Institute of Plant BiochemistryA Leibniz Institute

Weinberg 306120 Halle (Saale)

Germany

Phone: +49 (0) 3 45 - 55 82 11 10Fax: +49 (0) 3 45 - 55 82 11 09

Email: pr@ipb-halle.dewww.ipb-halle.de

2000 - 2002ANNUAL REPORT

Department: Stress and Developmental Biology 45Head: Prof. Dierk Scheel

Research Groups:Signal Perception in Plant-Pathogen Interactions 46Head: Thorsten Nürnberger

Cellular Signaling 48Head: Dierk Scheel

Induced Pathogen Defense 50Heads: Sabine Rosahl & Dierk Scheel

Metal Homeostasis 52Heads: Dieter Neumann & Stephan Clemens

Publications, Books and Bookchapters, Publications in press,Patents, Doctoral Theses, Diploma Theses 54

Department: Secondary Metabolism 57Head: Prof. Dieter Strack

Research Groups:Molecular Physiology of Mycorrhiza 58Head: Michael H. Walter

Cell Biology of Mycorrhiza 60Head: Bettina Hause

Biochemistry of Mycorrhiza (since 2002) 62Head: Willibald Schliemann

Glycosyltransferases 64Head: Thomas Vogt

Biochemistry of Betalains (until 2001) 66Head: Willibald Schliemann

Hydroxycinnamic Acids 68Head: Dieter Strack

Publications, Books and Bookchapters, Publications in press,Patents, Doctoral Theses, Diploma Theses 70

Department:Administration and Technical Services 73Head: Lothar Franzen

Resources and Investments 74

Staffing Schedule 75

Use of Funds from External Sources 76

Guest Researchers and Fellows 80

Press and Public Relations 83Head: Sylvia Pieplow

Map & Impressum 86

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Table of Contents

Presentation of the Institute 4

Departmental Organization 7

Board of Directors,Foundation Council, Scientific Advisory Board 8

Scientific Institute Council, Persons with SpecialResponsibilities, Personnel Committee 9

Department: Natural Product Biotechnology 11Head: Prof. Toni M. Kutchan

Research Groups:Alkaloid Biosynthesis 12Head: Toni M. Kutchan

Opium Poppy Biotechnology 14Head: Susanne Frick

Plant Cell Cultures 16Head: Gabriele Herrmann

Alkaloid Functional Genomics 18Head: Jonathan Page

Mode of Action of Jasmonates 20Heads: Claus Wasternack & Otto Miersch

Papaver-Gene Expression Analysis 22Head: Jörg Ziegler

Publications, Books and Book Chapters, Publications in press,Patents, Doctoral Theses, Diploma Theses 24

Hops Secondary Metabolism 26Departments of Natural Product Biotechnology and Bioorganic Chemistry - joint projectHeads: Jonathan Page, Jürgen Schmidt, Frederick Stevens (until September 2002)

Department: Bioorganic Chemistry 29Head: Prof. Ludger Wessjohann

Research Groups:Synthesis & Method Development 30Heads: Ludger Wessjohann & Brunhilde Voigt

Biocatalysis & Design of Ligands 32Head: Ludger Wessjohann

Plant and Fungal Metabolites / Microanalytics 34Heads: Norbert Arnold, Jürgen Schmidt, Ludger Wessjohann & Gernot Schneider (until June 2001)

Structural Analysis & Computational Chemistry 38Heads: Wolfgang Brandt & Andrea Porzel

Publications, Books and Bookchapters, Publications in press,Patents, Doctoral Theses, Diploma Theses 40

Searching for Signals: Stress-Induced Changes inArabidopsis Secondary Metabolite, Peptide andProtein Patterns (GABI) 43Departments of Bioorganic Chemistry and Stress and Developmental Biology - joint projectHeads: Stephan Clemens, Jürgen Schmidt, Ludger Wessjohann, Dierk Scheel

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The large manifold of plant spe-cies is reflected in the enormousdiversity of their natural products.This content of natural com -pounds is made more complex bythe change in metabolite patternsduring development as well aswhen a plant is responding to itsenvironment. Knowledge of thestructure and function of naturalproducts is requisite to under-standing plant diversity, develop-mental and adaptation processes.New resources can then becomeavailable for innovative applicationin plant production, plant protec-tion, biotechnology and in thedevelopment of biologically activecompounds. Furthermore, the rea - lization of genome sequencingand the growing availability of ex -pressed sequence tags of variousspecies is of fundamental impor-tance to functional genome analy-sis.

The comprehensive analysis ofplant and fungal natural productsis a priority in the research mis-sion of the Institute of PlantBiochemistry. Structure analysis,synthesis and derivatization ofnatural products contribute to anunderstanding of their functionand to an increase in their struc-tural diversity. This also forms thebasis for investigation of their bio-synthesis and for discovering newbiologically active compounds. Aqualitative and quantitative analy-sis of natural products in biologi-cal materials requires the deve-lopment of suitable analyticalmethods. Subsequent identifi-cation and isolation of biosynthe-tic enzymes can provide access tothe encoding genes, which in turnenables study of the regulation ofthe biosynthesis. The use of mu -tants and transgenic plants ultima-tely makes possible the analysis of

biological function as well as thegeneration of plants with alterednatural product profiles.

Molecular interactions form thebasis of cellular function. Aninterdisciplinary analysis of theseinteractions is therefore of cen-tral importance to the researchmission of the Institute of PlantBiochemistry. The optimal adap-tation of plants to their habitatdepends upon receptor-mediatedperception of biotic and abioticenvironmental parameters. Ex ter -nal signals are evaluated, compa-red and converted into physiolo-gical responses via altered geneexpression patterns that are con-trolled by cellular and systemicsignal transduction networks. Themolecular basis of these proces-ses, receptor/ligand, en zyme/li -gand and protein/protein inter-actions, have application in thedevelopment of new biologicallyactive agents. From this perspec-tive, the mechanisms of communi-cation between plants and theirsymbionts and pathogens are in -vestigated as are biosynthetic andsignal transduction pathways.Chemical struc tures of these in -teracting components are alsomodified using gene technologicalmethods, directed evolution andchemical derivatization. The ef -fects of these changes can be mo -nitored in model systems or withactivity screens until a moleculewith the desired characteristics(e. g. a drug, a signal compound oran enzyme) is achieved. The deve-lopment of new syntheses, scree-ning tests, assays and analyticalmethods is supported by visuali-zation of molecular interactionsvia computer modelling.

A nexus of natural product re-search and the study of molecular

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Presentation of the Institute

The Institute of Plant Bio -chemistry (IPB) in Halle wasfounded on 1 January 1992

as a non-university research insti-tute of the so-called "Blue List". In1995, the union of the Blue ListInstitutes formed the Blue ListScience Association (Wis sen -schaftsgemeinschaft Blaue Liste),which was subsequently restruc-tured and renamed the LeibnizAssociation (Leibniz Ge -meinschaft) in October 1997. TheIPB belongs to the life sciencessection of the Leibniz Asso ciation.The original institute was foundedas "Arbeits stelle für Bio chemieder Pflanzen" on 1 January 1958by Prof. Dr. Dr. h.c. mult. KurtMothes by order of the Ger manAcademy of Science in Berlin. In1960 it was renamed Institute forBiochemistry of Plants.

The IPB consists of four scientificdepartments and the administra-tion and central services depart-ment. Currently 112 employeswork at the IPB paid from theregular budget and another 47fun ded by third-party funds. Theresearch profile of the institute isunique within the German scien-tific community. The comprehen-sive analysis of natural productsfrom plants and fungi, the investi-gation of the interaction of plantswith pathogens, symbionts andabiotic stresses, studies of mole-cular interactions as part of com-plex biological processes, andmetabolic engineering are at thecenter of research activities.Excellent basic research is regar-ded as the indispensable basis forthe successful implementation ofapplication-oriented research pro - jects. The institute benefits, in par-ticular, from the fact that thescientific departments of the IPBcomplement each other in terms

of their methodical approachesand the equipment at their dispo-sal. This allows interdisciplinaryresearch using the latest chemical,physiological, cell-biological, bio-chemical, molecular-biological andgenetic methods for comprehen-sive analysis of complex subjects.

The IPB is located on theWeinberg Campus, which hoststhe natural science departmentsof the Martin Luther University,several non-university institutesand biotechnology companies.Close relationships and coope-rations exist between the insti-tute, the university and industries.Beside extensive scientific colla-boration with several universitydepartments, the institute's de -partment heads are full profes-sors at the university and, there-fore, involved in teaching andsupervision of undergraduate andgraduate students. Together withthe Institute of Plant Genetics andCrop Plant Research (IPK) inGatersleben and the Max PlanckInstitutes for Chemical Ecology inJena and of Molecular PlantPhysiology in Golm the IPB formsthe Plant Metabolism Network,PlantMetaNet. This network linksthe plant metabolomics compe-tence that has been developed toan excellent level in these fourplant research institutes inCentral Germany.

Research mission statementFour thematically, methodologi-cally and organisationally overlap-ping research priorities form thebasis of the research missionstate ment of the Institute of PlantBiochemistry - plant natural pro-ducts, molecular interactions,information technology and meta-bolic engineering.

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Departmental Organization

6

Presentation of the Institute

interactions is the storage andevaluation of the large amount ofdata that is generated. In particu-lar, high through put processesused in metabolome and pro-teome analysis and in the produc-tion of combinatorial librariesmake necessary the developmentof new methods in informationtech nology. To this end, a newjunior group in information tech-nology is being established at theInstitute of Plant Bio chemistry.

Metabolic engineering is an over-lapping priority in three areas ofbasic research - natural products,molecular interactions and infor-mation technology. Model plantsare generated that have potentialfor various types of application.More specifically, de signer plantswith tailored natural product pro-files, containing new health-pro-moting metabolites or showingimproved adaptation to habitatare being developed. Plants withthese characteristics could servefor the sustainable production of

valuable chemicals, as biologicaltest systems or could be ofimportance to plant breeders.

Within four departments withdis tinct, but complementaryresearch di rections and state-of-the-art equipment, the Instituteof Plant Bio chemistry providesoptimal conditions with whichto execute multidisciplinary re -search in the areas of chemistry,physiology, cell biology, bioche-mistry, molecular biology andgenetics. The analysis of topicscentral to modern plant biologyand chemistry using this widearray of methodologies enablesa meaningful interpretation of thecomplex interactions in plantdevelopment and diversity thatwould otherwise not be pos-sible. The ultimate transfer ofthese results to practical appli-cations could make ecologicallycompatible uses of plant bio-technology a reality. <

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Scientific Institute Council, Persons with Special Responsibilities,Personnel Committee

Dr. Jürgen Schmidt ChairmanDepartment Bioorganic Chemistry

Scientific Institute Council

Dr. Otto Miersch Vice-ChairmanDepartment Natural Product Biotechnology

Dr. Bettina Hause Department Secondary Metabolism

Dr. Dieter Neumann Department Stress and Developmental Biology

Dr. Thorsten Nürnberger Department Stress and Developmental Biology

Dr. Thomas Vogt Department Secondary Metabolism

Dr. Brunhilde Voigt Department Bioorganic Chemistry

Dr. Michael H. Walter Department Secondary Metabolism

Persons with Special ResponsibilitiesDr. Gabriele Herrmann Disabled Persons’ Affairs

Hans-Günter König Energy

Dr. Robert Kramell,Dr. Thorsten Nürnberger Radiation Protection

Kerstin Manke Equal Opportunity

Sylvia Pieplow Public Relations

Dr. Sabine Rosahl Biological Safety

Prof. Dierk Scheel,Prof. Claus Wasternack Gene Technology (GenTG)

Dr. Willibald Schliemannn Personal Data Privacy

Dr. Hans-Jürgen SteudteSecurity engineerDr. Brunhilde VoigtEberhard Warkus

Workplace Safety

Dr. Susanne Frick,Martina Lerbs,Angelika Weinel

Further Members

Andrea Piskol Chairwoman

Peter Schneider Vice-Chairman

Personnel Committee

MinisterialratThomas Reitmann

Senior Superior CounsellorMinistry of Education and Cultural Affairs of theState of Saxony Anhalt

Prof. Dierk Scheel Managing Director and Head of the Departmentof Stress and Developmental Biology

Board of Directors

Foundation Council

Lothar Franzen Head of the Department ofAdministration and Technical Services

Prof. Toni M. Kutchan Head of the Department ofNatural Product Biotechnology

Prof. Dieter Strack Head of the Department ofSecondary Metabolism

Prof. Ludger Wessjohann Head of the Department ofBioorganic Chemistry

NN Superior CounsellorFederal Ministry of Education and Research

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

Prof. Alfons Gierl Technical University of Munich,Vice-Chairman of Scientific Advisory Board

Prof. Reinhard Neubert Vice-Rector for Research and PostgraduateStudents of the University of Halle

Dr. Wolfgang Rechner Ministry of Education and Cultural Affairs of theState Saxony Anhalt

Prof. Jörg Stetter Bayer AG, Leverkusen

Dr. Hans-Jürgen Strunck Federal Ministry of Education and Research

Scientific Advisory Board

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Board of Directors, Foundation Council, ScientificAdvisory Board

Prof. Lutz F. Tietze University of Göttingen

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

Prof. Wilhelm Boland ChairmanMax Planck Institute for Chemical Ecology, Jena

Prof. Alfons Gierl Vice-ChairmanTechnical University of Munich

Prof. Thomas Boller University of Basel

Prof. Horst Kunz University of Mainz

Prof. Birger Lindberg MØller Royal Veterenary and Agricultural University,Copenhagen, Denmark

PD Dr. habil.Günter Strittmatter KWS SAAT AG, Einbeck

Prof. Ulrich Wobus Institute of Plant Geneticsand Crop Plant Research, Gatersleben

Within the Department ofNatural Product Biotech -

nology, the central theme of researchis the analysis of the biosynthesis ofplant natural products at the molecu-lar genetic level. Of particular inte-rest is the isolation of genes enco-ding enzymes and re g u latory pro-teins involved in the formation ofphysiologically ac tive, small molecu-les derived from L-tyrosine or L-tryptophan (alkaloids) and acetylCoenzyme A (polyketides).

Alkaloids are pharmacologicallyactive, nitrogen-containing,basic compounds producedin ap pro x i mately 20 % offlowering plants. Eachspecies ac cu mulatesalkaloids in a u niqueand de fined pattern.The role of alkaloidsin plants has been alongstanding ques - tion, but a pictureemerges that sup-ports an ecochemicalfunction for thesecompounds. Al ka loid -con taining plants werealso mankind's original“materia medica”.Many of these plants arestill used today as sourcesof prescription drugs.These biosynthetic pathways

are attractive targets for mole-cular biology because of their role

in plant chemical ecology and thebiotechnological potential for theproduction of commercially im -portant compounds.

The biosynthesis of polyketides pre-sent in medicinal plants is also underinvestigation. The com pounds areprominent in tropical traditionalmedicine and are used to treat a widevariety of ailments, with particularem pha sis on pa ra sites. Plant polyke-

tide synthases are en -coded by a multi-genefamily that has chal-conesynthase as a pro to type.Gene fa mily evo lution inplants ap pears to occurby gene duplication follo-wed by nucleotide sub -stitution that can lead tobiochemical diversity.Plant polyketide syntha-ses are presu-mably deri-ved from a commonancestor that diverged toperform different reac-tions. The identification and char ac te -ri za tion of polyketide synthasesinvolved in the formation a variety ofnatural products should lead to abetter understanding of the evolu-tion of these secondary metabolites.

The techniques that are used to iso-late and identify these genes arewide-ranging, from enzyme purifica-tion followed by amino acid sequen-ce determination to EST-sequencingand macro / mi cro array analysis.Both plant cell cultures and nativeplant material serve as a source ofenzymes and genes. The characteri-zation of the gene products is carriedout after over-expression in aheterologous expression systemsuch as bacteria, yeast, insect cells orplants. A part of gene product cha-racterization is the localization of theprotein in a plant tissue or cell. Tothis end, antibodies are raised againstthe he terologously-expressed bio-synthetic proteins and immunolocali-zation techniques are used to identi-fy the cell type in which the biosyn-thetic enzymes accumulate. Thispotentially provides insight into theregulation of natural product biosyn-thesis and yields information essenti-al to the metabolic engineering ofsecondary pathways. Ulti ma te ly, thebiosynthetic genes are transformedback into the na-tive plant as sense,

antisense or RNAi vector constructsand the influence of the transgene onmetabolic pathways is deter mined byHPLC-MS. In this manner, plants withtailored natural product profiles canbe generated for industrial and rese-arch use.

In addition, the signalling propertiesof jasmonates and octadecanoids instress-induced and developmentalprocesses con-tinues to be investiga-ted. In particular, the spatial and tem -poral expression of allene oxide cycla-se alleles (a jasmonic acid biosynthe-tic gene) and the physiolo gical role ofjasmonic acid me ta bolites, such as12-hydroxy-jasmonate, are beingdeter mined. <

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Department: Natural Product BiotechnologyHead: Prof. Toni M. Kutchan

Secretary: Christine Dietel

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proteomic analysis of P. somniferumlatex (Decker et al. Electrophoresis 21,3500-3516 [2000]). The cDNA wasamplified from P. som niferum RNA byreverse transcription PCR using primersbased on the internal amino acid sequen-ces. The recombinant protein wasexpressed in Spodoptera frugiperda Sf9cells in a baculovirus expression vector.Steady state kinetic measurements withthe heterologously expressed enzymeand mass spectrometric analysis of theen zymic products suggest that the enzy-me is capable of carry through multipleO-me thylations, on the isoquinoline- andon the benzyl moiety of several sub -strates. The tetrahydrobenzylisoquino -lines (R)-reticuline (4.20 s-1mM-1), (S)-reticuline (4.50), (R)-protosinomenine(1.67), and (R,S)-isoorientaline (1.44) aswell as guaiacol (5.87) and isovanillic acid(1.21) are O-me thylated by the enzymewith the ratio kcat/Km shown in paren -theses. A phylogenetic comparison ofthe amino acid sequence of this O-methyltransferase to those from 16other plant species suggests that thisenzyme groups more closely to isoqui-noline biosynthetic O-me thy -ltransferases from Coptis japonica thanto those from Thalictrum tuberosum.

It is known that morphine and otheralkaloidal biosynthetic intermediatessuch as dopamine accumulate in smoothvesicles within P. somniferum laticifer cells.In the mature plant, laticifers form a reti-culated system that extends throughout

aerial parts of the plant.Exuded latex is the cyto-plasm and vesicles of thesereticulated laticifer cells. Inorder to localize within thiscomplex system the pro-teins for which we haveisolated cDNAs, antibodieshave been raised againsthe terologously ex pressedreticuline 7-O-me thyl -trans ferase, salutaridinol 7-O-acetyltransferase, co dei -none reductase, the berbe-rine bridge enzyme and thecytochrome P-450-depen-dent mono oxy ge nase (S)-N-methylcoclaurine 3'-hy - droxylase. Immu no lo cali -zation studies with each ofthe antibody preparationsis being carried out withsections of P. somniferumcapsule. Initial results indi-cate that multiple celltypes are involved in al -kaloid biosynthesis in thisplant. A heterologously ex -pressed major latex proteinhas been used as a latexmarker protein. Of the pro -teins thus far analyzed, onlycodeinone reductase canbe localized to laticifer cells. Theseresults imply that intercellular transportof either intermediates or enzymes playsa role in isoquinoline alkaloid biosynthesisin P. som niferum. <

The figure shows the immunolocalization of anenzyme of alkaloid biosynthesis in cross-sections ofPapaver somniferum capsule. The expression of (S)-reticuline 7-O-methyltransferase, an enzyme in -volved in the biosynthesis of tetrahydrobenzyliso-quinoline alkaloids in opium poppy, occurs in thephloem of the bundle sheath (green fluorescence).The red fluorescing cells are laticifers, stained by anantibody raised against a major latex protein.Laticifers are the site of alkaloid accumulation inaerial plant parts. These results imply a transport ofintermediates from phloem to laticifers during bio-synthesis.

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In recent years, we have isolated andcharacterized cDNAs encoding severalenzymes of tetrahydrobenzylisoquinoli-ne alkaloid biosynthesis from P. somnife-rum. The first enzyme in the biosyntheticpathway for which we have isolated acDNA is norcoclaurine 6-O-methyl-transferase. The next is the cytochromeP-450-dependent monooxygenase (S)-N-me thyl coclaurine 3'-hydroxylase.These en zymes are common to the mor-phine, noscapine and sanguinarine bio-synthetic pathways. Specific to the san-guinarine pathway is the berberinebridge enzyme that oxidatively cyclizes

the N-methyl moiety of (S)-reticuline tothe bridge carbon C-8 of (S)-scoulerine.Specific to noscapine biosynthesis is reti-culine 7-O-methyltransferase. Finally,specific to mor phine biosynthesis aresalutaridinol 7-O-acetyltransferase andcodeinone reductase, the penultimateenzyme of the morphine pathway thatreduces co deinone to codeine.

Reticuline 7-O-methyltransferase con-verts reticuline to laudanine in tetrahy-drobenzylisoquinoline biosynthesis in P. somniferum. This new enzyme of al ka -loid biosynthesis was identified during a

Group members

Kum-Boo Choi(Humboldt Fellow since October 2002)

Torsten Grothe(PhD student until April 2002)

Robert Kramell(postdoctoral position since July 2001)

Monika Krohn(technician since July 2001)

Tobias Kurz(PhD student since April 2002)

Birgit Ortel(technician since April 2002)

Anan Ounaroon(PhD student until September 2002)

Khaled Sabarna(PhD student since May 2002)

Marion Weid(PhD student since December 2000)

Collaborators

Wanchai De-EknamkulChulalongkorn University, Bangkok, Thailand

Tony FistTasmanian Alkaloids, Tasmania, Australia

Phil LarkinsScientific and Industrial Research Organisation PlantIndustry, Canberra, Australia

Friedrich LottspeichMax Planck Institute for Biochemistry, Martinsried,Germany

Werner RoosUniversity of Halle, Germany

Joachim StöckigtUniversity of Mainz, Germany

Research Group: Alkaloid BiosynthesisHead: Toni M. Kutchan

The opium poppy Papaver somniferum is still today one of our most impor-tant medicinal plants. Among the 80 alkaloids produced by this plant, three aremedicinally important. These are the narcotic analgesic morphine, the analge-sic and antitussive codeine and the antitussive noscapine. The biosynthesis ofcodeine and morphine is almost completely elucidated at the enzyme level.Relatively little is understood, however, concerning the biosynthesis of nosca-pine. We also understand very little of how alkaloid biosynthesis is regulatedand of the biological role of these compounds in the plant. We are systema-tically isolating cDNAs that encode the unique en-zymes of alkaloid biosyn-thesis in opium poppy. These cDNAs are functionally heterologously expres-sed in bacterial and insect cell cultures and characterized. The seven cDNAsthat we have isolated to date from P. somniferum will be used in in situ hybri-dization and the encoded heterologous proteins in immunolocalization stu-dies in order to identify the cellular sites of biosynthesis of some of thevarious classes of isoquinoline alkaloids (morphinan, benzo[c]phenanthridine,phthalideisoquinoline) produced by this plant. Initial results already providethe first insight as to how biosynthesis and accumulation of the various classesof these alkaloids are regulated in this plant.

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The pictures show incised capsules of opiumpoppy with protruding latex.

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somatic embryogenesis from twelve celllines containing six different cDNA con-structs. After the isolation of DNA andRNA, we analyzed these plants fromwhich 150 F0 have been proven to betrans genic and their seeds have been via-ble. Seeds from 17 transgenic F0 plantshave not been able to germinate and 23transgenic F0 plants did not contain anyseeds at all.

The F0 plants were analyzed by PCR orby dot blots and brought to flower andseed set. The alkaloid pattern of the firstgeneration was determined from leafextracts by HPLC and showed alteredalkaloid concentrations compared tocontrol plants. Molecular and chromato-graphic analysis of the F1 generation isunderway for all constructs and celllines. The alkaloid pattern in the secondgeneration is always analyzed in latexand in selected plants, also in leaves androots.

Latex from wild type plants of P. somni-ferum L. inbred parent line showed ahigh concentration of morphine, theba -ine and codeine. Another alkaloid, whichis present in this extract, is oripavine. Inthe roots of the wild type, the majoralka-loid is the benzophenanthridine san-guinarine.

From the 150 transgenic F0 plants, 43 areharboring the S4S4::antiBBE construct.We confirmed the presence of the trans-gene of the T1 plants with the samemethods described for the first gene-ration. Additionally, we examined theseplants with Southern- and Northernblotting. The alkaloid pattern was ana-lyzed in latex as well as extracts fromroots.

We found ten F1 plants containingS4S4::antiBBE with a different alkaloidpattern compared to the wild type. Themajor alkaloids in the bbe antisenseplants are morphine, codeine and the ba-ine. In contrast, a peak corresponding to

oripavine was not always detected inthese ten transgenic plants mentionedabove. One out of these ten plants show -ed an increased total amount of reticu -line instead. The pattern of the HPLCchromatogram of the T1 and T2 plants isalmost identical. These results are a firstevidence that an alkaloid pattern in bbeantisense plants is an hereditary trait. Atthe moment, we are working to confirmthe heredity of the other alkaloid pat-terns observed in bbe antisense plantsand are measuring the HPLC profiles ofplants containing the remaining fivecDNA constructs (full-length cor sense,partial cor sense, cpr sense, cyp80b1sense, cyp80b1 antisense).

Since we have not been able to silenceben zylisoquinoline biosynthesis in transge-nic poppy plants containing S4S4::antiBBEor S4S4::antiCYP80B1, we constructedplasmids containing partial sequences thatare potentially able to trigger RNA inter-ference in P. somniferum. Explants ofopium poppy were transformed withseven different constructs: bbe RNAi,cor RNAi, cpr RNAi, cyp80b1 RNAi, 6-omt RNAi, 7-omt RNAi and salat RNAi.All the explants have started to developcalli.

Last year three new genes became avail -able from benzylisoquinoline biosynthe-sis. These encode salutaridinol 7-O-ace-tyltransferase (SALAT), (S)-norcoclau -rine 6-O-methyltransferase (6-OMT)and (S)-reticuline 7-O-methyltransferase(7-OMT). Both methyltransferases havebeen cloned in sense orientation in ourbinary vector and have been transfor-med into opium poppy. All cultures deve-loped calli and have started to differen-tiate. <

Somatic regeneration of P. somniferum. Explantsfirst give rise to a type I callus (A), which starts todifferentiate after a certain time to type II callus(B). After the transfer to a hormone free mediumthis type II callus develops small embryos (C) andfinally little plantlets (D). The whole regenerationprocess is shown in picture E.

B

C

D

E

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During the last years, several genes fromthe biosynthetic pathways for reticuline,sanguinarine and morphine have beencloned. Although the biosynthesis is wellunderstood at the enzymatic level, themolecular and biochemical mechanismsthat regulate these pathways are notknown. The goal of this project is to de -ve lop a stable transformation and rege-neration method for opium poppy, whichwill make the metabolic engineering ofthe above mentioned compounds possible.Poppy seed oil finds use in chemicalindustry for the production of pigmentsand lacquer, but its residual morphinelevels prevents more widespread appli-cations. As well, because opium is theraw material for the illicit production ofheroin, cultivation of poppy is restricted.By completely suppressing morphinebio synthesis, opium poppy could becomea "harmless" crop plant. So far, there hasbeen no success with breeding programsand mutations to obtain a morphine-freepoppy. In the best case, a reduction ofmor phine biosynthesis has been achieved.The transformation of opium poppycould be an alternative to circumventthese problems.

We have used an Agrobacterium-media-ted approach to introduce differentcDNAs encoding enzymes of morphineand sanguinarine biosynthesis in sense or

antisense orientation into explants toattempt to alter their alkaloid profile.Alkaloid-free plants developed in thismanner will be used to test the chemicalecological function of morphinan andben zo phenanthridine alkaloids in plants.

With a transgenic cell line expressing theantisense construct of berberine bridgeenzyme (BBE) we hope to reduce themetabolic flux through sanguinarinepathway and to enhance the concentra-tion of papaverine and / or morphineinstead.We are interested if a transgeniccell line overexpressing codeinone re -duc tase (COR) leads to a poppy plant,which contains more morphine or wherethe concentration of morphine is lowereddue to a possible feedback inhibition ofthis pathway. We have also producedpoppy transformants where we influenceall cytochrome P450 enzymes of thebenzylisoquinoline pathways by introdu-cing a NADPH:cytochrome P450-oxido-reductase (CPR). Finally, we are trying toreduce or silence the complete alkaloidbiosynthetic pathway with a transgeniccell line containing the antisense con-struct of (S)-N-methylcoclaurinehydroxy lase (CYP80B1). With a cell lineoverexpressing CYP80B1, we are tryingto stimulate all three pathways together.The last three years we have been ableto regenerate 190 F0 poppy plants via

Group members

Sandra Barth(technician until December 2001)

Kathleen Gutezeit(technician since March 2002)

Stefanie Haase(PhD student since May 2002)

Katja Kempe(diploma student since May 2002)

Anja Zeuner(technician)

Collaborators

Tony FistTasmanian Alkaloids, Westbury, Australia

Phil LarkinScientific and Industrial Research Organisation PlantIndustry, Canberra, Australia

Jürgen SchmidtInstitute of Plant Biochemistry, Halle, Germany

Research Group: Opium Poppy BiotechnologyHead: Susanne Frick

Opium poppy (Papaver somniferum L.), which contains more than 80 differentalkaloids, remains one of the most important industrial medicinal plants.Poppy serves as a renewable resource of a number of medicinally relevantalkaloids. These include the analgesic and nar cotic drug morphine, the coughsuppressant codeine, as well as the muscle relaxant papaverine, the antitumo-ric agent noscapine and the antimicrobial sanguinarine. We are developingtransfor mation systems for opium poppy that will allow us:

to investigate the regulation and ecological function of these alkaloids inplants,

and to alter the alkaloid metabolism in commercial poppy varieties inorder to obtain varieties lacking alkaloids or with tailored alkaloid pro-files for industrial and pharmaceutical use.

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A

Biosynthetic pathway from L-tyrosine to sanguinarine,papaverine and morphine in Papaver somniferum. Enzymesare highlighted in red.

regeneration of transfected protoplaststo a new suspension culture. From anumber of methods we tested, the onlysuccessful one was the alginate-method.Fresh prepared protoplasts are mixedwith an alginate solution (a polyuronicacid from Macrocystis pyrifera) and dro-plets of this mixture are solidified inCaCl2. Alginate clumps are then culti-vated in 24-well plates and treated withchanging hormone media for cell divisionand growth. After two weeks first celldi visions are visible and after six to eightweeks, minicalli are produced. The wholeprocess of regeneration takes approxi-mately three months, which is much toolong.

During our experiments with suspensioncultures of Eschscholzia californica weobserved that also the protoplasts ofthis culture can be elicitated with jasmo-nates or a yeast elicitor (table 1). This eli-citation is visible already after 24 or 48hours due to increasing amounts of san-guinarine and chelirubine. With help ofelicitation, transfected protoplasts canbe checked during 48 hours concerningtheir changes in the alkaloid content. Pro -

to plasts with a re -duced alkaloid con-tent (determinedspec troscopically orby HPLC analysis)would than be di -rectly the materialfor localization andcharacterization ofthe block in the bio-synthetic pathway.For the transfec-tion of protoplasts, we used two diffe-rent meth ods - both PEG-mediatedtransfection and electroporation is pos-sible with protoplast preparations.

To start with the search for a viral vec-tor, which is active in our system, weused a TMV-derived viral vector, whichshould have a broad host specificity. Fora simple detection under UV light, wecloned a Green Fluorescent Proteingene into it, but we were not able todetect any green fluorescence. The maintask now will be the search for a vector,which can be transfected into Esch -scholzia protoplasts and expressed. <

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Table 1: Alkaloid content in protoplasts ofEschscholzia californica after elicitation withmethyl jasmonate after two days.

Figure 3: Protoplasts of Eschscholzia californica

Molecular genetic methods should beused in the investigation of biosyntheticpathways in secondary metabolism ofplants. One possibility to bring newgenetic information into cells such asplant protoplasts could be the use ofviral or bacterial vectors. Such infor-mation will be clones of a cDNA library,which should cause gain of function /loss of function effects. Some cultures ofour cell culture collection contain colo-red alkaloids visible under UV and nor-mal light. We selected Eschscholzia cali-fornica suspension culture because oftheir production of a red colored mixtu-re of sanguinarine and chelirubine. Infigure 2, the changes in the color of thissuspension during one growing cycle(seven days) are shown. Vectors contai-ning antisense cDNA of biosyntheticenzymes should stop alkaloid productionafter successful transfection and the cellsshould show reduced color.

The first steps in the project were thedevelopment of a protocol for the forma-tion of protoplasts, a search for cultiva-tion or regeneration methods and a testof transfection methods for protoplastsfrom suspension culture. The formationof protoplasts from Eschscholzia califor-nica was done by use of cellulase andpectolyase as cell wall degrading enzymesand a purification with a Ficoll gradient(figure 3). We are now able to produceenough protoplasts of good quality and aviability of at least six to nine days.The next step normally should be the

Group membersDomenika Arndt(technician)

Ingeborg Reeh(technician)

CollaboratorsGreg PogueLarge Scale Biology, Vacaville, California, USA

Werner RoosUniversity of Halle, Germany

Research Group: Plant Cell CulturesHead: Gabriele Herrmann

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Figure 1: A view to one part of the plant cell culture collection of the department "Natural ProductBiotechnology": suspension cultures of different plant species.

Figure 2: The development of a suspension culture of Eschscholzia californica during one week.

