampczynska, Poland Prof. Tran Van Sung, Vietnam 2006-10-1 - 2006-11-30 July, August and December 2005 Dr. Stefanie Wetzel, Germany Dr. Trinh Thi Thuy, Vietnam Graduiertenprogramm 2005-6-1 – 2006-7-31 2006-1-1 - 2006-9-30

Marcio Weber Paixao, Brazil Dr. Michael Weber, Germany DAAD Fellow 2006-10-1 - 2006-11-30 2005-10-4 - 2006-3-30 Dr. Esther van der Zalm, Dr. Heike Wilhelm, Germany The Netherlands Bio Service GmbH, EU and the State of Graduiertenprogramm Saxony Anhalt 2005-12-16 – 2008-12-31 2003 -8-1 - 2006-4-30 DEPARTMENT OF SECONDARY METABOLISM DEPARTMENT OF STRESS AND Dr. Kirill Demchenko, Russia DEVELOPMENTAL BIOLOGY 2006-4-24 – 2006-6-30 Albor Dobón Alonso, Spain 2006-9-1 - 2006-11-30 Dr. Zakir Hossain, Bangladesh Humboldt Fellow Dr. Jolly Basak, India 2006-4-1 – 2008-3-31 Humboldt Fellow 2006-8-1 - 2007-7-31 Jakub Grzegorz Kopycki, Poland since January 2005 Annegret Bährecke, Germany Graduiertenprogramm 2004-6-1 - 2007-5-31

83 PRESS AND PUBLIC RELATIONS Head: Sylvia Pieplow

Assistant: Susanne Kubenz

PUBLIC EVENTS Day of Chemistry Long Night of Sciences 2005 and 2006 On September 23, 2006, the IPB participated With about 390 and 300 visitors, respectively, for the first time in the Open day of Chemistry the fourth and fifth Long Nights of Sciences, (Schautag der Chemie), a public performance each on the first Fri day in July, proved to be very organized by the German Chemical Industry As sociation (Verband der Chemischen Indus - trie) allover Germany. At this event, especially our chemists from the Department of Bio or - ganic Chemistry presented their work by guided tours through the laboratories and lectures on their scientific projects. Ap pro xi ma tely 100 guests visited the institute and found out about many possibilities of vocational training in chemical industry.

Leibniz Association meets Science and Policy Long Night of Sciences 2006 On November 3, 2006, the Leibniz Association organized a panel discussion with specialists successful for the Leibniz Institute of Plant Bio - from science and policy, who discussed in front chemistry (IPB). Curious guests enthusiastically of more then 200 members of the public cur- participated in guided tours through the la bora - rent knowlegde of brain plasticity. Within this to ries and greenhouses. In addition, our scientists event in the Johanniskirche in Magdeburg all displayed experiments and exhibitions in the five institutes of the Leibniz Association located foyer. Special lectures were fa vored by the pub lic. in the state of Saxony-Anhalt were present In 2005 Ste phan Cle mens spoke about chances with an information booth displaying topics of for and risks of green gene technology, whereas their own scientific projects. The IPB presented Jür gen Schmidt gave an overview about the the isolation and characterization of antibiotics history of mass spectrometry. In the next year from resident fungi, which catched the interest Andrea Porzel presented interesting aspects of by politicians, press, and the public. chirality in nature, technology and art. Simultaneously with this pub lic event in 2006, the institute opened as visual highlight an exhibition with in teresting pictures and mi - croscopic exposures of re sear chers own scientific projects.

Public discussions on plant biotechnology Within the “6. Jugendumwelttage”, a public discussion event concerning political and ecology-

minded education of young peoples, in May Professor Wolfgang Böhmer, Premier of Saxony-Anhalt 2006 in Leipzig, Dierk Scheel gave an insight in (2. from left), Professor Ernst Theodor Rietschel, Pre si - dent of the Leibniz Assocoation (3. from left) and Pro - new technologies of plant science with his lec- fessor Jan Hendrik Olbertz, Minister of Cultural Affairs of ture named “Effective genes – how to make Saxony-Anhalt (right) were very interested in scientific new plants”. topics of the IPB at the panel discussion, organized by the Leibniz Association in November 2006 in Magdeburg.

