Scientific Report 2003-2004

SCIENTIFIC REPORT 2003-2004

Leibniz Institute of Plant Biochemistry Weinberg 3 06120 Halle Germany

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

Email: [email protected] www.ipb-halle.de 2

Table of Contents

Message from the Managing Director 4 Departmental Organization 5 Research Mission Statement 6

Department of Natural Product Biotechnology 8 Head: Toni M. Kutchan, Professor Research Groups: Alkaloid Biosynthesis 10 Toni M. Kutchan Opium Poppy Biotechnology 12 Susanne Frick Mode of Action of Jasmonates 14 Claus Wasternack & Otto Miersch Papaver-Gene Expression Analysis 16 Jörg Ziegler Publications and Other Scientific Activities 2003 18 Publications and Other Scientific Activities 2004 19

Department of Bioorganic Chemistry 20 Head: Ludger Wessjohann, Professor Research Groups: Plant and Fungal Metabolites/Microanalytics 22 Norbert Arnold & Jürgen Schmidt Structural Analysis & Computational Chemistry 24 Wolfgang Brandt & Andrea Porzel Research Group: Isoprenoids 26 Ludger Wessjohann Research Group: Synthesis and Method Development 28 Ludger Wessjohann & Bernhard Westermann Publications and Other Scientific Activities 2003 32 Publications and Other Scientific Activities 2004 34

Department of Stress and Developmental Biology 36 Head: Dierk Scheel, Professor Research Groups: Molecular Communication in Plant-Pathogen Interactions 38 Wolfgang Knogge Cellular Signaling 40 Dierk Scheel & Justin Lee Induced Pathogen Defense 42 Dierk Scheel & Sabine Rosahl Metal Homeostasis 44 Stephan Clemens Metabolite Profiling in Arabidopsis and Crop Plants 46 Stephan Clemens, Jürgen Schmidt, Ludger Wessjohann, Dierk Scheel Publications and Other Scientific Activities 2003 48 Publications and Other Scientific Activities 2004 49 3

Department of Secondary Metabolism 50 Head: Dieter Strack, Professor Research Groups: Molecular Physiology of Mycorrhiza 52 Michael H. Walter Cell Biology of Mycorrhiza 54 Bettina Hause Biochemistry of Mycorrhiza 56 Willibald Schliemann Glycosyl- and Methyltransferases 58 Thomas Vogt Hydroxycinnamic Acids 60 Dieter Strack Publications and Other Scientific Activities 2003 62 Publications and Other Scientific Activities 2004 63

Administration and Technical Services 64 Head: Lothar Franzen Administration Employees 66 Employee Statistics 67 Involvement of the IPB in National and International Scientific Networks 68 Resources and Investments 69 Funds from External Sources 70 Seminars, Workshops and Colloquia 2003 74 Seminars, Workshops and Colloquia 2004 76 Guest Researchers and Fellows 78

Press and Public Relations 80 Head: Sylvia Pieplow Publications and Press Releases 2003 84 Publications and Press Releases 2004 85 Map and Impressum 87 4

Message from the Managing Director

Board of Directors Dierk Scheel, Professor Managing Director until November 2004 Head of the Department of Stress and Developmental Biology Toni M. Kutchan, Professor Managing Director since November 2004 Head of the Department of Natural Product he Leibniz Institute of Plant Biochemistry (IPB) is a research center that is Biotechnology rich in contemporary German history. Founded as the Institute for Bio che - Lothar Franzen T Head of Administration and Technical mistry of Plants in 1958 by Kurt Mothes, this former research institute of the East Services German Academy of Sciences was unique in emphasizing a multidisciplinary ap - Dieter Strack, Professor proach to studying plant natural products and hormone-regulated processes. Head of the Department of Secondary Scientists from around the globe were regularly invited to visit and the institute Metabolism enjoyed international recognition. The IPB, as we know the institute today, was Ludger Wessjohann, Professor Head of the Department of Bioorganic founded in 1992 and has developed into a thriving center of modern plant re - Chemistry search. The multidisciplinary style of research introduced by Kurt Mothes conti- nues and has been expanded upon. Within four research departments, more than Board of Trustees 170 scientific staff, doctoral and diploma students and guests from the world-over Ministerialrat Thomas Reitmann contribute to the exciting bustle of state-of-the-art research. Chairman of the Board of Trustees, Ministry of Education and Cultural Affairs of the State of Saxony Anhalt The fundamental research programs at the IPB center on, in a broad sense, natu- Ministerialrat ral product research and molecular interactions. From natural product isolation Jürgen Roemer - Mähler, Dr. and structure determination through to investigations of biosynthesis and regu- Vice Chairman of the Board of Trustees, Federal Ministry of Education and Research lation, new biologically active molecules are sought and production sources optimized through chemical synthesis and genetic engineering. Cellular func tion as Wilhelm Boland, Professor Max Planck Institute for Chemical Ecology, regulated by molecular interactions are investigated at the level of receptor- Jena, ligand, enzyme-substrate and protein-protein interactions using computer mode- Chairman of Scientific Advisory Board ling and a variety of biochemical and genetic techniques. The years 2003-2004 Alfons Gierl, Professor Technical University of Munich, were very productive for us and intriguing new research developments within the Vice Chairman of Scientific Advisory Board departments at the IPB in these areas are highlighted with in this scientific report. Reinhard Neubert, Professor Vice Rector for Research and Postgraduate Scientists at the institute are participating in an increasing number of local, natio- Students of the University of Halle nal and international research networks. A new Priority Program of the German Jörg Stetter, Professor Bayer AG, Leverkusen Research Foundation (DFG, SPP 1152, Evolution of Metabolic Diversity) was ini - tia ted and is coordinated by scientists at the IPB. In 2004, members of the insti - Scientific Advisory Board tute contributed to the successful application to the German Research Foun - Wilhelm Boland, Professor dation for a new Collaborative Research Center (SFB 648, Molecular mechanisms Chairman of Scientific Advisory Board, of in for mation processing in plants) at the Martin Luther University of Halle-Wit - Max Planck Institute for Chemical Ecology, Jena ten berg. Alfons Gierl, Professor Vice Chairman of Scientific Advisory Board, We have further improved upon our research facilities during the past two years; Technical University of Munich, the institute dedicated a new 500 square meter shared-use building in Spring Department of Genetics 2004. The construction provides space for a mass spectrometry center, nucleo - Raoul J. Bino, Professor University of Wageningen, The Netherlands tide sequence analysis, fermentation and synthetic organic chemistry laborato- Laboratory of Plant Physiology ries. Our greenhouses are also being expanded; we began in November 2004 Thomas Boller, Professor with the construction of an additional 346 square meters of floor space that will University of Basel, Institute of Botany meet the growing demand of the IPB's research programs with transgenic plants. Horst Kunz, Professor These new plant growth facilities will be ready for occupancy in Spring 2005. University of Mainz, Institute of Organic Chemistry We hope that in this report, we are able to share our excitement for plant bio- Birger Lindberg MØ ller, Professoer chemistry and its manifold applications with you. We are proud of how our insti- University of Copenhagen, tute has developed over the years and if you are planning on being in the Halle Department of Plant Biology (Saale) area, please do stop by to learn more about us. Günter Strittmatter, Dr. KWS SAAT AG, Einbeck Lutz F. Tietze, Professor University of Göttingen, Toni M. Kutchan Institute of Organic Chemistry Lothar Willmitzer, Professor Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm Ulrich Wobus, Professor Institute of Plant Genetics and Crop Plant Research, Gatersleben 5

Departmental Organization

Board of Trustees Ministerialrat T. Reitmann Chairman Ministerialrat J. Roemer-Mähler, Dr. Vice Chairman Scientific Advisory Board W. Boland, Professor Chairman A. Gierl, Professor Board of Directors Vice Chairman Public Relations D. Scheel, Professor Managing Director until 31.10.2004 S. Pieplow T. M. Kutchan, Professor Personal Assistent to the Managing Director Managing Director since 01.11.2004 L. Franzen Administratitive Director D. Strack, Professor L. Wessjohann, Professor

Department of Department of Department of Department of Administration and Technical Natural Product Bioorganic Chemistry Stress and Developmental Secondary Metabolism Services Biotechnology Head: L. Wessjohann, Biology Head: D. Strack, Professor Head: L. Franzen Head: T. M. Kutchan, Professor Head: D. Scheel, Professor

Plant and Fungal Meta - Molecular Commu ni- Molecular Accounting Alkaloid Biosynthesis bolites /Microanalytics cation in Plant-Pathogen Physiology of Mycorrhiza Human Resources Toni M. Kutchan Norbert Arnold & Interactions Michael H. Walter General Administration Jürgen Schmidt Wolfgang Knogge

Structural Analysis & Opium Poppy Cell Biology Library Services Computational Chemistry Cellular Signaling Biotechnology of Mycorrhiza Stockroom Wolfgang Brandt & Dierk Scheel & Justin Lee Susanne Frick Bettina Hause Graphic Arts Andrea Porzel

Mode of Induced Biochemistry Facilities Management Action of Jasmonates Isoprenoids Pathogen Defense of Mycorrhiza Computer Support Claus Wasternack & Ludger Wessjohann Dierk Scheel & Sabine Willibald Schliemann Horticulture Otto Miersch Rosahl

Synthesis and Papaver Gene Expression Glycosyltransferases & Method Development Metal Homeostasis Analysis Methyltransferases Ludger Wessjohann & Stephan Clemens Jörg Ziegler Thomas Vogt Bernhard Westermann

Bioinformatics & Hydroxycinnamic Acids Mass Spectrometry Dieter Strack Dierk Scheel

Metabolite Profiling in Arabidopsis and Crop Plants Part of the German Plant Genome Project Stephan Clemens, Jürgen Schmidt, Ludger Wessjohann & Dierk Scheel 6

Research Mission Statement

our thematically, methodologically and organisationally overlapping re search pri- Forities form the basis of the research mission statement of the Institute of Plant Biochemistry - plant natural products, molecular in teractions, information technology and metabolic engineering.

The large manifold of plant species is reflected in the enormous diversity of their natural products. This content of natural compounds is made more complex by the change in metabolite patterns during development as well as when a plant is responding to its environment. Knowledge of the structure and function of natural products is requisite to understanding plant diversity, developmental and adaptation processes. New resources can then become available for innovative application in plant production, plant protection, biotechnology and in the development of biologically active compounds. Furthermore, the realization of genome sequencing and the growing availability of expressed sequence tags of various species is of fundamental importance to functional genome analysis.

The comprehensive analysis of plant and fungal natural products is a priority in the research mission of the Institute of Plant Biochemistry. Struc ture analysis, synthesis and derivatization of natural products contribute to an understanding of their function and to an increase in their structural di versity. This also forms the basis for investigation of their biosynthesis and for discovering new biologically active compounds. A qualitative and quantitative analysis of natural products in biological materials requires the de velopment of suitable analytical methods. Subsequent identification and isolation of biosynthetic enzymes can provide access to the encoding genes, which in turn enables study of the regulation of the biosynthesis. The use of mutants and transgenic plants ultimately makes possible the analysis of biological function as well as the generation of plants with al tered natural product profiles.