The main working task of the group is the maintenance of the plant cell cul-ture collection of our department. This collection includes about 250 differentplant species of 45 different plant families. About 40 species are cultivated inthe form of suspension cultures and all others as callus cultures on varioussolid media. In figure 1 a look to a part of our suspension culture collectionis to be seen. The culture collection contains a number of plants producinginteresting secondary metabolites such as alkaloids and represents the mainsource of biological material for coworkers and interested colleagues. In addi-tion, we work in the field of alkaloid biosynthesis in the DFG project"Functional genomics in plant cell cultures under use of viral vectors".

cannabis, we have constructed a tri-chome-specific cDNA library from puri-fied trichome secretory cell clusters.More than 1.200 ESTs (expressedsequence tags) from this library havebeen sequenced and assigned putativegene function using bioinformatic com-parisons. Through this approach we haveidentified candidate cDNA clones oftype III polyketide synthases, which maypar ticipate in cannabinoid biosynthesis,and an oxidocyclase, D9-tetrahydrocan-nabinolic acid synthase that is a key en-zyme in the cannabinoid biosyntheticpathway. Heterologous expression and in

vitro enzymatic assay are being used tofunctional characterize these genes.

Stemming from the group's interest inthe chalcone synthase superfamily oftype III polyketide synthases, the role ofthese enzymes in forming medicinal plantcompounds in Rheum tataricum L.(Polygonaceae), and Cassia alata L.(Fabaceae) was studied. A new resvera-trol-forming stilbene synthase was clo-ned from the former (Samappito et al, inpress), while a series of chalcone syntha-ses was characterized from the latter(Samappito et al, 2002). <

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Figure 2: Cannabinoids and terpenoids accumu-late in glandular trichomes of Cannabis sativa.Photo: J. Page

Plants respond to virus infection bysilencing (turning off) viral genes andthereby blocking viral replication. By clo-ning plant genes into viruses, plants canbe made to direct this antiviral defenseagainst their own genes, leading to a loss-of-function phenotype for the targetedgene. Fast-forward genetic methodsusing viruses promise to both speed upplant gene discovery and allow for thecloning of novel genes inaccessible tocurrent techniques. The targets of our

VIGS efforts are enzymesinvolved in tropane al-kaloid (nicotine) biosyn-thesis and transcriptionfactors controlling meta-bolite synthesis and accu-mulation in glandular tri-chomes of Nicotiana ben-thamiana Domin. Glan -dular trichomes are resi-nous hairs that cover lea-ves and flowers in manyplant species. Their pri-mary function is defen-sive, although they alsoplay a role in detoxifi-cation, and therefore theyare a major site of natural

product production and storage. In theSolanaceae, they are readily targeted byvirus constructs (Figure 1). We are buil-ding a catalog of MYB transcription fac-tors from N. benthamiana trichomes andtesting the effect that silencing theseregulatory proteins has on metabolitecontent and trichome morphology. Ex -periments with known enzymes of nico-tine biosynthesis, such as putrescine-N-methyltransferase (PMT) and quinolatephospho-ribosyltransferase, have shownthat gene silencing can reduce nicotinelevels to about 30 % of control levels. Ba -sed on these results, we are constructingcDNA libraries in viral vectors for use inhigh-throughput VIGS approaches toalkaloid biosynthesis.

Cannabis is grown worldwide for indu-strial purposes, yielding fibre and seeds,and for its content of psychoactive can-nabinoids (e. g. D9-tetrahydrocannabinol,THC). The biosynthetic pathway leadingto cannabinoids is not completely under-stood at the biochemical or geneticlevel. Cannabinoid biosynthesis occursmainly in glandular trichomes (Figure 2)that cover female cannabis flowers at ahigh density. Using a high-THC strain of

Group members

Verona Dietl(technician)

Annegret Flier(technician until February 2002)

Nils Günnewich(student since October 2002)

Ursula Schäfer(PhD student)

Vincent Spelbos(student until March 2002)

Collaborators

Valery DoljaOregon State University, Corvallis, Oregon, USA

Jürgen SchmidtInstitute of Plant Biochemistry, Halle, Germany

J.-Frederick StevensOregon State University, Oregon, USA

Research Group: Alkaloid Functional GenomicsHead: Jonathan Page

We are tapping the biosynthetic potential of the plant kingdom by studyingthe metabolic pathways leading to complex natural products. Our group isusing functional genomic approaches to dis-cover genes encoding enzymesand transcription factors involved in natural product biosynthesis. This rese-arch focuses on biosynthetic processes occurring in tissues or organs, such asglandular trichomes that secrete natural products.We are using virus-inducedgene silencing (VIGS) to identify genes involved in alkaloid metabolism and tri-chome development in Nicotiana benthamiana. Biochemical genomics, whichcombines transcriptome and metabolite analysis, is being applied to uncoverenzymes catalyzing the formation of terpenophenolic chemicals in Cannabissativa L. (hemp, marijuana) and Humulus lupulus L. (hops), see Research Group“Hops Secondary Metabolism”.

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Figure 1: Plant virus targeting of glandular trichomes.Nicotiana benthamiana trichomes exhibit GFP expressionafter infection with a tobacco mosaic virus containing a GFPreporter gene. Photo: U.Schäfer

Oxylipin profiling and expression analy-ses in WT, 35S::AOCsense and35S::AOCan ti sense lines revealed regula-tion of JA bio synthesis by substrate avai-

lability, an activity control of preexistingenzymes and a feed forward regulation.This was substantiated by analysis ofArabidopsis and mutants affected in JAbiosynthesis. Here, four different non-redundant AOCs are tissue-specificallyactive, thus allowing control of the oxyli-pin signature of different organs.

Using knockout lines of AOC1-AOC4,AOC1-4RNAi-lines as well as antisenseapproaches, individual functions ofAOC1-AOC4 in stress responses andduring development of Arabidopsis isunder study and will allow us, to analyzethe mode of action of JA.

Previously, 12-hydroxy-JA was onlyknown as tuber-inducing compound inSolanaceae. We could identify 12-hy -droxy-JA and its sulfated derivative in A. thaliana as a signaling compound inflower development. <

Biosynthesis of jasmonic acid catalyzed by a lip -oxygenase (LOX), an allene oxide synthase (AOS),an allene oxide cyclase (AOC), an OPDA reduc -tase (OPR3) and b-oxidative steps.

21

Phylogenetic tree analysis for AOC's. (fromStenzel et al. 2003b)

Amplification in wound-signaling by co-localizationof AOC, JA generation and systemin formation aswell as systemin-dependent AOC expression andJA-dependent prosystemin expression. The ampli-fication is compromised in 35S::AOCsense plans(cf. Stenzel et al. 2003a).

Previous work on stress responses andcloning of JA biosynthetic enzymes inbarley was finished by analyses of three13-Lipoxygenases, three allene oxidesynthases (AOS’s) and one AOC, all ofthem located in chloroplasts. Since 2000,we are working with tomato, Ara bi dop -sis and tobacco. The first AOC was clo-ned from tomato. This single copy gene isspecifically expressed in ovules of youngflowers and all vascular bundles, accom-panied by a specific pattern of variousjasmonate and octadecanoid compounds(oxylipin signature) in distinct flowerorgans. In leaves the vascular bundle-spe-cific occurrence of AOC attributes to apreferen tial generation of jasmonates inmain veins. Based on a co-localization ofthe AOC, the JA-generation, the locationof the wound signal systemin in vasculartissues and the data from various trans-genic tomato plants, an amplification mo -del on wound signaling is proposed.

The capacity of the phloem to respondrapidly in wound signaling was furthersupported by detection of JA biosynthe-tic enzymes including AOC in sieve ele-ments. The importance of JA in signalingwas strengthened by grafting experi-ments between 35S::AOCanti senseplants and wild type plants.

Group members

Carolin Delker(PhD student since June 2002)

Tobias Kurz(PhD student until March 2002)

Claudia Kutter(student until August 2001)

Helmut Maucher(postdoctoral position until March 2002)

Lydia Müller(student since October 2002)

Jana Neumerkel(student since December 2002)

Birgit Ortel(technician until March 2002)

Andrea Pitzschke(student until May 2000)

Diana Schmidt(student until August 2001)

Ulrike Schubert(student until July 2001)

Irene Stenzel(postdoctoral position)

Carola Uhlig(technician since February 2002)

Sabine Vorkefeld(technician since July 2002)

Collaborators

Guillermina AbdalaUniversidad Nacional de Rio Cuarto, Argentina

Klaus ApelUniversity of Zurich, Switzerland

Wilhelm BolandMax Planck Institute of Chemical Ecology, Jena, Germany

Udo ConradInstitute of Plant Genetics and Crop Plant Research,Gatersleben, Germany

Bettina Hause, Sabine Rosahl,Dierk Scheel, Jürgen SchmidtInstitute of Plant Biochemistry, Halle, Germany

Gerd HauseUniversity of Halle, Germany

Harry KleeUniversity of Florida, Gainesville, USA

Thomas RoitschUniversity of Würzburg, Germany

John TurnerUniversity of East-Anglia, Norwich, UK

Luc VarinConcordia University, Montreal, Canada

Research Group: Mode of Action of JasmonatesHeads: Claus Wasternack & Otto Miersch

Jasmonates and their precursors, the octadecanoids, are signals in plant stressresponses and in plant development. A mechanistic analysis of the mode ofaction of jasmonates is performed by a reverse genetics approach using the alle-ne oxide cyclase (AOC)-catalyzed step in jasmonate biosynthesis. "Gain of func-tion" and "Loss of function" studies with transgenic tomato plants revealedmodulation of jasmonates and allowed to inspect the role of jasmonates inresponse to biotic and abiotic stresses as well as flower and seed development.In order to use genetic approaches, functional analysis of AOC and jasmonic acid(JA) is also performed in Arabidopsis thaliana. Analytics of jasmonates and otheroxylipins including chemical synthesis of standards and labeled substrates is anessential part of this work.

20

Occurrence of AOC protein in plastids of compa-nion cells (big arrows) and sieve elements (smallarrows)of tomato flower stalks (A, B) and petioles(C-E). Longi tudinal sections were probed with ananti-AOC-antibody (A, C) or with the pre-im -mune serum (B). D: differential interference con-trast image of C. E: DAPI staining to visualizenuclei. The sieve plate of a sieve element is markedby an asterisks (cf. Hause et al. 2003).

Immunocytological localization of AOC pro-tein in leaves of Arabidopsis thaliana. (A)preimmune serum,(B) location of AOC in chlo-roplasts (Stenzel et al. 2003b)

in the data-bases. The largest groups ofcDNAs coding for proteins with knownfunction are invol-ved in tran s criptionaland trans lational control, in responses tostress, and in redox control. A notherhighly re presented group codes forproteins participating in meta bolism,mainly pri mary metabolism. To 20sequences, a role in secondary metabo-lism could be ascribed. Five sequencescode for proteins with known functionin the benzyl isoquinoline pathway, andfor one cDNA, showing high homologyto an enzyme involved in another alkalo-id pathway that does not occur inPapaver, its possible role in thebenzylisoquino line pathway is currentlyunder investigation. The EST-sequencingproject still continues, but additionally, toaccess cDNAs implicated in benzyliso-quinoline biosynthesis at a higher fre-quency, the construction of a P450-monoxygenase specific EST-collectionhas been initiated. These enzymes play amajor role in the modification of thebenzylisoquinoline core structures lea-ding to the high structural diversity.

The expression of these cDNAs in fourPapaver species differing in their ability toperform the last steps in the biosynthesisof morphine was examined and correla-ted with the occurrence of morphine inthe respective alkaloid profiles. By combi-nation of all possible datasets, the num-ber of cDNAs possibly responsible forthe accumulation of morphine could bereduced to 39 candidates, most of themcoding for unidentified proteins. Furthercomparisons are in progress to decreasethe number of cDNAs far enough, that afunctional characterization is feasible.

Another project uses the cDNA-AFLPtechnique to isolate cDNAs differentiallyexpressed dependent on an alkaloid pro-file. In this approach, we compare a wildtype P. somniferum plant with a mutantplant accumulating thebaine, which issituated four steps upstream of morphinein the biosynthetic pathway. More than100 differentially expressed fragmentswere found. Their specificity for the mor-phine-free phenotype is currently beingexamined by macroarray analysis. <

23

Interloping diagram of the number of genes diffe-rentially expressed between P. bracteatum and P.somniferum grown in the field (red circle), P. brac-teatum and P. somniferum grown in the greenhouse(blue circle) and P. bracteatum and P. somniferumNoscapine (green circle). The number in the over-lapping areas indicates the number of genes thatshow differential expression in the combination ofthe respective comparisons.

22

Currently, more than 70 different poppyspecies belonging to the genus Papaverhave been described. Roughly, they areable to synthesize about 2.500 differentbenzylisoquinolines, which can be groupedinto nine classes. The profiles of benzyl-isoquinolines produced by the plants arespecies-specific, however they are alsodependent on growth conditions. Thesame holds true for gene expression.This variability requires sensitive methodsto record all needed parameters in oneindividual plant. HPLC methods wereemployed to detect the main com-pounds of poppy alkaloids and LC-MScoupling will be used for the low-abun-dance compounds. For gene expressionanalysis, a protocol for macroarray pro-

duction was developed and the esta-blishment of microarray technology hasstarted. These methods are sensitiveenough to record the alkaloid profile andthe gene expression pattern from oneindividual plant. For 60 Papaver speciesand ten varieties and mutants of theopium poppy Papaver somniferum, themain alkaloids could be identified byHPLC. For low-abundant and not yetidentified alkaloids, LC-MS analysis is inprogress. As probes for the arrays, weuse PCR fragments derived from an ESTproject of P. somniferum stems. Amongall Papaver species, this plant synthesizesthe largest number of different benzyli-soquinolines and the stem has beenshown to possess the highest biosynthe-

tic activity. Up tonow, we se -quenced more than2.000 ESTs and ob -tained 1.100 uni-que sequences. A -bout 40 % ei thercode for proteinswith unknown func -tion or have no ho -mology to entries

Group membersAndreas Gesell(PhD student since July 2002)

Silvia Wegener(technician)

CollaboratorsBirgit DrägerUniversity of Halle, Germany

Research Group: Papaver-Gene Expression AnalysisHead: Jörg Ziegler

Poppies of the genus Papaver produce a large variety of benzylisoquinolinealkaloids. Some of them are of pharmaceutical impor-tance such as the analge-sic morphine, the antitussive noscapine or the vasodilator papaverine. The bio-synthesis to (S)-reticuline, the cen tral intermediate to all monomeric benzyli-soquinoline alkaloids is well understood on the molecular level, knowledge onthe later steps, which lead to the diversity of this class of compounds, is stillincomplete. Similarly, the regulatory steps leading to the accumulation of thesesubstances are unknown. To approach cDNA clones coding for the enzymes ofthese biosynthesis processes, we make use of the close genetic relationship, butthe diversity in the alkaloid pro-file, between Papaver species or varieties,respectively. We examine and correlate the gene expression profiles on EST-arrays (expressed sequence tag) with specific alkaloid profiles. By the combi-nation of many different datasets of alkaloid profile-gene expression corre-lations, we want to reduce the number of candidate cDNAs to a manageablenumber to start their functional characterization.

Functional classification of P. somniferum stem ESTs. Abbreviations: PS: photosynthesis, MLP: major latex proteins, protein syn / deg: pro-tein synthesis and degradation, G-prot: G-proteins, AA: amino acids, alk: alkaloids, phenyl: phenylpropanoids, isopr: isoprenoids.

25

Kutchan, T. M. Aromatic and pyrone polyketides syn-

thesized by a stilbene synthase from Rheum tataricum.Phytochemistry 62, 313-323 (2003).

Stenzel, I., Hause, B., Maucher, H., Pitzschke, A., Miersch,O., Ziegler, J., Ryan, C. & Wasternack, C. Allene oxidecyclase dependence of the wound response and vascu-lar bundle specific generation of jasmonate -Amplification in wound-signalling. The Plant J. 33, 577-589 (2003a).

Stenzel, I., Hause, B., Miersch, O., Kurz, T., Maucher, H.,Weichert, H., Ziegler, J., Feussner, I. & Wasternack, C.Jasmonate biosynthesis by substrate availability andthe allene oxide cyclase family of Arabidopsis thaliana.Plant Mol. Biol. 51, 895-911 (2003b).

Stenzel, I., Ziehte, K., Schurath, J., Hertel, S. C., Bosse, D.& Köck, M. Differential expression of PSI14, a phospha-tase gene family, in response to phosphate availability,pathogen infection and during development. Physiol.Plant. (2003c).

Vigliocco, A., Bonamico, M. B., Alemano, S., Miersch, O.& Abdala, G. Activation of jasmonic acid production inZea mays L. infected by the maize rough dwarf virus-Río Cuarto. Reversions of symptoms by salicylic acid.Biocell 26 (3), 369-374 (2002).

Books and Book Chapters in pressStenzel, I., Maucher, H., Hornung, E., Wasternack, C. &Feussner, I. Transcriptional activation of jasmonate bio-synthesis enzymes is not reflected at protein level. In:Advanced Research on Plant Lipids. (Murata, N.,Yamada, M., Nishida, I., Okuyama, H., Sekuja, J. &Haijime, W., eds.) Kluwer Academic Publishers,Dordrecht, pp. 267-270 (2003).

Stumpe, M., Stenzel, I., Weichert, H., Hause, B. &Feussner, I. The lipoxygenase pathway in mycorrhizalroots of Medicago truncatula. In: Advanced Researchon Plant Lipids. (Murata, N.,Yamada, M., Nishida, I.,Okuyama, H., Sekuja, J. & Haijime, W., eds.) KluwerAcademic Publishers, Dordrecht.

Wasternack, C. & Abel, S. Plant hormones. In:Molecular Plant Physiology. chapter 15 (Sharma, R.,ed.) Harword Press, Binghamton.

Wasternack, C. Jasmonates - Biosynthesis and role instress responses and developmental processes. In:Programmed Cell Death and Related Processes inPlants. (Nooden, L.D., ed.) Academic Press Inc., NewYork.

Weichert, H., Maucher, H., Hornung, E., Wasternack, C.& Feussner, I. Shift in fatty acid and oxylipin pattern oftomato leaves following overexpression of the alleneoxide cyclase. In: Advanced Research on Plant Lipids.(Murata, N., Yamada, M., Nishida, I., Okuyama, H.,Sekuja, J. & Haijime, W., eds.) Kluwer AcademicPublishers, Dordrecht, pp. 275-278 (2003).

PatentsKutchan, T. M., Zenk, M. H. & Grothe, T. Salutaridinol 7-O-acetyltransferase and derivatives thereof in thenames of Institut für Pflanzenbiochemie and MeinhartH. Zenk. European patent 01114122.3 (2001).

Kutchan, T. M., Zenk, M. H. & Grothe, T. Salutaridinol 7-O-acetyltransferase and derivatives thereof in thenames of Institut für Pflanzenbiochemie and MeinhartH. Zenk. US patent application No. PCT/EP 02/07455(2002).

Kutchan, T. M., Zenk, M. H. & Grothe, T. Salutaridinol 7-O-acetyltransferase and derivatives thereof in thenames of Institut für Pflanzenbiochemie and MeinhartH. Zenk. patent application No. PCT/WO 02/101052A2 (2002).May, C., Kindl, H., Rentz, A. & Feußner, I. The b-barrelstructure of lipid body lipoxygenase. PCT/WO0129227 (2001).

Ziegler, J., Stenzel, I., Hause, B. & Wasternack, C.Allenoxidcyclasegen und dessen Verwendung zumHerstellen von Jasmonsäure. German patent10004468.9 (2000).

Ziegler, J., Stenzel, I., Hause, B. & Wasternack, C.Allenoxidcyclasegen und dessen Verwendung zumHerstellen von Jasmonsäure. Japanese patent applica-tion based on PCT/EP O1/01148, No. 100102978(2001).

Ziegler, J., Stenzel, I., Hause, B. & Wasternack, C.Allenoxidcyclasegen und dessen Verwendung zumHer stellen von Jasmonsäure. US patent application(application number pending) based onPCT/EPO1/01148 (2002).

Doctoral ThesesBalkenhohl, Thomas: Abbau von Speichertriglyceridenin keimenden Samen der Gurke (Cucumis sativus).University of Halle-Wittenberg, Faculty ofMathematics, Natural Sciences and Technology,February 2000.

Fong-Chin Huang: Molecular cloning and heterologousexpression of Papaver somniferum cytochrome P450genes involved in secondary metabolism. University ofMunich, Department of Chemistry and Pharmacy,15/5/2000.

Grothe, Torsten: Untersuchungen zur Morphin -biosynthese im Schlafmohn: Klonierung, heterologeExpression und Charakterisierung der Salutaridinol-7-O-Acetyltransferase sowie Reinigung der Thebain-Synthase aus dem Milchsaft von Papaver somniferumL. University of Halle-Wittenberg, Faculty of Mathe -matics, Natural Sciences and Technology, 25/4/2002.

Jennewein, Stefan: Klonierung und heterologeExpression von Cytochrom-P450-Enzymen derNADPH:Cytochrom-P450-Reduktase, des Cytochrom b5und der Taxadiensynthase aus Taxus chinensis.University of Munich, Department of Chemistry andPharmacy 2000.

Anan Ounaroon: Molecular cloning and functionalexpression of three O-methyltransferases fromPapaver somniferum L. University of Halle-Wittenberg, Faculty of Mathematics, Natural Sciencesand Technology, 11/9/2002.

Samappito, Supachai: Cloning and expression of poly-ketide synthase genes from Cassia alata, Plumbagoindica and Rheum tataricum. Chulangkorn University,Bangkok, 30/10/2002.

Diploma ThesesKutter, Claudia: Funktionelle Analyse der Allen -oxidcyclase in Lycopersicon esculentum Mill.University of Halle-Wittenberg, Department ofBio chemis- try / Bio technology, 23/9/2001.

Pitzschke, Andrea: Funktionelle Analyse einer Allen -oxidcylase-cDNA durch homologe Transformation inTomate. University of Halle-Wittenberg, Departmentof Biochemistry / Biotechnology, 5/5/2000.Rüder, Constantin: Untersuchungen zu Interaktions -partnern Jasmonat-induzierter Proteine mit Hilfe desHefedihybridsystems. University of Halle-Wittenberg, Department of Biochemistry / Biotechnology, 19/9/2000.Schilling, Stephan: Isolierung und Charakterisierung vonGlutaminyl-Cyclase aus tierischem und pflanzlichemMaterial. University of Halle-Wittenberg, Departmentof Biochemistry / Biotechnology, 18/8/2000.

Schmidt, Diana: Analyse zur Regulation der Allenoxid-cyclase-Promotoren aus Arabidopsis thaliana mittelstransgener Ansätze. University of Halle-Wittenberg,Department of Biochemistry / Biotechnology, 23/8/2001.

Spelbos, Vincent: Prenyltransferasen in Humulus lupu-lus. University of Utrecht, November 2002.

Schubert, Ulrike: Untersuchungen zur funktionellenAnalyse von Allenoxidcyclase mittels Sense- undAntisense-Ansätzen. University of Halle-Witten berg,Department of Biochemistry / Biotechnology, 28/6/2001. <

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53, 555-564 (2000).Kramell, R., Miersch, O., Atzorn, R., Parthier, B. &Wasternack, C. Octadecanoid-derived alteration ofgene expression and the 'oxylipin signature' in stressedbarley leaves - implications for different signalling path-ways. Plant Physiol. 123, 177-186 (2000).

Kutchan, T. M. Ecological arsenal and developmentaldispatcher - The paradigm of secondary metabolism.Plant Physiol. 125, 58-60 (2001).

Maucher, H., Hause, B., Feussner, I., Ziegler, J. &Wasternack, C. Allene oxide synthases of barley(Hordeum vulgare cv. Salome) - tissue specific regula-tion in seedling development. Plant J. 21, 199-213(2000).

Miersch, O. & Wasternack, C. Octadecanoid and jas-monate signaling in tomato leaves (Lycopersicon escu-lentum Mill.): Endogenous jasmonates do not inducejasmonate biosynthesis. Biol. Chem. 381, 715-722(2000).

Nibbe, M., Hilpert, B., Wasternack, C., Miersch, O. &Apel, K. Cell death and salicylate- and jasmonate-dependent stress responses in Arabidopsis are con-trolled by single cet genes. Planta 216, 120-128 (2002).

Oven, M., Grill, E., Golan-Goldhirsh, A., Kutchan, T. M. &Zenk, M. H. Increase of free cysteine and citric acid inplant cells exposed to cobalt ions. Phytochemistry 60,467-474 (2002).

Oven, M., Page, J. E., Zenk, M. H. & Kutchan, T. M.Molecular characterization of the homo-phytochelatinsynthase of soybean Glycine max. J. Biol. Chem. 277,4747-4754 (2002).

Oven, M., Raith, K., Neubert, R. H. H., Kutchan, T. M. &Zenk, M. H. Homophytochelatins are synthesized inresponse to cadmium in azuki beans. Plant Physiol. 126,1275-1280 (2001).

Samappito, S., Page, J. E., Schmidt, J., De-Eknamkul, W. &Kutchan, T. M. Molecular characterization of root-spe-cific chalcone synthases from Cassia alata. Planta 216,64-71 (2002).

Schilling, S., Hoffmann, T., Wermann, M., Heiser, U.,Wasternack, C. & Demuth, H.-U. Continuous spectro-metric assays for glutaminyl cyclase activity. AnalyticalBiochemistry 303, 49-56 (2002).

Schilling, S., Hoffmann, T., Rosche, F., Manhart, S.,Wasternack, C. & Demuth, H.-U. Heterologousexpression and characterization of human glutaminylcyclase: evidence for a disulfide bond with importancefor catalytic activity. Biochemistry 41, 10849-10857(2002).

Warzecha, H., Gerasimenko, I., Kutchan, T. M. &Stöckigt, J. Molecular cloning and functional bacterialexpression of a plant glucosidase specifically involvedin alkaloid biosynthesis. Phytochemistry 54, 657-666(2000).

Wasternack, C. & Hause, B. Jasmonate - Signale zurStressabwehr und Entwicklung in Pflanzen. Biologie inunserer Zeit 30, 312-319 (2000).

Wasternack, C. & Hause, B. Jasmonates and octadeca-noids: Signals in plant stress responses and plant deve-lopment. Progr. Nucleic Acid Res. Mol. Biol. 72, 165-221(2002).

Weichert, H., Kohlmann, M., Wasternack, C. &Feussner, I. Lipids and signalling: oxylipins 3 - functionalaspects. Biochem. Soc. Trans. 28, 861-862 (2001).

Weichert, H., Kolbe, A., Kraus, A., Wasternack, C. &Feussner, I. Metabolic profiling of oxylipins in germina-ting cucumber seedlings - lipoxygenase-dependentdegradation of triacylglycerols and biosynthesis ofvolatile aldehydes. Planta 215, 612-619 (2002).

Weichert, H., Kolbe, A., Wasternack, C. & Feussner, I.Formation of 4-hydroxy-2-alkenals in barley leaves.Biochem. Soc. Trans 28, 850-853 (2000).

Weichert, H., Kolbe, A., Wasternack, C. & Feussner, I.Formation of 4-hydroxy-1-alkenals in barley leaves.Biochem. Soc. Trans. 28, 850-851 (2001).

Ziegler, J., Keinänen, M. & Baldwin, I.T. Herbivore-indu-ced allene oxide synthase transcripts and jasmonicacid in Nicotiana attenuata. Phytochemistry 58, 729-738 (2001).

Ziegler, J., Stenzel, I., Hause, B., Maucher, H., Hamberg,M., Grimm, M., Ganal, M. & Wasternack, C. Molecularcloning of allene oxide cyclase: The enzyme establis-hing the stereochemistry of octadecanoids and jasmo-nates. J. Biol. Chem. 275, 19132-19138 (2000).

Books and Book ChaptersKutchan, T. M. Sequence-based approaches to alkaloidgene identification. In: Phytochemistry in the Genomicsand Postgenomics Era. Recent Advances inPhytochemistry, Vol. 36. (Romeo, J.T. & Dixon, R. A., eds.)Pergamon Elsevier Science Ltd. Kidlington, Oxford, pp.163-178 (2002).

Kutchan, T. M. & Schröder, J. Selected cell cultures andinduction methods for cloning and assaying cytochro-mes P-450 in alkaloid pathways. In: Cytochrome P450Part C. Methods Enzymol. 357 (Johnson, E.F., ed.)Academic Press, Amsterdam, Boston London, NewYork, Paris, San Franciso, San Diego, Oxford, pp. 370-381 (2002).

Scheel, D. & Wasternack, C. (eds.) Plant SignalTransduction. Oxford University Press, Oxford (2002).

Scheel, D. & Wasternack, C. Signal transduction inplants: cross-talk with the environment. In: Plant SignalTransduction. (Scheel, D. & Wasternack, C., eds.)Oxford University Press, Oxford, pp. 1-5 (2002).

Publications in pressAbdala, G., Miersch, O., Kramell, R., Vigliocco, A.,Agostini, E., Forchetti, G. & Alemano, S. Jasmonate andoctadecanoid occurrence in tomato hardy roots.Endogenous level changes in response to NaCl. PlantGrowth Regul. (2003).

Bailey, N. J. C., Oven, M., Holmes, E., Nicholson, J. K. &Zenk, M. H. Metabolomic analysis of the consequencesof cadmium exposure in Silene cucubalus cell culturesvia 1H NMR spectroscopy and chemometrics.Phytochemistry 62, 851-858 (2003).

Färber, K., Schumann, B., Miersch, O. & Roos, W.Selective desensitization of jasmonate- und pH-depen-dent signalling in the induction of benzophenanthridi-ne biosynthesis in cells of Eschscholzia californica.Phytochemistry 62, 491-500 (2003).

Monostori, T., Schulze, J., Sharma, V. K., Maucher, H.,Wasternack, C., & Hause, B. Novel plasmid vectors forhomologous transformation on barley (Hordeum vul-gare L.) with the JIP23 cDNA in sense and antisenseorientation. Cereal Res.

Samappito, S., Page, J. E., Schmidt, J., De-Eknamkul, W. &

PublicationsAbdala, G., Castro, G., Miersch, O. & Pierce, D. Changesin jasmonate and gibberellin levels during developmentof potato plants (Solanum tuberosum). Plant GrowthReg. 36, 121-126 (2002).

Bachmann, A., Hause, B., Maucher, H., Garbe, E., Vörös,K., Weichert, H., Wasternack, C. & Feussner, I.Jasmonate-induced lipid peroxidation in barley leavesinitiated by distinct 13-LOX forms of the chloroplast.Biol. Chem. 383, 1645-1657 (2002).

Berger, S., Weichert, H., Porzel, A., Wasternack, C.,Kühn, H. & Feussner, I. Enzymatic and non-enzymaticlipid peroxidation in leaf development. Biochim.Biophys. Acta 1533, 266-276 (2001).

Chaissaigne, H., Vacchina, V., Kutchan, T. M. & Zenk, M. H.Identification of phytochelatin-related peptides inmaize seedlings exposed to cadmium and obtainedenzymatically in vitro. Phytochemistry 56, 657-668(2001).

De-Eknamkul, W., Suttipanta, N. & Kutchan, T. M.Purification and characterization of deacetylipecosidesynthase from Alangium lamarckii Thw. Phytochemistry55, 177-181 (2000).

Ellis, C., Karafyllidis, I., Wasternack, C. & Turner, J. G. TheArabidopsis mutant cev1 links cell wall signaling to jas-monate and ethylene responses. Plant Cell 14, 1557-1566 (2002).

Feussner, I., Kühn, H. & Wasternack, C. The lipoxygena-se dependent degradation of storage lipids. TrendsPlant Sci. 6, 268-273 (2001).

Feussner, I. & Wasternack, C. The lipoxygenase pathway.Annu. Rev. Plant Biol. 53, 275-297 (2002).

Frick, S., Ounaroon, A. & Kutchan, T. M. Combinatorialbiochemistry in plants: the case of O-methyltransfera-ses. Phytochemistry 56, 1-4 (2001).

Grothe, T., Lenz, R. & Kutchan, T. M. Molecular charac -terization of the salutaridinol 7-O-acetyltransferaseinvolved in morphine biosynthesis in opium poppyPapaver somniferum. J. Biol. Chem. 276, 30717-30723(2001).

Haider, G., von Schrader, T., Füßlein, M., Blechert, S. &Kutchan, T. M. Structure-activity relationships of syn-thetic analogs of jasmonic acid and coronatine oninduction of benzo[c]phenanthridine alkaloid accumu-lation in Eschscholzia californica cell cultures. Biol.Chem. 381, 741-748 (2000).

Hause, B., Maier, W., Miersch, O., Kramell, R. & Strack, D.Induction of jasmonate biosynthesis in arbuscularmycorrhizal barley roots. Plant Physiol. 130, 1213-1220(2002).

Hause, B., Stenzel, I., Miersch, O., Maucher, H., Kramell,R., Ziegler, J. & Wasternack, C. Tissue-specific oxylipinsignature of tomato flower - The allene oxide cyclaseis highly expressed in distinct flower organs and vascu-lar bundles. Plant J. 24, 113-126 (2000).

Hilpert, B., Bohlmann, H., Den Camp, R. O., Przybyla, D.,Miersch, O., Buchala, A. & Apel, K. Isolation and charact-erization of signal transduction mutants of Arabidopsisthaliana that constitutively activate the octadecanoidpathway and form necrotic microlesions. Plant J. 26,435-446 (2001).