As supporter of genetic engineering in plants he acted again in December 2006 on a similar Guided tours for Pupils and Seniors public discussion forum, organized by students As in the years before, the IPB organized a cou- of Halle’s university. ple of guided tours through the institute for school classes, students, and senior groups.

84 SCIENTIFIC REPORT 2005 - 2006 PRESS AND PUBLIC RELATIONS

These tours were always coupled with a other scientific institutes of Saxony-Anhalt presentation on genetic engineering in in Biotechnika, Germany’s most important plants. international biotechnology fair. Car sten Milkowski from the Department of Secon - Untitled. Painting by Alexandra Fröb. VISITORS FROM ECONOMY AND POLICY dary Metabolism presented a seminar on Matt Buist, vice president of the Toronto transgenic rapeseed within the special Peißnitz – 1 Jahr – 1x täglich Bio technology Initiative and member of congress Wirtschaftskraft Pflanze – Zu - Photos by Beate Nixdorf, December 2005 the Business Development of Toronto visi- kunft durch Innovationen, whereas Clau dia and January 2006 ted our institute in November 2005. The Bo bach and Axel Teichert from the De - aim of the visit in frame of a cooperation part ment of Bioorganic Chemistry demon- Spuren im Einerlei, agreement between Halle and Toronto was strated the isolation and characterization Paintings by Alexandra Fröb, January and an exchange of information about the of powerful antibiotic substances from re - February 2006 scientific topics of and a possible exten sion sident fungi in the exhibition hall. of an already existing cooperation bet- Betrachtungen ween the IPB and Ca na dian resear chers NEW INTERNET PRESENCE Paintings by Beate Gödecke, March and and breeders. During 2005 the Internet presence of the April 2006 IPB was updated. The new websites are de - The scientific and economic situation of veloped in state-of-the art-style with the Impressioni mediterranee the state of Iowa displays a strong focus on content management system TYPO3. This Paintings by Eckart Haupt, May and June green biotechnology and renewable prima- sys tem allows fast and simple updating of 2006 ry products, which is similar to the busi- multiple contents by several re sponsible ness environment in Saxony-Anhalt. For persons in an autonomously manner. The IPB in voller Blüte this reason, members of the Business De - websites were provided with da tabases for Scientific photos, July - September 2006 velopment of Iowa and of the Iowa State staff members and publica tions, which can University visited our and other institutes also be updated by several authorized per- Stadtansichten with comparably research priorities in the sons, avoiding problems of updating in the Paintings by Iris Band, October and No - whole state of Saxony-Anhalt. The vistit of future. Equipped with a modern layout and vember 2006 the IPB in December 2005 was accompa- more user-friendly sites of the intranet, the nied by lively discussion about basic local new web presence started at the beginning Fernweh nach blauen Welten parameters in scientific policy and patent of 2006. Photos by Gert Edler, December 2006 and law in Germany. The project was organized January 2007 by the BIO Mittel deutsch land GmbH. ART EXHIBITIONS With four art exhibitions in 2005 and six In March 2006 breeders and scientists of in 2006 we attracted the public attention the Saaten-Union Resistenzlabor GmbH to the IPB in another way. At every vernis- used the opportunity to gain insight in IPB’s sage there was also the possibility to visit scientific topics, especially those of the De - the laboratories and greenhouses, which partment of Stress and Developmental was enjoyed by our guests. The following Biology. The visits aim was also the discus - art exhibitions could be seen at the insti- sion of collaboration projects in the future. tute in the past two years:

About 60 politicians of the German Bun - Photos by Ralf Kummer destag and the Parliament of Saxony-An - January and February 2005 halt visited our laboratories and greenhouses in November 2006. Representatives of Die Natur am Brocken all political parties discussed with Sylvia Photos by Thomas Fester, September and Pieplow current problems of plant bio- October 2005 technology. Hortus Botanicus PARTICIPATION IN TRADE SHOW Photos by Gudrun Hensling, October and In October 2005 the IPB participated to - November 2005 gether with universities, companies, and Marktansichten. Painting by Iris Band.