Molecular interactions form the basis of cellular function. An interdisciplinary analysis of these interactions is therefore of central importance to the research mission of the Institute of Plant Biochemistry. The optimal adaptation of plants to their habitat depends upon receptor-mediated perception of biotic and abiotic environmental parameters. External signals are evaluated, compared and converted into physiological responses via al tered gene expression patterns that are controlled by cellular and systemic signal transduction networks. The molecular basis of these processes, re - ceptor-ligand, enzyme-substrate and protein-protein interactions, have ap plication in the development of new biologically active agents. From this perspective, the mechanisms of communication between plants and their symbionts and pathogens are investigated as are biosynthetic and signal transduction pathways. Chemical structures of these interacting components are also modified using gene technological methods, directed evolution and chemical derivatization.The effects of these changes can be mo nitored in model systems or with activity screens until a molecule with the desired characteristics (e.g. a drug, signal compound or an enzyme) is achieved. The development of new syntheses, screening tests, assays and analytical methods is supported by visualization of molecular interactions via computer modeling. A nexus of natural product research and the study of molecular interactions is the 7

storage and evaluation of the large amount of data that is ge nerated. In particular, high throughput processes used in metabolome and proteome analysis and in the production of combinatorial libraries make necessary the development of new methods in information technology. To this end, a new junior group in information technology is being established at the Institute of Plant Biochemistry.

Metabolic engineering is an overlapping priority in three areas of basic research - natural products, molecular interactions and information technology. Model plants are generated that have potential for various types of application. More specifically, designer plants with tailored natural product profiles, containing new health-promo- ting metabolites or showing im proved adaptation to habitat are being developed. Plants with these cha racteristics could serve for the sustainable production of valuable chemicals, as biological test systems or could be of importance to plant bree - ders.

Within four departments with distinct, but complementary research directions and state-of-the-art equipment, the Institute of Plant Biochemistry provides optimal conditions with which to execute multidisciplinary re search in the areas of chemistry, physiology, cell biology, biochemistry, molecular biology and genetics. The analysis of topics central to modern plant biology and chemistry using this wide array of methodologies enables a meaningful interpretation of the complex interactions in plant development and diversity that would otherwise not be possible. The ultimate transfer of these results to practical applications could make ecologically compatible uses of plant biotechnology a reality. 8

Department of Natural Product Biotechnology Head: Toni M. Kutchan, Professor Secretary: Christine Dietel

Department of Natural Product Biotechnology 9

or much of human history, plant extracts have been used as ingredients Fin potions and poisons. In the eastern Mediterranean, use of opium poppy latex (Papaver somniferum) can be traced back at least to 1400 to 1200 B.C. Ancient folks used medicinal plant ex tracts as purgatives, anti- tussives, sedatives and treatments for a wide range of ailments from sna - kebites to fever. Use of medicinal plants then spread westward across Eu - rope. Over the centuries, one of the most important medicinals was opium. Analysis of the individual components of opium led to the identification of morphine. The isolation of morphine in 1806 by the German pharmacist Friedrich Sertürner gave rise to the study of alkaloids. The term "alkaloid" was, in fact, coined by another pharmacist, Carl Meissner, in 1819 in Halle, Germany. Alkaloids were originally defined as pharmacolog- ically active, nitrogen-containing basic compounds of plant origin. Al kaloid- containing plants were mankind's original materia medica. Ma ny alkaloids are still in use today as prescription drugs; one of the best known and widely used is the antitussive codeine from the opium poppy. After 198 years of alkaloid research, this class of natural products still finds significance in the medical field in the treatment of diseases ranging from cancer potentially through to malaria.

The Department of Natural Product Biotechnology emphasizes research on the elucidation of formation of plant natural products at the molecular genetic level. Our research centers on understanding the formation of se - lected natural products at several levels and then exploiting this knowledge using ge netic engineering. We are just beginning to metabolically engineer alkaloid metabolism in plants and in in vitro culture. Multicellular compart- mentation of alkaloid pathways and transport of intermediates must be considered if meaningful metabolic engineering experiments are to be designed. We also need to analyze and use promoters that drive transgene expression in the correct cell types to effect biosynthesis. Re gulation of these pathways at the gene and enzyme level is complex and there is still also much to be learned about metabolite levels and pathway interconnec- tion as we systematically over-express and suppress gene transcription. Today, pathway engineering in plants re mains highly variable.When we per- turb cellular physiology, metabolite homeostasis and intra- and intercellular partitioning can be effec ted in unpredictable ways. Another aspect that needs attention is the development of efficient transformation and rege neration protocols for alkaloid- producing plant outside of the So la - naceae. All told, there is still much to be done in this field of research. 10

Research Group: Alkaloid Biosynthesis Head: Toni M. Kutchan

The opium poppy Papaver somniferum is one of mankind's oldest medicinal plants. Opium poppy today is the commercial source of the narcotic analgesics morphine and codeine. Along with these two morphinans, opium poppy produces approximately 80 alkaloids belonging to various tetrahydrobenzylisoquinoline-derived classes. It has been known for over a century that morphinan alkaloids accumulate in the latex of opium poppy.With identification of many of the enzymes of alkaloid biosynthesis in this plant, biochemical data suggested involvement of multiple cell types in alkaloid biosynthesis in poppy. It has also been suggested, based upon the failure to produce morphine in undifferentiated P. somniferum cell cultures, that morphine accumulation is related to cytodifferentiation - noting that laticifers are absent from these cultures, but present in tissue cultures. We now have addressed the question of cell-specific localization of the enzymes of alkaloid biosynthesis and the site of gene transcript accumulation. We have carried through the immunolocalization of five enzymes of alkaloid formation in opium poppy: (R,S)-3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase central to the biosynthesis of tetrahydroisoquinoline-derived alkaloids, the berberine bridge enzyme of the sanguinarine pathway, (R,S)-reticuline 7-O-methyltransferase specific to laudanosine formation, and salutaridinol 7-O-acetyltransferase and codeinone reductase that lead to morphine.

Group Members The opium poppy Papaver somnife- Nor coclaurine is O-methylated by (R,S)-norco- rum is the source of the narcotic clau-rine 6-O-methyltransferase. (S)-Coclaurine Kum-Boo Choi analgesics codeine and morphine, is next N-methylated by (R,S)-coclaurine N- Humboldt-Fellow until September 2003 which accumulate in specialized in - methyltransferase and the cDNA encoding this Maria Luisa Diaz-Chavez DAAD PhD Student since October 2003 ternal secretory cells called latici- enzyme has been characterized. (S)-N- Nils Günnewich fers. In the aerial parts of the plant, Methylcoclaurine is then hydroxylated by the PhD Student since December 2003 the laticifer cells are anastomosed, cytochrome P-450 de pendent monooxygenase Aphacha Jindaprasert forming an articulated network. La - CYP80B [(S)-N-methylcoclaurine 3'-hydroxlya- DAAD PhD Student since December 1 2004 ticifers are found associated with se].The cDNA en coding the corresponding cyto- Robert Kramell the vascular bundle in all plant parts. chrome P-450 re ductase has been isolated as Postdoctoral Position The morphinan alkaloids morphine, well. (S)-3'-Hydroxy-N-methylcoclaurine is Monika Krohn Technician until October 2004 codeine and thebaine are found methylated to (S)-reticu-line by (R,S)-3'-hydroxy- Kristin Krukenberg both in roots and in aerial plant N-methylcoclaurine 4'-O-methyltransferase Fulbright-Fellow until July 2003 parts and specifically accumulate in (4'OMT).The cDNA 4'omt has been isolated and Tobias Kurz vesicles within laticifers. The ben zo - PhD Student [c]phenanthridine alkaloid sanguina- Alfonso Lara DAAD PhD Student since April 2003 rine is found in root tissue.The syn- Natsajee Nualkaev theses of sanguinarine and of the DAAD PhD Student until December tetrahydrobenzylisoquinoline alka- 2004 loid laudanine are completely un - Christin Richter PhD Student since August 2004 derstood at the enzyme level. Near - Khaled Sabarna ly all enzymes of morphine biosyn- PhD Student thesis have also been described. In Karin Springob more recent years, cDNAs enco- Postdoctoral Position since July 2003 ding ten enzymes of alkaloid biosyn- Marion Weid PhD Student until March 2004 thesis in P. somniferum have been isolated and characterized. The Immunolabeling of of SalAT (salutaridinol 7-O-acetyltransfe- pathway starting from the first tetrahydrobenzy- rase) of morphine biosynthesis in a cross-section of capsule of P. somniferum. The panels show a cross-section lisoquinoline alkaloidal intermediate (S)-norco- through a small vascular bundle of the capsule wall. The clau-rine to the central isoquinoline alkaloid bio- green arrows indicate the position of anti-biosynthetic enzyme antibody; the red arrows indicate the po sition of synthetic intermediate (S)-reticuline is under- anti-MLP 15 antibody and is indicative of laticifer cells. stood at both the enzyme and gene level. (S)- 11

characterized from P. somniferum. (S)-Reticuline curring before and after a central Collaborators is central intermediate of isoquinoline alkaloid branch point is analyzed. In additi- Gerhard Bringmann biosynthesis, which leads to a plethora of alkaloi- on, in situ localization of 7omt University of Würzburg, Germany dal structures. In P. somniferum, (R,S)-reticuline and cor was performed to cor- Wanchai De-Eknamkul 1 Chulalongkorn University, Bangkok, can be methylated by (R,S)-reticuline 7-O- relate the site of gene transcripti- Thailand methyltransferase, for which the cDNA 7omt has on to enzyme ac cumulation. In Tony Fist been described, to the tetrahydrobenzylisoquino- capsule and stem, both O-methyl- Tasmanian Alkaloids, Tasmania, Australia line laudanine. The N-methyl group of (S)-reticu- transferases and the O-acetyl- Phil Larkins Scientific and Industrial Research line can alternatively be oxidatively cyclized by transferase are found predomi- Organisation Plant Industry, Canberra, the berberine bridge enzyme (BBE) to C-8 of (S)- nantly in parenchyma cells within Australia Friedrich Lottspeich scoulerine. (S)-Scoulerine is then fur-ther conver- the vascular bundle and co - Max Planck Institute for Biochemistry, ted in these plants to the antimicrobial deinone reductase is localized to Martinsried, Germany benzo[c]phenanthridine alkaloids sanguina-rine. laticifers, the site of morphinan al - Markus Piotrowski University of Bochum, Germany Along the pathway on which (S)-reticuline is spe- kaloid accumulation. In developing Joachim Stöckigt cifically converted to morphine, cDNAs encoding root tip, both O-me thyltrans fera- University of Mainz, Germany two biosynthetic enzymes have been identified. ses and the O-acetyltransferase Salutaridinol 7-O-acetyltransferase, encoded by are found in the pericycle of the stele and the ber- salat, transfers an acetyl moiety from acetyl-CoA berine bridge enzyme is localized to parenchyma to the 7-hydroxyl group of salutaridinol. cells of the root cortex. Laticifers are not found Codeinone reductase is encoded by cor and in developing root tip and, likewise, codeinone catalyzes the penultimate step in morphine bio- reductase was not detected. These results provi- synthesis, the NADPH-dependent reduction of de cell-specific localization that gives a co herent the keto moiety of codeinone to the 6-hydroxyl picture of the spa-tial distribution of alkaloid bio- group of codeine. synthesis in opium poppy.