Huang, F.-C. & Kutchan, T. M. Distribution of morphinanand benzo[c]phenanthridine alkaloid gene transcriptaccumulation in Papaver somniferum. Phytochemistry

Publications, Books and Book Chapters, In press,Patents, Doctoral Theses, Diploma Theses

27

Figure: A mature cone from hops (Humulus lupulus) with lupulin glandsvisible as yellow structures at the base of cone scales. Inset:Magnification of lupulin glands. (Photo: Annett Kohlberg)

26

Hops (Humulus lupulus L., Cannabaceae)are the principal flavor ingredient in beer,contributing phytochemicals with bothtaste (e.g. the bitter acid humulone) and'nutraceutical' (e.g. the prenylflavonoidxanthohumol) properties. These terpe-nophenolic metabolites are of mixedbiosynthetic origin, with precursors de-rived from terpenoid and phenolic (poly-ketide) pathways. Bitter acids and pre-nylflavonoids are mainly made and sto-red in specialized glandular trichomes,termed lupulin glands, found on hopcones (s. Figure). A key step in the bio-synthesis of terpenophenolics is thetransfer of isoprenoid unit(s) to the aro-matic ring of the phenolic moiety by aro-matic prenyltransferase enzymes. A col-laborative project between the Depart -

ments of Natural Product Biotechnology(Jonathan Page) and Bioorganic Che -mistry (J. Frederick Stevens and JürgenSchmidt) aimed at clarifying the prenyl-transferase reactions in hops was initia-ted in 2002. We developed a sensitivemass spectrometric assay for in vitroprenyltransferase activity and were ableto detect the enzyme-mediated trans ferof dimethylallyl diphosphate (DMAPP) tothe aromatic rings of precursor com-pounds. This industry-supported re -search will continue with a biochemicalgenomics project aimed at identifyinggenes encoding the enzymes of terpeno-phenolic biosynthesis. At a later stage wehope to characterize the enzymes andutilize them as biocatalysts in chemicaltransformations. <

Group members

Marco Dessoy(PhD student since May 2002)

Verona Dietl(technician)

Martina Lerbs(technician)

Raik Löser(research scientist until December 2001)

Vincent Spelbos(student until March 2002)

Research Group: Hops Secondary MetabolismHeads: Jonathan Page, Jürgen Schmidt, Frederick Stevens (until September 2002)

siderable action in the planning of the newfunctional building (house R) in close coopera-tion with the administration. House R is plan-ned to become the new home of our screeningfacilities and of the biocatalysis group. In addi-tion, it will contain cross-departmental installa-tions like a night lab, solvent distillation, a fer-mentation room, and laboratories of otherdepartments. Also, in 2002 Phytobase was ini-tiated as a central information database plat-form for chemical constituents from plants,fungi, or valuable synthetic compounds.Phytobase is planned to be the cornerstone ofour future information integration and partlywill be made available within a larger contextto all groups dependent on phytochemical data,including e. g. food industry, government andlegislation, and research groups in the fields ofnatural products, nutrition, ecology, bioinforma-tics, metabolomics or pharmaceutical develop-ment.

Despite the scientific and organizational unrest,and increasingly difficult access to outsideresources for phytochemical projects, a conti-nuous increase in publication output was achie-ved from 2000 to 2002. Several diploma/M.Sc.-titles (one in Halle), and six PhDs were grantedto group members. Finally, Fred Stevens, whostarted his habilitation in the de part ment in2000, in late 2002 accepted a call for a profes-sorship at Oregon State University in Corvallis(USA). <

Department: Bioorganic ChemistryHead: Prof. Ludger Wessjohann

Secretary: Elisabeth Kaydamov

28

Plants and fungi provide a rich source ofhighly diverse natural products and enzy-mes. The department focuses on the isolation,characterization, and modification of the che-mical constituents, thereby trying to shedsome light on their function in nature. The ana-lytical work is backed by an extensive synthesisprogram, designed to increase compound avai-lability and molecular diversity by combinatori-al chemistry, method development, and denovo synthesis. Applications of this researchinclude the use of metabolites as lead structu-res for drugs, cosmetics, or as re search tools,and the use of enzymes as screening targets, oras catalysts for synthesis.

In late 2000 the former department head,Günther Adam, and the interim head, GernotSchneider, transferred the responsibility toLudger Wessjohann, who moved from the VrijeUniversiteit Amsterdam to Halle in early 2001.At the same time, the department translocatedinto house D and into the northern part of thenow fully modernized house C. In parallel tothe physical move, a scientific reorganizationwas started. Four working groups were formedin 2001, three of them in totally new areas, andwere established in the following year:

In addition, one interdepartmental group(GABI), working on the profiling of se con darymetabolites ("me ta bo lomics") from Arabi dop -sis thaliana, was continued in cooperation withthe department of stress- and developmentalbiology (Dierk Scheel). A second interdepart-mental group (Humulus), studying hop con -stituents and secondary metabolism, was initi-ated with the department of plant biotechno-logy (Jonathan Page and Toni M. Kutchan).Finally, some members of the group remainedactive in Ams ter dam until 2003 with projectsin total synthesis, especially towards new pro-drug concepts and terpenoid modifications.

It should be mentioned that despite this formalseparation in research groups, most projects of

the department are in te grated, i. e. they spantwo or even three of these research groups.Thus e. g. the projects on isoprenoids and pre-nyltransferases involve contributions from thegroups Biocatalysis & Computational Chemi -stry, and to a minor extent from Synthesis &Method Development, Mi cro ana lytics andHumulus.

The present heads of research, Brun hildeVoigt, Andrea Porzel and Jürgen Schmidt wereenforced by two new head scientists, themycologist Norbert Ar nold (Plant and FungalMetabolites), who is also the new substitutehead of the department, and the biochemistWolf gang Brandt (Compu tational Che mis try).During the re port period, the group grew fromsome ten to about 35 members of about tennationalities. Nu merous guest researchers,exchange students and probationers, fromaround the world as well as from local institu-tes and schools, visited the department. Theclosest relationships exist with colleagues inBrazil, Vietnam, Hungary, and The Nether lands.

As part of the reorganization, valuable researchequip ment was newly in stalled, or re-installedand totally overhauled, among these threeNMR and six mass spectrometer, including oneFT-ICR-MS (v.i.), a glovebox, a synthetic robot,a pipetting ro bot, and a computational che -mistry network. A fungal strain collection anda computerized chemical stockroom systemwere started. Also, the department took con-

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Synthesis & Method Development

Biocatalysis & Design of Ligands

Plant and Fungal Meta bolites &Microanalytics

Structural Analytics & ComputationalChem istry

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31

wards dipeptides and their derivatives provedto be the most successful. The reaction runs inenvironmentally benign solvent like ethanol,but also reactions in water or without solventare possible. No waste is produced but oneequi valent of water.

In our approach to 14-membered cyclopeptidealkaloids, MCRs were successfully applied forthe rapid, atom-economic construction oflinear precursors. For even larger, highly func-tionalized macrocycles, a further extension of"simple" MCRs like U-4CRs towards multiple

interlocked MCRs with bifunctional compo-nents is necessary (e. g. the 1.5-fold U-5CR,depicted in figure 2). Hereby control of themultiplication factor is crucial in order to avoidpolymer formation. Towards this end, newcom plex components have been developedwith two active attachment-points (amine, al -dehyde, isonitrile or carboxylic acid).

Starting with bile acids suitable bifunctiona-lized steroid components were synthesizedand two of such steroid units were cyclized viapeptide bridges by Ugi-multicomponent reac-

tions (U-MCRs) leading to steroid cyclopep-tides. Using different reaction components, ma -crocycle libraries were synthesized with ultima-te efficiency, i. e. in one step. These ma crocyclesare the first members of a class of host com-pounds, where the rigid backbone and lipophilicsurface of two steroid moieties is connected viapeptide groups allowing variable diameters, con-formations and chemical functions. Molecularmodeling showed that the ca vities of the synthe-sized macrocycles are large enough to encapsu-late small organic substrates (figure 3).Modification of Macrocycles

Medicinally active macrocycles are commonly"decorated" with side-chains like lipids, phos-phates, and heterocycles like sugars. We are acentral partner in the EU-project ComBioCat,which aims at the enzymatic modification ofpolymer bound natural and natural-product-like macrocycles, their release, "decoration"and screening with self-selecting methods. Wecould successfully establish a traceless linkerfor aldehydes on a polymer suitable for enzy-matic reactions. This was exemplified with rifa-mycin, a macrocycle with sensitive enolether,acetal, acetate, and dienoate moieties, whichcould be modified and released as its antibioticderivative rifampicin (figure 4).Chromium and Selenium Mediated

Selective Organic TransformationsChromium(II) mediated reactions are highlychemoselective and allow transformations incomplex molecules without additional protec-tion and deprotection. They also show un -common selectivity. We could demonstratethat the Hiyama-reaction to homoallylalcoholscan give allylketones, depending on the reac-tion conditions. These findings also suggestsolutions to the problem of low enantioselec-tivity of the reaction with chiral ligands. Theresults are used to further the development ofan iterative process towards polyketide sub-structures.

Selenium compounds are very versatile rea-gents in natural product synthesis, unavoidablefor some transformations like the w-oxidationof terpenoids. However, they are toxic, odo-rous and in some cases not as efficient as de-sired. We are developing selenium reagentswith new characteristics: chiral, selectivelyremovable, or solid phase bound. A fluorousphase selective reagent with improved selen-oxide-elimination properties was successfullytested. The synthesis of chiral and solid phasebound versions is under investigation. In a DFGResearch Focus Program we are investigatingthe chemical properties of selenocystein.

Synthesis of Benzopyran Natural ProductsBenzopyrans include such common plantsecondary metabolites as coumarins, flavo -noids, anthocyanins etc. We are developingnew synthetic routes towards these com-pounds, especially to derivatives with newsubstitution patterns. <

Figure 2:A 6-component macrocyclization reaction constructed from an interlocked 1.5xUgi-4-component-reaction withtwo bifunctional building blocks with compatible functional groups F (F = e.g. hydroxy-, sugar- or ester-moieties).Pseudodilution is achieved by slow addition of one component. Head-head and head-tail cyclization and the two formedstereocenters will provide a library of eight isomers.

Figure 3: Calculated conformation of macrocyclus XSA192, C88H152N8O8, synthesized in one pot, shown withglucose as a guest molecule.

Figure 4: The macrocyclic antibiotic rifamycin bound viahydrazon linker to an enzyme penetrable polymeric bak-kbone (globe).

30

MacrocyclesTotal Synthesis of MacrocyclesThe most exciting polyketide macrocyclesdiscovered in recent years are the epothilones.They are antimitotic compounds with taxol-like activity, which are also active against mul-tiple drug resistant cancer cell lines. Currentlythese compounds are in phase II clinical trials.One of the shortest routes to epothilones wasdeveloped by us in Amsterdam. This approachis continued towards new analogues, which pro -mise improved properties.

The Fast Track to Macrocycles:Multi Component Reactions (MCRs)A synthesis of designed macrocycles of high

functionality by the traditional total synthesisapproach is extremely wasteful in all resources:chemicals, manpower and time. It is only usefulfor valuable compounds like epothilones. Theproblem will potentiate if compound librarieswill have to be designed, either for quantitativestructure-activity relationship (QSAR) or evo-lutionary adaptation studies. Three problemswill have to be solved for a sustainable routeto highly functionalized asymmetric macro-cycles and are addressed by our MCR-approach:

We concentrated on the synthesis of naturalproduct-like macrocycles inspired by the14-membered ansa-cyclopeptides from plantsand the bis-aryl-ether antibiotics (e. g. vanco-mycin). Another series is based on bifunctionalbuilding blocks derived from plant metabolites,especially terpenoids and steroids. Of the va -rious multi component reactions, the Ugi-four-component-reaction (U-4CR) to -

Group members

John Bethke(postdoctoral position since June 2002)

Tran Van Chien(visiting PhD student since October 2002)

Uwe Eichelberger(postdoctoral position since July 2001)

Dirk Michalik(postdoctoral position until December 2001)

Lars Ostermann(postdoctoral position until June 2002)

Eelco Ruijter(PhD student since March 2001)

Angela Schaks(technician)

Günther Scheid(postdoctoral position until June 2002)

Gisela Schmidt(technician)

Henri Schrekker(PhD student since January 2001)

Tran Thi Phuong Thao(PhD student since November 2001)

Mieke Toorneman(PhD student, based at Vrije Universiteit Amsterdamsince February 1999)

Mingzhao Zhu(PhD student since October 2001)

Friederike Ziethe(research scientist until December 2002)

Collaborators

Jan AndreesenUniversity of Halle, Germany

Uwe BornscheuerUniversity of Greifswald, Germany

Antonio Luiz BragaFederal University of Santa Maria, Brazil

Alexander Dömling,Wolfgang Richter, Lutz WeberMorphoChem AG Munich, Germany

Sabine FlitschUniversity of Edinburgh, UK

Lucia GardossiUniversity of Trieste, Italy

Thomas Hjertberg, Bertil HelgeeChalmers University of Technology, Sweden

Udo KraglUniversity of Rostock, Germany

Rob Leurs, Martine SmitFree University of Amsterdam, The Netherlands

Graham MargettsPolymerlabs UK

Karoly Micskei, Tamas PatonayUniversity of Debrecen, Hungary

Romano OrruFree University of Amsterdam, The Netherlands

Research Group: Synthesis & Method DevelopmentHeads: Ludger Wessjohann & Brunhilde Voigt

The targets of our synthetic efforts are natural products, their derivatives, and naturalproduct-like libraries, mostly of polyketide, isoprenoid or small peptoid structure, andto a limited extent designer molecules, e. g. for pro-drug concepts. A crucial prere-quisite for an efficient access to such complex molecules is the availability of newmethods with improved selectivity. The group developes these, based on our exper-tise in chromium and selenium reagents, biocatalytic methods (s. also dedicated rese-arch group) and multi component reactions. Selective reactions also offer the toolsfor creating chemical diversity from the modification of natural products.Combinatorial approaches in liquid as well as on solid phase are used to obtain smalldedicated libraries, which help to find substances with im-proved biological activity profiles. However, the access to libraries of structurallycomplex, natural product like molecules is usually limited because of lengthy multistepprocedures. Multicomponent one-pot reactions, multiple catalytic systems, and self-selecting (evolutionary) procedures are possible solutions to improve the accessibili-ty of structurally complex enti ties. These processes can be applied in chemistry, e. g.for selective separation and catalysis, or for pharmaceutical lead structure develop-ment with an emphasis on molecules with anticancer, antibiotic, phytoestrogenic, orcosmetic properties. Especially macrocycles are of interest to us, because their con-formational design is poorly understood. They exhibit more flexibility than classicalaromatic drugs but have less entropy loss upon binding than open chain forms.

The structural formula of natural epothilone D, a druglead compound for cancer therapy. The differentcolors signify structural elements originating from thebuilding blocks used for the total synthesis byWessjohann et al.

1.

2.3.

Rapid access to polyfunctional building blok-ks.The fast and efficient connection of these.Efficient macrocyclization strategies andcatalysts not based on the dilution principle.

33

and a first chromogenic assay was deve-loped. The important factors, whichgovern the enzyme stability were identi-fied, and consequently the yield of pro-duct could be improved to almost 99 %for the natural substrate, reducing at thesame time the amount of enzyme requi-red.

HennaHenna is a powder from dried leaves ofthe henna-plant Lawsonia inermis. Sincesome 3000 years, henna is used for thetemporary dyeing of hair or skin.2-Hydroxynaphthoquinone (lawsone,figure 3) was considered the main ingre-dient responsible for the dyeing. How -ever, distinct differences were found in

hair colored with henna-paste compared to pure law-sone (figure 4). Thus, the firstsevere analysis of the consti-tuents of henna was underta-ken. This revealed not onlythe absence of lawsone infresh plant material, but alsothe presence of several newconstituents, some of whichproved cru cial for the dyingcapability of the plant materi-al. For the first time we couldprove that the liberation ofdye precursors proceeds

enzymatically, and that a complex pro-dye concept is active in the plant powder.Apart from studying the enzymatic trans-formation, we also started to look at theconjugation of the final dye lawsone topeptides and proteins.

OtherWe could achieve the regioselective enzy-matic resolution of epothilone acyloin buil-ding blocks as part of our on-going efforttowards an effective synthesis of epothilo-ne derivatives (cf. research group "synthe-sis"). The best suitable lipases were identi-fied. We also tested the selective enzyma-tic acylation /deacylation of macrocyclices ter and hydroxy side-chains in solid phasegels. <

Figure 3: The chemical structure of the henna-dye lawsone

Figure 4: Fluorescence microscopy of cross-sec -tioned hairs dyed by various preparations. It isclear ly visible that henna leaf-paste, still containingactive enzyme, dyes hair throughout and fully,where as a water extract and synthetic lawsonepossess greatly reduced almost identical dyeingproperties (Photo by Bettina Hause).

Henna,leaf paste

Henna,water extract

Lawsone

Figure 2: The influence of co-solvents and their concentration onthe geranylation yield of 4-hydroxybenzoate catalyzed by ubiA-oli-goprenyltransferase from E. coli.

32

Prenyltransferases and IsoprenoidCompoundsThe synthesis of isotope labeled tenta-tive metabolites of the new non-mevalo-nate (dxp-) pathway was achieved. In acollaboration, some compounds wereutilized to prove for the first time, that4-hydroxy-dimethylallyldiphosphate(4-OH-DMAPP) is an intermediate ofthe new pathway in plants. A modifiedPoulter-procedure for the synthesis ofvery sensitive aldehyde-pyrophosphateswas developed, in addition to our newsynthesis of organic diphosphates andcyclic phosphates. Furthermore, we stu-died the physico-chemical properties of

the intermediates of the latter synthesis.UbiA-prenyltransferase is a membranebound enzyme that catalyzes the oligo-prenylation of 4-hydroxybenzoic acid(PHB) in 3-position as part of the bio-synthesis of ubiquinones (figure 1).Previously we could demonstrate theuse of the E. coli - enzyme in vitro andelaborate a model of the aromatic sub-strate. This model was extended andimproved, and for the first time also onefor the prenyl component was developed.Both substrate models were incorpora-ted in the first protein models of thisclass of transferases, based on homologycalculations (cf. research group "compu-tational chemistry"), which form thebasis for future verification by site direc-ted mutagenesis and mechanism-basedinhibitors. The synthesis of several suchinhibitory compounds was successfullystarted.

For the better production of prenylatedhydroxybenzoates, the influence ofvarious parameters and modifiers likecosolvents (figure 2), additives and metalions on the reaction was studied.Improved assay-conditions were found,

Group members

Marco Dessoy(PhD student since May 2002)

Michael Fulhorst(PhD student since January 2001)

Gudrun Hahn(technician)

Andrea Köver(PhD student since November 2001)

Martina Lerbs(technician)

Raik Löser(research scientist until December 2001)

Lech Luczak(postdoctoral position since August 2002)

Fred Stevens(postdoctoral position until October 2002)

Svetlana Zakharova(postdoctoral position since November 2002)

Collaborators

Jürgen AllwohnWella AG, Darmstadt, Germany

Han AsardUniversity of Nebraska-Lincoln, USA

Uwe BornscheuerUniversity of Greifswald, Germany

Bettina HauseInstitute of Plant Biochemistry, Halle, Germany

Lutz HeideUniversity of Tübingen, Germany

Udo KraglUniversity of Rostock, Germany

Romano OrruFree University of Amsterdam, The Netherlands

Markus PietzschUniversity of Halle, Germany

Kazufumi YazakiUniversity of Kyoto, Japan

Meinhart ZenkUniversity of Halle, Germany

Research Group: Biocatalysis & Design of LigandsHead: Ludger Wessjohann

The isoprenoid metabolism pathways provide insight into the predominantmechanisms and routes, nature uses to build up carbon skeletons.Understanding these, will provide new enzymes for in vitro C-C-couplingreactions, e. g. biocatalysts for the production of prenylated and terpenoidcompounds, as well as new targets for inhibitors of important metabolic pro-cesses in plants, most pathogenic bacteria, and many parasites. Of special inte-rest to us is the transfer of prenyldiphosphates onto aromatic substrates, asneither the mechanism nor structural information about these mostly mem-brane bound enzymes is available. We hope to elucidate mechanistic andstructural details, provide better access to probes and substrates, developmechanism-based inhibitors and finally achieve access to a set of enzymesenabling a multitude of enzymatic C-C-coupling reactions.

But also other enzymes, e. g. hydrolyases and oxidoreductases are used forthe efficient synthesis of building blocks, especially for enantio- or regioselec-tive transformations. In some cases, enzymatic reactions can also be used forpro-drug like systems, or such systems are discovered in nature.

Figure 1: The enzymatic oligoprenylation (n > 1, OPP = diphosphate) of p-hydroxy benzoate (PHB).

35

(Moraceae) eleven new furanocouma-rins, especially with oxygenated geranylchains, as well as one benzofuran deriva-tive could be structurally elucidated byhigh-resolution MS and 2D-NMR-analy-sis. With a series of new cardanols apotential cancerostatic activity wereidentified by GC-MS in Rhus thyrsiflora(Anacardiaceae). Moreover, besidessome known piperidine alkaloids a newchlorinated amide from Aloe sabaea(Aloeaceae) as well as a new 5-methyl-chromone glycoside from Commi phorasocotrana (Burseraceae) could be isola-ted. The first finding of two coumarin-flavonoid hybrid compounds from Gnidiasocotrana (Thymelaeceae), representinga new type of compounds, was an impor-tant topic in the report period. A phyto-chemical investigation of Eulophia peter-sii (Orchidaceae) led to the identificationof structurally known phenanthrenederivatives (cooperation with MohamedMasaoud, Sanáa).

In cooperation with Luay Ra shan (Am -man) the trail for a chemical basis forthe anticancer properties of a regionalplant was followed. The work on consti-tuents of African species in Antidesma,which led to the discovery of com-pounds highly active against trypano-somes (Chagas' disease) was concluded.A new project based on plants fromMadagascar was initiated. However, aswith other projects in this world region,the cooperation was halted for the timebeing.

We hope to re-establish active projects,when the safety, political and financial

situation has stabilized, because the pastcooperation was highly successful withrespect to our local partners, structuresor biological activities found.

The AmericasThough the political development inLatin America is very favorable, naturalproduct research is increasingly ham-pered by bureaucracy for both, the localpartners as well as the internationalones. Together with a veterinary insti-tute in Southern Brazil we looked atpampas plants toxic for cattle. Consti -tuents, which are potentially responsiblefor the observed bone deformation oreven death, have been identified.

Rather than being guided by ethnophar-mocological, phytochemical or observa -tive selection of plants, in a differentapproach we concentrate on the tissuespecific profiling of constituents fromsecretive plant organs. The emphasis is onelaiophores, floral glands that produceoils as rewards for pollinators of predo-minantly neotropic plants. The oil flowersyndrome was not discovered be fore theseventies. The chemical composition offloral oils is mostly unknown; the analyti-cal methods for their profiling and relia-ble analyses were not yet established. Incooperation with the Botanical GardenMunich-Nymphenburg (Günter Gerlach)and several international partners (e. g.Beryl B. Simpson), we gained access tovarious species of oil flower plants. Theanalytical work included the collection ofthe floral oils by microscopic techniques,development of micro-derivatizationmethods, and analysis of the volatile deri-vatives by GC/MS. The underivatized oilswere analyzed by electrospray tandemmass spectrometry. Structures ofuncommon chain oxidized lipids could beelucidated, e. g. novel dihydroxylated fattyacids and their glycerides in the floral oilfrom species of the families Malpi ghi -aceae and Orchidaceae. The investigationof the secretions from seven different,non-related plant families supported the

Oil flower of Ennelophus euryandrus (Iridaceae,photo by Günter Gerlach), and formula of a typi-cal floral oil component with a 2-acetoxy fattyacid residue.

Centris bee visiting the oil flower of Malpighiaemarginata. Photo by Günter Gerlach

6-(8''-umbelliferyl)-apigenin from Gnidia socotrana(Thymelaeceae)

34

Plant metabolites: Southeast AsiaHEA(N)TOS is a drug used for an effec-tive detoxification treatment of drugaddiction. It is based on the traditionalherbal medicine of Vietnam. The abbre-viation HEA(N)TOS is derived from"heat of the sun", and was originallydeveloped by the Vietnamese herbalistTran Khuong Dan. It is now produced inan improved formula at the Institute ofChemistry of the National Center forNatural Sciences and Technology inHanoi. It is composed of 13 medicinalplants and natural products grown inVietnam. As part of an UNESCO pro-ject concerning the international scienti-fic development and standardization ofthe anti-drug medication HEA(N)TOS,we perform phytochemical studies of theconstituents in close cooperation withSung Tran from the Institute of Chemis -try in Hanoi. The aim of our work is theisolation and structural elucidation ofthe compounds with potential biologicalactivities and the compilation of litera-ture data on the components alreadyknown. These investigations contributeto the botanical identification of theused plant species and provide thenecessary prerequisites for further deve-lopment and a future global use ofHEA(N)TOS. Until now, our group inve-stigated the constituents of seven of the13 components. So far, the phytochemi-cal investigations resulted in the identifi-

cation of approximately 150 substances.Besides numerous known compoundsfrom ma ny classes, several new com-pounds were detected.

Apart HEA(N)TOS, the investigation ofmedicinal plants from Southeast Asia,especially Vietnam and Myanmar (Birma),is continued. A PhD-thesis on consti-tuents of Fissistigma spec. from Vietnamwas concluded and numerous new flavo-noid-terpenoid-hybrid compounds couldbe published.

Africa, Mediterranean, and Middle EastIn the course of a phytochemical projectwith the Sanáa University (Yemen), aseries of new natural compounds werefound. Thus, from Dorstenia gigas

Group members

Nguyen Hoang Anh(postdoctoral position until August 2001)

Torsten Blitzke(postdoctoral position until May 2000)

Katrin Franke(postdoctoral position since September 2000)

Gudrun Hahn(technician)

Tobias Herzfeld(PhD student until September 2002)

Myint Myint Khine(PhD student since September 2002)

Christine Kuhnt(technician)

Monika Kummer(technician)

Martina Lerbs(technician)

Tilo Lübken(PhD student since March 2001)

Jana Mühlenberg(PhD student since August 2002)

Ernst Plaß(postdoctoral position until August 2001)

Lars Seipold(PhD student since January 2000)

Trinh Thi Thuy(postdoctoral position until October 2002)

Nguyen Hong Thi Van(guest scientist since April 2002)

Research Group: Plant and Fungal Metabolites / MicroanalyticsHeads: Norbert Arnold, Jürgen Schmidt, Ludger Wessjohann & Gernot Schneider(until June 2001)

Defined natural substances find numerous applications in society and indu-stry, e .g . as chemical raw materials, food additives, cosmetics, in agriculture,and especially in medicinal chemistry where they form the basis of more thanone third of all currently approved drugs. The research group focuses on theisolation and chemical characterization of pure compounds from plants andfungi, which are a rich and diverse source of secondary metabolites. In addi-tion, techniques for the improved analysis and profiling of plant metabolitesare developed, especially through the application of mass spectrometry. Thebiological activities of extracts, fractions and especially pure substances aretested in bioassays. These are designed to elucidate the function of the testedcompounds in na ture or to screen for medicinally or otherwise useful pro-perties.

The traditional Vietnamese herbalist Dan and hisHea(n)tos drug preparation.

37

7-Tesla-Fourier-transform ion cyclotron reso -nance mass spectrometer (FT-ICR-MS) with anelectrospray (ESI) ion source

tabacum (Otto Miersch, Halle; LucVarin, Montreal). Betalains, resveratrolgluco side, sinapic acid derivatives, flavo-noids and other secondary metabolitescould be also evaluated by MS/MS tech-niques (Willi bald Schliemann, AlfredBaumert, Thomas Vogt, Dieter Strack,Halle). In collaboration with the group ofLutz Heide (Tübingen) especially the LC-ESI-selected reaction monitoring (SRM) asa sensitive and effective method for traceanalysis, has led to the identification of aseries of new aminocoumarin antibiotics ofthe novobiocin-, chlorobiocin - and cou-mermycin type. Pro ducts of the polyketidesynthesis were identified by LC-ESI-MS/MS(Toni M. Kutchan, Jonathan Page, Halle).Using both, positive and nega tive ion elec-trospray, perlatolic acid derived depsides

and depsidones from the lichen Lecideainops were analyzed (Siegfried Huneck).Phytosterols as marker for specific mu-tations during the embryogenesis ofArabidopsis could be identified by GC-MS(collaboration with Katrin Schrick andGerd Jürgens, Tübingen).

In 2001, the mass spectrometry facilitieswere improved by establishing the elec-trospray Fourier-transform ion cyclo-tron resonance technique. This newtechnique allows mass spectral analyseswith very high resolution and mass ac -cacy. This allowed the solution of somediffi cult problems with synthetic com-pounds (e. g. macrocycles) and for theidentification of natural products. <

36

fact, that plant diversity is reflected in dif-ferent chemical compo-sitions of the flo-ral oils, and that the syndrome evolvedindependently. In a related project, thefloral glands of hops (Humulus lupulus)are studied in a cooperative effort withthe department of plant biotechnology(see separate chapter).

Fungal metabolites: The kingdom of fungi is composed of anestimated 106 specimen and forms on ofthe biggest group of organismsin our world. Many of them areliving in symbiosis with plants(mycorrhizal fungi), others arepathogenic. At this time only5 % (75.000) of all fungi arewell described. In our researchon fungal metabolites, we aremain ly focused on compoundsfrom fruitbodies of Basidio -mycetes (e. g. Hygro phorus,Cor tinarius). Species in thegenus Hygro phorus Sect.Oliva ce oum brini are well cha-racterized by a yellow reaction after tre-ating the stem with base like potassiumhydroxide. The responsible constituents

could be isolated and their structureselucidated as cyclopentenon derivatives.Information about their biosynthesis isexpected from feeding experiments with13C-labeled precursors. In addition, somecompounds show remarkable antifungalactivity in our bioassay.

The fruitbodies of Cortinarius bolaris, aspecies described in the literature aspoisonous, are staining yellow when bru-ised or cut. The yellow stained areas

show a bright golden fluorescence in UV-light. The chemical principle underlyingthese phenomena could be isolated andwas characterized as a new benzofuranglycoside. Further research is directed atthe chemical constituents of Sepe doni -um (Fungi imperfecti), which live as para-sites on boletes and bolete relatives (Bo le -ta les).

Microanalytics:The coupling of HPLC and electrospray(ES) tandem mass spectrometric methodswas successfully applied to the micro-analysis of a series of natural compoundsin collaboration with all departments ofthe IPB and external groups. Thus, a LC-ESI-MS/MS method for the determi-nation of 5-methylchromone glycosideswas developed and some new com-pounds of this type from Aloe speciescould be identified. 12-hydroxysulfonyl-oxyjasmonic acid was identified byselected reaction monitoring inArabidopsis thaliana and Nicotiana

Collaborators

Joe AmmiratiUniversity of Washington, Seattle, USA

Marta AndriantsiferanaUniversity of Antananarivo, Madagascar

Helmut BeslUniversity of Regensburg, Germany

Manfred BinderClark University, Worcester, USA

Joao Braga de MelloUniversidade Federal de Rio Grande do Sul,Porto Alegre, Brazil

Günter GerlachBotanical Garden Munich, Germany

Lutz Heide, Shu-Ming LiUniversity of Tübingen, Germany

Jochen KopkaMax Planck Institute of Molecular Plant Physiology,Golm, Germany+

Toni M. Kutchan, Jonathan Page, DierkScheel, Stephan Clemens, Dieter Strack,Alfred Baumert, Willibald SchliemannInstitute of Plant Biochemistry, Halle, Germany

Kurt MerzweilerUniversity of Halle, Germany

Luay RashanApplied Science University, Amman, Jordan

Joachim SchröderUniversity of Freiburg, Germany

Beryl B. SimpsonUniversity of Texas, Austin, USA

Wolfgang SteglichUniversity of Munich, Germany

Tran Van SungNRCS, Institute of Chemistry, Hanoi, Vietnam

Meinhard H. ZenkBiocenter Halle, Germany

Research Group: Plant and Fungal Metabolites / MicroanalyticsHeads: Norbert Arnold, Jürgen Schmidt, Ludger Wessjohann & Gernot Schneider(until June 2001)

Cortinarius bolaris (day light) Cortinarius bolaris (UV 365nm)

Feeding experiment on Hygrophorus latitabundus

39

test of the Federal Institute for MaterialsResearch and Testing (BAM, Berlin) for thevalidation of 1H NMR spectroscopy as a reliablyquantitative analysis method. The main taskof the NMR-subgroup was the structural elu-cidation of natural products and syntheticcom pounds in collaboration with the othergroups of the department. As an example ofa successful structural elucidation, figure 2shows two new aurones (collaboration withFred Stevens). The constitution as well as theconfiguration of the double bonds could beelucidated by one- and two-dimensionalNMR experiments and NOE investigations.

In late 2001, the subgroup "computationalchemistry" was started. A powerful computer-cluster of altogether four UNIX-workstationsand six LINUX and WINDOWS-PCs wasinstalled as basic prerequisite for the perfor-mance of molecular modeling calculations.For this purpose, software program packages,

such as SYBYL, MOE (MolecularOperating Environ ment) SPAR-TAN, JAGUAR and GAUSSIANwere installed (figure 3).

The first project aims at thehomology modeling of aromaticprenyltransferases, an importantgroup of enzymes of which neither3-D structural information normechanistic details of the cata-

lysis mechanism were known. Most aromaticprenyltransferases are membrane bound, like4-hydroxybenzoate oligoprenyltransferase(ubiA), a key enzyme in the biosynthesispathway of ubiquinone. It catalyzes the preny-lation of 4-hydroxybenzoate in the 3-positionwith an oligoprenyldiphosphate and is one ofthe best-characterized examples, which wasalso available to us for experimental verifica-tion (cf. biocatalysis group). By using homo -logy modeling and multiple alignments,secondary structure prediction, moleculardynamics simulations and energy optimiza-tions, two first models with two possible acti-ve sites could be created and refined (figure4).

Other investigations concerned the analysisof structure activity relationships of epothi -lones accompanied by conformational inve-stigations based on NMR data, conformatio-nal studies of macrocycles, and calculations

on the reaction mecha-nism of the late enzymesof the non-mevalonatepathway (MEP-pathway)of isoprenoid biosynthe-sis, especially the conver-sion of 2-methyl-D-ery -thritol-2,4-cyclodiphos-phates.

Within the EU-project"Opioid Treatment ofChro nic Pain and Inflam -mation of the Loco motorSystem", 3D-models ofthe opioid receptors havebeen developed. Basedon these models and cor-

Figure 3: Configuration of the workstations and PC cluster of the modelinggroup.

responding docking studies the structure-activity

re lation ships of a multitude of opioids couldbe explained and new derivatives with impro-ved properties could be proposed for synthe-sis. Further more, the unusual long duration ofaction of a new class of kappa selectiveopioids could be clarified based on mechani-stic investigations and ab inito DFT-calcula-tions.