85 MEDIA PRESENCE OF THE IPB IN 2005 AND 2006

ARTICLES AND PRESS RELEASES 2005 BRANNT. PRESS RELEASE. DECEMBER 1 JANUARY 8 Ausstellung am IPB. Mit teldeutsche Zeitung, p. Bank, M. Zusammenarbeit mit Kanada. Mittel - SEPTEMBER 13 12. deutsche Zeitung, p. 18. PIEPLOW, S. VORTRAG ZUR NATUR AM BROC KEN AM IPB. PRESS RELEASE. DECEMBER 1 JANUARY 26 Bank, M. Kanadier zu Besuch. Mitteldeutsche PIEPLOW, S. FOTOAUSSTELLUNG VON RALF KUMMER SEPTEMBER 14 Zei tung, p. 12. AM LEIBNIZ-INSTITUT FÜR PFLAN ZENBIOCHEMIE, Krause, I. Lern-CD zeigt Natur des Brockens. PRESS RELEASE. Mitteldeutsche Zeitung, p. 10. DECEMBER 7 Pieplow, S. Samen ohne Bitterstoffe aus trans- JANUARY 27 OCTOBER 7 genem Raps. EAST Magazin 1/12/2005, p. 10- Bank, M. Fotoausstellung am IPB, Mit tel deut - PIEPLOW, S. HORTUS BOTANICUS - GRÜNE OASE 11. sche Zeitung, p. 8. INMITTEN DER STADT. PRESS RELEASE. DECEMBER 7 OCTOBER 12 FEBRUARY 5 Wingert, N. Pilze gegen Killer-Bakterien. EAST Krause, I. Fotos im Institut. Mitteldeut sche Waldpilze liefern Antibiotika, Die Na turheil - Magazin 1/12/2005, p. 6-8. kun de 2/2005, p. 36. Zei tung, p. 8.

DECEMBER 28 MARCH 15 OCTOBER 12 Höhn, T. D. Leibniz-Professor bricht eine Lanze Aus dem Dschungel in die Apotheke, Apothe - Grüne Oase in der Stadt. Sonntags nach - für Gentechnik. Deutsche Presse-Agentur ken Umschau, p. 70-73. richten. GmbH Halle.

MAY 19 OCTOBER 18 Köhler, M. Auch Pflanzen kommunizieren. Sto - Krause, I. Vom Hilfsarbeiter zum Wissen - Press releases were depending on their topics ry Service der Wirtschafts förde rung Halle, schaftler. Mitteldeutsche Zeitung, p. 14. also published by: www. wifoe.halle.de . - www. allpr.de OCTOBER 19 - www.biomitteldeutschland.de MAY 20 Hortus botanicus. AmtsBlatt, S. 1. - www.bista.de Hoffmann, R. Schlaflos in Halle. Scientia Halen - - www.chemlin.de sis 5/2005, p. 3. OCTOBER 24 - www.halle.de Bank, M. Volles Wachstum nicht nur im Ge- - www.innovations-report.de MAY 23 wächshaus. Story Service der Wirt - - www.interconnections.de PIEPLOW, S. NEUES DOMIZIL FÜR TABAK, MOHN schaftsförderung Halle,www.wifoe.halle.de. - www.lehrer-online.de UND RAPS. PRESS RELEASE - www.mdr.de OCTOBER 27 - www.mz-web.de MAY 25 Krause, I. Den Brocken auf CD-Rom gebannt. - www.planeterde.de Pommert, S. Bestes Klima für Tomaten. Mittel - Mitteldeutsche Zeitung, p. 13. - www.pressrelations.de deut sche Zeitung, p.9. - www.uni-protokolle.de NOVEMBER 15 - www.sachsen-anhalt.de JUNE 10 Mäder, A.Analysieren im Akkord. Ber li ner Zei - - www.studieren-im-netz.de Smiljanic, M. Blei fressende Blümchen. Financial tung, p. 12. Times Deutschland, www.ftd.de. RADIO TRANSMISSION NOVEMBER 29 November 30 JUNE 22 PIEPLOW, S. NEUER GESCHÄFTSFÜHRENDER DI REK- Kersten, C. Raps und Rost als Giftkiller- neue PIEPLOW, S. CHEMIE IM GRÜNEN BE REICH. PRES S TOR AM LEIBNIZ-INSTITUT FÜR PFLAN ZEN BIOCHE - Techniken der Altlastensa nie rung. Bayerischer RELEASE. MIE. PRES S RELEASE. Rundfunk, IQ - Wis senschaft und Forschung.