Fluorescence immunocytological localization was In summary, two cell ty - carried out with the 4’OMT (reticuline- ), the pes, parenchyma within the 7OMT (laudanine- ), BBE (sanguinarine- ), SalAT vascular bundle and la tici - and COR (morphine-pathways) with P. somnife- fers, are sites of the bio- rum capsule, stem and root tissue sections. In this synthesis of isoquinoline manner, the spatial organization of enzymes oc - al kaloids in P. somniferum. This implies spatial regulation of the biosynthesis of the different classes of P. somniferum alkaloids in that the early stages of Dissected capsule of P. somniferum, white arrow indi- mor-phine biosynthesis cates the position of the large vascular bundle; the small occur in parenchyma cells vascular bundle is located closer to the capsule wall. surrounding laticifers and then at late stages, possibly at the level of either salutaridinol-7-O-acetate or thebaine, moves into the laticifer, which is the storage site of the morphinans thebaine, codeine and morphine. The role of intercellular transporters of alkaloidal intermediates as well as intracellular transport into vesicles within laticifers adds an additional potential level of regulation to morphine biosynthesis Schematic drawing of a longitudinal section through the that still needs to be investigated. vascular bundle of P. somniferum stem. xy, xylem; cc, companion cell; la, laticifer; ph, phloem; sp, sieve plate. 12

Research Group: Opium Poppy Biotechnology Head: Susanne Frick

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

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

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

During the last years several genes chemical-ecological func tion of morphinan and Group Members from the biosynthetic pathways for benzophenanthridine alkaloids in plants. After the Kathleen Gutezeit reticuline, sanguinarine and morphi- regeneration of plants, the qualitative and quanti- Technician ne have been cloned (Fig. 1).- Al tative determination of alkaloids is analyzed by Stefanie Haase PhD Student though the biosynthesis is well un - HPLC and LC-MS in latex, leaves and roots of Elke Hillert derstood at the enzyme level, the opium poppy. Finally the heredity of the alkaloid Technician molecular and biochemical mecha- concentration/pattern is confirmed.With a trans- Katja Kempe nisms that regulate these pathways genic cell line expressing the antisense construct PhD Student are not known. A major goal of this of berberine bridge enzyme (BBE) we hoped to Heike Riegler Diploma Student since October 2004 project was the development of a reduce the metabolic flux through sanguinarine stable transformation and regenera- pathway and to enhance the concentration of Collaborators tion method for opium poppy, which morphine instead. We are interested whether a Tony Fist, would make the metabolic enginee- transgenic cell line over expressing codeinone re - Tasmanian Alkaloids, Westbury, Australia ring of the above-mentioned com- ductase (COR) leads to a poppy plant that con- Philip Larkin, Scientific and Industrial Research pounds possible. Poppy seed oil finds tains more morphine or where the concentrati- Organisation Plant Industry, Canberra, use in chemical industry for the pro - on of morphine is lowered due to a possible Australia duc tion of pigments and lacquer, but Jürgen Schmidt, Institute of Plant Biochemistry, Halle, its residual morphine levels prevents Germany more widespread applications. Be - cause opi um is the raw ma terial for the illicit production of heroin, cultivation of poppy is re stric - ted. By completely sup pressing morphine biosynthesis, opium pop py could be come a "harmless" crop plant. So far there has been no success with breeding programs and mu tations to obtain an alkaloid-free poppy. In the best case a re duction of alkaloid biosynthesis has been achieved. The transforma tion of opium poppy could be an alternative to circumvent these problems.

We have used an Agrobac te rium-mediated ap - proach to introduce different cDNAs encoding enzymes of reticuline, morphine and sanguinarine biosynthesis as sense-, antisense- or RNAi-con-

structs into explants to attempt to alter the alka- Figure1: Biosynthetic pathway from L-tyrosine to sanguinarine, lau- loid profile (Fig. 2, A-E ). Alka loid-free plants de - danosine and morphine in P. sonmniferum. Enzymes are highlighted veloped in this manner will be used to test the in red. 13

feedback inhibition of this pathway. We have The overexpression and antisense expressi- also produced poppy transformants, in which on of cyp80b1 cDNA produced poppy we potentially influenced all cytochrome plants with altered alkaloid concentrations. P450 enzymes of the benzylisoquinoline The in crease or decrease of alkaloid concen- pathways by introducing a NADPH:cyto- tration in the transgenic cell lines was herita- chrome P450-oxidoreductase (CPR). Finally ble at least to the T3 generation. Compared B we are trying to reduce or silence the com- to cyp80b1 the overexpression of cpr cDNA plete alkaloid biosynthetic pathway with a did not give rise to an increased alkaloid transgenic cell line containing the antisense concentration in transgenic opium poppy. construct of (S)-N-methylcoclaurine hydro- xylase (CYP80B1). With a cell line overex- Because we have not been able to silence pressing cyp80b1 we are trying to stimulate benzylisoquinoline biosynthesis in transgenic all three pathways to gether. poppy plants containing S4S4:antiBBE or S4S4:antiCYP80B1, we constructed plasmids With the antisense expression of bbe cDNA containing partial sequences that are poten - C we have been able to produce transgenic tially able to trigger RNA interference in Pa - opium poppy plants with altered alkaloid profi- pa ver somniferum. Explants of opium poppy les. The transformation of these plants was eva- were transformed with different constructs. luated by PCR, northern and Sou thern hybri- Meanwhile we have been able to produce se - disation. The transgenic plants contained one veral T0 plants. The analysis of these plants is additional copy of the bbe gene. We observed in progress. an in creased concentration of several pathway in termediates from all biosynthetic branches To study the cell- and tissue specific expres- e.g. reticuline, laudanine, laudanosine, dehydro- sion of benzylisoquinoline biosynthetic ge nes, reticuline, salutaridine and (S)-scoulerine. The we started to isolate their endogenous pro- D transformation altered the ratio of morphinan moters. With promoter/reporter gene con- and tetrahydrobenzylisoquinoline alkaloids in structs we want to analyze how biosynthesis latex but not the benzophenanthridine alka- and accumulation of alkaloids are regulated. loids in roots. The altered alkaloid profile was heritable at least to the T2 generation.

Figure 2: Somatic re generation of P. somniferum. Ex plants first give rise to a type I callus (A), which starts to differentiate after a certain time (B). After the transfer to a hormone free medium this type II callus deve- lops small em bryos (C) and fi nal - ly little plant lets (D). The whole regeneration process is shown in picture E. 14

Research Group: Mode of Action of Jasmonates Heads: Claus Wasternack & Otto Miersch

Group Members Jasmonates (JA) and their precursors, the octadecanoids, are signals in plant stress responses and in plant development. A mechanistic analysis of the mode of action of Carolin Delker PhD Student since June 2002 jasmonates is performed by a reverse genetic approach using the allene oxide cycla- Conrad Dorer se (AOC)-catalyzed step in jasmonate biosynthesis. "Gain of function" and "Loss of Diploma Student until February 2004 function" studies with transgenic tomato plants revealed modulation of jasmonates Yvonne Freyer and allowed to inspect role of jasmonates in response to biotic and abiotic stresses Diploma Student until October 2004 as well as flower and seed development. Functional analysis of AOC using genetic Lydia Müller Diploma Student until July 2003 approaches is also performed in Arabidopsis to ask on specific versus redundant Jana Neumerkel functions of the four AOCs in this plant. These data are linked to metabolite profiles Diploma Student until November 2003, on jasmonates and octadecanoids under various stress conditions and in different afterwards PhD Student developmental stages. In addition to this analytical work, chemical synthesis of stan- Birgit Ortel Technician dards and labeled substrates is an essential part of this work Katrin Rehagel Diploma Student since October 2004 Irene Stenzel Postdoctoral Position The role of JA biosynthesis is bio synthetic enzymes in clu ding AOC in sieve e - Carola Uhlig Technician analyzed in tomato and Arabi - lements of to ma to leaf veins (Fig. 1). dopsis. Preferentially, the AOC- Sabine Vorkefeld Technician until May 2004 catalyzed step and the profile of Using transgenic tomato plants over-expressing or jasmonates and other oxylipins re pres sing con sti tutively the AOC or expressing a Collaborators are studied to elucidate specific reporter gene dri ven by the AOC promoter (coop. T. functions in plant stress respon- Roitsch, Wue r z burg, Germany), role of AOC and Guillermina Abdala Universidad Nacional de Rio Cuarto, Argentina ses and development. generation and func tion of jasmonates could be in - Wilhelm Boland spected.Tissue specific expression of AOC in seed- Max Planck Institute of Chemical Ecology, Jena, The first AOC was cloned from ling development, preferentially in roots (Fig. 3) and Germany tomato (Ziegler et al. 2000, pa - during flower and embryo development points to Udo Conrad Institute of Plant Genetics and Crop Plant tent No. DE 1000 4468.9). This new functions of JA and its metabolites. This AOC Research, Gatersleben, Germany single copy gene is specifically ex - gene activity and JA function differs from that of Ivo Feussner University of Goettingen, Germany pressed in ovules of young flo- Arabidopsis suggesting the plasticity of different wers and all vascular bundles. In plant species to use signaling compounds. These dif- Bettina Hause, Sabine Rosahl, Dierk Scheel, Jürgen Schmidt leaves, the vascular bundle-speci- ferences together with data from the literature Institute of Plant Biochemistry, Halle, Germany fic occurrence of AOC attributes point to role of JA in female fertility of tomato, whe- Gerd Hause to a preferential ge neration of reas in Arabidopsis male fertility is strictly JA depen- University of Halle, Germany jas monates in main veins, which dent. Gregg Howe University of Michigan, East Lansing, USA led together with data from va - Harry Klee rious transgenic tomato plants to Interestingly, the vascular bundle- and ovule-specific University of Florida, Gainesville, USA a proposed amp lification model occurrence of AOC protein in flowers is accompa- Thomas Roitsch in wound signaling with preferen- nied with a specific profile of octadecanoids and jas- University of Würzburg, Germany tial signaling properties of JA.This monates as well as JA amino acid conjugates being Luc Varin Concordia University, Montreal, Canada is supported by de tection of JA different in the various flower organs. Constitutive over-expression of AOC led to qualitative and quantitative changes in these profiles (Fig. 2) as well as that of the free and esterified fatty acids and their peroxidation products (coop. I. Feussner, University of Goettingen). The data suggest different regulation of JA biosynthesis in flowers and leaves and indicate that the different branches of the lipoxygenase pathway can be altered depending of the terminal pro-

Figure1: AOC protein occurs in companion cells (CC) and sieve elements (SE), but AOC mRNA is exclusively ducts. located in companion cells. AOC protein is detected by immunolabeling indicated by green fluorescence (left). In situ hybridisation to detect AOC mRNA was performed by probing with an AOCantisense probe (middle), note that the triangle-shaped sieve elements are free of label. As a control an AOCsense probe was used indicating no cell-specific labelling (right) (taken from Hause et al., Plant Cell Physiol. 44, 643-648, 2003). 15