In 2002, "Phytobase" was started in collabo-ration with Volkmar Vill (Hamburg) as a long-term development project. The compoundbased reference and spectroscopy collectionwill include biological information and willdirectly link all available data in an object-oriented database. Phyto base will be the cen-tral information system of the departmentand is a cornerstone for future developmentof the IPBs natural product research. It will beavailable to the other departments, and parti-ally to external partners (PlantMetaNet, fun-ding partners) and the public. <

Figure 4: The most likely model of 4-hydroxyben-zoate oligoprenyltransferase with docked substra-tes octaprenyl-diphosphate (OP-PP), 4-hydroxy-benzoate (4-HB) and magnesium (Mg) at theexpected active site.

Figure 2: Constitution and configuration of two new aurones

38

In 2000, the DFG supported project"Conformation and structure-activity re-lationship of brassinosteroids" was finishedwith investigations of the side-chain confor-mations of brassinosteroids in aqueous solu-tion with and without the presence of micelleforming agents. Using the sophisticated WETsolvent signal suppression technique (watersuppression enhanced through T1 effects),spectra with sufficient signal-to-noise ratioscould be recorded, even if the solubility ofbrassinosteroids in water is less than 0.2 mmol/l. A highly conserved solutionstruc ture of the steroidal side-chain wasfound in case of brassinolide whereas the lessbioactive 24-epi-brassinolide showed diffe-rent conformations dependent on themedium.

The NMR equipment was largely modernizedand expended with the different focus of thedepartment in 2001. The 500 MHz and the

300 MHz NMR spectrometer were equippedwith new radio-frequency consoles and newprobe heads. Inter alia, for the first time high-resolution MAS (magic angle spinning) protonspectra, deuterium decoupled 13C spectra andDOSY (diffusion ordered spectroscopy) spec-tra could be recorded. A new 400 MHz NMRspectrometer equipped with a four nucleiauto-switchable probe was installed in thatyear (figure 1). Since March 2002, this spec-trometer is operated as an open-access rou-tine NMR for trained graduate students, post-docs and technicians of the department. Inpreparation for the open access use, a set ofmacro programs was developed, which allowthe easy set-up of experiments and data pro-cessing. This included solvent de pend ent shimsets and parameter files, which were adjustedat regular intervals. All users were trained inthe operation of the NMR spectrometer andhad to pass an "NMR driving test" beforeusing the instrument unsu per vised.

Service measurements ofNMR spectra as well asopto-analytical (IR, UV,CD and ORD) spectrawere carried out forscientists of this and otherdepartments of the IPB.Since 2000, the samplevolume increased severaltimes. Currently some5000 spectra are recor-ded per annum. The NMRlaboratory of the IPB par-ticipated successfully in anational interlaboratory

Group membersMonika Bögel(research scientist since August 2001)

Lars Bräuer(student until June 2002, afterwards PhD student)

Alexander Buske(research scientist until December 2000)

Susanne Drosihn(research scientist until February 2002)

Dubravko Jelic(guest scientist until March 2002)

Olaf Ludwig(system administrator since March 2001)

Maritta Süße(technician)

Larisa Vasilets(guest scientist until December 2002)

CollaboratorsAPOGEPHA GmbHDresden, Germany

Horst BögelUniversity of Halle, Germany

Dieter BrömmeThe Mount Sinai School of Medicine, New York, USA

Volker Christoffel,Barbara SpenglerBionorica AG, Neumarkt, Germany

Ivo FeußnerUniversity of Göttingen, Germany

Susanna FürstSemmelweis University of Budapest, Hungary

Lutz Heide, Shuming LiUniversity of Tübingen, Germany

Ulrike HolzgrabeUniversity of Würzburg, Germany

Andris KreicbergsKarolinska Institute, Stockholm, Sweden

Volker LipkaMax Planck Institute of Plant Breeding Research,Cologne, Germany

Klaus NeubertUniversity of Halle, Germany

PLIVA AGZagreb, Kroatien

Dierk Scheel, Dieter Strack,Thomas Voigt, Judith HansInstitute of Plant Biochemistry, Halle, Germany

Helmut SchmidhammerUniversity of Innsbruck, Austria

Sungene GmbHGatersleben, Germany

Volkmar VillUniversity of Hamburg, Germany

Meinhard H. ZenkUniversity of Halle, Germany

Research Group: Structural Analysis & Computational ChemistryHeads: Wolfgang Brandt & Andrea Porzel

The research group is investigating three-dimensional molecular structures ofsmall molecules and proteins as well as reaction mechanisms in the field ofbioorganic chemistry by means of molecular modeling, semi-empirical calcu-lations, nuclear magnetic resonance (NMR) and optical spectroscopy. Thegroup is also respon sible for the development of a database, designed to solveproblems involved with phytochemical investigations such as fast derepli-cation (Phytobase). The collected information together with data mining andnew data from the other research groups forms the basis for chemoinforma-tic analyses, which will enable new insights in the biological significance ofplant and fungal metabolites.

Figure 1:The new 400 MHz NMR spectrometer used by Dr. Trinh Thi Thuyin open-access mode

41

Maier, W., Schmidt, J., Nimtz, M., Wray, V. & Strack, D.Secondary products in mycorrhizal tobacco andtomato roots. Phytochemistry 54, 473-479 (2000).

Nguyen Thi Hoang Anh & Tran Van Sung, Someresults on Phytochemical study of Rehmannia gluti-nosa rhizomes. Proceeding of national conferenceon organic chemistry, 329-332 (2001).

Nguyen Thi Hoang Anh, Tran Van Sung, Porzel, A.,Franke, K. & Wessjohann, L. Homoisoflavonoidsfrom Ophiopogon japonicus Ker-Gawler.Phytochemistry 62, 1153-1158 (2002).

Porzel, A. & Huneck, S. Acaranoic acid and acareno-ic acid: Confirmation of structure by modern NMRMethods. Bibliotheca Lichenologica 78, 365-368(2001).

Porzel, A., Trinh Phuong Lien, Schmidt, J., Drosihn, S.,Wagner, C., Merzweiler, K., Tran Van Sung & Adam,G. Fissistigmatins A-D: Novel type natural productswith flavonoid-sesquiterpene hybrid structure fromFissistigma bracteolatum. Tetrahedron 56, 865-872(2000).

Samappito, S., Page, J., Schmidt, J. De-Eknamkul, W. &Kutchan, T. M. Molecular characterization of root-specific chalcone synthases from Cassia alata.Planta 216, 64-71 (2002).

Schliemann, W., Cai, Y., Degenkolb, T., Schmidt, J. &Corke, H. Betalains of Celosia argentea.Phytochemistry 58, 159-165 (2001).

Schmidt, J., Blitzke, T. & Masaoud, M. Structural inve-stigations of 5-methylchromone glycosides fromAloe species by liquid chromatography / electro-spray tandem mass spectrometry. Eur. Mass.Spectrom. 7, 481-490 (2001).

Schmidt, J., Richter, K., Voigt, B. & Adam, G.Metabolic transformation of the brassinosteroid24-epicastasterone by the cockroach Periplanetaamericana. Z. Naturforsch. 55c, 233-239 (2000).

Schmidt, J. & Wessjohann, L. Studies in natural pro-ducts chemistry. Book reviews in Phytochemistry61, 880 (2002).

Schneider, G., Fuchs, P. & Schmidt, J. Evidence for thedirect 2b- and 3b-hydroxylation of [2H2]GA20-13-O-[6'-2H2]glucoside in seedlings of Phaseolus cocci-neus L. Physiologia Plantarum 116, 144-147 (2002).

Schneider, G., Koch, M., Fuchs, P. & Schmidt, J.Identification of metabolically formed glucosyl con-jugates of [17-D2]GA34. Phytochem. Anal. 11, 232-239 (2000).

Schrekker, H., de Bolster, M., Orru, R. &Wessjohann, L. In Situ Formation of Allyl Ketonesvia Hiyama-Nozaki Reactions Followed by aChromium-Mediated Oppenauer Oxidation. J. Org.Chem. 67, 1975-1981 (2002).

Schrick, K., Mayer, U., Horrichs, A., Kuhnt, C., Bellini,C., Dangl, J., Schmidt, J. & Jürgens, G. FACKEL is asterol C-14 reductase required for organized celldivision and expansion in Arabidopsis embryogene-sis. Genes & Development 14, 1471-1484 (2000).

Schrick, K., Mayer, U., Martin, G., Bellini, C., Kuhnt,C., Schmidt, J. & Jürgens, G. Interactions betweensterol biosynthesis genes in embryonic develop-ment of Arabidopsis. Plant J. 31, 61-73 (2002).

Schulz-Lang, E., Burrow, R. A., Braga, A. L., Appelt, H.R., Schneider, P. H., Silveira, C. C. & Wessjohann, L. A.R,R-(+)-Bis[(3-benzyloxazolan-4-yl)methyl]disulfi-de. Acta Cryst. E57, 41-42 (2001).

Shu-Ming Li, Westrich, L , Schmidt, J., Kuhnt, C. &.Heide, L. Methyltransferase genes in Streptomycesrishiriensis: new coumermycin derivatives fromgene inactivation experiments. Microbiology 148,3317-3326 (2002).

Smagghe, G., Decombel, L., Carton, B., Voigt, B.,Adam, G. & Tirry, L. Action of brassinosteroids inthe cotton leafworm Spodoptera littoralis. InsectBiochemistry and Molecular Biology 32, 199-204(2002).

Stano, J., Micieta, K., Neubert, K., Luckner, M. &Adam, G. A simple method for the identificationand assay of extracellular plant b-galactosidase.Pharmazie 57, 176-177 (2002).

Tierens, K. F. M.-J., Thomma, B. P. H. J., Brouwer, M.,Schmidt, J., Kistner, K., Porzel, A., Mauch-Mani, B.,Cammue, B. P. A. & Broekaert, W. F. Study of theRole of Antimicrobial Glucosinolate-DerivedIsothio cyanates in Resistance of Arabidopsis thalia-na to Microbial Pathogens. Plant Physiol. 125, 1688-1699 (2001).

Tran Van Sung, Trinh Thi Thuy, Thach Thi Dan, Adam,G. & Merzweiler, K. Isolation and structure of iso-corydin and corydalmin from the rhizome ofStephania rotunda. J. of Chemistry (Vietnamesisch)40, 35-40 (2002).

Trinh Phuong Lien, Kamperdick, C, Schmidt, J., TranVan Sung & Adam, G. Apotirucallane triterpenoidsfrom Luvunga sarmentosa (Rutaceae).Phytochemistry 60, 747-754 (2002).

Trinh Phuong Lien, Porzel, A. & Schmidt, J., Tran VanSung & Adam, G. Chalconoids from Fissistigmabracteolatum. Phytochemistry 53, 991-995 (2000).

Trinh Phuong Lien, Tran Van Sung & Adam, G.Phytochemische Untersuchungen überInhaltsstoffe der vietnamesischen HeilpflanzeTabernaemontana corymbosa (Nghien Cúu ThànhPhàn Hóa Hoc Cay Lài Trau Tu TánTabernaemontana Corymbosa). Zeitschrift fürChemie Vietnam 39, 39-44 (2001).

Trinh Thi Thuy, Tran Van Sung & Adam, G. Limonoideaus der vietnamesischen Heilpflanze Clausenaexcavata (Các Hop Chát Limonoit Tù Cay Hòng BiDai Clausena excavata). Zeitschrift für ChemieVietnam 39, 27-33 (2001).

Vasilets, L. A., Brandt, W., Postina, R., Kirichenko, S.&Anders, A. (2000) Molecular mechanisms of PKC-mediated inhibition of cation transport by theNa+/K+-ATPase: sitedirected mutagenesis andmolecular modelling studies. Pflügers Arch. 439,R321 (2000).

Voigt, B., Porzel, A., Adam, G., Golsch, D., Adam, W.,Wagner, C. & Merzweiler, K. Synthesis of 2,24-die-picastasterone and 3,24-diepicastasterone aspotential brassinosteroid metabolites of the cok-kroach Periplaneta americana. Collect. Czech.Chem. Commun. 67, 91-102 (2002).

Voigt, B., Whiting, P. & Dinan, L. The ecdysteroid ago-nist/antagonist and brassinosteroid-like activities ofsynthetic brassinosteroid/ecdysteroid hybrid mole-cules. Cell. Mol. Life Sci. 58, 1133-1140 (2001).

Wessjohann, L. A. Synthesis of natural-product-based compound libraries. Curr. Opin. Chem. Biol.4, 303-309 (2000).

Wrenger, S., Kähne, T., Faust, J., Mrestani-Klaus, C.,Fengler, A., Stöckel-Maschek, A., Lorey, S., Brandt, W.,Neubert, K., Ansorge, S. & Reinhold, D.Downregulation of T cell activation following inhi-bition of dipeptidyl peptidase IV/CD26 by the N-terminal part of the thromboxane A2 receptor. JBiol Chem. 275, 22180-22186 (2000).

Books and Book ChaptersBrandt, W. Development of a tertiary-structuremodel of the C-terminal domain of DPPIV. In: Adv.Exp. Med. Biol., Vol 477, Cellular Peptidases inImmune Functions and Diseases (2), (Langner, J. &Ansorge, S., eds.) Kluwer Academic/PlenumPublishers, Dordrecht, pp. 97-102 (2000).

Bühling, F., Nägler, D., Fengler, A., Brandt, W., Welte, T.& Ansorge, S. The growing family of mammalianpapain-like cysteine proteinases. In: Adv. Exp. Med.Biol., Vol 477, Cellular Peptidases in ImmuneFunctions and Diseases (2), (Langner, J. & Ansorge,S., eds.) Kluwer Academic/Plenum Publishers,Dordrecht, pp. 241-254 (2000).

Fengler, A. & Brandt, W. Development and validationof homology models of human cathepsins K, S, H,and F. In: Adv. Exp. Med. Biol., Vol 477, CellularPeptidases in Immune Functions and Diseases (2)(Langner, J. & Ansorge, S., eds.) KluwerAcademic/Plenum Publishers, Dordrecht, pp. 255-260 (2000).

Mrestani-Klaus, C., Fengler, A., Faust, J., Brandt, W.,Wrenger, S., Reinhold, D., Ansorge, S. & Neubert, K.N-terminal HIV-1 Tat nonapeptides as inhibitors ofdipeptidyl peptidase IV. Conformational characteri-zation. In: Adv. Exp. Med. Biol., Vol. 477, CellularPeptidases in Immune Functions and Diseases (2),(Langner, J. & Ansorge, S., eds.) KluwerAcademic/Plenum Publishers, Dordrecht, pp. 125-130 (2000).

Schmidt, J., Spengler, B., Voigt, B. & Adam, G.Brassinosteroids - Structures, Analysis andSynthesis. In: Evolution of Metabolic Pathways,Recent Advances in Phytochemistry 34 (Romeo, J.T., Ibrahim, R., Varin, L. & DeLuca, V., eds.) Pergamon,Amsterdam, pp. 385-407 (2000).

Vasilets, L. A., Brandt, W., Postina, R., Fotis, H.,Tatjanenko, L. V. & Gvozdev, A. R. Molecular mecha-nisms of covalent regulation of the Na+/K+-ATPaseby protein kinases. In: The Sodium Pump (Taniguchi,K. & Kaya, S. eds.) Elsevier, Amsterdam, pp. 507-572.

Wessjohann, L. A. & Scheid, G. Synthetic Access toEpothilones - Natural Products with ExtraordinaryAnticancer Activity. In: Organic ChemistryHighlights IV (Schmalz, H.-G., ed.) Wiley-VCH,Weinheim, pp. 251-267 (2000).

Wrenger, S., Reinhold, D., Faust, J., Mrestani-Klaus,C., Brandt, W., Fengler, A., Neubert, K. & Ansorge, S.Effects of nonapeptides derived from the N-termi-nal structure of human immunodeficiency virus-1(HIV-1) Tat on suppression of CD26-dependent Tcell growth. In: Adv. Exp. Med. Biol., Vol 477, CellularPeptidases in Immune Functions and Diseases (2),(Langner, J. & Ansorge, S. eds.) KluwerAcademic/Plenum Publishers, Dordrecht, pp. 161-166 (2000).

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Buske, A., Schmidt, J. & Hoffmann, P. Chemo -taxonomy of the tribe Antidesmeae (Eu -phorbiaceae): antidesmone and related com-pounds. Phytochemistry 60, 489-496 (2002).

Buske, A., Schmidt, J., Porzel, A. & Adam, G.Alkaloidal, Megastigmane and Lignane Glucosidesfrom Antidesma membranaceum (Euphorbiaceae).Eur. J. Org. Chem. 2001, 3537-3543 (2001).

Cassán, F., Bottini, R., Schneider, G. & Piccoli, P.Azospirillum brasiliense and Azospirillum lipoferumhydrolyse conjigates of GA20 and metabolize theresultant aglycones to GA1 in seedlings of ricedwarf mutants. Plant Physiol. 125, 2053-2058(2001).

Clemens, S., Schroeder, J. I. & Degenkolb, T.Caenorhabditis elegans expresses a functional phy-tochelatin synthase. Eur. J. Biochem. 268, 3640 -3643 (2001).

Drosihn, S., Porzel, A. & Brandt, W. Determinationof preferred conformations of brassinosteroids bymeans of NMR investigations and Boltzmann stati-stical analysis of simulated annaeling calculations. J.Mol. Model. 7, 34-42 (2001).

Eichelberger, U., Mansourova, M., Hennig, L.,Findeisen, M., Giesa, S., Müller, D. & Welzel, P. A crossmetathesis-based synthesis of analogues of the 2-O-alkyl glycerate part of the moenomycins.Tetrahedron 57, 9737-9742 (2001).

Eichelberger, U., Neundorf, I., Hennig, L., Findeisen,M., Giesa, S., Müller, D. & Welzel, P. Synthesis of ana-logues of the 2-O-alkyl glycerate part of the moe-nomycins. Tetrahedron 58, 545-559 (2002).

Ettrich, R., Brandt, W., Kopecky Jr., V., Baumruk, V.,Hofbauerova, K. & Pavlicek, Z. Study of chaperone-like activity of human haptoglobin: Localisation ofchaperone binding sites on the three-dimensionalstructure of the H-chain deduced by knowledge-based modeling. Biol. Chem., 383, 1667-1676(2002).

Franke, K., Kuhnt, C., Schmidt, J. & Munoz, O. 24-epi-castasterone and phytosterols from seeds ofMaytenus boaria (Celastraceae). Rev. Latinoamer.Quim. 27, 111-115 (2000).

Franke, K., Masaoud, M. & Schmidt, J. Cardanolsfrom Rhus thyrsiflora. Planta Medica 67, 477-479(2001).

Franke, K., Porzel, A., Masaoud, M., Adam, G. &Schmidt, J. Furanocoumarins from Dorstenia gigas.Phytochemistry 56, 611-621 (2001).

Franke, K., Porzel, A. & Schmidt, J. Flavone-coumarinhybrids from Gnidia socotrana. Phytochemistry 61,873-878 (2002).

Galm, U., Schimana, J., Fiedler, H.-P., Schmidt, J., Shu-Ming Li & Heide, L. Cloning and analysis of thesimocyclinone biosynthetic gene cluster ofStreptomyces antibioticus Tü 6040. Arch Microbiol178, 102-114 (2002).

Gao, W., Löser, R., Raschke, M., Dessoy, M., Fulhorst,M., Alpermann, H., Wessjohann, L. A. & Zenk, M. H.(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate: Anintermediate in the formation of terpenoids inplant chromoplasts. Angew. Chem. Int. Ed. 41, 2604-2608 (2002).

Gräther, O. & Schneider, B. The metabolic diversityof plant cell and tissue cultures. Physiology,Progress in Botany 62, 266-304 (2001).

Holzgrabe, U., Cambareri, A., Kuhl, U., Siener, T.,Brandt, W., Straßburger, W., Friderichs, E.,Englberger, W., Kögel, B. & Haurand, M.Diazabicyclononanones, a potent class of kappaopioid analgesics. Il Farmaco 57, 531-534 (2002).

Holzgrabe, U., Friderichs, E., Englberger, W., Kögel,B., Haurand, M., Strassburger, W., Brandt, W.,Cambareri, A., Kuhl, U. & Siener, T.Diazabicyclononanones, a new class of opioid-typeanalgesics. Science and Culture 68, 11-18 (2002).

Hui Xu, Zhao-Xin Wang, Schmidt, J., Heide, L. &Shu-Ming Li. Genetic analysis of the biosynthesis ofthe pyrrole and carbamoyl moieties of coumermy-cin A1 and novobiocin. Mol. Genet. Genomics 268,387-396 (2002).

Irmler, S., Schröder, G., St-Pierre, B., Crouch, N. P.,Hotze, M., Schmidt, J., Strack, D., Matern, U. &Schröder, J. Indole alkaloid biosynthesis inCatharanthus roseus: new enzyme activities andidentification of cytochrome P450 CYP72A1 assecologanin synthase. Plant J. 24, 797-804 (2000).

Kamperdick, C., Nguyen Minh Phuong, Tran VanSung & Adam, G. Guaiane dimers from Xylopia viel-ana. Phytochemistry 56, 335-340 (2001).

Kobayashi, N., Schmidt, J., Nimtz, M., Wray, V. &Schliemann, W. Betalaines from Christmas cactus.Phytochemistry 54, 419-426 (2000).

Kobayashi, N., Schmidt, J., Wray, V. & Schliemann, W.Metabolic formation and occurrence of dopamine-derived betacyanins. Phytochemistry 56, 429-436(2001).

Kolbe, A., Fuchs, P., Porzel, A., Baumeister, U., Kolbe,A. & Adam, G. Synthesis and crystal structure of[26,27-2H6] 24-epicathasterone. J. Chem. Soc.,Perkin Trans. 1, 2022-2027 (2002).

Kolbe, A., Kramell, R., Porzel, A., Schmidt, J.,Schneider, G. & Adam, G. Synthesis of dexametha-sone conjugates of the phytohormones gibberellinA3 and 24-epicastasterone. Collect. Czech. Chem.Commun. 67, 103-114 (2002).

Landtag, J., Baumert, A., Degenkolb, T., Schmidt, J.,Wray, V., Scheel, D., Strack, D. & Rosahl, S.Accumulation of tyrosol glucoside in transgenicpotato plants expressing a parsley tyrosine decar-boxylase. Phytochemistry 60, 683-689 (2002).

Lecaille F., Choe Y., Brandt W., Li, Z, Craik, C.S. &Bromme, D. Selective inhibition of the collagenoly-tic activity of human cathepsin K by altering its S2subsite specificity. Biochemistry 41, 8447-8454(2002).

Loc, Tran Van, Tran Van Sung, Kamperdick, C. &Adam, G. Synthesis of amino acid conjugates andfurther derivatives of 3b-hydroxylup-20(29)ene-23,28-dioic acid. J. für praktischeChemie/Chemiker-Zeitung 342, 63-71 (2000).

Lübken, T., Kraus, A. & Lorenz, W. Polyphenole inWeinen aus Sachsen und Sachsen-Anhalt.Lebensmittelchemie 56, 103 (2002).

PublicationsAdam, G. 25 Jahre deutsch-vietnamesischeZusammenarbeit auf dem Gebiet der pflanzlichenNaturstoffchemie - ein Resümee. Viet Nam Info 1,5-6 (2000).

Amaral, A. C. F., Kuster, R. M., Bessa, W. d. S., Barnes,R. A., Kaplan, M. A. C. & Wessjohann, L. A. Flavonoidsand other phenolics from leaves of two Marlieraspecies (Myrtaceae). Biochemical Systematics andEcology 29, 653-654 (2001).

Baumert, A., Mock, H.-P., Schmidt, J., Herbers, K.,Sonnewald, U. & Strack, D. Patterns of phenylpro -panoids in non-inoculated and potato virus Y-inocu-lated leaves of transgenic tobacco plants expressingyeast-derived invertase. Phytochemistry 56, 535-541 (2001).

Berger, S., Weichert, H., Porzel, A., Wasternack, C.,Kühn, H. & Feussner, I. Enzymatic and non-enzyma-tic lipid peroxidation in leaf development. Biochim.Biophys. Acta 1533, 266-276 (2001).

Berlich, M., Menge, S., Bruns, I., Schmidt, J., Schneider,B. & Krauss, J. Coumarins give misleading absorban-ce with Ellman's reagent suggestive of thiol conju-gates. Analyst 127, 333-336 (2002).

Blitzke, T., Baranovsky, A. & Schneider, B. Synthesisand protein binding of (4-carboxybutyl)carbamoyl-sustituted Taxoids. Helv. Chim. Acta 84, 1989-1995(2001).

Blitzke, T., Masaoud, M. & Schmidt, J. Constituents ofEulophia petersii. Fitoterapia 71, 590-591 (2000).

Blitzke, T., Masaoud, M. & Schmidt, J. Constitutentsof Aloe rubroviolacea. Fitoterapia 72, 78-79 (2001).

Blitzke, T., Porzel, A., Masaoud, M. & Schmidt, J. Achlorinated amide and piperidine alkaloids fromAloe sabaea. Phytochemistry 55, 979-982 (2000).

Blitzke, T., Schmidt, J. & Masaoud, M. 7-O-Methylaloeresin A - a new chromone glycosidefrom Commiphora socotrana. Nat. Prod. Letters15, 27-33 (2001).

Braga, A. L., Appelt, H. R., Schneider, P. H., Rodrigues,O. E. D., Silveira, C. C. & Wessjohann, L. A. New C2-symmetric chiral disulfide-ligands derived from (R)-cysteine. Tetrahedron 57, 3291-3295 (2001).

Braga, A., Silva, S., Lütke, D., Drekener, R., Silveira, C.,Rocha, J. & Wessjohann, L. Chiral diselenide ligandsfor the asymmetric copper-catalyzed conjugateaddition of Grignard reagents to enones.Tetrahedron Letters 43, 7329-7331 (2002).

Brandt, W. Struktur-Wirkungsbeziehungen vonOpioiden. Pharmazie in unserer Zeit 31, 60-66(2002).

Brandt, W., Anders, A. & Vasilets, L. A. Predicted alte-rations in tertiary structure of the N terminus ofNa+/K+-ATPase alpha subunit caused by phosphory-lation or acidic replacement of the PKC phospho-rylation site Ser-23. Cell Biochemistry andBiophysics 37, 83-95 (2002).

Bringmann, G., Schlauer, J., Rischer, H., Wohlfarth, M.,Mühlbacher, J., Buske, A., Porzel, A., Schmidt, J. &Adam, G. Revised structure of antidesmone, anunusual alkaloid from tropical Antidesma plants(Euphorbiaceae). Tetrahedron 56, 3691-3695(2000).

Publications, Books and Bookchapters, In press,Patents, Doctoral Theses, Diploma Theses

43

The profiling of stress-induced metabolicchanges in Arabidopsis plants grownunder sterile conditions in a hydroponicsystem has been established. A standardi-zed extraction procedure for root andleaf (secondary) metabolites is intro-duced. The methanolic extracts are analy-zed by Cap-LC-ESI-Q-TOF-MS, hep-tane extracts for the more hy - dro phobic com pounds areana lyzed by GC-MS. Cap-LC-ESI-Q-TOF-MS represents anew profiling approach that isto complement the more esta-blished GC-MS techniques. Becausevery few tools are available for datadeconvolution and data extraction wedeveloped respective procedures for theautomatic data analysis. Several samplescan now be processed per day. In leafextracts about 1200 mass signals areresolved and detected, in root signalsabout 1000 mass signals. Mass data can bedirectly compared to an Arabidopsis lite-rature database. The exceptional massaccuracy of ESI-Q-TOF-MS together withits tandem MS option allows tentativeidentification or classification of intere-sting compounds. An extensive evaluationof the whole Cap-LC-ESI-Q-TOF-MS-based profiling approach is now complete.Changes in response to the stress causedby exposure to elevated heavy metallevels have been analyzed. In the course ofcollaborations with other groups, severalArabidopsis mutants with defects in, forinstance, signal transduction or de fense,are being profiled. Similarly, in order to beable to use metabolite profiling for studies

on natural di ver sity, data sets for a num-ber of Ara bidopsis ecotypes are being ge -nerated.

The profiling of proteins and peptides isbased on two-dimensional gel electropho-resis, MALDI-TOF-MS and nano-spray-

ESI-MS. Patterns of soluble leaf,root or seed proteins areresolved in large-formattwo-dimensional gels. Gelimages are carefully ana-lyzed. Interesting protein

spots showing stress-relatedchan ges in abundance are picked, di -

gested and subjected to MALDI-TOFmass spectrometry for identificationbased on peptide mass fingerprints. Imageanalysis, which represents the bottleneckof searches for changes within the pro-teome, has been optimized by adoptingnew ima ging software. A number of Ara -bidopsis mutants have been analyzed un -der different stress conditions. For some ofthe identified proteins that show stress-re lated changes functional characterizationhas been initiated by isolating Arabidopsisinsertion lines for the respective genes.

The intercellular washing fluid of Ara -bidopsis leaves is analyzed for peptidesand metabolites by nanospray-ESI-Q-TOF-MS. In principle, the detection ofmolecules in this compartment is possi-ble. Progress, however, has so far beenslow due to limitations in MS capacityas the same machine is used for cap-LC-coupled metabolite profiling andnanospray-MS. <

Group members

Thomas Degenkolb(postdoctoral position since June 2000)

Claudia Horn(technician)

Kerstin Körber(technician)

Edda von Röpenack-Lahaye(postdoctoral position since May 2000)

Udo Roth(postdoctoral position since May 2000)

Collaborators

Thomas AltmannUniversity of Potsdam, GermanyGABI-Arabidopsis-Verbund III

Paul Schulze-Lefert,Bernd WeisshaarMax Planck Institute of Plant Breeding Research,Cologne, Germany

Research Group:Searching for Signals: Stress-Induced Changes in ArabidopsisSecondary Metabolite, Peptide and Protein Patterns (GABI)

Heads: Stephan Clemens, Jürgen Schmidt, Ludger Wessjohann, Dierk Scheel

Our project is aiming at contributing to the "post-genomic" ana-lysis of themodel organism Arabidopsis thaliana by establishing an extensive profiling ofproteins, peptides, and metabolites. These profiles are to be used for thedetection and identification of early stress responses and novel signalingmolecules. Eventually, they will provide valuable tools for the analysis ofvarious developmental and stress-induced changes as well as for the bioche-mical phenotyping of mutants and the exploration of natural diversity.Exemplary biotic and abiotic stresses under investigation are pathogen attackand toxic metal exposure, respectively.

figures: Reproducible plant growth of Arabidopsis

42

Nguyen Thi Hoang Anh, Tran Van Sung, Wessjohann,L. & Adam, G. The iridoids and iridoid glucosid fromthe Rehmannia glutinosa rhizome. J. of Chemistry(Vietnamesisch), in press (2002/2003).

Samappito, S., Page, J., Schmidt, J., De-Eknamkul, W.& Kutchan, T. M. Aromatic and pyrone polyketidessynthesized by a stilbene synthase from Rheumtataricum. Phytochemistry 62, 313-323 (2003).

Doctoral ThesesBuske, Alexander: Phytochemische Unter -suchungen der afrikanischen EuphorbiaceenAntidesma membranaceum und Antidesma veno-sum. Martin-Luther-University of Halle-Wittenberg, 19/10/2000.

Frutos-Höner, Annabelle: Methodology studies onin situ generated Fischer carbene complexes ofgroup VI transition metals, on the Chromium-Reformatsky reacion of nitrogen based com-pounds, and analytical studies of the Vogel collec-tion, Sandwich between Ludwig-Maximilians-Universität München, Dept. of Organic Chemistryand University of California in San Diego - UCSD(USA), November 2000.

Scheid, Günther: A New Synthesis of EpothiloneMacrocycles, Free University of Amsterdam (NL),Bioorganic Chemistry, 11/04/2002.

Sinks, Udo Eckard: New Applications of Sulfoxidesand Synthesis of Asymmetric PhenylselenideReagents, Sandwich between Free University ofAmsterdam (NL), Bio-organic Chemistry, andLudwig-Maximilians-Universität München, Dept. ofOrganic Chemistry, 15/05/2001.

Trinh Thi Thuy: Phytochemische Untersuchungender vietnameschen Heilpflanzen Clausena excavata

und Zanthoxylum avicennae (Rutaceae), NationalCenter for Natural Scientific and Technology(Vietnam), Institute of Chemistry and Martin-Luther-University of Halle-Wittenberg,01/11/2001.

Wild, Harry: Neue Anwendungen der Chrom-Reformatsky-Reaktion, Ludwig-Maximilians-Uni -versität München, Dept. of Organic Chemistry,10/10/2000.

Diploma ThesesBelting, Claudia: Terpene derived enediyes aspotential anti-tumor drugs. Hochschule Enschede,The Netherlands, 31/01/2002 (Sandwich).

Bräuer, Lars: Modellierung der 4-HydroxybenzoatOligoprenyltransferase und Charakterisierungpotentiell aktiver Zentren. Martin-Luther-University of Halle-Wittenberg, 25/06/2002.

Spelbos, Vincent: Prenyltransferasen in Humuluslupulus. University of Utrecht, The Netherlands,November 2002.

Schültingkämper, Heike: The combinatorial diaste-reoselective synthesis of highly functionalizedtetrahydropyrans. Hochschule Enschede, TheNetherlands, 18/01/2002 (Sandwich).

Wesseling, Claudia: Synthesis of a building block forthe natural product cis-gigantrionenine.Hochschule Enschede, The Netherlands,31/01/2002 (Sandwich).

In the report period, another nine students recei-ved their diploma-degree (dutch: Drs.) at the FreeUniversity of Amsterdam, Bio-organic Chemistry,under the supervision of Prof. Wessjohann.<

Publications in PressEckermann, C., Schröder, G., Eckermann, St., Strack,D., Schmidt, J., Schneider, B. & Schröder, J. Stilbenecarboxylate biosynthesis: a new function in thefamily of chalcone synthase related proteins.Phytochemistry 62, 271-286 (2003).