JULY 1 NOVEMBER 29 TELEVISION TRANSMISSIONS Bank, M. Forschung für tausende Be sucher. PIEPLOW, S. PEISSNITZ -1JAHR -1X TÄGLICH. PRESS JANUARY 28 Mittel deutsche Zeitung, p. 15. RELEASE. Simon, S. Gesundmacher wieder entdeckt: Kohl, Rüben, Wirsing & Co. mdr Fernsehen, JULY 1 NOVEMBER 30 Hauptsache Gesund. Wiederhold, B. In der Iod-Stärke-Uhr wird es Neuer Direktor. Wochen spiegel blau. Mitteldeutsche Zeitung, Photo, p. 15. AUGUST 31 NOVEMBER 30 Neue Exzellenzforschung in Sachsen-Anhalt. JULY 2 Neuer Direktor am IPB. Mitteldeutsche Zei - mdr Fernsehen, Nachrichten in Hier ab vier. Bank, M. Wie kommt es zum Zelltod? Mittel - tung. deut sche Zeitung, p. 18. DECEMBER 15 NOVEMBER 30 Struff, K., Antibiotische Wirkstoffe aus heimi- SEPTEMBER 6 PIEPLOW, S. BESUCH AUS KANADA AM LEIB NIZ- schen Pilzen. mdr Fernsehen, Hier ab vier. PIEPLOW, S. EGON STAHL AWARD FÜR PRO FESSOR INSTI TUTFÜR PFLANZEN BIOCHE MIE. PRESS RE LEASE. DETLEF GRÖGER. PRESS RELEASE. DECEMBER 1 SEPTEMBER 13 Wilhelm, K. Strippenzie her der Unterwelt. PIEPLOW, S. NATUR AM BROCKEN AUF CD GE- Bild der Wissenschaft 12/2005, p. 28-33. 86 SCIENTIFIC REPORT 2005 - 2006 PRESS AND PUBLIC RELATIONS

ARTICLES AND PRESS RELEASES 2006 JANUARY 18 ACRYL UND PASTELL VON ALEXANDRA FRÖB AM LEIBNIZ-INSTITUT FÜR PFLANZENBIO CHEMIE. PRESS RELEASE.

JANUARY 25 Blick ins Herz der Blüte. Mit teldeutsche Zeitung, photo, p. 13.

FEBRUARY Hupfer, A. Vorhang auf für die kleinen Moleküle. Laborjournal 1-2/2006, p. 20-23.