3-d-old seedling 10-d-old seedling

Data on JA and 12-oxo-phytodienoic acid (OPDA) regulation by spatial level in tomato mutants and in our 35S::AOC anti - and temporal sense tomato plants following infection with Xan - differen ces in the tho monas campestris cv. vesicatori supported a expression of the anti AOC

model of sequential action of jasmonate, ethylene four AOCs. In con- Figure 3: AOC promoter activity in tomato seedling development. Seedlings and salicylate in this plant pathogen interaction trast, the four of a trans genic AOC(1000 bp)::uidA line expressing the b-glucorunidase, whose activity is indicated by blue staining, were inspected after 3 and 10 (coop. H. Klee, Gainesville, USA). Also in collabora- recombinant AOCs days, respectively, after sawing (das). Note the AOC promoter activity in the root tip and hypocotyl 3 das (left) and the root elongation zone as well as tive work (M. Köck, Biocenter, University of Halle, exhibited similar en - the vascular bundles of cotyledons 10 das (right). AOC promoter activity Germany), data were found which support a new JA zymatic properties corresponds to location of AOC protein probed with an anti-AOC antibody and visualized by violet staining (left/right) (photos by I. Stenzel and B. signaling pathway. In tomato the wound-responsive and substrate speci- Hause). RNaseLE gene is expressed only locally and in a JA- ficities. and systemin-independent manner. Previously, 12-hydroxy-JA was only known as tuber- In Arabidopsis the AOC is encoded by four genes. inducing compound in So la naceae species. We could Inspection of transgenic Arabidopsis plants expres- identify 12-hydroxy-JA and its sulfated derivative in sing a reporter gene driven by each AOC promoter, A. thaliana and tomato. From Arabi dop sis a sulfo- revealed non-redundant functions of the AOCs. Pre - transferase (AtST2a) was cloned which converts ferentially, in root growth and seedling development specifically 12-hydroxy-JA (coop. L. Varin, Montreal, as well as in flower development (Fig. 4) there are Canada). In transgenic plants over-expressing or re - tissue and organ specific promoter activities, non- pressing constitutively AtST2a a shift in flowering redundantly for AOC1, AOC2, AOC3 and AOC4. time depending on the 12-OH-JA level was detec- These data and analyses of knockout-lines indicate ted. In tomato 12-OH-JA is a permanent constitu- that Ara bi dopsis is able to perform a fine-tuning of ent of flower organs. In to mato leaves 12-OH-JA is formed upon wounding in a JA-dependent manner. The role of 12-OH-JA in a photoperiod-dependent plant and a day-neutral plant is in the focus of future work.

National and international cooperation is done in the above-mentioned aspects of tomato and Ara bidopsis. The know-how of the group on JA and oxylipin analy- tics is used and requested by many groups. A minor ac tivity is linked to collaboration with the Probiodrug AG in Halle.The company is working on role of gluta- minylcyclases (QCs) of human, animal and plant sour - ces. QCs attribute in mammalian sys - tems to a functionalization of peptide hormones and proteins by pyrogluta- mate formation at the N-ter minus. Our effort is to clone plant ho mo lo gues of the QC as a tool for de sign of potent QC

Figure 2: Constitutive over-expression of AOC in tomato leads to elevat- inhibitors. ed levels of jasmonates and octadecanoids in flowers but not in leaves. The Figure 4: Promoter activity of the AOC4 gene of Arabidop sis thaliana. 14-d-old ratio among the compounds being specific for each flower organ is diffe- seed lings (above) and flowers (below) of transgenic lines carrying a AOC4::uidA con- rentially altered by over-expression of AOC. The diameter of each circle struct were inspected on b-glucuronidase activity visualized by the blue staining. is indicative for the total amount of all compounds, the sector within a cir- Note AOC4 promoter activity in root tips, side root primordia and vascular bund- cle is indicative for a distinct compound given in % of the total amount les of the seedlings, as well as in sepals, petals, filaments and stigmas of open flo- (taken from Miersch et al., Phytochem. 65, 847-856, 2004). wers (photos by I. Stenzel). 16

Research Group: Papaver-Gene Expression Analysis Head: Jörg Ziegler

Poppies of the genus Papaver produce a large variety of benzylisoquinoline alkaloids. Some of them are of pharmaceutical importance such as the analgesic morphine, the antitussive noscapine or the vasodilator papaverine. The biosynthesis to (S)-reticuline, the central intermediate to all monomeric benzylisoquinoline alkaloids is well understood on the molecular level, the knowledge of the later steps, which lead to the diversity of this class of compounds, is still incomplete. Similarly, the regulatory steps leading to the accumulation of these substances, is unknown. To approach cDNA clones coding for these processes we make use of the close genetic relationship but the diversity in the alkaloid profile between Papaver species or varieties. We examine and correlate the gene expression profiles on EST-arrays with specific alkaloid profiles. By the combination of many different datasets of alkaloid profile-gene expression correlations, we could re-duce the number of candidate cDNAs with possible functions in benzylisoquinoline accumulation to eleven.

Group Members Currently, more than 70 different all other alkaloids are available now and their in - poppy species belonging to the terpretation with respect to their identification is Andreas Gesell genus Papaver have been described. in progress. PhD Student Roughly, they are able to synthesise Susan Voigtländer Diploma Student since November about 2,500 different benzylisoqui- As probes for the arrays we use PCR fragments 2004 nolines, which can be grouped into derived from two EST-projects of Papaver somni- Silvia Wegener nine classes. The profile of benzyl - ferum stems and seedlings. Among all Papaver Technician isoquinolines produced by the plants species, this plant synthesizes the largest number are species specific; it is, however also dependent of different benzylisoquinolines. Up to now we on growth conditions. The same holds true for sequenced about 3,700 ESTs and obtained 2,000 gene expression. This variability requires sensi- UniGenes. About 50 percent either code for pro- tive methods to record all needed parameters in teins with unknown function or have no homolo- one individual plant. HPLC methods were em - gy to entries in the databases. The largest groups ployed to detect the main compounds and LC- of cDNAs coding for proteins with known func- MS coupling will be used for the low abundant tion are involved in transcriptional and translatio- compounds. For gene expression analysis, a pro- nal control, in responses to stress, and in redox tocol for macroarray production was developed. control. Another highly represented group codes These methods are sensitive enough to record for proteins participating in metabolism, mainly the alkaloid profile and the gene expression pat- primary metabolism. To 20 sequences, a possible tern from one individual plant. For ten Papaver role in secondary metabolism could be ascribed. species and five varieties and mutants of the Six sequences code for proteins with known opium poppy Papaver somniferum, the alkaloid function in the benzylisoquinoline pathway. Up to profile was recorded by HPLC and HPLC-MS. now, the expression of these cDNAs was exa- Some of the major compounds of each plant mined in nine Papaver species and P. somniferum could be identified by MS-MS. The MS-MS data of varieties, respectively. These expression profiles

Functional classification of ESTs from P. somniferum stems and seedlings. Abbreviations: PS: photosynthesis, MLP: major latex proteins, protein syn/deg: protein synthesis and degradation, G-prot: G-proteins, AA: amino acids, alk: alkaloids, phenyl: phenylpropanoids, isopr: isoprenoids. 17

were compared with the alkaloid profile of each bone. Up to now, 33 ESTs Collaborators plant. In a first approach, we looked at cDNAs coding for these enzymes Christian Ammer showing differential expression in morphinan al - could be isolated. One of these Institute of Plant Biochemistry, Halle, Germany kaloid producing P. somniferum plants compared showed high homology to a Birgit Dräger to other Papaver species, which do not produce previously characterized P-450 University of Halle, Germany this class of benzylisoquinolines. From 2,000 ESTs enzyme, which catalyses the Sylvestre Marillonnet examined, only eleven cDNAs showed morphi- formation of a me thyl ene dioxy Icon Genetics AG, Biozentrum Halle, Germany nan specific expression. Mostly, these showed no bridge in benzylisoquinoline Jürgen Schmidt homology at all or homology to proteins with biosynthesis. The full-length Leibniz Institute of Plant Biochemistry, Halle, unknown function or to putative proteins when cDNA of this EST was isolated Germany compared to the NCBI database. One of the dif- and its substrate specificity is under investigation. ferentially expressed cDNAs showed homology In order to a chie ve high expression levels of this to an O-methyltransferase.The full-length cDNA en zyme, an overexpression sys tem in Nicotiana was isolated and the protein was overexpressed benthamiana using viral vectors is currently in bacteria. Functional characterization of the re - estab lished. combinant protein identified this cDNA as 3 - hydroxy-N-methylcoclaurine 4 -O-methyltrans- Recent localization studies indicated, that the en - ferase. The enzyme showed a strict substrate zymes of benzylisoquinoline biosynthesis are lo - specificity in that it only accepted tetrahydroben- cated in different cell types, suggesting that in ter - zyl-isoquinolines as substrates, and not, as other mediates have to be transported between cells. class II-O-methyltransferases, phenolic substan- The large group of ABC-transporters have been ces. shown to transport many secondary metabolites, among them also alkaloids. In our EST da tabase, A second approach to identify cDNAs implicated we found ten sequences coding for ABC-trans- in the biosynthesis of benzylisoquinolines and the porters. Currently, we are isolating the full-length regulation of alkaloid accumulation implies the cDNAs for three of these transporters. For func - functional characterization of ESTs showing ho - tional characterization of the transporters, com- mology to proteins, where such a role can be as - plementation of yeast strains deficient in ABC- sumed. transporter will be used. In contrast to the wild- type yeast, the mu tants are not able to transport The enzyme family of the P450-monooxygenases benzylisoquinolines. Overexpression of an ABC- are known to catalyze many reactions in the struc- transporter with specificity towards benzyliso- tural modification of the benzylisoquinoline back- quinolines in the mutants should restore wild