Huneck, S., Lumbsch, H. Th., Porzel, A. & Schmidt, J.Die Verteilung von Flechteninhaltsstoffen inLecanora muralis und Lecidea inops und dieAbhängigkeit der Usninsäure-Konzentration vomSubstrat und von den Jahreszeiten bei Lecanoramuralis. Herzogia.

Kolbe, A., Porzel, A., Schmidt, J. & Adam, G. A newsynthesis of [26,28-2H6]brassinolide and [26,28-2H6]castasterone via unusual methyl migration. J.Lab. Comp. Radiopharm.

Mrestani-Klaus, C., Brandt, W., Faust, J., Wrengler, S.,Reinhold, D., Ansorge, S. & Neubert, K. New resultson the conformations of potent DP IV (CD26) inhi-bitors bearing the N-terminal MWP structuralmotif. Int. Conf. "Dipeptidyl aminopeptidases: Basicscience and clinical applications", Berlin, 26.-29.09.

Münzenberger, B., Hammer, E., Wray, V., Schauer, F.,Schmidt, J. & Strack, D. Detoxification of ferulic acidby ectomycorrhizal fungi. Mycorrhiza.

Nguyen Thi Hoang Anh, Tran Van Sung, Wessjohann,L & Adam, G. Some homoisoflavonoidal compoundsfrom Ophiopogon japonicus Ker - Gawler. J. ofChemistry (Vietnamesisch), in press (2002/2003).

Nguyen Thi Hoang Anh, Tran Van Sung, Wessjohann,L. & Adam, G. Some hydroxycinamic acid esters ofphenylethyl alcohol glycosides from Rehmannia glu-tinosa Libosh. J. of Chemistry (Vietnamesisch), inpress (2002/2003).

Publications, Books and Bookchapters, Publications in press,Patents, Doctoral Theses, Diploma Theses

Plant development, although gene-tically determined, is largelymodulated by biotic and abiotic envi-ronmental factors. In this way, deve-lopmental programs are adapted tospecific lo cal con ditions and protecti-v e

as well asd e f e n s ereac tionsare initia-

ted du ring stress situations - anadvantageous situation for sedentaryliving plants.

The basis for those processes is theability of plants to perceive environ-mental factors and ini-tiate signaltransduction networks that modifygene expression patterns. The inve-stigation of the molecular mecha-nisms underlying this course of

events is the main topic of thedepartment of "Stress andDeve lop mental Bio logy".

Plant patho gens playa major role in bio-tic stress. The

work ofseveral rese-

arch groups ofthe departmentfocuses on theana-lysis of recogni-

tion, signal transduc-tion and gene activation

processes in plant-pathogeninteractions. The work on abio-

tic environmental factors centersaround metal homeostasis in plants,using hyperaccumulating model orga-nisms. <

45

Department: Stress and Developmental BiologyHead: Prof. Dierk Scheel

Secretary: Ruth Laue

44

plants.In addition, all Phytophthora speciestested possess a 24-kDa protein (NPP1)that triggers defense responses in pars-ley very similar as does Pep-13. NPP1-mediated activation of pathogen defensein parsley does not employ the Pep-13receptor. However, early-induced cellularresponses implicated in elicitor signaltransmission (increased levels of cyto-plasmic calcium, production of reactiveoxygen species, MAP kinase activation)were stimulated by either elicitor, sugge-sting the existence of converging signa-ling pathways in parsley. Infiltration ofNPP1 into leaves of Arabidopsis thalianaresulted in transcript accumulation ofpathogenesis-related (PR) genes, produc-tion of reactive oxygen species and ethy-lene, callose apposition, and hypersensi-tive-like cell death. NPP1-mediated in -duction of the PR1 gene is salicylic acid-dependent, and, unlike the P. syringae pv.tomato DC3000(avrRpm1)-induced PR1gene expression, required both func -tional NDR1 and PAD4. Importantly,Ara bi dopsis plants infiltrated with NPP1constitute an experimental system that isamenable to forward genetic approachesaiming at the dissection of signaling path-ways implicated in the activation of non-cultivar-specific plant defense.

The HrpZ gene product from the beanhalo-blight pathogen, Pseudomonassyringae pv. phaseolicola (HrpZPsph), issecreted in an Hrp-dependent mannerby this bacterium, and exported by thetype III se cretion system when expres-sed in the mammalian pathogen Yersiniaenterocolitica. HrpZPsph was found to

stably asso ciate with liposomes and syn-thetic bilayer membranes. Under sym-metric ionic conditions, addition of 2 nMpurified recombinant HrpZPsph to the cis-compartment of planar lipid bilayers pro -voked an ion current with a large uni taryconductivity of 207 pS. HrpZPsph-relatedproteins from P. s. pv. tomato or syringaetriggered ion currents similar to thosestimulated by HrpZPsph. The HrpZPsph-mediated ion-conducting pore was per-meable for cations but did not mediatefluxes of Cl¯. Such pore-forming activitymay allow nutrient release and / or deli-very of virulence factors during bacterialcoloni zation of host plants. In addition,HrpZ has been shown to trigger a com-plex defense response in parsley andtobacco. Ligand / receptor interactionstudies revealed the presence of a high-affinity binding site for HrpZPsph in plasmamembranes of both plants. Series oftruncated HrpZPsph proteins were analy-zed with respect to their abilities toinduce plant defense as well as to formion-conducting pores in liposomes. Thepore-forming activity of HrpZPsph wasfound to require the intact protein, whiledefense responses were stimulated by a

C-terminal fraction of the protein inboth plants. Thus, pore-forming activityof HrpZPsph does not determine the acti-vation of plant defense, but may reflectthe role of the protein during (attemp-ted) bacterial infection of plants. <

NPP1 induces a complex defense response inArabidopsis thaliana Col-0. Infiltrations were per-formed with 2.5 µM of each recombinant NPP1, amutant derivative of NPP1 with reduced activity,or Glutathione-S-Transferase as control. Necroticlesion formation 48 h upon elicitation (a), produc-tion of reactive oxygen species 3 h upon elicita-tion (b), and callose apposition 24 h upon elicita-tion (c).

47

(a) (b)

(c)

A calcium-dependent transglutaminase(TGase) present in the cell wall of asmany as ten species of the genusPhytophthora serves as recognitiondeterminant for the activation of non-

cultivar-specific defense responses inparsley and potato.An evolutionarily high-ly conserved peptide fragment of thisprotein (Pep-13) was identified within asurface-exposed loop structure of the

protein. Pep-13 wasshown to be neces saryand sufficient for re -ceptor-mediated activa-tion of de fense responsesin both plants. Mutationswithin the Pep-13 motif ofthe P. sojae TGase, whichreduced or abolished theelicitor activity of the in -tact protein, similarly af -fected its enzyme activity.Apparently, during evolu-tion plants have acquiredreceptors for the recog -nition of stable and func-tionally indispensable sur-face epitopes of micro bialpatho gens, suggesting thatsuch perception mo dulesmay form the molecularbasis of durable pathogenresistance in non-host

Group membersFrédéric Brunner(postdoctoral position)

Jutta Elster(technician)

Stephan Engelhardt(student since November 2002)

Guido Fellbrich(PhD student until July 2002)

Yvonne Gäbler(student until 2001, afterwards PhD student)

Claudia Horn(technician)

Birgit Kemmerling(postdoctoral position since April 2002)

Justin Lee(postdoctoral position)

Annette Romanski(PhD student until July 2001)

Christel Rülke(technician)

CollaboratorsGuy CornelisUniversity of Brussels, Belgium

Georg FelixFriedrich Miescher Institute, Basel, Switzerland

Jane Glazebrook, Tong ZhuTorrey Mesa Research Institute (Syngenta),San Diego, USA

Heribert HirtUniversity of Vienna, Austria

Sakari Kauppinen,Grete RasmussenNOVO NORDISK A/S, Bagsvaerd, Denmark

Harald KellerInstitut National de la Recherche Agronomique (INRA),Antibes, France

Birgit Klüsener, Elmar WeilerUniversity of Bochum, Germany

John MansfieldImperial College at Wye, University of London, UK

Nicholas PanopoulosUniversity of Crete, Greece

Steffen PanznerNovosom AG, Halle, Germany

Martin RomantschukUniversity of Helsinki, Finland

Dietmar StahlKleinwanzlebener Saatzucht AG, Einbeck, Germany

Zhongmin WeiEDEN Bioscience, Bothell, USA

46

Research Group:Signal Perception in Plant-Pathogen InteractionsHead: Thorsten Nürnberger

NPP1-induced cell death in parsleyViability of parsley protoplasts treated with 20 nM NPP1 or water(control) was determined 24 h upon elicitation (upper panel). Viabilityof parsley protoplasts (5x105/ml) was determined by double-stainingwith 50 µg/ml fluorescein diacetate and 10 µg/ml propidium iodide 24 hafter treatment (Jabs et al., 1997).

NPP1 (2.5 µM) or water (control) infiltrated into parsley leaves for 48 h(lower panel).

Innate immunity is well described for animals and is also suggested to beimportant for plants. In vertebrates and insects, microbial pathogen sensingrelies on the recognition of pathogen-specific structures, which are not foundin hosts and which are indispen-sable for the lifestyle of the microorganism.Receptor-mediated signal perception by the host gives rise to the activationof specific immune responses, such as the synthesis of antimicrobial com-pounds. We investigate whether pathogen recognition by animals and plantsshare similar characteristics. Our data suggest that the evo lution of pathogenperception systems in plants is likely to be si mi lar to that described foranimals. Microbial surfaces constitute com plex patterns for the activation ofplant pathogen defense. Recognition of microbial pattern by plants appears toresult in more sensitive perception of pathogens and synergistically enhancedplant defense. Phytopathogenic bacteria of the genus Pseudomonas produceand secrete the effector protein HrpZ during (attempted) infection of plants.HrpZ was shown to insert into lipid bilayer mem branes and to form cation-conducting channels. This ion channel-forming activity, however, appears notto be the molecular basis for the activation of defense responses in plants tre-ated with HrpZ.

49

While the activation of MAPKs and PR1,PR2 and WRKY transcription factorgenes was found to be independent ofthe oxydative burst, superoxide anionradicals, the primary reactive oxygenspecies formed during the Pep-13-stimu-lated oxidative burst, are necessary andsufficient for phytoalexin production andactivation of those genes encoding theirbiosynthetic and additional phenylpropa-noid pathway enzymes. The formation ofsuperoxide anion radicals is catalyzed byNADPH oxidases, which are structurallysimilar to the catalytic subunit of themammalian respiratory burst oxidase.Two NADPH oxidase-encoding geneswere isolated from parsley. In compari-son to the catalytic subunit of the respi-

ratory burst oxidase, these proteins areN-terminally extended by a region har-boring two Ca2+-binding EF hands. Oneof the NADPH oxidase transcripts accu-mulates rapidly and transiently upon eli-citation. In addition, transcripts encodingenzymes with and without EF handswere found to be generated by alterna-tive splicing. Heterologous expression ofboth type of proteins in yeast resulted inproduction of active NADPH oxidasesembedded in microsomal membranes.Only the larger protein with the EFhands required Ca2+ for activity.

The oxidative burst is necessary but notsufficient for Pep-13-stimulated produc-tion of the oxylipins, jasmonate and its

precursor 12-oxo-phytodienoic acid. Si -multaneous treatment of the cells withlipoxygenase inhibitors completely blok-ked the accumulation of both oxylipins,but did not affect Pep-13-mediated phy-toalexin synthesis, suggesting that jasmo-nate and/or 12-oxo-phytodienoic acidre present the starting point of yet anot-her signal transduction branch. Salicylicacid, a plant defense signaling compoundinvolved in signaling pathways that initia-te programmed cell death, does notaccumulate in Pep-13-treated parsleycells. Interestingly, parsley cells and leavesdo not undergo programmed cell deathin response to Pep-13 treatment. <

Elicitor treatment induces nucleartranslocation of MPK3Cultured parsley cells were treatedwith Pep13 (100 nM; C, D) or H2O(A, B) and fixed in 4% paraformaldehy-de 15 min. after initiation of treat-ment. Cells were embedded in paraf-fin, cut into 6 µm sections and stainedwith PcMPK3 antiserum (A, C). Goatanti-rabbit secondary antibody conju-gated with Alexa 488 was used tovisualize the primary antiserum boundto MPK3; nuclei were also counter-stained with DAPI (B, D). After treat-ment with Pep13 most nuclei werestained by PcMPK3 antiserum, where-as no or little nuclear staining wasdetectable in control cells.

48

Pep-13 treatment of suspension-culturedparsley cells rapidly stimulates Ca2+ influxresulting in a characteristic sustainedincrease in cytosolic Ca2+ levels, which isessential for all the other known elicitorresponses. At least four mitogen-activa-ted protein kinase (MAPK) cascades areactivated downstream of this Ca2+ tran-sient. Four MAPK-encoding genes have

been isolated from parsley, designatedPcMPK3a, 3b, 4 and 6 according to theirsequence similarities to MAPK-encodinggenes of Arabidopsis thaliana. Upon elici-tation PcMPK3a, 3b, 6 and a fourth so farunknown MAPK were found to be activa-ted by phosphorylation of the conservedTEY motif and translocated to thenucleus, whereas PcMPK4 was not affec-

ted. Transient co-expres-sion of dominant inactiveversions of PcMPK3a, 4and 6 with reporter genefusions of the PR1 (patho-genesis-related) and PR2pro moters has demonstra-ted that elicitor activationof these genes is regulatedby PcMPK3a (and possiblyalso 3b) and/or PcMPK6,but not by PcMPK4.MAPKs are themselves ac -ti vated through phospho-rylation by MAPK kinases.Two MAPK kinase-encodinggenes, PcMEK1 and 2, havebeen isolated from parsley.Only PcMEK2 was foundto be activated in Pep-13-treated cells and was thenable to phosphorylatePcMPK3a, 3b and 6, nothowever PcMPK4.

Group members

Reetta Ahlfors(guest scientist since July 2002, PhD student)

Barbara Degner(technician)

Magdalena Krzymowska(postdoctoral position until June 2002)

Violetta Macioszek(postdoctoral position since September 2002)

Anja Nickstadt(PhD student until May 2002)

Jason Rudd(postdoctoral position since April 2000)

Rita Schlichting(PhD student since July 2002)

Heidi Zinecker(PhD student until December 2000)

Collaborators

Thomas BollerFriedrich Miescher Institute, Basel, Switzerland

Jeff DanglUniversity of North Carolina, Chapel Hill, USA

Jerome GiraudatInstitut des Sciences du Végétal, CNRS,Gif-sûr-Yvette, France

Heribert HirtUniversity of Vienna, Austria

Jonathan JonesThe Sainsbury Laboratory, Norwich, UK

Chris LambJohn Innes Centre, Norwich, UK

John MundyUniversity of Copenhagen, Denmark

Teun MunnikUniversity of Amsterdam, The Netherlands

Karsten NiehausUniversity of Bielefeld, Germany

Jane Parker, Imre SomssichMax Planck Institute for Plant Breeding Research,Cologne, Germany

Jose Sanchez-SerranoAutonomous University, Madrid, Spain

Research Group: Cellular SignalingHead: Dierk Scheel

Parsley is not a host plant for the soybean pathogen, Phytophthora sojae, but ifgerminating zoospores of this oomycete try to invade the plant, it respondswith a multifaceted defense response that terminates the infection process.Theoligopeptide elicitor Pep-13, originating from a hyphal cell wall transglutaminaseof P. sojae, is one of the pathogen-associated molecular patterns (PAMPs)recognized by the plant cell via a plasma membrane-localized receptor (see pre-ceding report). Upon binding of Pep-13, this receptor initiates a cellular signaltransduction cascade that causes dramatic alterations of the gene expressionpattern, primarily resulting from activation of defense-related genes. The cellu-lar signaling elements linking the Pep-13 receptor to specific activation ofdefense-related genes include plasma membrane-located ion channels, proteinkinases, an NADPH oxidase and jasmonate. Together with additional unknowncomponents, these elements form a modular signaling network tightly regula-ting the temporal and spatial activation of defense reactions.

Pep-13-initiated signal transduction processes in parsley.

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dies against jasmonic and 12-oxo-phyto-dienoic acid.Jasmonate-dependent expression of theproteinase-inhibitor-II-genes is reducedin the transgenic plants indicating thatthe levels of physiologically active jasmo-nic acid are reduced due to binding bythe antibodies. The effect on defensegene expression and on the response topathogen infection is presently beinganalyzed.

Since P. infestans is not able to success-fully infect A. thaliana, the analysis ofthis nonhost pathogen interactionshould elucidate mechanisms of defenseagainst the infectious agent of late blightdi-sease. Microscopic analyses revealedthat P. in-festans spores germinate onArabidopsis leaves and attempt topene trate cells. However, successful

penetration is only observed in rarecases. The plant cell reacts with thedeposition of callose, accumulation ofautofluorescent material and localizedhypersensitive cell death. The Arabi -dopsis mutant pen2 (collaboration withVolker Lipka and Paul Schulze-Lefert,MPI Cologne), identified as allowingenhanced penetration of Blumeriagramnis f. sp. hordei, reacts similarly toP. in-festans infection with higher pene-tration frequencies and increased celldeath. Although the first layer of de-fense in nonhost resistance appears tobe affected in the mutant, pen2 is stillable to contain the pathogen. To iden-tify further components involved innonhost resistance, pen2 seeds weremutagenized and are presently beingscreened for alterations in theirresponse to P. infestans infection. <

Immunolocalization of single chain antibodiesdirected against 12-oxo-phytodienoic acid in chlo-roplasts of transgenic potato plants. In contrast tountransformed control plants (upper panel), trans-genic plants express single chain antibodies in chlo-roplasts as indicated by the green fluorescence(middle and lower panel; B. Hause).

Growth of P. infestans in potato.The oomycete P. infestans, the cau-sal agent of late blight disesase ofpotato, spreads intercellularly insusceptible potato leaves (leftpanel) and is able to sporulate(right panel). Infected cells reactwith cell death as indicated by thewhite autofluorescence (middlepanel).

The Phytophthora sojae-derived oligo-peptide elicitor Pep-13, originally identi-fied as an inducer of plant defense inparsley and shown to act as a pathogen-associated molecular pattern (PAMP) inevoking innate immune responses, alsotriggers defense responses in potato. Incultured potato cells, Pep-13 treatmentresults in the formation of hydrogenperoxide, alkalinization of the culturemedium, accumulation of 9-lipoxygena-se-derived oxylipins and activation ofdefense genes. Similarly, accumulation oftranscripts encoding enzymes of thephenylpro-panoid pathway, lipoxygenasesand pa tho genesis-related proteinsoccurs in po tato leaves in response toPep-13 infiltration. Derivatives of Pep-13show similar elicitor activity in parsleyand potato, suggesting a receptor-media-ted induction of defense response inpotato analogous to that observed inparsley. Interestingly, unlike in parsley,infiltration of Pep-13 into leaves leads torapid cell death in potato. Using transge-nic plants with modulated levels of jas-monic and salicylic acid, the dependenceof Pep-13-induced defense reactions onthese signaling compounds is being stu-

died.Oxylipins play an important role in theplant's reaction to pathogen attack. Inpotato, 9-lipoxygenase-derived oxylipinsaccumulate in response to Pep-13 andelicitor treatment as well as after patho-gen infection. To analyze the role of 9-lip oxy genase-derived oxylipins, trans-genic potato plants expressing RNAinterference constructs, targeted at thepathogen-induced 9-lipoxygenase of po -tato, were generated and are being ana-lyzed for alterations in their response topathogen infection. Whether oxylipinsfrom solanaceous plants like potato canalso be effective against pathogens inother plants is being tested by transfer-ring the respective genes from potatointo A. thaliana.

The 13-lipoxygenase products jasmonicacid and its precursor 12-oxo-phyto-dienoic acid accumulate in potato inresponse to infiltration of the phytopa-thogenic bacteria Pseudomonas syringaepv. maculicola. This nonhost pathogeninteraction leads to local and systemicde-fense gene expression and to increa-sed resistance against subsequent patho-gen attacks. 12-oxo-phytodienoic acid,but not jasmonic acid accumulates alsosystemically. To analyze the role ofthese 13-lipoxygenase products for defenseresponses, transgenic plants were gene-rated which express single chain antibo-

Group members

Carola Geiler(student until August 2001)

Cornelia Göbel(PhD student until August 2001)

Anja Grohnert(student until February 2001)

Vincentius A. Halim(PhD student since October 2002)

Astrid Hunger(PhD student until June 2002)

Martina Kausch(student until February 2002)

Jörn Landtag(student until June 2001,PhD student since September 2001)

Claudia Reh(student until February 2000)

Grit Rothe(postdoctoral position since March 2002)

Angelika Weinel(technician)

Lore Westphal(postdoctoral position since April 2002)

Collaborators

Udo Conrad, Patrick SchweizerInstitute of Plant Genetics and Crop Plant Research,Gatersleben, Germany

Ivo FeussnerUniversity of Göttingen, Germany

Markus FrankBASF Plant Science, Ludwigshafen, Germany

Bettina Hause, Dieter Strack,Claus WasternackInstitute of Plant Biochemistry, Halle, Germany

Volker Lipka, Jane Parker,Paul Schulze-LefertMax Planck Institute of Plant Breeding Research,Cologne, Germany

Mats HambergKarolinska Institute, Stockholm, Sweden

Research Group: Induced Pathogen DefenseHeads: Sabine Rosahl & Dierk Scheel

To elucidate defense mechanisms against the oomycte Phytoph-t hora infestans, the causal agent of late blight disease of potato, we are study-ing the interaction of P. infestans with its host plant potato and with the non-host plant Arabidopsis thaliana. For potato, analysis of the recognition of thepathogen, signal transduction and characterization of the pathogen defenseare our major interests.

50

Arabidopsis thaliana plants are grown under con-trolled conditions in a phytochamber. They are sub-sequently screened for alterations in their respon-se to infection with P. infestans, the causal agent oflate blight disease of potato. Thousands of plantsmust be screened to obtain one mutant.

na. A number of specifically metal-regula-ted putative signal transduction compo-nents have been identified both in A. hal-leri and A. thaliana. Five of them are stu-died in detail. A. thaliana knock-out lineshave been obtained. Their metal responsesand possible metal-related phenotypesare studied using a variety of techniquesincluding microarrays. Since recently itwas shown that Zn and Cd hyperaccumu-lation by A. halleri is a con stitutive pheno-menon found also on soil with normalmetal content, the molecular analysis wasextended to constitutive differences bet-ween the two Ara bi dopsis species. Geneexpression profiles were obtained forroots of hydroponically grown plants byusing Affymetrix GeneChips. They revea-led a number of about 20 genes which aresignificantly more active in A. halleri.Among them are genes encoding severalknown metal homeostasis factors such asmetal transporters and enzymes involvedin metal chelator synthesis. These genesrepresent prime candidates for determi-nants of Zn/Cd hyperaccumulation andare therefore studied in detail.

Several putative metal tolerance factorsare investigated in S. pombe, the modelfor PC-forming cells. The analysis of atransporter belonging to the CationDiffusion Facilitators and of the only S.pombe me tallothionein has led to new

insights into mechanisms of Zn homeosta-sis, Cd toxi city, and intracellular metaldistribution.Plant metal tolerance is an element-speci-fic process. For cell cultures of Silenejenissiensis it was shown that Zn and Cuare de toxified by distinct mechanisms. Zntolerance is mediated by two different Si-dependent processes. Exposure to Znresults in elevated Si content of cells. Znand Si containing precipitates are detec-table in the cytosol and the mitochondria.They were identified as incompletely sub-stituted Zn silicate. Such silicates are un -stable and decompose to SiO2, detec-table in the cytosol as an electron trans-parent structure and identified by EELspectra. Zn silicate is hypothesized tofunc tion as a temporary storage form ofZn, which prevents toxic effects withinthe cytosol. A second, unusual mechanismmay contribute to the Zn tolerance ofsome plants. Apparently, a large fractionof Zn is directly taken up into the va cuoleas Zn silicate without membrane passage.Transport occurs in vesicles formed byplasma membrane and tonoplast. Cu ex -posure, on the other hand, does not resultin ultrastructu-ral changes.Highest Cu con - centrations arefound in themito chon dr iaand there is noco-localizationwith Si. <

Zn-silicate in mitochondria and cytoplasm ofSilene jenessiensis (ESI).

53

Growth of two Arabidopsis species on normal (-Cd) and Cadmium-contaminated (+Cd) soil. Arabidopsis thaliana (left) growth is affec-ted, whereas Arabidopsis halleri (right) is able to tolerate highCadmium concentrations.

52

The formation of phytochelatins (PCs) is aprinciple response of plants, many fungiand algae to toxic metal exposure. Weshowed that invertebrates such as

Caenorhabditis elegans also express func-tional PC synthases (PCS) by expressingthe respective protein in a PCS-deficientS. pombe strain. In a similar way, the exi-stence of a second PCS in Arabidopsis

tha liana was demonstrated. PCS genes areconstitutively expressed. PC synthesis isdirectly activated by the binding of a heavymetal ion or the corresponding glutathione

chelate to the enzy-me. In order to fur -ther elucidate thisac tivation process andto establish a tech -nique for the cha-racterization of me -tal-binding sites weused "peptide scans",i.e. spotted peptidelibraries re presentingPCS proteins. Cd2+-bin ding sites could belo ca lized and functio-nally characterized bysite-directed mu ta - genesis.

The molecular ana -lysis of plant metalresponses and deter-minants of metal hy -per accumulation isbeing pursued in themodel sys temsArabi dopsis thaliana

and Arabi dopsis halleri. Ex pression profi-ling in A. halleri by cDNA-AFLP has beencon tinued. The focus is on metal sensingand metal signal transduction since virtual-ly noth ing is known about these phenome-

Group members

Clarice de Figuereido(PhD student)

Marina Häußler(technician)

Emiko Harada(postdoctoral position since April 2002)

Elke Hillert(technician)

Sylvia Krüger(technician)

Thomas Maier(postdoctoral position until May 2002)

Claudia Simm(PhD student since October 2000)

Pierre Tennstedt(PhD student since August 2002)

Christoph Vess(PhD student)

Susan Wassersleben(PhD student since July 2000)

Michael Weber(PhD student since March 2001)

Uta zur Nieden(research scientist)

Collaborators

Udo Conrad, Renate ManteuffelInstitute of Plant Genetics and Crop Plant Research,Gatersleben, Germany

Klaus KloppstechUniversity of Hannover, Germany

Ute KrämerMax Planck Institute of Molecular Plant Physiology,Golm, Germany

Gerhard KüllertzMax Planck Research Unit for Enzymology of ProteinFolding, Halle, Germany

Olaf LichtenbergerInstitute of Plant Biochemistry, Halle, Germany

Enrico MartinoiaUniversity of Neuchatel, Switzerland

Dietrich NiesUniversity of Halle, Germany

Uwe SchmidtFederal Research Centre for Forestry and ForestProducts, Hamburg, Germany

Julian SchroederUniversity of California at San Diego, La Jolla, USA

Wilhelm SchwiegerUniversity of Erlangen, Germany

Research Group: Metal HomeostasisHeads: Dieter Neumann & Stephan Clemens

Plants - like all other organisms - are able to tightly regulate the intracellularconcentration and the distribution of essential heavy metals such as zinc andcopper. Also, the cytosolic concentrations of non-essential toxic heavy metals(e.g. cadmium, lead) have to be minimized. Some plant species (so-called metal-lophytes) can tolerate otherwise toxic concentrations and grow on heavymetal contaminated soil. Main objective of the group is to elucidate the mecha-nisms underlying plant metal homeostasis and metal hyperaccumulation. Weare using analytical electron microscopy and a range of biochemical and mole-cular techniques. Plants under investigation are Arabidopsis thaliana, its closerelative Arabidopsis halleri, and other metallophytes (Silene vulgaris, Minuartiaverna and Armeria maritima). In addition, we are working withSchizosaccharomyces pombe as a cellular model for metal homeostasis.

The phytochelatin pathway and possible other mechanisms of Cd2+ detoxifica-tion. PC synthesis from GSH is activated by several metal and metalloid ions.Cd2+ ions enter the cell via Fe2+, Zn2+ or Ca2+ transporters. Upstream of GSHbiosynthesis are sulphate assimilation and cysteine biosynthesis. In S. pombe,PC-Cd complexes (LMW) are transported into the vacuole by the ABC-typetransporter Hmt1. In plant cells , this transport is hypothesized to be media-ted by a protein of the same family. Inside the vacuole HMW complexes areformed by addition of sulphide, which apparently is derived from cysteine.Other mechanisms of Cd2+ detoxification discussed for plants and other orga-nisms are vacuolar sequestration dependent on either CDF proteins orCd2+/H+ antiporters, binding to metallothioneins or efflux mediated by CPx-type ATPases.

55

Varet, A., Parker, J., Tornero, P., Nass, N., Nürnberger,T., Dangl, J. L., Scheel, D., Lee, J. NHL25 and NHL3,two NDR1/HIN1-like genes in Arabidopsis thalianawith potential role(s) in plant defense. Mol. PlantMicrobe Interact. 15, 608-616 (2002).

Veit, S., Wörle, J. M., Nürnberger, T., Koch, W. & Seitz,H. U. A novel protein elicitor (PaNie) from Pythiumaphanidermatum induces dual defense responses incarrot and Arabidopsis. Plant Physiol. 127, 832-841(2001).

Books and Book chaptersBruns, I., Sutter, K., Neumann, D. & Krauss, G.-J.Glutathione accumulation - a specific response ofmosses to heavy metal stress. In: Sulfur Nutritionand Sulfur Assimilation in Higher Plants (Brunold,C., ed.) Haupt, Bern, pp. 389-391 (2000).

Clemens, S., Thomine, S. & Schroeder, J. I. Molecularmechanisms that control plant tolerance to heavymetals and possible roles towards manipulatingmetal accumulation. In: Plant Biotechnology andTransgenic Plants (Oksman-Caldentey, K.-M. &Barz, H.W., eds.) Marcel Dekker, Inc., New York, pp.665-691 (2002).

Hirt, H. & Scheel, D. Receptor-mediated MAP kina-se activation in plant defense. In: Results andProblems in Cell Differentiation, Vol. 27. MAPKinases in Plant Signal Transduction (Hirt, H., ed.)Springer-Verlag, Heidelberg, pp. 85-93 (2000).

Scheel, D. Parasitismus im Pflanzenreich. In:Parasitismus als Lebensform. Nova Acta LeopoldinaNF. 316, Nr. 83, Barth, Heidelberg, S. 25-31 (2000).

Scheel, D., Blume, B., Brunner, F., Fellbrich, G.,Dalboge, H., Hirt, H., Kauppinen, S., Kroj, T.,Ligterink, W., Nürnberger, T., Tschöpe, M., Zinecker,H. & zur Nieden, U. Receptor-mediated signaltransduction in plant defense. In: Biology of Plant-Microbe Interactions, Vol. 2 (de Wit, P. J. G. M.,Bisseling, T. & Stiekema, W. J., eds.) InternationalSociety for Molecular Plant-Microbe Interactions,St. Paul, pp. 131-135 (2000).

Scheel, D. Oxidative burst and the role of reactiveoxygen species in plant-pathogen interactions. In:Oxidative Stress in Plants (Inzé, D. & van Montagu,M., eds.) Taylor & Francis, London, pp. 137-153(2002).

Scheel, D. Signal transduction elements. In: PlantBiotechnology and Transgenic Plants (Oksman-Caldentey, K.-M., Barz, H. W., eds.) Marcel Dekker,Inc., New York, pp. 427-444 (2002).

Scheel, D. & Wasternack, C., eds. Plant SignalTransduction., Oxford University Press, Oxford,(2002).

Scheel, D. & Wasternack, C. Signal transduction inplants: cross talk with the environment. In: PlantSignal Transduction (Scheel, D., Wasternack, C.,eds.) Oxford University Press, Oxford, pp. 1-5(2002).

Books and Book chapters in pressClemens, S., Simm, C. & Maier, T. Heavy metal bin-ding proteins and peptides. In: Biopolymers, Vol. 7Polyamides and complex proteinaceous MaterialsPart A, (Fahnestock, S. R., ed.) Wiley-VCH, NewYork (2003).

Lee, J. & Nürnberger, T. Pseudomonas syringaepathovars and related pathogens. In: Developmentsin Plant Pathology, Vol. 10, (Mansfield, J.W. & Vivian,

A., eds.) Kluwer Academic Publishers Dordrecht.Neumann, D. Silicon in plants. In: Progress inMolecular and Subcellular Biology. SiliconBiomineralization. Springer-Verlag, Wien-New York.

Nürnberger, T. Elicitor-mediated signal transductionin the activation of plant pathogen defense. In: PlantHormone Research, Vol. 13, (Bisseling, T. & Schell, J.,eds.) Springer-Verlag, Wien-New York.

PatentsFeussner, I., Hornung, E. & Rosahl, S. 11-Ara -chidonat-Lipoxygenase-Mutante. German patent19931819.0 (2001).

Scheel, D., Rosahl, S., Strack, D. & Schmidt, A. Trans -gene Pflanzen mit erhöhter Resistenz gegen denBefall durch Phytopathogene. German patent19846001 C2 (2000).

Doctoral ThesesBau, Stephan: Untersuchungen zur Jasmonat-Signaltransduktion in Arabidopsis thaliana anhanddes Jasmonat-regulierten Gens Atjrg21. Universityof Halle-Wittenberg, Department ofBiochemistry/Biotechnology, 26/01/2001.

De Figueiredo, Clarice: Physiologisch-biochemischeMechanismen der Schwermetalltoleranz beiArmeria mari tima (Mill.) Willd. ssp. halleri (Wallr.).University of Halle-Wittenberg, Department ofBiochemistry/Bio technology, 24/09/2002.

Fellbrich, Guido: Interaktionen zwischen Pflanzenund phytopathogenen Oomyceten. Isolierung,Sequenzierung und partielle Charakterisierungeines Proteinelicitors aus Phytophthora parasitica.University of Halle-Wittenberg, Department ofBiochemistry/Biotechnology, 08/07/2001.