FEBRUARY 9 Blüten in der Kunst. Mitteldeutsche Zeitung, JULY 4 OCTOBER 24 pho to, p. 14. Krause, I. Hitzezuschlag für die Bauleute. Mit - Iris-Band-Schau. Mitteldeutsche Zeitung, p. 14. tel deutsche Zeitung, p. 9. FEBRUARY 23 NOVEMBER 3 Schierholz, A. Eine Stadt muss sich entdecken. JULY 7 Scholtyseck, W. Schau von Iris Band zeigt Mitteldeutsche Zeitung, p. 3. BILDERAUSDER FORSCHUNGZUR LANGEN Stadt-Träume und Traum-Städte. Mittel deut - NACHT DER WISSENSCHAFT. PRESS RELEASE. sche Zeitung, photo, p. 17. MARCH 8 PROFESSOR TONI M. KUTCHAN FOLGT RUF NACH JULY 8 DECEMBER 11 AMERIKA. PRESS RELEASE. Krause, I. Ein Mord zur Langen Nacht. Mittel - FERNWEH NACH BLAUEN WELTEN AM LEIB NIZ-IN- deutsche Zeitung, p. 11. STITUT FÜR PFLANZENBIOCHEMIE. PRESS RE LEASE. MARCH 9 Krause, I. Mohn-Expertin zieht nach Amerika JULY 14 DECEMBER 12 um. Mitteldeutsche Zeitung, p. 16. Pieplow, S. Virtuellen Doktorhut erwerben. Pohle, H. Fotograf zeigt Bilder von Kreta. Mit - East Magazin 2/2006, p. 38. teldeutsche Zeitung, p. 11. MARCH 9 DER MOHN ZIEHT NACH AMERIKA. PRESS RE LEASE. JULY 15 DECEMBER 13 Deutsch, M. Wissenschaftliche Kost pro be, Mit - Schau im Institut. Mitteldeutsche Zeitung, p. MARCH 14 teldeutsche Zeitung, p. 13. 12. BEATE GÖDECKE STELLT AM LEIBNIZ-INSTI TUT FÜR PFLANZENBIOCHEMIE AUS. PRESS RELEASE. AUGUST 31 Press releases were depending on their topics METALLTOLERANTE PFLANZEN - EINE FRAGE DER also published by: MARCH 16 GENAKTIVITÄT? PRESS RELEASE. - www.allpr.de Krause I. Kunst von Kunstpädagogin. Mittel - - www.bionity.com deut sche Zeitung, p. 18. SEPTEMBER 18 - www.bista.de LEIBNIZ-INSTITUT FÜR PFLANZENBIOCHEMIE ÖFF- - www.chemieonline.de MARCH 16 NET SEINE PFORTEN ZUM SCHAUTAG DER CHEMIE. - www.chemiereport.de SAMEN OHNE BITTERSTOFFE AUS TRANSGENEM RAPS. PRESS RELEASE. - www.chemlin.de PRESS RELEASE. - www.halle.de SEPTEMBER 20 - www.innovationsreport.de APRIL Schautag der Chemie. BILD-Zeitung. - www.interconnections.de Bünnagel, D. Ob Weizenmehl, Tofu, Reis. Leib - - wwwjobvector.de niz-Journal 1/2006, p. 10-11. SEPTEMBER 20 - www.juraforum.de Krause, I. Schautag der Chemie. Mittel - - www.kompetenznetze.de APRIL deutsche Zeitung, p. 13. - www.pressrelations.de Scheel, D. Nutzen der “Grünen” Gen technik. - www.sachsen-anhalt.de Leibniz-Journal 1/2006, p. 14. SEPTEMBER 20 - www.scienzz.de Institut öffnet seine Pforten zum Tag der Che - - www.uni-protokolle.de APRIL mie. Wochenspiegel, p. 4. - www.vdbiol.de Prof. Toni M. Kutchan verlässt das IPB. Labor pra - - www.wissenschaft24.de xis, April 2006, p. 18., Process, April 2006, p. 16. SEPTEMBER 25 Möbius, J. Chancen in der Chemie - Leib niz- RADIO TRANSMISSION MAY 11 Institut öffnete Pforten für Besucher. Mittel - JANUARY 12 MEDITERRANE IMPRESSIONEN AUS DER SICHT DES deutsche Zeitung, p. 8. Für stärkere Nutzung der Grünen Gentech - MUSIKERS. PRESS RELEASE. nik. Interview with Dierk Scheel. Deutschland - OCTOBER 23 radio Kultur. MAY 12 IRIS BAND ZEIGT STÄDTE AUS DER VOGEL PER SPEK- Städter, A. Ein Alleskönner unter den Pflanzen. TIVE. PRESS RELEASE. Mitteldeutsche Zeitung, p. 13.

87 MAP & IMPRESSUM

SCIENTIFIC REPORT 2005-2006 LEIBNIZ INSTITUTE OF PLANT BIOCHEMISTRY

PUBLISHER: Leibniz Institute of Plant Biochemistry Weinberg 3 06120 Halle (Saale), Germany www.ipb-halle.de

EDITOR: Sylvia Pieplow Press and Public Relations Phone: +49 (0) 345-55 82 11 10 Fax: +49 (0) 345-55 82 11 19 email: [email protected]

TYPOGRAPHY & LAYOUT: Sylvia Pieplow

GRAPHICS & PICTURES: Leibniz Institute of Plant Biochemistry 88 Christine Kaufmann, Annett Kohlberg, Bettina Hause and others Copyright © 2007, all rights reserved Leibniz Institute of Plant Bio chemistry (IPB), Halle, Germany. No parts of this publication may be reproduced by any mechanical, photographic, electronic process, or in form of a photographic recording, nor may be stored in a retrieval system, transmitted or otherwise copied for public or private use, without written permission from the publisher.

Halle, August 2007