Expression level of selected cDNAs in different Papaver species. 18

Publications and Other Scientific Activities 2003

Publications Ounaroon, A., Decker, G., Schmidt, J., Lott - Stumpe, M., Stenzel, I, Weichert, H., Hau se, Abdala, G., Miersch, O., Kramell, R., Vi g li oc - speich, F. & Kutchan, T. M. (R,S)-Re ti culine B. & Feussner, I. The lipoxygenase path way co, A., Agostini, E., Forchetti, G. & Alemano, 7-O-methyltransferase and (R,S)-nor co- in mycorrhizal roots of Medica go trunca- S. Jasmonate and octadecanoid occurrence clau rine 6-O-methyltransferase of Papaver tula. In: Advanced Research on Plant Lipids in tomato hairy roots. Endogenous level somniferum-cDNA cloning and characte- (Murata, N., Yamada, M., Ni shi da, I., Oku ya - chan ges in response to NaCl. Plant rization of methyl transfer enzymes of ma, H., Sekija, J. & Hajime, W., eds.) Kluwer Growth Regul. 40, 21-27. alkaloid biosynthesis in opium pop py. Plant Academic Publishers, Dordrecht, pp. 287- J. 36, 808-819. 290. Bailey, N. J. C., Oven, M., Holmes, E., Ni - cholson, J. K. & Zenk, M. H. Meta bolo mic Pedranzani, H., Racagni, G., Alemano, S., Weichert, H., Maucher, H., Hornung, E., Was - analysis of the consequences of cadmium Miersch, O., Ramírez, I., Peña-Cortés, H., Ta - ternack, C. & Feussner, I. Shift in fat ty acid exposure in Silene cucubalus cell cultures leisnik, E., Machado-Domenech, E. & Abdala, and oxylipin pattern of tomato leaves follo- via 1H NMR spectroscopy and chemome- G. Salt tolerant tomato plants show increa- wing overexpression of the alle ne oxide cyc - trics. Phytoche mis try 62, 851-858. sed levels of jasmonic acid. Plant Growth lase. In: Advanced Research on Plant Lipids Re gul. 41, 149-158. (Mu rata, N., Yama da, M., Nishida, I., Okuyama, Färber, K., Schumann, B., Miersch, O. & H., Sekija, J. & Hajime, W., eds.) Kluwer Aca - Roos, W. Selective desensitization of jas- Samappito, S., Page, J.E., Schmidt, J., De-Ek - demic Pub lishers, Dordrecht, pp. 275-278. monate- and pH-dependent signalling in namkul, W. & Kutchan, T. M. Aromatic and the induction of benzophenanthridine bio- pyrone polyketides synthesized by a stilbe- Patents synthesis in cells of Eschscholzia californica. ne synthase from Rheum tataricum. Phyto - Kutchan, T. M., Ounaroon, A., Haase, S. & Phytochemistry 62, 491-500. che mistry 62, 313-323. Frick, S. O-Methyltransferases of tetrahydrobenzylisoquinoline alkaloid bio syn the - Gidda, K. S., Miersch, O., Schmidt, J., Was ter - Schilling, S., Manhart, S., Hoffmann, T., Lud - sis in Papaver somniferum. Euro pean nack, C. & Varin, L. Biochemical and molecu- wig, H.-H., Wasternack, C. & De muth, H.- Patent Application No. 03020023.2. lar characterization of a hydroxy-jasmo nate U. Sub strate specificity of glutaminyl cyc - sulfotransferase from Arabidop sis thaliana. J. lases from plants and animals. Biol. Chem. Varin, L., Miersch, O., Maucher, H. & Was - Biol. Chem. 278, 17895-17900. 384, 1583-1592. ternack, C. Methods and genetic se quences for producing male sterile plants, and plants Hause, B., Hause, G., Kutter, C., Miersch, O. & Schilling, S., Niestroj, A. J., Rahfeld, J.-U., genetically modified to alter anther deve- Wasternack, C. Enzymes of jasmonate bio- Hoffmann, T., Wermann, M., Zunkel, K., lopment. USA/Canada Patent Ap pli cation synthesis occur in tomato sieve elements. Wasternack, C. & Demuth, H.-U. Iden ti fi - No. N/R: 004807-0007. Plant Cell Physiol. 44, 643-648. ca tion of human glutaminyl cyclase as a metalloenzyme-Potent inhibition by imida- Diploma Theses Hause, B., Stenzel, I., Miersch, O. & Waster - zole derivatives and heterocyclic chela- Dorer, Conrad: Enzymatische Eigen schaf - nack, C. Occurrence of the allene oxide cyc - tors. J. Biol. Chem. 278, 49773-49779. ten der vier Allenoxidcyclasen aus Arabi - lase in different organs and tissues of Arabi - dopsis thaliana und Untersuchungen zur dopsis thaliana. Phytoche mistry 64, 971-980. Stenzel, I., Hause, B., Maucher, H., Pitzsch - AOC-Aktivität transgener Pflanzen sowie ke, A., Miersch, O., Ziegler, J., Ryan, C. A. & Arabidopsis-Mutanten, University of Halle- Monostori, T., Schulze, J., Sharma, V. K., Mau - Wasternack, C. Allene oxide cyc lase de - Wittenberg, Department of Bio chemist ry/ cher, H., Wasternack, C. & Hause, B. No vel pendence of the wound res ponse and vas- Biotechnology, 24 /3/2004. plasmid vectors for homologous transfor- cular bundle spe cific generation of jasmo- mation on barley (Hordeum vulgare L.) with nates in tomato - amplification in wound- Günnewich, Nils: Expression und Cha rak - the JIP23 cDNA in sense and antisense signalling. Plant J. 33, 577-589. terisierung von Terpensynthasen und Pre - orienta tion. Cereal Res. Comm. 31, 17-24. nyltransferasen aus Cannabis sativa L., Uni - Stenzel, I., Hause, B., Miersch, O., Kurz, T., versity of Halle-Wittenberg, Depart ment O'Donnell, P. J., Schmelz, E., Block,A., Miersch, Maucher, H., Weichert, H., Ziegler, J., Feuss - of Bioche mistry/ Biotech no logy, 23/09/2003. O., Wasternack, C., Jones, J. B. & Klee, H. J. ner, I. & Wasternack, C. Jasmonate biosyn- Multiple hormones cooperatively control a thesis and the allene oxide cyclase family Kempe, Katja: Herstellung von Vektoren zur susceptible tomato pa tho gen de fense re - of Ara bidopsis thaliana. Plant Mol. Biol. 51, "RNA interference" für Gene der Ben zyliso - sponse. Plant Physiol. 133, 1181-1189. 895-911. chinolin-Alkaloid-Biosynthese aus Pa pa ver somniferum L. Technical Uni ver sity, Berg aka - Stenzel, I., Ziehte, K., Schurath, J., Hertel, S. demie Freiberg, 18/02/2003. C., Bosse, D. & Köck, M. Differential ex - pression of PSI14, a phosphatase gene fa - Müller, Lydia: Untersuchungen zur Regu la - mily, in response to phosphate availability, tion der Promotoren von AOC1, AOC2, pathogen infection and during develop- AOC3 und AOC4 in Arabidopsis thaliana, ment. Physiol. Plant 118, 138-146. University of Halle-Wittenberg, De part - ment of Biochemistry/Biotech nology, Books and Book Chapters 16/07/2003. Stenzel, I., Hause, B., Feussner, I. & Was ter - nack, C. Transcriptional activation of jas- Neumerkel, Jana: Gewebsspezifische Ana - monate biosynthesis enzymes is not re - lyse der Jasmonatwirkungsweise in der lo- flected at protein level. In: Advanced Re - kalen Wundantwort der Tomate, Univer si - search on Plant Lipids (Murata, N., Ya mada, ty of Halle-Wittenberg, Depart ment of Bio - M., Nishida, I., Okuyama, H., Se ki ja, J. & Ha - chemistry/Biotechnology, 26/11/2003. jime, W., eds.) Kluwer Aca demic Pu bli - shers, Dordrecht, pp. 267-270. Department of Natural Product Biotechnology 19

Publications and Other Scientific Activities 2004

Publications rus -induced gene silencing. Plant Physiol. quinoline biosynthesis. Planta. Bücking, H., Förster, H., Stenzel, I., Miersch 134, 1401-1413. O., & Hause, B. Applied jasmonates accu- Ziegler, J & Kutchan, T. M. Differential gene mulate extracellularly in to ma to, but intra- Schüler, G., Mithöfer, A., Baldwin, I.T., Ber- expression in Papaver species in compari- cellularly in barley. FEBS Lett. 562, 45-50. ger, S., Ebel, S., Santos, J. G., Herrmann, G., son with alkaloid profiles. Acta Horti cul - Hölscher, D., Kramell, R., Kutchan, T. M., Mau - turae. Frick, S., Chitty, J. A., Kramell, R., Schmidt, J., cher, H., Schneider, B., Stenzel, I., Was ternack, Allen, R. S., Larkin, P. J. & Kutchan, T .M. C., & Boland, W. Co ro nalon: a pow erful tool Book Chapter Transformation of opium poppy (Papa ver in plant stress physiology. FEBS Lett. 563, 17- Wasternack, C. Jasmonates - Biosynthe sis somniferum L.) with antisense berberine 22. and role in stress responses and de ve lop - bridge enzyme 1 (anti-bbe1) via so ma tic mental processes. In Programmed Cell em bryogenesis results in an altered ratio of Weid, M., Ziegler, J. & Kutchan, T .M.The roles Death and Related Processes in Plants. al kaloids in latex but not in roots. of latex and the vascular bundle in morphine (Noo den, L.D., ed.) Academic Press, New Transgenic Res. 13, 607-613. biosynthesis in the opium poppy, Papaver York, pp. 143-154. somniferum. Proc. Nat. Acad. Sci. USA 101, Groß, N., Wasternack, C. & Köck, M. 13957-13962. Books and Book Chapters in press Wound induced RNaseLE expression is Kutchan, T. M., Frick, S. & Weid, M. Engi nee - jasmonate and systemin independent and Publications in Press ring plant alkaloid biosynthetic pathways - oc curs only locally in tomato (Lyco - Frick, S., Kramell, R., Larkin, P. J. & Kut chan, Progress and prospects. In: Ad van ces in persicon esculentum cv. Lukullus). Phyto - T. M. Studying morphine biosynthesis using Plant Biochemistry and Mole cular Biology che mistry 65, 1343-1350. transgenic opium poppy (Papa ver somnife- (Le wis, N. & Nes, D. W., eds.) Vol. 1, Bio en - rum L.). Acta Hortic. gi neering and Molecular Biology of Plant Halitschke, R., Ziegler, J., Keinänen, M. & Pathways (Bohnert, H.J. & Ngu yen, H.T., Baldwin, I. T. Silencing of hydroperoxide Frick, S., Kramell, R., Schmidt, J., Fist, A. J. & eds.) Elsevier Science Ltd., Ox ford. lyase and allene oxide synthase reveals Kutchan, T. M. Comparative qualitative and substrate and defense signaling crosstalk in quantitative determination of alkaloids in Wasternack, C. & Abel, S. Plant hormones. Nicotiana attenuata. Plant J. 40, 35-46. narcotic and condiment Papaver somnife- In: Molecular Plant Physiology. Chap ter 15, rum cultivars. J. Nat. Prod. (Sharma, R., ed.) Harward Press, Bingham - Köck, M., Groß, N., Stenzel, I., & Hause, G. ton. Phloem-specific expression of the wound- Gerhard, B., Fischer, K., Balkenhohl, T. J., inducible ribonuclease LE from tomato (Ly - Pohnert, G., Kühn, H., Wasternack, C. & Patent copersicon esculentum cv. Lu kul lus). Planta Feussner, I. Lipoxygenase-mediated me ta - Kutchan, T. M., Frick, S. & Kempe, K. Mo du - 219, 233-242. bolism of storage lipids in germinating sun- lation of alkaloid biosynthesis in plants and flower cotyledons and beta-oxidation of plants having altered alkaloid bio synthesis. Ma, X.-Y., Koepke, J., Fritzsch, G., Diem, R., (9Z, 11E, 13S)-13-hydroxy-octadeca-9, 11- European Patent Kutchan, T. M., Michel, H. & Stöckigt, I. Crys - dienoicacid by the cotyledonary glyoxyso- Application No. 04019154.6. talization and preliminary X-ray crystallo- mes. Planta. graphic analysis of strictosidine synthase Diploma Theses from Rauwolfia - the first member of a no - Kutchan, T. M. A role for intra and intercel- Freyer, Yvonne: Phänotypische Charak te ri - vel enzyme family. Biochim. Biophys. Acta lular translocation in natural product bio- sierung einer Allenoxidcyclase-Knock out- 1702, 121-124. synthesis. Curr. Opin. Plant Biol. Linie (AOC3-KO) von Arabidopsis tha liana. University of Halle-Wittenberg, Depart- Maucher, H., Stenzel, I., Miersch, O., Stein, Kutchan, T. M. Predicitve plant metabolic ment of Biochemistry/Biotech no logy, N., Prasad, M., Zierold, U., Schwei zer, P., engineering - still full of surprises. Trend 24/11/2004. Dorer, C., Hause, B. & Waster nack, C. The Biotechnol. allene oxide cyclase of barley (Hordeum Pienkny, Silke: Jasmonatbiosynthese durch vulgare L.) - cloning and organ-specific ex - Ounaroon, A., Frick, S. & Kutchan T. M. Mo- Allenoxidcyclasen (AOCs) in Ara bidopsis pression. Phytochemis try 65, 801-811. le cu lar genetic analysis of an O-methyl- thaliana -Unterdrückung der AOC2-Ex - transferase of the opium poppy Papaver pression durch RNA-Inter fe renz. Universi - Miersch, O., Weichert, H., Stenzel, I., Hau se, somniferum. Acta Horticulturae. ty of Halle-Wittenberg, De partment of B., Maucher, H., Feussner, I. & Was ternack, C. Biochemistry/Biotech no logy, 17/09/2004. Constitutive overexpression of allene oxide Rudus, I., Kepczynska, E. Kepczynski J., Was - cyclase in tomato (Lyco persicon esculen- ternack C. & Miersch, O. Distinct changes Doctoral Theses tum cv. Lukullus) elevates levels of jasmona- in jasmonate and 12-oxophytodienoic acid Schilling, Stephan: Charakterisierung der tes and octadecanoids in flower organs but content of Medicago sativa L. du ring soma- humanen Glutaminylcyclase im Vergleich not in lea ves. Phyto chemistry 65, 847-856. tic embryogenesis. Acta Physiol. Plant. mit dem analogen Enzym aus Carica pa pa - ya. University of Halle-Wittenberg, De - Millgate, A. G., Pogson, B.J., Wilson, I. W, Wasternack, C. Jasmonates - Overview on partment of Biochemistry/Biotech no logy, Kutchan, T. M., Zenk, M. H., Gerlach, W. L., biosynthesis and diversity in actions. In: Jas - 05/05/2004. Fist, A. J. & Larkin, P. J. Morphine pathway monates, Special Issue of J. Plant Growth block in top1 poppies. Nature 431, 413- Re gul. (Wasternack, C., ed.). Weid, Marion: Gewebe- und zellspezifische 414. Lokalisation der Alkaloidbiosyn the se in Pa - Ziegler, J., Diaz-Chávez, M. L., Kramell, R., paver somniferum L., University of Halle- Page, J. E., Hause, G., Raschke, M., Gao, W., Ammer, C., & Kutchan, T. M. Comparative Wittenberg, Department of Bio che mis - Schmidt, J., Zenk, M. H. & Kutchan, T. M. ma croarray analysis of morphine contai- try/Biotechnology, 15/09/2004. Functional analysis of the final steps of the ning Papa ver somniferum and eight mor - 1-deoxy-D-xylulose 5-phosphate (DXP) phine free Papaver species identifies an O- pathway to isoprenoids in plant using vi - methyltransferase involved in benzyliso- 20