Göbel, Cornelia: Untersuchungen zur Funktion vonOxylipinen bei der Pathogenantwort in Solanumtuberosum L. University of Halle-Wittenberg,Department of Biochemistry/Biotechnology,05/12/2001.

Kemmerling, Birgit: Identifizierung und Charak -terisierung systemisch responsiver Gene derKartoffel (Solanum tuberosum L.) nach Inokulationmit dem nichtpathogenen Bakterium Pseudomonassyringae pv. maculicola. University Halle-Wittenberg, Department of Biology, 06/09/2001.

Lubaretz, Olga: Non-stress induced small headshock proteins in higher plants. University of Halle-Wittenberg, Department ofBiochemistry/Biotechnology, 14/06/2001.

Patzlaff, Astrid: Untersuchungen zur Expression vonPeroxidase Ca aus Arabidopsis thaliana undGenerierung von Mutanten mit veränderterExpression. University of Halle-Wittenberg,Department of Biochemistry/Bio technology,22/06/2001.

Petters, Julia: Isolierung und Charakterisierungpathogen- und stressresponsiver Gene derKartoffel (Solanum tuberosum L.). University ofHalle-Wittenberg, Department of Pharmacy,23/11/2001.

Romanski, Annette: Das Elicitorprotein NPP1 -Isolierung und Charakterisierung der korrespon-dierenden cDNA, heterologe Expression desProteins und Studien zur Signalperception.University of Halle-Wittenberg, Department of

Biochemistry/Biotechnology, 21/11/2001.

Zinecker, Heidi: Reaktive Sauerstoffspezies in derpflanzlichen Pathogenabwehr - Isolierung undCharakterisierung von Genen aus Petroselinum cri-spum L., die für putative NADPH-Oxidasen kodie-ren. University of Halle-Wittenberg, Department ofBiochemistry/Biotechnology, 09/01/2001.

Diploma ThesesGäbler, Yvonne: Anlage von cDNA-Microarrays derPetersilie (Petroselinum crispum). University ofHalle-Wittenberg, Department of Biochemis try/Bio technology, 05/09/2001.

Geiler, Carola: Induktion von Abwehrreaktionen inSolanum tuberosum L. durch den Oligopeptid-Elicitor Pep-13 aus Phytophthora sojae. Universityof Halle-Wittenberg, Department of Biology,17/08/2001.

Haase, Stefanie: Untersuchung putativer Schwer -metalltransporter aus Schizosaccharomycespombe. University of Halle-Wittenberg,Department of Biochemistry/Biotechnology,28/01/2002.

Landtag, Jörn: Transformation von Kartoffel- undTabakpflanzen mit der Tyrosin-Decarboxylase-2cDNA aus Petersilie und Untersuchung derExpression des Transgens. University of Halle-Wittenberg, Department of Biology, April 2001.

Simm, Claudia: Phytochelatinsynthase aus Schizo -saccharomyces pombe. Untersuchungen zurLokalisierung, Regulation und biochemischerFunktionalität. University of Halle-Wittenberg,Department of Biochemistry/Biotechnology,12/07/2000.

Tennstedt, Pierre: Untersuchungen zur Abwehr -expression in Arabidopsis-Mutanten. University ofHalle-Wittenberg, Department of Biochemistry/Biotechnology, 29/07/2002. <

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induction and modulation of plant defense respon-ses by bacterial lipopolysaccharides. Annu. Rev.Phytopathol. 38, 241-261 (2000).

Fellbrich, G., Blume, B., Brunner, F., Hirt, H., Kroj, T.,Ligterink, W., Romanski, A. & Nürnberger, T.Phytophthora parasitica elicitor-induced reactionsin cells of Petroselinum crispum. Plant Cell Physiol.41, 692-701 (2000).

Fellbrich, G., Romanski, A., Varet, A., Blume, B.,Brunner, F., Engelhardt, S., Felix, G., Kemmerling, B.,Krzymowska, M. & Nürnberger, T. NPP1, aPhytophthora-associated trigger of plant defense inparsley and Arabidopsis. Plant J. 32, 375-390 (2002).

Göbel, C., Feussner, I., Schmidt, A., Scheel, D.,Sanchez-Serrano, J., Hamberg, M. & Rosahl, S.Oxylipin profiling reveals the preferential stimula-tion of the 9-lipoxygenase pathway in elicitor-trea-ted potato cells. J. Biol. Chem. 276, 6267-6273(2001).

Göbel, C., Feussner, I., Hamberg, M. & Rosahl, S.Oxylipin profiling in pathogen-infected potato lea-ves. Biochim. Biophys. Acta 1584, 55-64 (2002).

Hornung, E., Rosahl, S., Kühn, H. & Feussner, I.Creating lipoxygenases with new positional specifi-cities by site-directed mutagenesis. Biochem. Soc.Trans. 28, 825-826 (2000).

Ichimura, K., Shinozaki, K., Tena, G., Sheen, J., Henry, Y.,Champion, A., Kreis, M., Zhang, S., Hirt, H., Wilson, C.,Heberle-Bors, E., Ellis, B. E., Morris, P. C., Innes, R. W.,Ecker, J. R., Scheel, D., Klessig, D. F., Machida,Y., Mundy, J.,Ohashi, Y. & Walker, J. C. Mitogen-activated proteinkinase cascades in plants: a new nomenclature. TrendsPlant Sci. 7, 301-308 (2002).

Kamphausen, T., Fanghähnel, J., Neumann, D., Schulz,B. & Rahfeld, J.-U. Characterization of Arabidopsisthaliana AtFKBP42 that is membrane bound andinteracts with HSP90. Plant J. 32, 263-276 (2002).

Kroj, T., Rudd, J. J., Nürnberger, T., Gäbler, Y., Lee, J. &Scheel, D. Mitogen-activated kinases play an essen-tial role in oxidative burst-independent expressionof pathogenesis-related genes in parsley. J. Biol.Chem. published November 7, 2002 as10.1074/jbc.M208200200.

Landgraf, P., Feussner, I., Hunger, A., Scheel, D. & Rosahl,S. Systemic accumulation of 12-oxo-phytodienoic acidin SAR-induced potato plants. Eur. J. Plant Pathol. 108,279-283 (2002).

Landtag, J., Baumert, A., Degenkolb, T., Schmidt, J.,Wray, V., Scheel, D., Strack, D. & Rosahl, S.Accumulation of tyrosol glucoside in transgenicpotato plants expressing a parsley tyrosine decar-boxylase. Phytochemistry 60, 683-689 (2002).

Lee, J., Klessig, D. F. & Nürnberger, T. A harpin bin-ding site in tobacco plasma membranes mediatesactivation of the pathogenesis-related gene HIN1independent of extracellular calcium but depen-dent on mitogen-activated protein kinase activity.Plant Cell 13, 1079-1093 (2001).

Lee, J., Klüsener, B., Tsiamis, G., Stevens, C., Neyt, C.,Tampakaki, A. P., Panopoulos, N. J., Nöller, J., Weiler, E.W., Cornelis, G. R., Mansfield, J. W. & Nürnberger, T.HrpZPsph from the plant pathogen Pseudomonassyringae pv. phaseolicola is exported by the type IIIsecretion pathway and forms an ion-conducting porein vitro. Proc. Natl. Acad. Sci. U.S.A., 98, 289-294(2001).Lee, J. & Rudd, J. J. Calcium-dependent protein kina-

ses: versatile plant signalling components necessaryfor pathogen defence. Trends Plant Sci. 7, 97(2002).

Li, J., Nass, N., Kusaba, M., Dodds, P., Treloar, N.,Clarke, A. E. & Newbigin, E. J. A genetic map of theNicotiana alata S-locus that includes three pollen-expressed genes. Theor. Appl. Genet. 100, 956-964(2000).

Lubaretz, O. & zur Nieden, U. Accumulation of plantsmall heat-stress proteins in storage organs. Planta215, 220-228 (2002).

Luderer, R., Rivas, S., Nürnberger, T., Mattei, B., Vanden Hooven, H. W., Van der Hoorn, R. A. L., Romeis,T., Wehrfritz, J.-M., Blume, B., Nennstiel, D.,Zuidema, D., Vervoort, J., De Lorenzo, G., Jones, J. D.G., De Wit, P. J. G. M. & Joosten, M. H. A. J. No evi-dence for binding between resistance gene productCf-9 of tomato and avirulence gene product AVR9of Cladosporium fulvum. Mol. Plant MicrobeInteract. 14, 867-876 (2001).

Nass, N. & Scheel, D. Enhanced luciferin entry cau-ses rapid wound-induced light emission in plantsexpressing high levels of luciferase. Planta 212, 149-154 (2001).

Neumann, D. & De Figueiredo, C. A novel mecha-nism of silicon uptake. Protoplasma 220, 59-67(2002).

Neumann, D. & zur Nieden, U. Silicon and heavymetal tolerance of higher plants. Phytochemistry56, 685-692 (2001).

Newman, M.-A., von Röpenack-Lahaye, E., Parr, A.,Daniels, M. J. & Dow, J. M. Induction of hydroxycin-namoyl-tyramine conjugates in pepper byXanthomonas campestris, a plant defense responseactivated by hrp gene-dependent and hrp gene-independent mechanisms. Mol. Plant MicrobeInteract. 14, 785-792 (2001).

Newman, M.A., von Roepenack-Lahaye, E., Parr, A.,Daniels, M. J. & Dow, J. M. Prior exposure to lipopo-lysaccharide potentiates expression of plant defen-ses in response to bacteria. Plant J. 29, 487-495(2002).

Noeringer, C., Scheel, D. & Blee, E. Lipoxygenaseisoforms in elicitor-treated parsley cell suspensioncultures. Biochem. Soc. Trans. 28, 2827-2829(2000).

Nürnberger, T. & Scheel, D. Signal transmission inthe plant immune response. Trends Plant Sci. 6,372-379 (2001).

Nürnberger, T. & Brunner, F. Innate immunity inplants and animals: emerging parallels between therecognition of general elicitors and pathogen-asso-ciated molecular patterns. Curr. Opin. Plant Biol. 5,318-324 (2002).

Petters, J., Göbel, C., Scheel, D. & Rosahl, S. A patho-gen-responsive cDNA from potato encodes a pro-tein with homology to a phosphate-starvationinduced phosphatase. Plant Cell Physiol. 43, 1049-1053 (2002).

Stumpe, M., Kandzia, R., Göbel, C., Rosahl, S. &Feussner, I. A pathogen-inducible divinyl ether syn-thase (CYP74D) from elicitor-treated potatosuspension cells. FEBS Lett. 507, 371-376 (2001).

PublicationsAbel, S., Nürnberger, T., Ahnert, V., Krauss, G.-J. &Glund, K. Induction of an extracellular cyclic nucle-otide phosphodiesterase as an accessory ribonu-cleolytic activity during phosphate starvation of cul-tured tomato cells. Plant Physiol. 122, 543-552(2000).

Berger, S., Weichert, H., Porzel, A., Wasternack, C.,Kühn, H. & Feussner, I. Enzymatic and non-enzyma-tic lipid peroxidation in leaf development. Biochim.Biophys. Acta 1533, 266-276 (2001).

Berger, S. Jasmonate-related mutants of Arabidopsisas tools for studying stress signaling. Planta 214,497-504 (2002).

Berger, S., Mitchell-Olds, T. & Stotz, H. U. Local anddifferential control of vegetative storage proteinexpression in response to herbivore damage inArabidopsis thaliana. Physiol. Plant. 114, 85-91(2002).

Bloss, T., Clemens, S. & Nies, D. H. Characterizationof the ZAT1p zinc transporter from Arabidopsisthaliana in microbial model organisms and reconsti-tuted proteoliposomes. Planta 214, 783-791 (2002).

Blume, B., Nürnberger, T., Nass, N. & Scheel, D.Receptor-mediated increase in cytoplasmic freecalcium required for activation of pathogen defensein parsley. Plant Cell 12, 1425-1440 (2000).

Brunner, F., Rosahl, S., Lee, J., Rudd, J. J., Geiler, C.,Kauppinen, S., Rasmussen, G., Scheel, D. &Nürnberger, T. Pep-13, a plant defense-inducingpathogen-associated pattern from Phytophthora.EMBO J. 21, 6681-6688 (2002).

Brunner, F., Wirtz, W., Rose, J. K. C., Darvill, A. G.,Govers, F., Scheel, D. & Nürnberger, T. A ß-glucosida-se/xylosidase from the phytopathogenic oomycete,Phytophthora infestans. Phytochemistry 59, 689-696 (2002).

Bruns, I., Sutter, K., Menge, S., Neumann, D. & Krauss,G.-J. Cadmium lets increase the glutathione pool inbryophytes. J. Plant Physiol. 158, 79-89 (2001).

Cazalé, A.-C. & Clemens, S. Arabidopsis thalianaexpresses a second functional phytochelatin syn-thase. FEBS Lett. 507, 215-219 (2001).

Clemens, S. Molecular mechanisms of plant metaltolerance and homeostasis. Planta 212, 475-486(2001).

Clemens, S. Developing tools for phytoremediation:Towards a molecular understanding of plant metaltolerance and accumulation. Int. J. Occup. Med.Environm. Health 14, 235-239 (2001).

Clemens, S., Bloss, T., Vess, C., Neumann, D., Nies, D.H. & zur Nieden, U. A transporter in the endoplas-mic reticulum of Schizosaccharomyces pombe cellsmediates zinc storage and differentially affects tran-sition metal tolerance. J. Biol. Chem. 277, 18215-18221 (2002).

Clemens, S., Palmgren, M. G. & Krämer, U. A longway ahead: understanding and engineering plantmetal accumulation. Trends Plant Sci. 7, 309-315(2002).

Clemens, S., Schroeder, J. I. & Degenkolb, T.Caenorhabditis elegans expresses a functional phy-tochelatin synthase. Eur. J. Biochem. 268, 3640-3643(2001).Dow, M., Newman, M.-A. & von Roepenack, E. The

Publications, Books and Bookchapters, Publications in press,Patents, Doctoral Theses, Diploma Theses

Work of the department is con-cerned with the molecular

regulation of plant secondary meta-bolism, evolution of the enzymesinvolved in the biosynthesis of secon-dary products and their role in inter-actions of plants with their environ-ment.

The work on metabolic regulationincludes isolation and characteriza-tion of the corresponding enzymesand the encoding genes, focu-sing on transferases.We cur -rently investigate ma lateand choline hy -

droxycinnamoyltransferasesas well as several hydroxy-cinnamate glucosyltrans-ferases from A ra bidopsis thali-ana and ra pe (Bras si ca na pus).In addition flavonoid andbetanidin glucosyltransfera-ses from be tacyanin-accumulating plants or fla-vonoid methyltransferasesfrom the ice plant (Me sem - bryan themum crys tallinum)are investigated.

The aim of the work on glu-cosyl- and hy -droxycinnamoyltransferases is to elu-cidate their evo lutionary origin andstructure-function re lations to pre-dict substrate specificity. Glu cosyl -trans ferases in vol ved in betacyaninbiosynthesis are considered to beoligophyletic and originate from dif-ferent clusters of flavonoid glucosyl-transferases. Hydroxy cinna moyl -trans fe ra ses, which are dependent onb-acetal esters as acyldonors, arevacuolar serine carboxypeptidase-like (SCPL) proteins as found for theenzyme involved in the formation ofsinapoylmalate in Ara bidopsis. Thegeneral existence of vacuolar b-ace-tal ester-de pen dent acyltransferaseswould prove a new concept of cellcom partmentation of plant se -condary metabolism.

Special emphasis is also placed onprograms focusing on the molecularinteractions of plants with arbuscularmycorrhizal fungi. The work of twogroups is concerned with fungus-induced alterations in plant isopre-noid metabolism, in particular caro-tenoid biosynthesis and degradation,accompanied by a dramatic reorgani-zation of plastid population in arbu-scule-harbouring root cells. Anothermain objective is the analysis of therole of phytohormones, in particular

jasmonates, in development and func-tional mainte-nance of mycorrhizalsymbiosis. These studies are suppor-ted by comprehensive analysis of pri-mary and secondary metabo-lites("metabolite profiling") in wild-typeand transgenic mycorrhizal plants. <

57

Department: Secondary MetabolismHead: Prof. Dieter Strack

Secretary: Heidemarie Stolz

56

59

with Glo mus mosseae and G. intraradi-ces. For the first time in plants the exi-stence of two distinct, only distantly rela-ted classes of DXS genes could be dedu-ced from the analysis of M. truncatulacDNAs. Only the expression of DXS2from M. truncatula is regulated by arbu-scular mycorrhizal fun gi. DXS1 is expres-sed in most tissues at a constitutive leveland appears to fulfill mainly housekee-ping functions. Similar complementaryexpression profiles and mycorrhiza-regulation of DXS2 transcript levelswere found in maize, tomato and tobac-co. Additional data suggest an involve-ment of DXS2 in the biosynthesis ofmany other secondary isoprenoids suchas leaf trichome monoterpenes of mintand solanaceous species, petal caroteno-ids, and terpenoid indole alkaloids. As aresult, a new concept of dedicated DXSenzymes for primary and secon-dary iso-prenoids can be introduced (see figure).Genomic sequences harbouring DXS2genes have been isolated. These materi-als will provide useful tools for gene sup-pression studies and promoter analyses.

A single class of cDNAs for DXR hasbeen isolated from a maize mycorrhizalroot library. DXR transcript levels areelevated in mycorrhizal maize roots butnot to the high extent as has been foundfor DXS2. Recombinant maize DXR pro-

tein from E. coli has been used to createspecific antibodies. Their use in immuno-localisation studies vi -sua lized an ex -ten sive net-work of in -

terconnectedplas tids a round ma -ture fungal arbuscules incolonized cortical cells. The most recentwork involves the carotenoid cleavagestep performed by carotenoid cleavingdioxygenases (CCDs). Several cDNAclones from both M. truncatula andmaize have been isolated. Initial analysesin maize indicate a mycorrhiza-media-ted regulation of this step as well.

Another project targeted moregeneral aspects of plastid develop-ment during the symbiosis. Useof transgenic tobacco plants ex pres sing aplastid-directed green fluorescent pro-tein has shown dramatic changes inmycorrhizal roots with a similar networkof plastids around arbuscules as seenwith the DXR antibody. These networksare highly dynamic structures appearingand disappearing concomi-tantly withformation and degradation of arbuscules

(see report B. Hause). <

Phylogenetic tree of DXS proteins from plants andthe photosynthetic bacterium Rhodobacter capsula-tus. Branches underlined in green indicate preferenti-al expression in green tissues. Conversely, orangeback ground indicates expression correlated with thebiosynthesis of secondary isoprenoids such as apoca-rotenoids of mycorrhizal roots, petal carotenoids ormonoterpenes of leaf trichomes. The data introducea concept of dedicated roles of DXS1 and DXS2 inprimary and secondary functions, respectively.

Visualization of plastid reorgani -zation in a mycorrhized root cell.A fluorescence-labeled antibodyspecific for DXR reacts with pla-stids covering a fungal arbuscule(right) or surrounding a nucleus(upper left).

Metabolite analysis of roots of variousplants including cereals, tobacco andlegumes colonized by the mycorrhizalfungus Glomus intraradices has led tothe identification of two classes of apo-carotenoids: (i) glycosylated C13 cyclohe-xenone derivatives and (ii) an acyclic C14po ly ene compound termed mycorradi-cin. Further biochemical work has nowprovided a facile and sensitive detectionmethod for mycorradicin. The wide-spread but not universal occurrence ofthis compound in mycorrhizal roots of

various plant families could be shown.The apocarotenoids are presumably in -tegrated into a complex mixture of es -ters between mycorradicin and glycosy-lated C13 cyclohexenone derivatives. Ac -cumulation of this complex in mycorrhi-zal roots correlates with degradation offungal arbuscules.

Early steps of apocarotenoid biosynthe-sis are catalyzed by the enzymes 1-deoxy-D-xylulose 5-phosphate synthase (DXS)and 1-deoxy-D-xylulose 5-phosphate re -

duc toisomerase (DXR)as part of the re -cently discoveredMEP pathway loca-ted to plastids.Strongly elevatedtrans cript levels forboth enzymes com - pared to controlswere shown for my -corrhizal roots ofse veral cereals. Amore detailed ana-lysis of DXS generegulation and or -ga nization was ini -ti ated for the mo -del legume Medi -cago truncatulaand its interaction

Group MembersThomas Fester(postdoctoral position until December 2001)

Kristine Halfmann(PhD student until February 2000)

Joachim Hans(PhD student)

Swanhild Lohse(PhD student until December 2001)

Kerstin Manke(technician)

Alexander Röhrig(student since August 2002)

Sudha Sahay(DAAD-fellow until August 2001)

Michael Stephan(postdoctoral position until September 2001)

Gerlinde Waiblinger(technician until December 2001)

CollaboratorsJörg DegenhardtMax Planck Institute for Chemical Ecology, Jena, Germany

Philipp FrankenMax Planck Institute for Terrestrial Microbiology,Marburg, Germany

Giovanni GiulianoEnte per le nuove tecnologie, l energia e l ambiente,ENEA, Rome, Italy

Bettina Hause, Jürgen SchmidtInstitute of Plant Biochemistry, Halle, Germany

Martin ParniskeJohn Innes Center, Norwich, UK

Andreas PerlickUniversity of Bielefeld, Germany

Ajit VarmaJawaharlal Nehru University, New Delhi, India

Victor WrayGerman Research Centre for Biotechnology,Braunschweig, Germany

Eleonore WurtzelCity University New York, Bronx, USA

Most herbaceous plants form symbiotic associations with a small number offungi in the rhizosphere in order to improve their water uptake and acquisi-tion of mineral nutrients. These interactions are called arbuscular mycorrhizas(AM), a term derived from the haustoria-like fungal arbuscules developing inthe root cortex. The work of the group focuses on alterations in plant isopre-noid metabolism induced by AM fungi, in particular on reactions located in pla-stids. Starting from metabolite analyses of various apocarotenoids, a numberof fungus-stimulated gene activities from their biosynthetic pathway could becharacterized. These include steps from the non-mevalonate methylerythritolphosphate (MEP) and from the carotenoid pathways. For the first reaction ofthe MEP pathway a diversification and specific expression of a 1-deoxy-D-xylu-lose 5-phos-phate synthase 2 (DXS2) gene in mycorrhizal roots was shown.Anew concept of dedicated roles of DXS1 and DXS2 in the biosynthesis of pri-mary and secondary isoprenoids has been introduced.

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Research Group: Molecular Physiology of MycorrhizaHead: Michael H. Walter

Isoprenoid biosynthesis and its compartmentation. Two separatepathways lead to the key intermediate isopentenyl diphosphate(IPP). The apocarotenoids blumenin (C13 cyclohexenone derivative)and mycorradicin accumulating in mycorhizal roots are highlighted.

osmotic stress or in defense against bioticstresses takes place. As a consequence,mycorrhizal roots may be more resistantagainst secondary infection and/or osmoticstresses.

To test this hypothesis we intend to per-form functional analyses by modulating jas-monate content in mycorrhizal roots of M.truncatula. A cDNA coding for the alleneoxide cyclase (AOC), the enzyme perfor-ming the crucial step in JA biosynthesis,was isolated from M. truncatula. Vectorswere constructed containing this cDNA insense or antisense orientation or theRNAi construct, all of them under controlof the CaMV35S promoter. After transfor-mation, plants are expected with increa-sed endogenous levels of JA (AOCsense)or de creased levels of JA (AOCantisense,AOC-RNAi). Assuming that altered JA-levels lead to altered mycorrhizal phenoty-pes, cell biological and biochemical appro-aches will be used to analyze these pheno-types. Additionally, gene expression studieswill be performed using cDNA microar-rays pro vided by the DFG Research FocusPro gram 1084 "Molecular Basis of Mycor -rhizal Symbioses". In a second approach,transgenic tobacco plants were used,which express a yeast invertase targetedto the apoplast. These plants exhibit alte-red source-sink relationships and will beanalyzed with respect to alterations in themycorrhizal phenotype expected on bio-chemical, molecular and cytological level.From both approaches, we hope to getinsights into the relationship of mycor-rhiza, jasmonate action and the sugar sta-tus within the mycorrhizal roots.

Concerning the activation of carotenoid

biosynthesis in AM roots, we have shownthat this process is virtually ubiquitous inthe plant kingdom. The extent of activa-tion, however, is variable regarding therespective plant species. One major cha-racteristic of the phenomenon is the fin-ding that carotenoid intermediates of thepathway are present only in very smallamounts, even in plants accumulating largeamounts of apocarotenoids (mycorradicinand glycosylated cyclohexenone derivati-ves). Two hypothetical functional reasonsfor the activation of carotenoid biosynthe-sis in AM roots are currently investigated:(i) The possible induction of carotenoidbiosynthesis by reactive oxygen species(ROS) produced during establishment ofthe AM symbiosis and a possible protec-tion of the plant cell against such ROS bycarotenoids; and (ii) a possible metabolicrole of the chlororespiratory activityinvolved in carotenoid biosynthesis.

The accumulation of apocarotenoids inAM roots might be part of a complexreorganisation of plastid structure andme tabolism in AM roots. Root corticalcell plastids are responsible for a numberof biosynthetic processes, which areessential for the formation and functio-ning of the symbiotic interface. In accor-dance with this functional importance,massive proliferation of plastids in colo-nized tobacco root cortical cells leadingto network-like structures covering thearbuscules have been observed. Currently,we are analyzing changes in the expres-sion levels of plastid-related genes usingDNA-arrays and Real-Time RT-PCR.These analyses will provide first infor-mation regarding the molecular and bio-chemical changes underlying the process

61

Confocal laser scanning micrographs of tobaccoroot cortex non-colonized (A) and colonized (B, C)by an AM fungus, respectively. The plastids are visu-alized by the green fluorescent protein targeted toplastids (transgenic tobacco plants courtesy of M.Hanson, Ithaca, New York, USA). In colonized cellsthe plastids formed a network-like structurearound the arbuscules. Bars represent 50 µm in A,B, and 25 µm in C.

Somatic regeneration of Medicago truncatula. Plant explants giverise to embryogenic callus (A), which then develops small embryos(B). After transfer of embryos to "Embryo-Developing-Media" smallplantlets are formed (C).

A possible role of jasmonates in themycorrhizal interaction is indicated by thefollowing data: Jasmonic acid (JA), appliedexogenously, promotes colonization andde velopment of mycorrhizalstruc tures, and the endogenousJA level of mycorrhizal roots isremarkably higher than that ofnon-mycorrhizal roots. The in -crease of JA content in barleyroots upon mycorrhization isaccompanied by the expressionof genes coding for enzymes ofJA biosynthesis (allene oxidesynthase, AOS) and for jasmona-te-induced proteins (JIP23). Inorder to record the kinetics ofJA accumulation during develop-ment of mycorrhizal structures,a system of "near-synchronous"mycorrhization was establishedby the use of nurse-pot cultures.Since JA levels increase later than the ini-tial steps of the plant-fungal interactionoccur, the development of mycorrhiza ratherthan the recognition of the interactingpartners may cause expression of JA-bio-synthetic genes and finally elevate JAlevels. In addition to the temporal pattern,the spatial pattern of gene expression ap -pearing during the development of thefungal organs within the root cortex (vesi-cles, arbuscules) was recorded. By use of

in situ-techniques (in situ-hybridization,im munocytochemistry) accumulation ofAOS and JIP23 mRNA and protein, respec-tively, could be shown to occur in cells

harboring arbuscules (see figure). From alldata ob-tained, the following hypotheticalscenario is suggested: The plant root sup-plies the fungus with carbohydrates Þ theplant root becomes a stronger sink organupon mycorrhization resulting in anenhanced accumulation of soluble sugarswithin the apoplast Þ expression of genescoding for enzymes of JA biosynthesisoccurs Þ level of jasmonates increases Þinduction of genes involved in response to

60

Research Group: Cell Biology of MycorrhizaHead: Bettina Hause

Group MembersThomas Fester(leader junior group since January 2002)

Ulrike Hintsche(technician)

Stanislav Isayenkov(PhD student until October 2002,afterwards postdoctoral position)

Swanhild Lohse(PhD student since January 2002)

Tamás Monostori(PhD student until March 2001)

Constantin Rüder(student until December 2000)

Sara Schaarschmidt(PhD student since May 2002)

Diana Schmidt(student until July 2001)

Carola Tretner(research scientist since September 2002)

Gerlinde Waiblinger(technician since January 2002)

CollaboratorsPeter Bramley, Paul FraserUniversity of London, UK

Ivo Feussner, Uwe SonnewaldInstitute of Plant Genetics and Crop Plant Research,Gatersleben, Germany

Philipp FrankenMax Planck Institute for Terrestrial Microbiology,Marburg, Germany

Giovanni GiulianoEnte per le nuove tecnologie, l energia e l ambiente,ENEA, Rome, Italy

Gerd HauseUniversity of Halle, Germany

Willy Peumans, Els Van DammeUniversity of Leuven, Belgium

Thomas RoitschUniversity of Würzburg, Germany

Claus Wasternack, Jürgen Schmidt,Otto MierschInstitute of Plant Biochemistry, Halle, Germany

Victor WrayGerman Research Centre for Biotechnology,Braunschweig, Germany

Plant hormones are believed to play a role in the establishment and develop-ment of symbiotic interactions between plants and arbuscular fungi (arbuscu-lar mycorrhiza, AM). Jasmonates, known as regulators in stress responses ofplants against various biotic and abiotic stresses, might be important regula-tors of this symbiosis. Therefore, the main objective of our group is the ana-lysis of the role of jasmonates during the interaction between Glomus intra-radices and barley (Hordeum vulgare) or barrel medic (Medicago truncatula).In a second project, the activation of carotenoid biosynthesis in AM roots isstudied. This activation is connected with a massive proliferation of the pla-stids of colonized root cortical cells. Cell biological phenomena as well asunderlying molecular changes of the plastid proliferation will be elucidated.

Figure: In situ-localization of AOS-transcripts within mycorrhizal barley roots. The detection performedwith the antisense probe (A, B) exhibits a clear staining of the cytoplasm of root cortex cells containingfungal structures (arrow head in B), whereas the negative control (sense, C, D) does not show labeling withinarbuscule-containing cells (arrow in C, arrow head in D). Bars represent 50 µm.

63

the levels are different for the individualcom ponents. In cooperation with theworking group of Thomas Fester (IPB)mycorrhiza-induced cyclohexenone de ri -vatives were detected in roots of Zeamays, Medicago truncatula and Orni tho -galum umbellatum by HPLC. In the lattermaterial ( see figure), besides the "yellowpigment" and cyclohexenones, a group ofhitherto unknown apocarotenoids withspectral properties similar to mycorradi-cin was observed suggesting that theymight be precursors of the "yellow pig-ment". Using lecaton from a leek/G.intraradices preculture for M. truncatulainoculation a fast and efficient mycorrhi-zation (ca. 90 % after 4 weeks) wasobserved which will be used in detailedstudies of mycorrhization kinetics.

To achieve an adequate handling of thequantitative metabolite profiling data, anefficient computing system with statisticsoftware was recently installed. In coor-dination with other groups of the institu-te dealing with similar bioinformatic pro-blems, these tools will be used for valida-tion, statistical evaluation and meaningfulpresentation of the results. In the futurematerial from transgenic plants providedby the collaborating groups will be analy-zed to determine the effect of the gene-tic alterations on the metabolite patternand to correlate the metabolite profileswith the gene ex pression profiles in afunctional genomic approach. <

The legume barrel medic (Medicago truncatula Gaertn. cv. Jemalong A17), the model plantfor functional genomic approaches to arbuscular mycorrhizal symbiosis.

Arbuscules of Glomus intraradices in the barrel medic root (trypan blue staining).

RP-HPLC-PDA, LC-ESI-MS and GC-TOF-MS are the methods used in ourmetabolite profiling approach. At thebeginning databases of reference com-pound were created using HPLC (flavo-noids, isoflavonoids, pterocarpans, cou-mestans and their glucosides), LC-MS(isoflavonoids, pterocarpans, coume-

stans) and GC-MS (amino acids, aliphaticacids, phenylpropanoids, in particular iso-flavonoids, sugars, sterols) to facilitatesubsequently the dereplication of theendogenous compounds. To reduce thechemical complexity of the metabolome,sequential extractions of lyophilized rootmaterial with dichloromethane, acetone

and 80% aqueous methanol wereperformed. In these extracts my -cor rhiza-specific alterations of morethan 300 root metabolites wereob served by HPLC. In LC-MS theaccumulation of different isoflavo-noid glucosides and their corre-sponding malonates as well as sapo-nins was detected. By GC-MS ofthe dichloromethane extract oftwelve weeks old mycorrhizal roots(M. truncatula / G. intraradices - 40% mycorrhization) a dramatic incre-ase of palmitelaidic and oleic acidwas detected, whereas other long-chained fatty acids decreased incomparison to non-mycorrhizalcontrols. In all ex tracts the levels ofsome acids of the primary metabo-lism (lactic, malic, malonic, succinic,citric, g-amino butyric and trihydro-xybu-tyric acids) were higher innon-mycorrhizal than in mycorrhi-zal roots. Sugars and inositol deri-va-tives are the predominatingcompounds in the acetone and 80% aqueous methanol extracts, but

Group MembersChristian Ammer(research scientist since September 2002)

Barbara Kolbe(technician)

Lars Seipold(PhD student since June 2002)

Collaborators

Thomas Degenkolb, Bettina Hause,Thomas Fester, Jürgen Schmidt,Michael H. WalterInstitute of Plant Biochemistry (IPB), Halle, Germany

Philipp FrankenMax Planck Institute for Terrestrial Microbiology,Marburg, Germany

Inna KuzovkinaTimiryasev Institute of Plant Physiology, RussianAcademy of Sciences, Moscow, Russia

Karsten NiehausUniversity of Bielefeld, Germany

Manfred Nimtz, Victor WrayGerman Research Centre for Biotechnology,Braunschweig, Germany

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Research Group: Biochemistry of Mycorrhiza (since 2002)Head: Willibald Schliemann

The research is focused on the comprehensive analysis of alte-rations of pri-mary and secondary metabolite patterns during the establishment of thearbuscular mycorrhizal symbiosis in the model system Medicago truncatula /Glomus intraradices with the aim to characterize the causal relationships bet-ween gene expression and metabolite profiles during the symbiosis. Metabolicprocesses that are essential for the functioning of this root-fungus systemhave to be elucidated that may be of general importance also in other mycor-rhizal systems. Furthermore, metabolite analysis of transgenic M. truncatulaplants is intended to evaluate the effect of gene transfer or knockouts on thekinetics of mycorrhizal symbiosis and phenotypical changes in plant develop-ment (in cooperation with projects of the DFG Research Focus Program1084 "Molecular Basics of Mycorrhizal Symbioses").