Department of Bioorganic Chemistry Head: Ludger Wessjohann, Professor Secretary: Elisabeth Kaydamov

Department of Bioorganic Chemistry 21

atural products play a key role in modern drug discovery. According to a recent survey, more Nthan 40% of all current trade drugs are natural products, or mostly derived or inspired by them, although they constitute less than 1% of the compounds screened for activity.This higher probability of success in essence has evolutionary causes. By our research we try to understand the structural and functional principles underlying this success, theoretically by chemo- and bioinformatic analyses and modelling, and in practice by isolation, characterization, modification, biosynthetic studies, and activity screens of secondary metabolites, and to some extent of proteins involved in their conversion and interaction. The analytical work is backed by an extensive synthesis program, designed to increase compound availability and molecular diversity by combinatorial chemistry, me thod development, and de novo synthesis. The principal compound classes covered are isoprenoids and lipids, phe- nolics and small cyclopept(o)ides. Applications of this research include the use of metabolites as lead structures for drugs, cosmetics, or as research tools, and the use of enzymes as screening targets, or as catalysts for synthesis.

The department comprises four major research units covering the following topics:

Isolation (Plant and Fungal Metabolites) Isoprenoids (Biosynthesis and Biocatalysis, Modification) Synthesis (incl. Method Development, Biocatalysis) Computational Chemistry (Cheminformatics, Modeling)

These are backed by the analytical units (NMR, MS, Screening, Databases). An excellent intradepart- mental cooperation ensures the success of projects, which are not located in one organisational group only, because they require-at different times-the whole range of expertise, from isolation to synthesis and biosynthesis competence, accompanied by analytical and computational methods. Interdepartmental research further broadens the basis of such projects. Thus, with the Department of Stress and Developmental Biology, a group dedicated to the profiling of secondary metabolites (metabolomics) and proteomics from Arabidopsis and ra pe seed (GABI) is continuously successful. Overall, interdepartmental research has increased considerably (Tubulinbinders and Glycosyl trans - ferases with Prof. Strack, Terpencyclases with Prof. Kutchan, Bioinformatics and Metabolomics with Prof. Scheel).

Two highlights of the year 2004 where the successful start of Prof. Bernhard Westermann as suc- cessor to Dr. Brunhilde Voigt as new group leader within the synthesis unit, and the inauguration of Building R with state of the art laboratories for combinatorial synthesis, biocatalysis, and specia lized rooms such as a night/stink lab, fermentation, GC-lab, DNA-sequencing etc. The laboratory sec tions dedicated to our department were fully occupied and functional in less than four months.

In the report period, the department hosted researchers of 17 nationalities from four continents, e.g. regularly from Brazil,Vietnam, and Hungary. The international quota of medium term non-permanent researchers (Ph.D.s/postdoctorals) was about 44%. 27% of the co-workers had a scholarship, e.g. from DAAD, CAPES, Daimler-Benz Foundation etc. Apart from diploma theses, six Ph.D.- titles were granted to co-workers, two of them with distinction marks. The department was successful in competitive DFG priority programs with five projects, graduate schools, European Research Council funds, BMBF- and four EU-projects, several DAAD-programs, HWP-program, and with industry financed projects.

The publication record was considerably increased. In the last two years, 69 contributions in re - viewed journals and books were published. But not only numbers increased, the publications were also placed in higher valued journals (averaged sum of impact points: 2001: 134, 2002: 152, 2003: 206, 2004: 292). The first patent applications based on IPB-research highlighted extremely potent anti- bacterial, antifungal and anticancer compounds.

The upcoming period will have to see improvements in the database-, IT- and screening facilities of the department as well as a further concentration and integration of research efforts along the major lines outlined hereafter. 22

Research Group: Plant and Fungal Metabolites/Microanalytics Heads: Norbert Arnold & Jürgen Schmidt

The isolation and structure elucidation of plant and fungal metabolites with modern analy- Group Members tical techniques is the basis for investigations of both the native function of natural products Sanela Bacinovic as well as for applications. The evolutionary selection undergone by these compounds, Diploma Student since November 2004 favour them as leads for drug candidates as well as for the identification of new targets, Danston Baraza which might be derived from un derstanding the native function. Also, ecological relations NAPRECA-DAAD Fellow until November 2004 or new enzymatic transformations can be deducted from the knowledge of an organism’s Kanchana Dumri constituents. DAAD-Leibniz Fellow since March 2004 Katrin Franke Postdoctoral Position Floral Oils - Evolution, lites from collected fruit bodies of higher fungi (Basi - Gudrun Hahn Analysis and Biological Significance diomycetes), preferentially mycorrhizal ones. In sim- Technician This project aims at the better under- ple bioassays, the isolated substances are tested for Myint Myint Khine standing of the chemical composition of fungicidal and bactericidal activity. D.-B. Fellow floral oils, of their biosynthesis and evo- Christine Kuhnt Technician lutionary origin. The oils serve as at trac - Species in Hygrophorus are rarely attacked by para- Monika Kummer tant for pollinators, for which they have sitic fungi, insects, or snails. 20 new 5-(hydroxyalkyl)- Technician different functions, most prominently nu - 2-cyclopentenone derivatives (hygrophorones) could Martina Lerbs tritional ones. The analysis of floral oils of be isolated from Hygrophorus latitabundus, H. oliva- Technician six plant families (Orchidaceae, Malpi - ceoalbus, H. persoonii, and H. pustulatus. These Tilo Lübken PhD Student ghiaceae, Krameriaceae, Iridaceae, Scro - hygrophorones have structural similarities to the Jana Mühlenberg phulariaceae and Solanaceae) re vealed antibiotic pentenomycin. Chemically, hygrophorones PhD Student until December 2003 acylated (3R)-hydroxy and dihydroxyla- are 2-cy clo pentenones with hydroxy or acetoxy Lars Seipold ted fatty acids, in free form and / or substituents at C-4 and/or C-5. An odd-numbered PhD Student until May 2004 bound as acylglycerols. The hydroxy 1'oxidized alkyl chain (C11, C13, C15, or C17) is attached Axel Teichert Diploma Student until May 2004 function is always located at odd numbe- at C-5. In addition, from H. persoonii the new g-buty- Nguyen Hong Thi Van red carbon atoms. The substitution pat- rolactone derivative (5-(E)-2-hydroxytetradexylide- Visiting Scientist until April 2003 terns of the hydroxylated fatty acids are ne-5H-fu ran-2-one) could be isolated. Some hygro- Alexander Voss in agreement with recently discove red phorones are responsible for the color reaction of Diploma Student since December 2004 hydroxylated aldehydes and alkyldiols the stipes of these fungi upon treatment with potas- from epicuticular waxes. This fact sug- sium hy droxide solution. In our bioassays, we could Collaborators gests a close relationship between the demonstrate fungicidal activity as well as a remarka- Joe Ammirati biosynthesis of plant epicuticular waxes ble bactericidal activity against gram-positive bacte- University of Washington, USA possessing a protective function for the ria. Strains of multiresistant Staphylococcus aureus Helmut Besl University of Regensburg, Germany plant and floral oils. The microanalytical (MRSA) and vancomycin-resistant Enterococcus fae- methods were refined further, based on cium (VRE) were inhibited in low concentration. A Günter Gerlach Botanical Garden Munich, Germany GC/EI-TOFMS of different derivatives pa tent has been filed. Lutz Heide (e.g. methyl es ters, trimethylsilyl esters University of Tübingen, Germany or ethers, pyrrolidides, dimethyldisulfide From fruit bodies of the basidiomycete Matthias H. Hoffmann Botanical Garden Halle, Germany adducts) and ESI-MS(/MS) of the non- Hygrophorus eburneus (Tricholomataceae) eight derivatized se cretions. This is used to fatty acids (C16, C18) with -oxocrotonate partial Christoffel Spengler g Bionorica AG Neumarkt, Germany extend the species basis. We could also structure could be isolated. Initial tests demonstrate Wolfgang Steglich revise that previous reports on their significant bactericidal and fungicidal activity. University of Munich, Germany Oncidinae flower oils (Orchidaceae). Tran Van Sung Institute of Chemistry Hanoi, Vietnam Cortinarius bolaris (subgenus Constituents of Higher Fungi Leprocybe) is described in the Fungi probably comprise the largest group of higher literature as poisonous.The cap organisms in our world with estimated 106 speci- and stem staining yellow when men. Therefore, fungi are an important resource of bruised. The underlying prin - new metabolites. Our research is mainly focused on ciple likely is a new benzofuran

the isola tion and characterization of fungal metabo- glycoside, which can be isola- Hygrophorus olivaceoalbus, the Shea thed Waxy Cap is growing in the Harz mountains. 23

ted from freeze-dried samples. This compound rative ac tivity pro - turns yellow after exposure to HCl or upon heating. files. Apo lar ex tracts The stereochemistry could not yet be established. of three plants (C. Surprisingly, fresh specimen of C. bolaris do not co mo sa, A. tagala, V. contain the benzofuran glycoside. Instead, a new yel- re pens) also show low-colored phthalimide derivative exhibiting a significant antifungal bright blue fluorescence appears. Its biosynthesis is activities. 1 assumed to originate from a benzofuran glycoside. Microanalytics Constituents of traditional medicinal plants In collaboration with the University of Halle the HEATOS - A Vietnamese opiate detoxification sym- mass spectral fragmentation of morphine and co - ptom medication deine was studied in The project HEATOS was originally coordinated by detail using LC- the United Nations (UNESCO and UNOPS). Dr. Dan electrospray MS/MS from Vietnam developed the original herbal aid for and high resolution opioid detoxification and called it HEATOS (heat of FT-ICR-MS combined sun). HEATOS consists of ca. 13 components and was with an IRMPD. La - constantly improved in composition, also with the aid beled codeine was of our findings. In cooperation with Vietnamese part- isolated and pu rified 2 ners our research is focused on the isolation and cha- from Papaver somni- racterization of the constituents of these plants. ferum seedlings, which were grown in the presence of Most species have been identified. So far some 180 [ring-13C6]-L-tyrosine, [ring-13C6]-tyramine and [1,2- compounds are isolated from eight plants. A sub- 13C2], [6-O-methyl 13C]-(R,S)-coclaurine. Using these stance based overview and extensive literature sur- labeled derivatives it was possible to vey was conducted. determine the origin of the carbons in the fragment ions. In collabora - Traditional South East Asian medicinal plants tion with the Department of Na - The aim of this project is to isolate and characte- tural Pro duct Biotechnology and the rize the phytoconstituents of the medicinal plants Biocenter of the Uni versity the LC- Streptocaulon tomentosum, Vitis repens, Curcuma ESI-MS/MS investigations of morphi- comosa, Aristolochia tagala and Spermacoce hispida nan-type and benzylisoquinoline al - from Myanmar, and to test their bioactivity. kaloids are continued. 3

The roots of Streptocaulon species are used medici - Furthermore, a se ries of new aminocoumarin anti- nally for the treatment of dysentery and stomach - biotics (2) produced in vi tro and in vivo by a combi- ache, and the leaves are used externally for the ned biochemical, genetic and synthetic ap proach from treatment of snake poisoning and abscesses. Cur cu - a coo pera tion of the NWC-Isoprenoids Group and ma comosa is a member of the economically impor- the University of Tübingen were structurally elucida- tant plant family Zingiberaceae. The extract of C. ted by using LC-ESI-SRM both under positive and co mosa is reported to be nematocidal and cholere- negative ionization. tic in rats. As well as Aristolochia tagala this species is used against malaria. Rhizomes of Vitis repens are The high-resolution FT-ICR-MS was successfully ap - used against different cancers. Spermacoce hispida is plied for the determination of the exact mass mea- an effective natural drug by treatment of hyperten- surements of rifamycin derivatives including their sion. fragments obtained by varying the capillary exit vol- tage (3). From S. tomentosum several triterpenoids, cardenolides, and oleanane-type saponins have been identi- In addition, the group is intensely involved in the fied. A new cardenolide (1) was isolated from the metabolic profiling and IT-projects at the IPB. roots. Some cardenolides had remarkable antipro life - 24

Research Group: Structural Analysis & Computational Chemistry Heads: Wolfgang Brandt & Andrea Porzel

The research group is investigating three-dimensional molecular structures of small molecules proteins and reaction mechanisms (e.g. enzyme catalysis) in the field of bioorganic chemistry by means of molecular modelling techniques, semi-empirical and ab initio calculations, bio- and cheminformatics, theoretical chemistry and NMR spectroscopy. The group is also involved in the development of molecular databases. The collected information together with data mining and new data from the other research groups form the basis for cheminformatic analyses which enables new insights into the biological significance of plant and fungal metabolites, and also in drug design.

Cheminformatics and NMR face. Analysis of macrocycles abundant in natural pro- In continuation of our cheminformatic activities, a ducts database of more than 500 experimental as well as Group Members A database containing the structures of calculated 1H/13C 1J shift correlated 2D NMR spectra Susanne Aust more than 120,000 compounds was ana- was designed. In collaboration with the Institute of Guest Research Scientist since March 2003 lyzed and data mining was performed for Computer Science, University of Halle, a database Monika Bögel macrocycles with more than 13 atoms management system was developed which provides Research Scientist until December 2003 in unbridged rings. The resulting data - similarity search and mixture analyses based on the base was analyzed for molecular weight 2D NMR database content. Lars Bräuer PhD Student distribution, the frequency of common Frank Broda substructural motifs, and biosynthetic Modern NMR spectroscopy experiments were used System Administrator since January 2004 origin in relation to ring size.The underly - for the structural elucidation of bioactive natural Stefanie Gulde ing principles of chemical diversity were products isolated from plants and higher fungi. Also, Diploma Student since October 2004 reviewed in terms na ture's strategies for the synthetic studies in the department were sup- Jana Selent diversity and complexity generation and ported by NMR investigations. Routine spectra PhD Student, Guest, until April 2004 with respect to biosynthetic origin. Fi - (1H/13C/19F/31P NMR, IR, CD, ORD, UV) were recor- Annett Siebenhüner Diploma Student since November nally, it was suggested that synthetic che- ded as a service for the Department of Bioorganic 2004 mists should not only use nature's mole- Chemistry and all research groups of the IPB. Maritta Süße cules, but also its strategies as a source Technician of inspiration. To illustrate this, the bio- Further steps towards an unified ACD-based plat- Bianca Wachsmuth Diploma Student until December 2004 synthesis of macrocycles by terpene and form of spectroscopy data were taken and the IT- polyketide cyclases and nonribosomal coordination within the IPB was improved. peptide synthetases, as well as recent advances in employing these strategies in an integrated synthe- Modelling and sis/biotechnology approach were briefly reviewed. Quantum Mechanical Calcu lations The functional role of selenocysteine (Sec) in the In house database, cheminformatic tools and spectros- catalysis mechanism of large thioredoxin reductases copy We investigated the redox mechanism of thiored- In collaboration with oxin reductases containing a selenocysteine in the Volkmar Vill, Univer sity ca talytically active site. Thioredoxin reductases ca - of Hamburg, the deve- ta lyze the reduction of thioredoxin disulfide and lopment of the in house some other oxidized cell constituents. They are ho - database project Phyto - modimeric proteins containing one FAD and accep- base has been continued. ting one NADPH per subunit as essential cofactors. Se ve ral hundred struc- Ba sed on the X-ray structure of rat thioredoxin re - tural formulas have been ductase, homology models of human thioredoxin added and in put/output re ductase were created, and subsequently docked program options and to thioredoxin to model the active complex. search tools were im - Figure1:The catalytically active site of thioredoxin reductase proved in order to de - The formation of a new type of a swapping catalytic in complex with the substrate thioredoxin. The red arrows indicate the proton transfer from Sec498 to His472 forming the sign a user-friendlier sur - triad between selenocysteine (Sec), histidine, and a catalytic triad together with Glu477. Carbon atoms of the substrate are colored in magenta. 25

glutamate could be detected in the protein struc - si milarities to the serine protease Collaborators ture. By means of DFT calculations we could show fa mily. APOGEPHA GmbH that the formation of such a triad is essential to sup- Selected amino acids suggested to Dresden, Germany port the proton transfer from selenol to a histidine be involved in substrate binding and Horst Bögel to stabilize a selenolate anion, which is able to inter- turnover, were substituted via site- University of Halle, Germany act with the disulfide of thioredoxin and catalyses directed mutagenesis of the functio- Susanna Fürst University of Budapest, Hungary the reductive disulfide opening (Fig. 1). nally expressed protein. Substi tu - Andris Kreicbergs tion of two highly conserved amino Karolinska Institute, Stockholm, Sweden 378 Whereas a simple proton transfer from selenocys - acids, Glu , located in the proposed ProBioDrug AG teine to histidine is thermodynamically disfa vo red it UDP-glucose binding site, and His22, Halle, Germany becomes favored when the carboxylic acid group of located close to the N-terminus, Ryszard Przewlocki Polish Academy of Sciences, Krakow, a glutamate stabilizes the formed imida zole cation. both led to a complete loss of en - Poland An identical process with a cysteine instead of sele- zyme activity, which considerably Helmut Schmidhammer nocysteine will require 4 kcal/mol more energy, supports the principal correctness University of Innsbruck, Austria which corresponds to a calculated equilibrium shift of the model. Volkmar Vill of ~ 1000:1 or a 103 rate acceleration a value close University of Hamburg, Germany 2 Prenyltransferases Wella AG to the experimental one of about 10 -times. Darmstadt, Germany A main project of the department is These results give new insights into the catalysis the investiga tion of C-C-coupling prenyltransfera- mechanism of thioredoxin reductase and for the ses, located in the research group Isoprenoids. This first time explain the advantage of the incorpora tion work is ac com pa nied by bioinformatic studies, of a selenocysteine instead of a cysteine residue in a homology mo delling, and quan tum mechanical calcu- protein. lations. Com putational chemistry work on p-hydroxybenzoate oligoprenyltransferase (ubi a) has been finis- The modeled protein structures of both the cytoso- hed for the moment, and re sults were pub lished. lic and mitochondrial thioredoxin reductase in com- Recently, homology protein modeling of other prenyl- plex with thioredoxin have been accepted by and transferases of other families was started. stored in the Protein Data Bank.

Protein 3D-homology modeling of UDP-glucose dependent betanidin 5-O-glucosyl-transferase from Dorothean thus bellidiformis This project was based on a close cooperation with the Department of Secondary Metabolism of our institute. The betanidin 5-O-glucosyltransferase (UGT73A5) from Livingstone daisy (Dorotheanthus bellidiformis) is involved in regiospecific glucosylation of betanidin and various fla- vonols. A three-dimensional model of this regiospecific betanidin and flavo noid glucosyltransferase has been constructed and the active site has been modeled (Fig. 2). To explain the observed inversion in the configuration of the bound sugar, semiempi- rical calculations favor an SN1-type reaction as a plausible alternative to the generally proposed SN2- mechanisms discussed for plant glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the ac - Figure 2: Stereo representation of the active site of the UGT73A5 from D. bellidiformis with beta- ceptor be tanidin, but also further imply that the re - nidin (magenta highlighted). Amino acids proposed to be involved in substrate binding and catalytic sugar transfer are displayed. Red-labeled residues belong to the PSPG-box, magenta ones are action mechanism may involve a ca talytic triad with essential for the catalysis and blue labeled ones for binding of the ligands. Hydrogen bonds are represented by green dotted lines. The red arrow indicates the initial step of the suggested catalysis mechanism. 26

Research Group: Isoprenoids Head: Ludger Wessjohann

Isoprenoid units can be detected in some 70,000 natural products, including steroids, terpenoids and conjugates with molecules of other major pathways. The study of isoprenoid compounds and metabolism provides insight into the predominant mechanisms and routes that nature uses to build up carbon skeletons. Understanding these, will provide new enzymes for in vitro C-C-coupling reactions as biocatalysts for the production of prenylated and terpenoid compounds, as well as new targets for inhibitors of important metabolic processes in plants, most pathogenic bacteria, and many parasites. Of special interest to us is the transfer of prenyldiphosphates onto sp2-carbons (aromatic and vinylic substrates). We hope to elucidate mechanistic and structural details, provide better access to probes and substrates, develop mechanism-based inhibitors and finally achieve access to a set of enzymes enabling a multitude of enzymatic C-C-coupling reactions. In addition, synthetic modifications of isoprenoids and diphosphates to increase the compound base are part of the program.