Star-of-Bethlehem (Ornithogalum umbellatum L.,Hyacinthaceae)Top: flowering plants; bottom: roots after mycorrhization (6months) with Glomus intraradices in comparison to non-mycorrhized controls (micrographs by courtesy of T. Fester,

specificity.

Besides our approach to correlate GTfunction with sequence information, ourresearch also contributes to the investi-gation of betacyanin biosynthesis in theCaryophyllales. Several lines of evidence,including cloning of highly homologous5- and 6-GT sequences from red beet(Beta vulgaris), suggest a conserved gly-cosylation of betacyanins at the betani-din level among all families within theCaryophyllales. GTs performing either5- or 6-glycosylation are phylogeneti-cally derived from two different classesof enzymes, involved in the position-specific glycosylation of flavonoids orother hydroxylated phenylpropanoids.The presence of a GT, glycosylatingcyclo-dopa, which has been proposed asthe glucose acceptor, cannot be ruledout, but is most unlikely.

Our second model system, the iceplant (Mesembryanthemum crystalli-num), is faced with extreme arid condi-tions in its na-tural habitat. Besides itswell-studied adap tation to droughtstress, the plant is capable of toleratingexposure to ex-treme light intensities,combined with a high dose of UV radi-ation, by a rapid ac cumulation of glyco-sylated and methylated flavonol andbetacyanin conjugates in leaf epidermallayers (see figure). At the molecularlevel a subtractive cDNA library of alight-in-duced versus non-induced lea-ves revealed the presence of severalinducible cDNAs possibly involved inthe adap-tive process of light tolerance.

No transcripts en codingGTs were found. Amonga va riety of inducedtranscripts ranging fromcatalase to JIP-23(Jasmonat Induced Pro -tein) or a salt to lerantpro tein, several O-me -thyl transferases (OMTs)were selected as putati-ve candidates to be in -volved in the methy-lation of the ob served(UV-) light-induced flavo-nol conjugates. One of the correspon-ding OMT-proteins showed the requi-red enzyme activities, and its presencewas consistent with the occurrence ofthe conjugated flavonol-6,3 -di-O-methylether derivatives in light-inducedbladder cells of the ice plant. This pro-tein was purified from leaves of the iceplant. Based on amino acid sequenceinformation, the cDNA was clonedfrom a cDNA library and ex pressed ina prokaryotic system. The recombinantenzyme displayed high position-specifi-city towards me thy lation of ortho-dihy-droxyl groups, with the ability tomethylate a variety of potential sub-strates, including flavo-noids, hydroxy-cinnamic acids and their correspondingCoA-esters. Protein sequence analysisindicates that this flavo noid-methylatingactivity most likely defines a new sub-group of small, Mg2+-dependent OMTs,previously shown to be involved only inthe methylation of the lignin precursorcaffeoyl coenzyme A. <

65

Red coloration of the ice plant due to epidermalaccumulation of betacyanins and flavonol conju -gates after five days of exposure to high light (1500µM/m2 x s) irradiation is observed only for plant A(UV-A/B radiation, cut-off filter 305 nm), but not forplant B (UV-A/B radiation, cut-off filter 360 nm).

Based on earlier work, our group started

on the molecular physiology of betalains,but lately as a result of the ongoingwork, the major focus has shiftedtowards enzymes involved in the modifi-cation of the two classes of plant naturalproducts under investigation, the betacy-anins and the flavonoids. Primarily, ourwork has been directed towards a de-tailed understanding of the biochemistryand molecular evolution of plant naturalproduct glycosyltransferases. By detailedsequence analysis and substrate specifi-city studies of two GTs, the betanidin 5-GT

and 6-GT from Dorotheanthus bellidi-

formis (Dbs), we were able to demon-strate an oligophyletic origin of the cor-responding glucosyltransferase genesfrom different clusters of flavonoid GTs.From this study, it is concluded thatregiospecificity and not substrate specifi-city ap pears to be the organizing princi-ple in cluster formation. This may haveimportant consequences for evaluatingthe structure/ function relationship wit-hin the rapidly growing genomic databa-ses for a variety of crop and non-cropspecies, like rice, corn or Arabidopsis,where a total of 110 GT-sequences withlargely unknown substrate specificitieshas al-ready been described. Site-direc-ted mu ta genesis, performed with theheterologously expressed 5-GT proteinfrom Dbs, among more than 20 conser-ved residues, indicate the presence ofseveral catalytically essential amino acidresi dues. They are probably involved inthe substrate binding of all GTs of the b-group, catalyzing enzyme reactions,which lead to an inversion of the sugarconfiguration). Although we were ableto modify and reduce the specific activi-ties of this en-zyme, changing one aminoacid only, this was apparently not suffi-cient to alter position specificity or lea-ding to a significant change in substrate

Group members

Stefan Ebert(student until July 2000)

Mwafaq Ibdah(PhD student until August 2002)

Judith Hans(PhD student since Mai 2000)

Dagmar Knöfel(technician)

Ute Vinzens(technician until September 2002)

Collaborators

Hans BohnertUniversity of Urbana, Illinois, USA

John CushmanUniversity of Reno, Nevada, USA

Patrik JonesChiba University, Chiba, Japan

Toni M. Kutchan, Sabine Rosahl,Jürgen SchmidtInstitute of Plant Biochemistry, Halle, Germany

Vladimir Kuznetsov, Inna KuzovkinaTimiryazev Institute of Plant Physiology, Russian Academyof Sciences, Moscow, Russia

Ullrich MaternUniversity of Marburg, Germany

Harald Seidlitz, Werner HellerNational Research Center for Environment and Health,Munich, Germany

Stabilisation and solubilisation of plant natural products are performed by awide range of glucosyltransferases (GTs) with often overlapping substratespecificities. These enzymes may also detoxify bioactive low-molecular weightcompounds, in particular those from exogenous sources. Sequence identitiescluster GTs according to regiospecificities rather than substrate specificitiesimply that this superfamily of proteins has evolved oligophyletically as one ofthe primary adaptive mechanisms of plants to meet the changing environmen-tal conditions in a timely and developmentally controlled manner. Similar obser-vations hold true for the superfamily of plant O-methyltransferases (OMTs),with a new subclass of enzymes involved in the modification of UV-inducedflavonoid conjugates discovered recently.

64

Research Group: GlycosyltransferasesHead: Thomas Vogt

Cladogram illustrating the distribution of selectedglucosyltransferases involved in betacyanin biosyn-thesis.

67

content increased in parallel with thefresh weight, the cyclo-dopa 5-O-glu-coside did not accumulate. The lowamount of cyclo-dopa 5-O-glucosidefound originates from betanin, which isin equilibrium with cyclo-dopa 5-O-glucoside and betalamic acid underslightly acidic conditions. This re sult isin contrast to previous data fromWyler et al. [Helv. Chim. Acta 67, 1348-1355 (1984)], but in accordance withrecent studies (Thomas Vogt, IPB) thatglucosyltransferases from red beetsaccept betanidin, but not cyclo-dopa assubstrate.

The structures of betalains occurringin inflorescences of two Celosia varie-ties (Celosia argentea var. cristata andCelosia argentea var. plumosa) wereelucidated in cooperation with part-ners of the IPB and from China. Threeyellow pigments were found to beimmonium conjugates of betalamicacid with dopamine, 3-me th oxy -tyramine and (S)-tryptophan.

In studies on vacuolar transport of beta-xanthins, the inhibition pattern of theMgATP-stimulated vacuolar uptake ofthe beet-specific miraxanthin V and vul-gaxanthin I (by 1 mM vanadate) and ofthe unnatural (R)-phe-nylalanine-betaxan-thin (by 0.1 µM bafilo-mycin A1 and 5 mMNH4Cl) suggests thepar ticipation of anABC-like directly-ener -gized transport mecha -nism and H+/antiportsystem, respectively.Both systems havebeen described in lite-rature for the vacuo-lar uptake of endoge-nous luteolin glucuro-nides in rye and flavo-noid glucosides in bar -ley, respectively. Theresearch on betalainswas terminated withthe end of 2001. <

Flowering Celosia argentea var. cristata (above), structures of two new end-ogenous betaxanthins and Celosia argentea var. plumosa (below).

A definite proof of the detection of dopa4,5-dioxygenase activity in plants has notbe achieved. In various enzyme assayswith [14C]dopa, no soluble betalamic acidcould be detected. However, radioacti-vity was released from the assay proteinsby alkaline hydrolysis; and by the additionof (S)-Phe, labeled (S)-Phe-betaxanthincould be identified. This proved the for-mation of betalamic acid, but in very lowamounts. For a molecular approach toidentify dopa dioxygenase using particlebombardment, a cell suspension cultureof Tinospora cordifolia (Menisperma -ceae) was selected which showed a 4-fold in crease in dopamine content after

treatment with 30 µM methyl jasmonate.However, feeding of betalamic acid tothe induced cells did not lead to the for-mation of yellow miraxanthin V, theessential prerequisite for the detectionof a transient expression of a dopa di-oxygenase cDNA.

To answer the question whether beta-nin biosynthesis in red beets proceedsexclusively via cyclo-dopa or via cyclo-dopa 5-O-glucoside, the contents ofbe tanin and cyclo-dopa 5-O-glucosidein red beet hypocotyls were moni-tored during eight weeks of plantdevelopment. Where as the betanin

Group MembersNaoko Kobayashi(PhD student until December 2001)

Barbara Kolbe(technician)

Shiming Liu(guest scientist until December 2001)

CollaboratorsHartmut BöhmGerman Institute of Human Nutrition, Bergholz-Rehbrücke, Germany

Yizhong Cai, Harold CorkeThe University of Hong Kong, Hong Kong, People'sRepublic of China

Inna KuzovkinaTimiryasev Institute of Plant Physiology, Russian Academyof Sciences, Moscow, Russia

Enrico Martinoia, Markus KleinUniversité de Neuchâtel, Switzerland

Jürgen Schmidt, Thomas DegenkolbInstitute of Plant Biochemistry (IPB), Halle, Germany

Victor Wray, Manfred NimtzGerman Research Centre for Biotechnology,Braunschweig, Germany

Betalains (red-violet betacyanins and yellow betaxanthins) are chromoalkalo-ids of chemotaxonomical importance. They functionally replace the anthocy-anins in members of most families of the Caryophyllales. Betacyanins are alsoof commercial interest as food colorants. The main objective of our researchis the unravelling of betalain biosynthesis. After the characterization of thebifunctional tyrosinase and spontaneously proceeding steps, experiments todetect the elusive dopa 4,5-dioxygenase are of particular interest as this enzy-me forms the chromophore betalamic acid, the key intermediate in betalainbiosynthesis.

66

Research Group: Biochemistry of Betalains (until 2001)Head: Willibald Schliemann

Time course of betanin and cyclo-dopa 5-O-glucoside accumulation during thedevelopment of red beets.

69

thesis and a physiological one trying todivert one of the sinapine precursors,choline, into a new metabolic sink. Thetwo enzymes in focus are the SGT andthe SCT. A cDNA encoding SGT wasisolated from cDNA libraries construc-ted from immature seeds and youngseed lings of rape. The deduced SGTamino acid sequence indicated that SGTbelongs to a distinct subgroup of gluco-syltransferases that catalyze the for-mation of 1-O-acylglucosides. The SGT-cDNA from rape was cloned and func-tionally expressed in E. coli. The recom-binant SGT carrying the His-tag at the C-terminus was purified. The enzyme sho-wed a molecular mass of 60 kDa (gel fil-tration) and 62 kDa (electrophoresis),respectively. It exhibited a broad sub-strate specificity, accepting cinnamate,4-coumarate, caffeate, ferulate and sina-pate. DNA cassettes for the dsRNAi-mediated seed-specific suppression ofSGT were constructed and cloned into abinary vector. Plant transformation wasperformed by collaborators of theUniversity of Goettingen (Ch. Moellers).

As a result of sequence comparison ana-lyses, four homologous genes encodinghydroxycinnamate glucosyltransferaseswere cloned from Arabidopsis. Thesegenes were functionally expressed in E.coli. According to the acceptor specifi-city, we identified one of them (AtSGT1)with high affinity to sinapate, whereasthe remaining three displayed broaderacceptor specificity. Based on the cDNAsequence of AtSGT1, DNA cassettes forthe dsRNAi-mediated suppression of theSGT, using a seed-specific (napine) and aconstitutive promoter (CaMV 35S), wereconstructed. Both suppression con-structs were used to transform Ara -

bidopsis. Homozygous transgenic linesare developed that will be used for quan-tification of sinapine and 1-sinapoylglu-cose in seeds.

As the SMT, the SCT belongs to thegroup of SCPL enzymes. By a "homolo-gy based cloning strategy", a full-lengthcDNA could be isolated from rapeseeds sharing about 85 % identity withthe SCT-cDNA from Arabidopsis. Afterexpression in E. coli, the recombinantprotein was shown to be in the inso-luble fraction.As this is one of the mainproblems with the class of SCPL pro-teins, the optimization of heterologousex pression has come into the focus ofour present work.

In the physiological approach, bacterialgenes (betA and betB) encoding choli-ne oxidase have been introduced inAra bidopsis and rape. It is assumedthat the glycine betaine pathway willcompete for choline as substrate insina-pine synthesis. Choline feedingexperiments using im matureArabidopsis and rape embryos revea-led that the level of free choline is limi-ted. Thus, to provide choline in a non-limiting concentration for glycinebetaine synthesis, we will suppressSGT activity in transgenic plants ex -pressing betA and betB. This strategywill hopefully not only im prove rape-seed used as healthy food but will en -hance by the accumulated oxidationproduct of choline, glycinebetain, thetolerance of rape to environmentalstresses, such as salt, low temperatureor drought that often affect seed ger-mination and plant productivity. <

Intracellular localization of SMT within Ara bidopsisrosette leaves. Cross sections were immunodeco-rated with polyclonal monospecific antibodies rai-sed against the recombinant SMT protein followedby fluorescence labelled secondary antibody. Agreen fluorescent label within the vacuoles ofmesophyll cells of wild-type leaves (A) is indicativeof the SMT protein (arrows). In contrast, in thevacuoles of mesophyll cells of the deletion mutantsng1 (B), defective in synthesis of sinapoylmalate,the fluorescent signals are absent (bar = 50 µm).

Cloning of the 1-sinapoylglucose:mala-te sinapoyltransferase (SMT) gene fromArabidopsis thaliana and immunolocaliza-tion of the SMT proteinSMT catalyzes the formation of sinapoyl-malate, one of the major phenylpro-panoid secondary metabolites accumu-lated by some members of the Brassi -caceae, e. g. Arabidopsis thaliana, rape(Bras sica napus) or red radish (Raphanussativus). In cooperation with Clint Chap -ple, we identified previously an Ara bi -dopsis mutant, sng1 (sinapoylglucoseaccumulator 1), which is defective in syn-thesis of sinapoylmalate. We have clonedthe corresponding gene and have foundthat it encodes a serine carboxypepti-dase-like (SCPL) protein. Expression ofSNG1 in E. coli demonstrated that itencodes the SMT. This finding suggeststhat SCPL proteins have acquired novelfunctions in plant metabolism and pro-vides an insight into the evolution of

secondary metabolic pathways in plants.

In an approach to immunolocalize theSMT protein, rabbit polyclonal anti-bodies were raised against the recom-binant SMT expressed in E. coli from thecorresponding Arabidopsis cDNA. Im -muno blot analysis of proteins from diffe-rent Arabidopsis tissues showed that theSMT is produced in all plant organs, ex -cept in the seeds and young seedlings.Immunofluorescent labeling of Arabi dop -sis leaf sections localized SMT to thecen tral vacuoles of mesophyll and epi-dermal cells (see figure). In accor-dancewith characteristics of SCPL proteins, weconclude that Arabidopsis SMT is syn-thesized as a precursor protein that istargeted to the endoplasmic reticulum.The protein is probably glycosylated inthe Golgi apparatus from where it is sub-sequently routed to the vacuole.

Cloning of the cDNAs encoding UDP-glu-cose:sinapate glucosyltransferase (SGT)and 1-sinapoylglu-cose:choline sinapoyl-transferase (SCT) from Arabidopsis thali-ana and Bras si ca napusThis work is part of the BMBF project"NAPUS 2000 - healthy food from trans-genic rape" and focuses on reduction ofthe antinutritive sinapine (sinapoylcholi-ne) content in rapeseed. We are fol-lowing two strategies. A molecular ap -proach (dsRNAi) aims to suppress thepivotal enzymatic steps of sinapine syn-

Group MembersAlfred Baumert(research scientist)

Claus Lehfeldt(PhD student until June 2001)

Carsten Milkowski(postdoctoral position)

Juliane Mittasch(PhD student since December 2002)

Lilian Nehlin(guest scientist until June 2002)

Ingrid Otschik(technician)

Diana Schmidt(PhD student since August 2001)

Collaborators

Diana BowlesDepartment of Biology, University of York, UK

Clint ChapplePurdue University, West Lafayette, USA

Martin Frauen, Gunhild LeckbandNordeutsche Pflanzenzucht, Hans Georg Lembke KG(breeder), Hohenlieth, Germany

Ernst HeinzUniversity of Hamburg, Germany

Knut Meyer, Paul V. ViitanenDuPont Central Research and Development, BiochemicalSciences and Engineering, Wilmington, Delaware, USA

Christian MöllersUniversity of Göttingen, Germany

José OrsiniSaaten Union Resistenzlabor GmbH, Leopoldshöhe,Germany

Jürgen Schmidt, Sabine RosahlInstitute of Plant Biochemistry, Halle, Germany

Joachim SchröderUniversity of Freiburg, Germany

Milton T. StubbsUniversity of Halle, Germany

Victor WrayGerman Research Centre for Biotechnology,Braunschweig, Germany

Higher plants accumulate a wealth of hydroxycinnamate (HCA) conjugates,mostly esters and amides. They are of prime ecological importance for plantsurvival. They protect plants against DNA-damaging UV light. Acylation ofanthocyanin pigments with HCAs results in an (intramolecular) copigmenta-tion effect, protecting these pigments against degradation. Soluble and cellwall-bound HCAs participate in plant defense against microbial attack. Withregard to their biosynthesis, HCAs are usually activated as coen-zyme A(CoA) thioesters or 1-O-acylglucosides (b-acetal esters), being the substratesof the HCA transferases involved in formation of various conjugates. Ourgroup is interested in structural and functional characterization of the UDP-glucose- and acylglucose-dependent glucosyl- and HCA transferases.

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Research Group: Hydroxycinnamic AcidsHead: Dieter Strack

71

Biosynthese pflanzlicher Farbstoffe. Angew. Chem.113, 3907-3911 (2001).Van Damme, E. J. M., Hause, B., Hu, J., Barre, A.,Rougé, P., Proost, P. & Peumans, W. J. Two distinctjacalin-related lectins with a different specificityand sub-cellular location are major vegetative sto-rage proteins in the bark of the mulberry (Morusnigra) tree. Plant Physiol. 130, 757-769 (2002).

Van Damme, E. J. M., Hu, J., Barre, A., Hause, B.,Baggerman, G., Rougé, P. & Peumanns, W. J.Purification, characterization, immunolocalizationand structural analysis of the abundant cytoplasmicbeta-amylase from Calystegia sepium (hedge bind-weed) rhizomes. Eur. J. Biochem. 268, 6263-6273(2001).

Vierheilig, H., Gagnon, H., Strack, D. & Maier, W.Accumulation of cyclohexenone derivatives in bar-ley, wheat and maize roots in response to inocula-tion with different arbuscular mycorrhizal fungi.Mycorrhiza 9, 291-293 (2000).

Vierheilig, H., Maier, W., Wyss, U., Samson, J., Strack,D. & Piché, Y. Cyclohexenone derivative- and phos-phate- levels in split-root systems and their role inthe systemic suppression of mycorrhization in pre-colonized barley plants. J. Plant Physiol. 157, 593-599 (2000).

Vogt, T. Substrate specificity and sequence analysisdefine a polyphyletic origin of betanidin 5- and 6-O-glucosyltransferase from Dorotheanthus bellidi-formis. Planta 214, 492-495 (2002).

Vogt, T. & Jones, P. Glycosyltransferases in plant natu-ral products synthesis: characterization of a super-gene family. Trends Plant Sci. 5, 380-386 (2000).

Walter, M. H., Fester, T. & Strack, D. Arbuscularmycorrhizal fungi induce the non-mevalonatemethylerythritol phosphate pathway of isoprenoidbiosynthesis correlated with accumulation of theyellow pigment and other apocarotenoids. Plant J.21, 571-578 (2000).

Walter, M. H., Hans, J. & Strack, D. Two distantlyrelated genes encoding 1-deoxy-D-xylulose 5-phosphate synthases: differential regulation inshoots and apocarotenoid-accumulating mycorrhi-zal roots. Plant. J. 31, 243-254 (2002).

Wasternack, C. & Hause, B. Jasmonate - Signale zurStressabwehr und Entwicklung in Pflanzen. Biologiein unserer Zeit 30, 312-320 (2000).

Wasternack, C. & Hause, B. Jasmonates and octade-canoids - signals in plant stress response and deve-lopment. Progr. Nucleic Acid Research 72, 165-221 (2002).

Yamamoto, K.-I., Kobayashi, N., Yoshitama, K.,Teramoto, S. & Komamine, A. Isolation and purifica-tion of tyrosine hydroxylase from callus cultures ofPortulaca grandiflora. Plant Cell Physiol. 42, 969-975 (2001).

Ziegler, J., Stenzel, I., Hause, B., Maucher, H.,Hamberg, M., Grimm, R., Ganal, M. & Wasternack,C. Molecular cloning of allene oxide syclase: Theenzyme establishing the stereochemistry of octa-decanoids and jasmonates. J. Biol. Chem. 275,19132-19138 (2000).

Books and Book chaptersFester, T. Leben aus dem Feuer? Eine Reise zu den

Anwohnern der Vulkane unseres Planeten, ShakerVerlag Aachen (2000).Fester, T., Peerenboom, E., Weiss, M. & Strack, D.Multimedia-Präsentation Mycorrhiza, (2001).

Strack, D. Enzymes involved in hydroxycinnamatemetabolism. In: Methods in Enzymology, Vol. 335,Flavonoids and Other Polyphenols (Packer, L., ed.)Academic Press, Sheffield, UK, pp. 70-81 (2001).

Varma, A. K., Singh, A., Sudha, Sahay, N. S., Sharma, J.,Roy, A., Kumari, M., Rana, D., Thakran, S., Deka, D.,Bharti, K., Hurek, T., Blechert, O., Rexer, K.-H., Kost,G., Hahn, A., Maier, W., Walter, M., Strack, D. &Kranner, I. Piriformospora indica - an axenically cul-turable mycorrhiza-like endosymbiotic fungus. In:The Mycota, IX, Fungal Associations (Hock, B., ed.),Springer-Verlag, Wien New York, pp. 125-150 (2001).

Vogt, T. Glycosyltransferases involved in plantsecondary metabolism. In: Evolution of MetabolicPathways. Recent Advances in Phytochemistry, Vol.34 (Romeo, J. T., Ibrahim, R., Varin, L., de Luca, V.,eds.) Elsevier Science, New-York, pp. 317-347(2000).

Publications in pressCacace, S., Schröder, G., Wehinger, E., Strack, D.,Schmidt, J. & Schröder, J. A flavonol O-methyltrans-ferase from Catharanthus roseus performing twosequential methylations. Phytochemistry 62, 127-138 (2003).

Eckermann, C., Schröder, G., Eckermann, S., Strack,D., Schmidt, J., Schneider, B. & Schröder, J.Stilbenecarboxylate biosynthesis: a new function inthe family of chalcone synthase-related proteins.Phytochemistry 62, 271-286 (2003).

Krajinski, F., Hause, B., Gianinazzi-Pearson, V. &Franken, P. Mtha1, an arbuscule cell-specific plasmamembrane H+-ATPase gene from Medicago trunca-tula. Plant Biol.

Opitz, S., Schnitzler, J.-P., Hause, B. & Schneider, B.Histochemical analysis of phenylphenalenone-rela-ted compounds in Xiphidium caeruleum(Haemodoraceae). Planta.

Peng, Z. F., Strack, D., Baumert, A., Subramaniam, R.,Goh, N. K., Chia, T. F., Tan, S. N. & Chia, L. S.Antioxidant flavonoids from leaves of Polygonumhydropiper L. Phytochemistry 62, 16-21 (2003).

Proels, R.K., Hause, B. & Roitsch, T. Novel mode ofhormone induction of tandem tomato invertasegenes in floral tissues. Plant Mol. Biol.

Stenzel, I., Hause, B., Maucher, H., Pitzschke, A.,Miersch, O., Kramell, R., Ziegler, J., Ryan C.A. &Wasternack, C. Allene oxide cyclase transgenespotentiate jasmonate biosynthesis and the wound-response of tomato leaves. Plant J. 33, 577-589(2003).

Stenzel, I., Hause, B., Miersch, O., Kurz, T., Maucher,H., Weichert, H., Ziegler, J., Feussner, I. &Wasternack, C. Jasmonate biosynthesis and theallene oxide cyclase family of Arabidopsis thaliana.Plant Mol. Biol.

Strack, D., Vogt, T. & Schliemann, W. Recent advancesin betalain research. Phytochemistry 62, 247-269(2003).

Books and Book chapters in pressStenzel, I., Hause, B., Feussner, I. & Wasternack, C.Transcriptional activation of jasmonate biosynthesisenzymes is not reflected at protein level. In:Advanced Research on Plant Lipids (Murata, N., ed.)Kluwer Academic Publishers, Dordrecht, 2002.

Stumpe, M., Stenzel, I., Weichert, H., Hause, B. &Feussner, I. The lipoxygenase pathway in mycorrhi-zal roots of Medicago truncatula. In: AdvancedResearch on Plant Lipids (Murata, N., ed.) KluwerAcademic Publishers, Dordrecht, 2002.

Thorson, J. & Vogt, T. Glycosylated natural products.In: Carbohydrate based Drug Discovery (Wong, C.-H., ed.).

PatentsHause, B., Bessler, K., Kogel, K. & Wasternack., C.Method of screening for agrochemicals. Europeanpatent 981245251.

Milkowski, C., Baumert, A. & Strack, D. Verfahren zurBeeinflussung des Sinapingehaltes in transgenenPflanzenzellen und Pflanzen. German patent10034320.1 (2000).

Rosahl, S., Scheel, D., Schmidt, A. & Strack, D.Transgene Pflanzen mit erhöhter Resistenz gegenBefall durch Phytopathogene. German patent19846001.5 (2000).

Ziegler, J., Stenzel, I., Hause, B. & Wasternack, C.Allenoxidcyclase-Gen und dessen Verwendung zumHerstellen von Jasmonsäure. German patent10004468.9 (2000).

Doctoral ThesesIbdah, Mwafaq: Lichtinduzierte Flavonoid- undBetacyanakkumulation in Mesembryanthemum cry-stallinum. University of Halle-Wittenberg,Department of Pharmacy, 15/5/2002.

Kobayashi, Naoko: Contributions to betalain bio-chemistry. New structures, condensation reactions,and vacuolar transport. University of Halle-Wittenberg, Department Biochemistry/Biotech -nology, 20/11/2002.

Lehfeldt, Claus-Ulrich: Das Gen derSinapoylglucose: L-Malat-Sinapoyltransferase vonArabidopsis thaliana (L.) Heynh.(Ackerschmalwand): Klonierung durch T-DNA-Tagging und Versuche zur Expression in Escherichiacoli. University of Halle-Wittenberg, Department ofBio chemistry/Bio tech nology, 11/4/2001. <

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(2001).Hause, B., Maier, W., Miersch, O., Kramell, R. &Strack, D. Induction of jasmonate biosynthesis inarbuscular mycorrhizal barley roots. Plant Physiol.130, 1213-1220 (2002).

Hause, B., Meyer, K., Viitanen, P. V., Chapple, C. &Strack, D. Immunolocalization of 1-O-sinapoylglu-cose:malate sinapoyltransferase in Arabidopsis tha-liana. Planta 215, 26-32 (2002).

Hause, B., Stenzel, I., Miersch, O., Maucher, H.,Kramell, R., Ziegler, J. & Wasternack, C. Tissue-speci-fic oxylipin signature of tomato flowers: allene oxidecyclase is highly expressed in distinct flower organsand vascular bundles. Plant J. 24, 113-126 (2000).

Hause, B., Weichert, H., Höhne, M., Kindl, H. &Feussner, I. Expression of cucumber lipid-body lipo-xygenase in transgenic tobacco: lipid-body lipoxy-genase is correctly targeted to seed lipid bodies.Planta 210, 708-714 (2000).

Ibdah, M., Krins, A., Seidlitz, H., Heller, W., Strack, D.& Vogt, T. Spectral dependence of flavonol and beta-cyanin accumulation in Mesembryanthemum cry-stallinum under enhanced UV radiation. Plant CellEnviron. 25, 1145-1154 (2002).

Irmler, S., Schröder, G., St-Pierre, B., Crouch, N. P.,Hotze, M., Schmidt, J., Strack, D., Matern, U. &Schröder, J. Indole alkaloid biosynthesis inCatharanthus roseus: new enzyme activities andidentification of cytochrome P450 CYP72A1 assecologanin synthase. Plant J. 24, 797-804 (2000).

Jones, P. & Vogt, T. Glycosyltransferases in seconda-ry plant product metabolism: tranquilizers and sti-mulant controllers. Planta 213, 164-174 (2001).

Kobayashi, N., Schmidt, J., Nimtz, M., Wray, V. &Schliemann, W. Betalains from Christmas cactus.Phytochemistry 54, 419-426 (2000).

Kobayashi, N., Schmidt, J., Wray, V. & Schliemann, W.Formation and occurrence of dopamine-derivedbetacyanins. Phytochemistry 56, 429-436 (2001).

Landtag, J., Baumert, A., Degenkolb, T., Schmidt, J.,Wray, V., Scheel, D., Strack, D. & Rosahl, S.Accumulation of tyrosol glucoside in transgenicpotato plants expressing a parsley tyrosine decar-boxylase. Phytochemistry 60, 683-689 (2002).

Lee, Y. K., Hippe-Sanwald, S., Jung, H. W., Hong, J. K.,Hause, B. & Hwang, B. K. In situ localization of chi-tinase mRNA and protein in compatible andincompatible interactions of pepper stems withPhytophthora capsici. Physiol. Mol. Plant Pathol. 57,111-121 (2000).

Lehfeldt, C., Amber, M. S., Meyer, K., Ruegger, M.,Cusumano, J. C., Viitanen, P. V., Strack, D. & Chapple,C. Cloning of the SNG1 gene of Arabidopsis reve-als a role for a serine carboxypeptidase-like pro-tein as an acyltransferase in secondary metabolism.Plant Cell 12, 1295-1306 (2000).

Lehmann, K., Hause, B., Altmann, D. & Köck, M.Tomato ribonuclease LX with the functional ERretention motif HDEF is expressed during pro-grammed cell death processes including xylem dif-ferentiation, germination and senescence. PlantPhysiol. 127, 436-449 (2001).

Maier, W., Schmidt, J., Nimtz, M., Wray, V. & Strack, D.Secondary products in mycorrhizal roots of tobac-

co and tomato. Phytochemistry 54, 473-479 (2000).Maucher, H., Hause, B., Feussner, I. & Wasternack, C.The allene oxide synthase of barley (Hordeum vul-gare cv. Salome) leaves is developmentally regula-ted. Plant J. 21, 199-213 (2000).

Mikkat, S., Milkowski, C. & Hagemann, M. The genesll0273 of the cyanobacterium Synechocystis sp.strain PCC6803 encodes a protein essential forgrowth at low Na+IK+ ratios. Plant Cell Environ. 23,549-559 (2000).

Milkowski, C., Baumert, A. & Strack, D. Cloning andexpression of a rape cDNA encoding UDP-gluco-se:sinapate glucosyltransferase. Planta 211, 883-886(2000).

Milkowski, C., Baumert, A. & Strack, D. Identificationof four Arabidopsis genes encoding hydroxycinna-mate glucosyltransferases. FEBS-Lett. 486,183-184(2000).

Milkowski, C., Krampe, S., Weirich, J., Hasse, V.,Boles, E. & Breunig. K. D. Feedback regulation of glu-cose transporter gene transcription inKluyveromyces lactis by glucose uptake. J.Bacteriol. 183, 5223-5229 (2001).

Nehlin, L., Möllers, C., Bergmann, P. & Glimelius, K.Transient beta-gus and gfp gene expression and via-bility analysis of microprojectile bombarded micro-spores of Brassica napus L. J. Plant Physiol. 156,175-183 (2000).

Pauk, J., Puolomatka, M., Tóth, K. L. & Monostori, T.In vitro androgenesis of triticale in isolated micro-spore culture. Plant Cell Tiss. Org. 61, 221-229(2000).

Peumans, W. J., Hause, B. & Van Damme, E. J. M. Thegalactose-binding and mannose-binding jacalin-rela-ted lectins are located in different sub-cellularcompartments. FEBS-Lett. 477, 186-192 (2000).

Riemann, D., Rontsch, J., Hause, B., Langner, J. &Kehlen, A. Cell-cell contact between lymphocytesand fibroblast-like synoviocytes induces lymphocy-tic expression of aminopeptidase N/CD13 andresults in lymphocytic activation. Adv. Exp. Med.Biol. 477, 57-66 (2000).

Roitsch, T., Ehneß, R., Goetz, M., Hause, B., Hofmann,M. & Sinha, A. K. Regulation and function of extra-cellular invertase from higher plants in relation toassimilate partitioning, stress responses and sugarsignalling. Aust. J. Plant Physiol. 27, 815-825 (2000).

Schliemann, W., Cai, Y., Degenkolb, T., Schmidt, J. &Corke, H. Betalains of Celosia argentea.Phytochemistry 58, 159-165 (2001).

Stephan, M., Bangerth, F. & Schneider, G. Transportand metabolism of exogenously applied gibberellinsto Malus domestica Borkh. cv. Jonagold. PlantGrowth Regul. 33, 77-85 (2001).

Strack, D., Fester, T., Hause, B. & Walter, M. H. Eineunterirdische Lebensgemeinschaft: Die arbuskuläreMykorrhiza. Biologie in unserer Zeit 31, 286-295(2001).

Strack, D. & Schliemann, W. Bifunctional polyphenoloxidases: novel functions in plant pigment biosyn-thesis. Angew. Chem. Int. Ed. 40, 3791-3794 (2001).

Strack, D. & Schliemann, W. BifunktionellePolyphenol oxidasen: neuartige Funktionen in der

PublicationsBachmann, A., Hause, B., Maucher, H., Garbe, E.,Weichert, H., Wasternack, C. & Feussner, I.Jasmonate-induced lipid peroxidation in barley lea-ves initiated by distinct 13-LOX forms of the chlo-roplast. Biol. Chem. 383, 1645-1657 (2002).

Back, K., Jang, S. M., Lee, B.-C., Schmidt, A., Strack, D.& Kim, K.-M. Cloning and characterization of ahydroxycinnamoyl-CoA:tyramine N-(hydroxycin-namoyl) transferase induced in response to UV-Cand wounding from Capsicum annuum. Plant CellPhysiol. 42, 475-481 (2001).

Baumert, A., Mock, H.-P., Schmidt, J., Herbers, K.,Sonnewald, U. & Strack, D. Patterns of phenylpropan-oids in non-inoculated and potato virus Y-inoculatedleaves of transgenic tobacco plants expressing yeast-derived invertase. Phytochemistry 56, 535-541(2001).

Binarová, P., Cenklová, V., Hause, B., Kubátová, E.,Lysák, M., Dolezel, J., Bögre, L. & Dráber, P. Nuclearg-tubulin during acentriolar plant mitosis. Plant Cell12, 433-442 (2000).

Breunig, K. D., Bolotin-Fukuhara, M., Bianchi, M. M.,Bourgarel, D., Falcone, C., Ferrero, I., Frontali, L.,Goffrini, P., Krijger, J. J., Mazzoni, C., Milkowski, C.,Steensma, H. Y., Wesolowski-Louvel, M. & Zeeman A.M. Regulation of primary matabolism inKluyveromyces lactis. Enzyme Microb. Technol. 26,771-780 (2000).

Cai, Y., Sun, M., Schliemann, W. & Corke, D. Chemicalstability and colorant properties of betaxanthin pig-ments from Celosia argentea. J. Agric. Food Chem.49, 4429-4435 (2001).

Chen, Y., Peumans, W. J., Hause, B., Bras, J., Kumar,M., Proost, P., Barre, A., Rougé, P. & Van Damme, E. J.M. Jasmonic acid methyl ester induces the synthesisof a cyto plasmic/nuclear chito-oligosaccharide bin-ding lectin in tobacco leaves. FASEB J. 16, 905-907(U225-251) (2002).

Ezcurra, I., Wycliffe, P., Nehlin, L., Ellerstrom, M. &Rask, L. Transactivation of the Brassica napus napinpromoter by AB13 requires interaction of the con-served B2 and B3 domains of AB13 with differentcis-elements: B2 mediates activation through anABRE, whereas B3 interacts with an RY/G-box.Plant J. 24, 57-66 (2000).

Fester, T., Hause, B., Schmidt, D., Halfmann, K.,Schmidt, J., Wray, V., Hause, G. & Strack, D.Occurrence and localization of apocarotenoids inarbuscular mycorrhizal plant roots. Plant CellPhysiol. 43, 256-265 (2002).

Fester, T., Kiess, M. & Strack, D. A mycorrhiza-responsive protein in wheat roots. Mykorrhiza 12,219-222 (2002).

Fester, T., Schmidt, D., Lohse, S., Walter, M. H., Giuliano,G., Bramley, P. M., Fraser, P. D., Hause, B. & Strack, D.Stimulation of carotenoid metabolism in arbuscularmycorrhizal roots. Planta 216, 148-154 (2002).

Fester, T., Strack, D. & Hause, B. Reorganization oftobacco root plastids during arbuscule develop-ment. Planta 213, 864-868 (2001).

Hao, Q., Van Damme, J. M., Hause, B., Barre, A.,Chen, Y., Rougé, P. & Peumans, W. J. Iris bulbs expresstype 1 and type 2 ribosome-inactivating proteinswith unusual properties. Plant Physiol. 125, 866-876

Publications, Books and Bookchapters, In press,Patents, Doctoral Theses, Diploma Theses

73

The departmentof Ad mi nis tra -

tion and Tech nicalSer vices re pre -sents the cen - tral infra struc -

tu ral unit within theinstitute. Ad mi n -istrative main focu-ses are personnel,legal, and financialmat ters. Main tasksof the central ser-vices are purcha-sing and accountma nagement, and maintenance of thescientific library and chemical store.Also the gardeners represent anessential component of the centralservices. The Technical Ser vices dealwith the buildings and properties.The technical co-workers in parti-cular take care of new construc-tions, the maintenance of theexis ting buildings and laborato-ries, and the technical andscientific equipment of thelaboratories.

The scientific library of theinstitute offers excellentpossibilities for literature-based research. The libra-ry has subscriptions to 83of international researchjournals and stocks

approximately 5.000 hardbackbooks. A reading hall with 18 in -ternet-connected computers andfive individual rooms are alsoavialable.

In addition to an experimen-tal field area, a series of fullyair-conditioned green hou-ses and phytochambers areavailable for the re searchprograms. In these areas

the gardeners take care of the expe-rimental plant material.

Since the reestablishment of theinstitute in the year 1992, most mainbuildings were re sto red completely.The main focus of the constructionworks concentrated on the labora-tory and technology areas, all ofwhich are now well equipped.

Beside the work the redeve lopmentof the existing buildings, new con-structions were undertaken in thelast years. Currently, a new buildingis under construction that willaccomodate new highly sensitiveanalytical instruments. In the nearfuture, additional greenhouse facili-ties and a central service buildingwill be constructed.

Altogether, the institute of fers abest possible infrastruc ture for itsdiverse research projects.<

Working Groups

FinanceHead: Barbara WolfAstrid Ortloff (until August 2002)Gudrun SchildbergBurgunde Seidl (since October 2002)Kerstin Wittenberg (since May 2002)

PersonnelHead: Kerstin BalkenhohlAlexandra BurwigCindy Maksimo (since April 2002)Rita StelzerKathleen Weckerle

General AdministrationHead: Rosemarie StraßnerAlexandra BurwigCindy Maksimo (since April 2002)Rita StelzerKathleen Weckerle

TraineesAntje OlschewskiClemens Schinke

LibraryHead: Andrea PiskolJessica Ackermann (Trainee)Antje Werner (Trainee)

Graphics & PhotographyHead: Christine KaufmannAnnett Kohlberg

Construction and MaintenanceHead: Matthias Böttcher (until December 2002)Detlef DieckmeyerCarsten Koth (since January 2002)Michael KrägeJörg LemnitzerKlaus-Peter SchneiderCatrin TimpelEberhard Warkus

ElectronicsHolger BartzHans-Günter KönigRonald Scheller

GardeningHead: Iris RudischMartina AllstädtNicole Mühlwald (Trainee)Christian Müller (since April 2002)Kristina Rejall (since June 2002)Steffen RudischKatja Scheming (Trainee since August 2002)Andrea Voigt (Trainee since August 2002)

Department: Administration and Technical ServicesHead: Lothar FranzenSecretary: Heide Pietsch

72

75

Staffing Schedule

Staffing schedule 2000

Number of members on annual average 166

Full-time employees in % 77

2001

167

77

2002

169

75

Total

502

229

Average

167

76

Part-time employees in % 23

Number of established posts 89

23

89

25

92

71

270

24

90

Temporary employees (budget) 19

Employess remunerated by third parties subsidies(avarage) 42

11

44

20

38

50

124

17

41

Employees remunerated by "Universitiy Special FundsProgramme III" (Hochschulsonderprogramm III / HSP III) 1

Employees remunerated by "University Science FundsProgramme" (Hochschulwissenschaftsprogramm / HWP) -

-

11

-

9

1

20

1

10

Proportion of female employess in % 60

Personnel fluctuation rate in % 16,4

59

10,4

61

13

180

39,8

60

13

Avarage age of employees 40

Scholarship/fellowship holders 10

39

6

39

5

118

21

39

7

Vocational training

Successfully completed vocational training 4 - - 4 1

Avarage number of apprentices 6 6 7 19 6

commercial areahorticultural arealibrary

231

222

232

685

232

74

Elsevier Elsevier Science Publisher

Research grants listed on this and thefollowing pages were given by

BMBF Bundesministerium für Bil dungund Forschung - Fede ralMinistry of Education andResearch

AFNG Arabidopsis FunctionalGenomics Network (DFG)

BML Bundesministerium für Ver -braucher schutz, Ernährung undLandwirtschaft - FederalMinistry of ConsumerProtection, Food and Agricultur

BPS BASF Plante Science GmBH

D-B Foundation Gottlieb Daimler and Karl BenzFoundation

DBU Deutsche BundesstiftungUmwelt

DFG DeutscheForschungsgemeinschaft

DAAD Deutscher AkademischerAustauschdienst - GermanAcademic Exchange Service

EU European Union

Firmenich

GABI Genom Analyse im BiologischenSystem Pflanze

GTZ Gesellschaft für TechnischeZusammenarbeit

Hopsteiner

HSP III Hochschulsonderprogramm III

HumboldtFoundation

Alexander von HumboldtFoundation

Icon genetics

KWS KWS SAAT AG

Probiodrug

Kultusministerium des LandesSachsen-Anhalt - Ministry ofEdu cation and Cultural Affairsof the State of Saxony Anhalt

PPP ProjektbezogenerPersonenaustausch (DAAD)

MK-LSA

Probiodrug AG

SFB 363 Sonderforschungsbereich 363 -Collaborative Research Centres

VW Foundation Volkswagen Foundation

Resources and Investments

in %in Mio. Euro

Personnel 32,413,0

Consumables 15,56,2

Grants / Subsidies 0,70,3

Investments 34,313,8

“University Science Funds Programme”(HWP) 3,01,2

Subtotal 34,5

Funds from external sourcesBMBF 3,21,3

MK-LSA 2,00,8

DFG 6,02,4

Industry 1,50,6

EU 1,20,5

other 0,20,1

Subtotal 14,15,7

InvestmentsEquipment

Building

Basic Financing Funds

Total 10040,2

Total

5,8

8,0

13,8

HWP Hochschulwissenschaftsprogramm

77

Project & Head of Project Financed byTotal durationAmount

2000 - 2002(in Euro)

Personnel postsfinanced

CRISP(Prof. D. Scheel) EU01/04 154.400 1

Heavy metal tolerance and silicon(U. zur Nieden) MK-LSA00/04 67.700 1

Non-host resistance(T. Nürnberger) KWS98/02 120.300 1

The role of jasmonates in pathogene defen-se (Prof. D. Scheel) DFG01/03 74.700 1

Jasmonate-insensitive mutant(Prof. D. Scheel, S. Berger) MK-LSA99/03 64.700 1

Arabidopsis halleri(S. Clemens) DFG00/03 60.300 1

Metallophytes(S. Clemens) EU01/03 104.100 1

Biomineralisation(D. Neumann) DFG01/03 49.100 1

Department of Stress and Developmental BiologyHeavy-metal tolerance (D. Neumann & S. Clemens) DFG / SFB 36302/04 96.400 1

Signal transduction (Prof. D. Scheel) DFG / SFB 36302/04 201.100 1

Chrom-(II)-mediated reactions(Prof. L. Wessjohann)

DAAD / PPPHungary02/03 5.700 0

Daimler Benz fellowship(Prof. L. Wessjohann) D-B Foundation2002 1.700 1

Subtotal: 1.000.800 8

Pathogen defense-related genes(Prof. D. Scheel) DFG98/00 30.000 1

Oxidative burst(Prof. D. Scheel)

DFG /Innovationskolleg99/00 25.500 1

Plant peptides(Prof. D. Scheel) DFG2000 460.200 1

Signal transduction(Prof. D. Scheel) DFG99/00 2.900 1

Chromatin and gene regulation(Prof. D. Scheel) DFG / SFB 36399/01 68.600 1

Elicitor receptors(T. Nürnberger) DFG99/01 54.100 2

Gene silencing(Prof. D. Scheel) MK-LSA00/01 91.400 1

Ozone signaling(Prof. D. Scheel)

DAAD / PPPFinnland2001 3000 0

Heat stress proteins(D. Neumann) DFG99/00 8.300 1

76

Use of Funds from External Sources

Project & Head of Project Financed byTotal durationAmount

2000 - 2002(in Euro)

Personnel postsfinanced

Jasmonate biosynthesis regulation(Prof. C. Wasternack & O. Miersch) DFG / SPP99/04 111.000 1

Glutamate cyclase(Prof. C. Wasternack) Probiodrug01/03 30.700 0

Allenoxidcyclase(Prof. C. Wasternack) Firmenich01/02 60.900 1

Papaver somniferum(Prof. T. Kutchan) DFG / SFB 36300/01 57.300 1

Functional genomics(G. Herrmann) DFG00/02 26.300 0

Analysis of genes(Prof. T. Kutchan) Icon Genetics00/02 230.900 1

Molecular genetics of isoquinolinealk.biosynth. (Prof. T. Kutchan) DFG01/04 67.600 2

Cellular signalling(Prof. T. Kutchan) DFG / MLU02/04 24.100 1

Transformation and regeneration of Papaversomniferum (S. Frick) DFG02/03 57.300 2

Modulation of jasmonates by transgenicplants (Prof. C. Wasternack & O. Miersch) DFG / SFB 36302/04 175.500 1

Subtotal: 973.350 12

Department of Bioorganic Chemistry

HEA(N)TOS(Prof. L. Wessjohann, Prof. G. Adam) BMBF00/03 264.700 2

COMBIOCAT(Prof. L. Wessjohann) EU01/04 142.100 2

EPILA(W. Brandt) EU01/03 29.800 2

MCR ligand synthesis(Prof. L. Wessjohann) DAAD / Probral02/03 8.800 0

Department of Natural Product Biotechnology

Papaver somniferum(Prof. T. Kutchan) DFG01/02 117.900 1

Salvia fragrances(Prof. T. Kutchan) DBU02/03 13.850 1

Conformation of brassinosteroids(A. Porzel, W. Brandt [MLU]) DFG99/00 6400 1

New bioactive natural products from endemicallyoccuring Yemenian plants (J. Schmidt, G. Adam) DFG / GTZ97/02 49.700 0

Structural elucidation and combinatorialchemistry (Prof. L. Wessjohann) MK-LSA / HWP2000 490.800 0

Fungi excursion(N. Arnold) DFG2001 1.100 0

79

Project & Head of Project Financed byTotal durationAmount

2000 - 2002(in Euro)

Personnel postsfinanced

Phytochemistry(Prof. D. Strack) Elsevier02/04 8.000 1

Stable transformation of Medicago truncatu-la (B. Hause) MK-LSA2002 47.300 0

Profiling of metabolites, proteins and peptidesDep. Stress and Developmental Biology andDep. Bioorganic Chemistry(S. Clemens)

BMBF / GABI00/04 588.200 4

HUMULUSDep. Bioorganic Chemistry andDep. Natural Product Biotechnology(F. Stevens, Prof. L. Wessjohann & J. Page)

Hopsteiner01/02 9.900 0

“Analytica 2000”(E. Peerenboom) MK-LSA2000 11.200 0

“Achema 2000”(E. Peerenboom) MK-LSA2000 1.300 0

Public Understanding of Sciences andHumanities (PUSH) - Multimedia projectabout the mycorrhiza (T. Fester & E. Peerenboom)

Donors Associationfor the Promotion ofSciences and Huma -nities in Germany

2001 6.200 0

Subtotal: 616.800 4

Joint projects

Subtotal: 967.700 13

Projects granted, total: 5.739.350 71

General view

MK-LSA

BMBF

Industry

DFG

EU

Other sources

785.200

1.331.000

573.300

2.401.700

555.300

92.850

78

Use of Funds from External Sources

Project & Head of Project Financed byTotal durationAmount

2000 - 2002(in Euro)

Personnel postsfinanced

Metabolite profiling(W. Schliemann) DFG02/04 24.400 1

Department of Secondary Metabolism

Betanidin-Glucosyltransferases(T. Vogt) DFG01/03 121.900 2

Metabolism of isoprenoids(M. Walter & T. Fester) DFG00/04 90.500 1

NAPUS 2000(Prof. D. Strack) BMBF99/04 378.100 2

The role of jasmonates during the establishmentof mycorrhiza (B. Hause & Prof. D. Strack) DFG00/04 83.500 1

Carotenoid biosynthesis in arbuscularmycorrhizal roots (T. Fester) DFG00/04 82.000 1

Humboldt fellowship(Prof. D. Scheel)

HumboldtFoundation01/02 5.600 0

Cooperation wtih South Africa(T. Nürnberger) VW Foundation01/04 40.000 0

NODO(S. Rosahl) EU02/04 50.700 1

Receptor kinases(T. Nürnberger) DFG / AFNG02/04 47.600 2

Arabidopsis thaliana interactions(Prof. D. Scheel) BPS01/02 10.900 0

Bioinformatics and Mass Spectronomy(Prof. D. Scheel) BMBF02/07 100.000 6

GABI-NONHOST(Prof. D. Scheel) BMBF02/06 109.700 4

Pathogene defense in Arabidopsis thaliana(S. Rosahl) MK-LSA2002 10.800 0

Subtotal: 2.180.700 34

Signals, delivery and response(T. Nürnberger) EU97/00 68.600 2

Betalains(W. Schliemann & Prof. D. Strack) DFG99/01 61.800 1

TIMBER(M. Walter) EU98/00 5.600 1

Endomycorrhiza(W. Maier & Prof. D. Strack) DFG98/01 24.000 1

Jasmonates in the development of barley(B. Hause & Prof C. Wasternack) DFG99/01 40.600 1

81

Dr. Oscar Dorneles Rodriguez(CAPES Fellow)

01.04.2002 - 20.09.2002Brazil

Lars Seipold 01.01.2002 - 31.05.2002Germany

Prof. Tran Van Sung 01.07.2002 - 18.12.2002Vietnam

Trin Thi Thuy 20.11.2001 - 19.11.2002Vietnam

Larissa Vasilets since 28.11.2002Russia

Dr. Svetlana Zakharova 30.10.2002 - 31.12.2002Russia

Department of Stress and Developmental Biology

Reetta Ahlfors(DAAD Fellow)

since 08.07.2002Finland

Dr. Susanne Berger(DFG Fellow)

01.04.2001 - 31.03.2002Germany

Anne-Claire Cazalé(Humboldt Fellow)

01.02.2000 - 31.12.2001France

Clarice de Figueiredo 01.11.1999 - 30.09.2001Brazil

Anna Drobek 01.09.2001 - 30.09.200126.02.2002 - 30.04.2002Poland

Dr. Emiko Harada(Humboldt Fellow)

since 22.02.2002Japan

Emma Jack 12.03.2001 - 20.04.2001Netherlands

Dr. Anano Dinakar Karve(Humboldt Fellow)

16.11.1999 - 14.02.2000India

Dr. Magdalena Krzymowska(Humboldt Fellow)

01.08.1999 - 30.06.2002Poland

Ma. Shaokang 06.05.2001 - 21.06.2001Singapore

Srpryia Paranthaman(Humboldt Fellow)

25.10.2002 - 20.12.2002India

Lizelle Piater 01.06.2002 - 29.07.2002South Africa

Claudia Simm(Fellow, Graduierten Kolleg)

since 01.10.2000Germany

Anne Varet 01.01.2002 - 30.04.2002France

Department of Secondary Metabolism

Stijn Jan Freddy Desmyter 10.01.2000 - 05.02.2000Netherlands

Dr. Shiming Liu 13.08.2001 - 13.06.2002China

Dr. Nirmal Sahay 10.01.2000 - 31.12.1999India

Dr. Sudha Sahay 24.09.1999 - 31.08.2001India

Diana Schmidt(Fellow, Bio Service GmbH, EU and the State ofSaxony Anhalt)

since 01.08.2001Germany

Prof. Luay Rashan(Humboldt Fellow)

01.07.2002 - 31.08.2002Jordan / Iraq

Joe Chou Hung Sim 06.05.2001 - 21.06.2001Singapore

Prof. Tamás Patony(DAAD Fellow)

13.10.2002 - 22.10.2002Hungary

Name PeriodCountry

80

Guest Researchers and Fellows

Prof. Guillermina Abdala 08.06.2001 - 16.07.2001Argentina

Dr. Maged Abou-Hashem 01.07.2002 - 22.10.2002Egypt

Arysyak Abrahamian(DAAD Fellow)

04.07.2000 - 31.12.2000Armenia

Nigel Bailey 19.11.2001 - 14.12.2001UK

Dr. Davide Berlanda 05.06.2001 - 21.06.2001Italy

Hubert Chassaigne(Humboldt Fellow)

15.01.2000 - 31.12.2000France

Dr. Kum-Boo Choi(Humboldt Fellow)

since 07.10.2002Korea

Predro Salvador de Rocha(FEBS Fellow)

10.04.2000 - 20.04.2000UK

Satinder Gitta 05.04.2000 - 04.06.2000Canada

Kristin Krukenberg(Fulbright Fellow)

23.09.2002 - 15.07.2003USA

Tamara Krupnova(DAAD Fellow)

01.10.1999 - 30.04.2000Kazakhstan

Anan Onaroon(DAAD Fellow)

26.07.1999 - 30.09.2002Thailand

Matjaz Oven 03.08.1999 - 31.07.2001Slovenia

Suppachai Samapitto(DAAD Fellow)

04.05.2000 - 03.05.2001Thailand

Anastasia Tkatcheva(SFB Fellow)

01.10.2001 - 28.02.2002Canada

Prof. Gülacti Topku(DAAD Fellow)

02.04.2000 - 02.07.2000Turkey

Prof. Luc Varin 01.10.2002 - 31.01.2003Canada

Dr. Ana Vigliocco 01.04.2002 - 31.05.2002Argentina

Dr. Bathany Zolman(SFB Fellow)

15.08.2002 - 31.10.2002USA

Prof. Antonio Luiz Braga(CAPES Fellow)

06.04.2002 - 21.04.2002Brazil

Tran Van Chien since 07.10.2002Vietnam

Marco Aurelio Dessoy(DAAD Fellow)

01.02.2001 - 30.06.2002Brazil

Csongor Hajdu(Erasmus + DAAD Fellow)

29.01.2001 - 31.07.200119.08.2002 - 13.12.2002Hungary

Dubravko Jelic 10.03.2002 - 22.03.2002Croatia

Myint Myint Khine(Daimler-Benz Fellow)

since 04.09.2002Myanmar (Burma)

Lazlo Merczs(DAAD Fellow)

10.11.2002 - 11.12.2002Hungary

Prof. Károly Micskei(DAAD Fellow)

17.06.2002 - 26.06.2002Hungary

Nguyen Hoang Anh 01.09.2000 - 31.08.2001Vietnam

Nguyen Hong Thi Van since 17.04.2002Vietnam

Department of Natural Product Biotechnology

Department of Bioorganic Chemistry

Name PeriodCountry

Participation in trade showsIn 2000, the institute exhibited severalpro jects at exhibitions and trade shows.These activities were planned and orga-nized by Ellen Peerenboom. In March,the IPB participated in one of the biggestinternational conventions for biotechno-logy, “Bio 2000” in Boston.

Together with the universities ofHalle and Magdeburg and severalother scientific institutes, our re -searchers presented their workat the Saxony Anhalt booth at“Ana lytika” (in 2000 and2002) in Munich and at“Achema” (in 2000) inFran k furt. In addition tothe projects on display,Claus Wasternack chai-red a workshop to thetopic "Plant Bio tech -nology - Novel Food"at a meeting held to -gether with “Analytika2000”.

Furthermore, in 2001the IPB participatedin “Biotechnika” inHan nover, Germany'smost important inter-national biotechnologyexhibition. At all the ex hibitions and tradeshows, visiting scientists

and journalists showed keeninterest in the institute'swork. As a result, several arti-cles were published in diffe-rent newspapers and journals.

Exhibitions at the frontierbetween science and art

Hosting the exhibition “Gene worldand nutrition” in June 2000, presentedby the Alimentarium Food Museum ofVevey, Switzerland, was a great successfor the IPB. More than 1000 guests visi-ted the institute to view and criticallydiscuss the interactive exhibits. This

exhibition pre-sented the history ofplant breeding and the increasing role ofgene technology for identifying and cre-ating new kinds of productive and resi-stant plants.

The exhibition “Life Science Art” in July2000 examined the theme of the humanbeing that lies behind the scientific re -searcher and his work. Silvia Stabel,painter and ex-scientist, displayed pain-tings with scientific themes and objectsfrom the artist's perspective. The pain-tings' message about the beauty and aes-thetic qualities of molecules, cells andscientific mo tifs was underlined by shortexplanations and quotations from re -searchers. Many visitors were veryimpressed by the exhibits, which wereshown under titles like “Alphabet of life”,“Hope” or “Orientation”.

Public events - a bridge to the peo-pleAs in the years before, the IPB participa-ted in 2000 and 2001 in the “Scienceday” on the market square in Halle. Ourresearchers presented the work of theinstitute by displaying posters and small-scale experiments. The event, organizedby the municipality and the University ofHalle, led to increased contact anddiscussion with interested citizens.

A similar presentation of scientific insti-tutes on the market square in Halle wascelebrated on the Uni versity’s 500thAnniversary in June 2002. Research ers ofthe IPB displayed and explained living mo -dels of mycorrhiza - a close partnershipbetween plants and fungi. In addition, vi -sitors had the possibility to see mycor-rhized plant cells under the microscope.A display of computer simulations aboutthe characteristics and behavior of pro-teins was also shown.

In February 2000, members of the insti-tute organized a charity concert for thecommunity kitchen of St. Elizabeth'sHospital in Halle. This community insti-

Group members

Gesine Krüger(Head until März 2002)

Jana Krupik(Assistent and Webmaster since November 2000)

Ellen Peerenboom(Head until Juli 2001)

Press and Public RelationsHead: Sylvia Pieplow

83

“Science day” 2001 on the market square in Halle

“Biotechnika” 2001 in Hannover

82

Research, the Ministry of EducationandCultural Affairs of Saxony Anhalt, theLeibniz Association, the city council ofHalle and scientists from all over theworld attended the official ceremonyand expressed their best wishes for thefuture. In a ceremonial address, Dierk

Scheel, director of the institute, spokeabout the successful scientific traditionof the IPB. Three scientific reports and aconcert by the chamber orchestra of theuniversity completed the program.After wards the institute members had aparty together with all invited guests. <

85

Congratulations for Günter Adam. His 70th birth-day was celebrated in December 2002

Benno Parthier celebrated his 70th birthday inAugust 2002

IPB’s 10th birthday in May 2002

PublicationsPeerenboom, E. Staffellauf in der Pflan zenzelle. In: Berichte aus derWissenschaft, Deutscher Forschungsdienst, Bonn, pp. 13-15 (2000).

Peerenboom, E. Zusatz für Farbindustrie bald aus Leinöl. WGL-Journal 1,p. 25 (2000).

Peerenboom, E. & Stabel, S. Life Science Art. Leibniz 4, Sonderbeilage(2000).

Pieplow, S. Pflanzliche “Staubsauger” ziehen Schwermetalle aus demBoden. Chemie.DE www.chemie.de/news/d/16725/ (2002).

Scheel, D., Frohberg, K., Peerenboom, E. & Wakenhut, U. Traditionenverbunden mit neuen wissenschaftlichen Potentialen. In:Wirtschaftsstandort Halle, Europäischer Verlag, Darmstadt, pp. 92-97(2000).

Press releasesLeckere Gene? Gen-Welten Ernährung Son derausstellung am IPB(E. Peeren boom, 06.06.2000)

Eine Symbiose aus Kunst und Wissen schaft: Sonder ausstel lung “LifeScience Art” am IPB (E. Peerenboom, 03.07.2000).

Prof. Dr. Ludger Wessjohann wird neuer Abteilungsleiter derAbteilung Natur stoff chemie am Leibniz-Institut für Pflan zen bio chemie(E. Peerenboom, 26.10.2000).

PlantMetaNet - neues Forschungs netz werk - Vier führendeInstitute auf dem Gebiet der Pflanzenforschung vereinbarenKooperation (E. Peerenboom, 06.06.2001).

Wissenschaftler entwickeln Lernmaterial für Schüler - Lern-CD fürdas Fach Bio logie: Mykorrhiza (G. Krüger, 12.12.2001).

IPB feiert sein 10jähriges Gründungs jubi läum (J. Krupik, 21.05.2002)

IPB ehrt langjährigen Direktor Prof. Dr. Benno Parthier (J. Krupik,27.08.2002)

Lange Nacht der Wissenschaften “Blick ins Innere der Pflanze(S. Pieplow, 18.09.2002)

Festveranstaltung zu Ehren von Pro fes sor Adam (S. Pieplow,09.12.2002)

Pflanzliche “Staubsauger” ziehen Schwer metalle aus dem Boden(S. Pieplow, 09.12.2002).

tution provides about 70 poor peoplewith a warm meal every day. As a resultof this classical concert, the IPB collea-gues proudly donated the money for 325meals to the hospital.

Under the motto "Green gene techno-logy - prospects and risks", 24 teachersfor chemistry and biology had the possi-bility to learn more about new methodsand molecular techniques of gene trans-fer into plants in May 2002. The training,held under the auspices of the CentralMarketing Organization of German Agri -cultural Industries, was organized by theIPB.

Instead of the annual "Science day", in2002 the university and the other re-search institutes of Halle celebrated a newevent - the "Long Night of Sciences". Atthis day in September, the institute'sdoors were open from 7 pm to midnightto welcome more than 300 members ofthe public. Visitors enthusiastically parti-cipated in guided tours through the labsand greenhouses of the IPB. In addition,experiments were displayed in the foyer,guests viewed the IPB's collection of co-l or ful cell cultures and had the possibi-lity to learn how a confocal laser-scan-ning microscope works. Be cause of thegreat success of this event, the "LongNight of Sciences" will be come an annualevent in Halle.

Public projectsThe IPB participated in the competition"Public Understanding of Sciences andHumanities" (PUSH) with a multimediaproject about mycorrhizal symbiosis.The interactive course about this fasci-nating biological interaction was produ-ced by Thomas Fester and Ellen Peeren -boom and was designed for students andinterested nonscientists alike. As one ofthe 22 final winners selected, the projectwas sponsored by the "Donors Asso -ciation for the Promotion of Sciencesand Human ities in Germany". InteractiveCD's were send away in 2001, at first to

all secondary schools of Saxony Anhalt,and afterwards as a result of numerousarticles in the regional and nationalpress, to many interested private citi-zens. This year an update and productionof an English version are planned.

As in previous years, the IPB organizedmany guided tours through the institutefor school classes and senior groups in2000 to 2002. In addition, all of the fourscientific departments sponsored severalperiods of practical training, which al-lowed many high-school students to gaininsight into lab work and to try outexperiments on the bench.

Since the beginning of 2001, the IPB hashad a new corporate design. The inter-nally designed logo was successfully in -troduced and promptly accepted by peo-ple from both within and outside theIPB. Since then, all of the letterheads onbusiness letters, flyers, publicity brochu-res and business cards display the newlogo. In addition, the institute's homepa-ge was completely reorganized and re-newed. The new version in German andEnglish has been online since May 2001.

CelebrationsThe year 2002 was a time of many cele-brations for the IPB. The institute hono-red two former members, each a cele-brity due to his personal qualities andscien tific lifework, with a splendid collo-quium. In August, Benno Parthier, theinstitute's former director and presidentof Germany's biggest and oldest acade-my, the German Academy of NaturalScien tists Leopoldina, celebrated his70th birthday together with his formercolleagues and the entire institute.Günter Adam, former head of thedepartment Natural Product Chemistry,also turned 70 in December.

In May 2002, the institute celebrated itsown birthday and foundation ten yearsago on a large scale. Representatives ofthe Federal Ministry of Education and

84

Uni versity’s 500th Anniversary in June 2002 on themarket square in Halle

"Long Night of Sciences" 2002. Prof. Dierk Scheelguided the visitors through the institute.

“Long Night of Sciences” 2002. Guests were verryinterested in the interactive CD about the mycor-rhiza.

Sylvia PieplowPress and Public Relations

+49 (0) 3 45 - 55 82 11 10+49 (0) 3 45 - 55 82 11 19spieplow@ipb-halle.depr@ipb-halle.de

Phone:Fax:email:

86

Map & Impressum

How to get to the IPB

Annual Report 2000 - 2002 of the Institute of Plant Biochem -istry Halle (Saale), July 2003

Publisher: Institute of Plant BiochemistryWeinberg 306120 Halle (Saale)Germanywww.ipb-halle.de

Editor:

Layout & Design: Jana KrupikSylvia Pieplow

Graphics & Pictures: Christine KaufmannAnnett KohlbergBettina Hauseand others

Copyright © 2003, 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 bestored in a retrieval system, transmitted or otherwise copied for public or privateuse, without written permission from the publisher.