Synthesis of isoprenoid substrates, inhibitors and pound triggered considerable commercial inte- natural products rest as mild menopause medication. However, the Group Members A large set of oligoprenyl diphos - total synthesis is lengthy, complicated and expen- phates was synthesized as substrates sive. Also, totally "artificial" synthetic compounds Lars Bräuer PhD Student and discovery tools, especially non- are less acceptable to the anticipated customer Marco Dessoy natural ones like homo-, nor- or hete- base. Based on the abundant natural hop com- PhD Student until July 2003 roderivatives, or dye labeled ones, to pound xanthohumol, we could de vise a simple, Michael Fulhorst probe substrate specificity. Interme - protection group free two step synthesis of 8-PN PhD Student until September 2003 diates to the non-mevalonate MEP- in over 90% yield, in which the natural carbon Gudrun Hahn Technician pathway, e.g. epoxidized and hydroxy- isotope distribution is re tained 100%. The most Roman Weber lated isopentenyl and dimethylallyldi- difficult task was the se lective cleavage of a me - PhD Student since September 2004 phosphate (DMAPP), were synthe - thyl ether in the presence of the acid sensitive Heike Wilhelm sized in iso tope labeled form. Further prenyl unit, which so far was only achieved with a Postdoctoral Position since August 2003 chain-mo dified derivatives were syn- few percent yield and conside rable byproduct Svetlana Zakharova thesized as part of a biocatalytic total formation. Postdoctoral Position synthesis program to wards prenylated aromatic natural products. ubi A Prenyltransferase 4-Hydroxybenzoate oligoprenyltransferase of E. In addition, several farnesyl and geranyl analo- coli, encoded in the gene ubiA, is an important gues with mimics of the pyrophosphate unit key enzyme in the biosynthetic pathway to have been prepared for an examination of their ubiqui none, an essential component of aerobic ability to in hibit prenyl transfer. 7,11-Dimethyl- life. It catalyzes the prenylation of 4-hydroxyben- 3-oxododeca-6,10-dienoic acid, 3-hydroxy-7,11- zoic acid in the 3-position using an oligoprenyl dimethyldodeca-6,10-dienoic acid, diphosphate as second substrate. An X-ray struc- 2-hydroxy-4,8-dimethyl-3,7-nona- ture is not available, not even of a related enzy- dienylphosphonic acid, and [[(4E)- me. The knowledge of the tertiary structure and 5,9-dimethyldeca-4,8-dienyl] phos - possible active sites is, however, essential for the phina to](difluo ro) me thyl phospho - understanding of the catalysis mechanism and the nate were synthesized from gera- substrate specificity. niol. w-2,w-1-dihydroxy lated farnesyl diphosphate was prepared from The substrate model known for the aromatic trans,trans-farnesol. part was extended. For the first time a minima - 1 lized model for the prenyl substrate could be 8-Prenylnaringenin (8-PN, 1) is the defined based on experiments with increasingly 8-Prenylnaringenin (8-PN) is the strongest strongest natural phytoestrogen wit- truncated GGPP-, FPP-, and GPP-derivatives (G = natural phytoestrogen within the flavonoids. hin the flavonoids. Recently the com- Geranyl, F = Farnesyl). 27

With respect to mechanistic studies, through cording to new protocol especial- modeling techniques, secondary structure pre- ly adapted to work without pro- Collaborators diction tools, molecular dynamics simulations, tection of the diphosphate moie- Martin Biendl and energy optimizations, an acceptable homolo- ty. In cooperation with Prof. Mein - Hallertauer Hopfenveredelungs GmbH, Mainburg, Germany gy model could be created. Multiple alignments of hart Zenk, these as well as the la - Uwe Bornscheuer a multitude of related proteins clearly showed beled cyc lic precursor MEcPP University of Greifswald 100% conservation of the amino acid residues were applied to chromoplast cul- Lutz Heide, Shuming Li forming the putative active site. An additional tures. For the first time, 4-hydro- University of Tübingen Germany highly conserved region could be detected, which xy-DMAPP was identified as the György Horvath University of Antwer, Belgium currently is considered to be a rudimentary ac - last intermediate towards DMAPP Toni M. Kutchan, Jon Page tive site, giving some hints regarding the evolutio- and IPP in plastids of higher IPB Halle, Germany nary origin of ubiA-enzyme. Semi-empiric quan- plants: Its isotope labeled form Markus Pietzsch, tum mechanical PM3 calculations have been per- was incorporated into the caro - Meinhart Zenk University of Halle, Germany formed to investigate the thermodynamics and tene precursor phytoene in vitro. SunGene GmbH & Co. KGaA kinetics of the catalysis mechanism. These results Gatersleben, Germany suggest a mechanism closer to SN1- than to SN2- This finding is in agreement with type for the cleavage of the diphosphate ion from our mechanistic and structural the isoprenyl unit. Most interesting is the depro - model of the fragment L271 to A375 of the enzyme tonation behavior of enzyme bound 4-hydroxy- GcpE of Streptomy ces coelicolor (transforming benzoic acid, of which formation of the pheno late MEcPP to 4-hydroxy-DMAPP), including NADPH, anion is favored to that of a benzoate anion. This the Fe4S4-cluster and MEcPP as ligand. Based on ideally aids the attack of the isoprenyl cation, this homology model, semi-empirical PM3 cal - which appeared to be the rate-limiting step of culations were used to analyze the likely catalysis the whole process according to quantum chemi- me chanism of the reductive ring opening of cal calculations. Probing this model by site direc- MEcPP. Our suggested mechanism is characte - ted mutagenesis failed so far, it proved extremely rized by a proton transfer (presumably from the difficult to obtain functional mutated protein. conserved arginine 286) to the substrate, accompanied by a ring opening without high energy bar- Finally, several analogues and mimics of farnesyl riers, followed by the transfer of two electrons and geranyl pyrophosphate were tested for en - delivered from the Fe4S4-cluster and finally pro- zyme inhibition in a competitive assay with GPP. ton transfer from a carboxylic acid side chain to The effect of these compounds on ubiA-prenyl- the hydroxyl group to be removed from the li - transferase activity varied substantially, ranging gand as water. The proposed mechanism is in from almost full competitive inhibition to, surpri- agreement with all known experimental findings singly, enhanced enzymatic activity at low concen- and the arrangement of the ligand within the trations by some compounds. The activity enhan- putative enzyme model. It differs especially from cing activity of some diphosphate mimics could proposals suggesting epoxide intermedia- be related to their magnesium ion complexing tes which, however, are highly unfavorable properties and is only effective at high ion/low in energy and could not be supported by enzyme concentration. It can be mimicked by theoretical or by experimental data. A EDTA-addition. principally si milar mechanism is also ex - pected for the reductive dehydroxylation Non-mevalonate of 4-hydroxy-DMAPP in the last step of (dxp- or mep-) Pathway in Plants this basic isoprenoid pathway. Experimental and theoretical investigations were performed concerning the two last steps of the dxp/mep-pathway in isoprenoid biosynthesis in plants. The proposed intrinsic or late interme - diates, 4-oxo- and 4-hydroxy-DMAPP were synthesized in deuterium or tritium labeled form ac - Hop cones 28

Research Group: Synthesis and Method Development Heads: Ludger Wessjohann & Bernhard Westermann

In order to obtain compounds of high biological relevance, the most recent development Group Members in organic synthesis favors the combination of (natural product) target-oriented and diver- Muhammad Abbas sity-oriented synthesis. The synthetic group is ideally placed in this context. Efforts are Postdoctoral Position since May 2004 undertaken to synthesize small natural products (i. a. such as provided by the Plant and John Bethke Fungal Metabolites Group) and mimics thereof, focussed natural product-like libraries, and Postdoctoral Position until April 2004 designer mole cules. The biggest problem with respect to natural product like and functio- Viktor Dick PhD Student since April 2004 nal structures is their complexity, which usually requires complicated multistep syntheses. Simon Dörner New synthetic tools, methods and concepts to improve the speed and selectivity of syn- PhD Student since April 2004 thetic reactions are the basis to obtain the desired chemical diversity of modified natural Tobias Dräger products or mimetics with improved or even new biological activity profiles, e. g., by multi PhD Student since October 2004 component reaction (MCR) strategies. One focus is on macrocyclic compounds, which Uwe Eichelberger Postdoctoral Position combine conformational preorganization with flexibility. Combinatorial approaches in Daniel Garcia-Rivera liquid as well as on solid phase have been used to obtain higher selectivity, improve reac- PhD Student since October 2003 tion conditions, and to form small focussed libraries demonstrating the power of our pro- Gergely Gulyas cedures. Applications in the chemical field include reactions in water, selective separation, PhD Student since August 2004 and catalysis; in the pharmaceutical field, compounds with anticancer, antibiotic, phytoestro- Alexander Gutsche Diploma Student since August 2004 genic and cosmetic properties have been studied. Nicole Hünecke Trainee since August 2004 Oliver Kreye PhD Student since July 2004 Christiane Neuhaus PhD Student since April 2004 Macrocycles Eelco Ruijter Even functionally very simple mole - Thus macrocycles, ideally even with adjustable PhD Student until June 2004 cules like host-molecules already re - conformational bias, are good candidates to mi - Angela Schaks quire considerable size and design. mic natural binders in a minimal fashion, e.g. to Technician Thus, in order to minimize entropy achieve hosts selective for binding in water, or for Gisela Schmidt Technician loss upon binding, a reduction of con- the design of protein-protein-interaction tools. Alex Schneider formational freedom compared to li - Synthetic approaches thus must have the flexibi- PhD Student since June 2004 near mo lecules is required. At the lity to introduce a large variety of structural ele- Henri Schrekker same time, some flexibility needs to ments of rigidity, flexibility, lipophilicity, and direc- PhD Student until February 2004 be retained in order to allow adap tive tional functionality. Tran Thi Phuong Thao PhD Student properties or different stages within a Tran Van Chien functional process, mostly a binding Access to libraries of such macrocycles requires PhD Student until September 2003 process. This can be achieved by self - the development of efficient, flexible, diversity- Katharina Wolf organization and folding (as in en - oriented synthesis routes. This still is a big chal- Trainee since August 2004 zymes), rigidifying elements (e.g. turn lenge for organic synthesis because of the excep- Mingzhao Zhu PhD Student inducers), or (macro-)cyclization. In na - tionally stringent requirements for reaction effi- ture, selec tive hosts or ligands of a MW- ciency, chemoselectivity, functional group tole- range below that of enzymes or me - rance, and stereoselectivity. Thus, diversity-orien - dium peptides often are macrocycles with rigidi- ted synthesis also provides an important en gine fying and flexible elements placed in the appro- for innovation in organic chemistry. Natural propriate positions, often of polyketide or peptide duct like macrocycles can combine rigidity and ori gin. Another important element for nature- flexibility at the same time, contradicting the pa - like selection and interaction properties, recogni- radigm that compounds of pharmaceutical in te - tion, and function is asymmetry. Besides asymme- rest have to be flat, and nitrogen containing. try, the overall functional group density and varia- Therefore, work in the group has been largely fo - bility distinguishes most natural hosts from e.g. cussed on the target- and diversity-oriented syn- crown ethers or calixarenes, which are rather ru - thesis of macrocycles and the development of dimentary hosts from a biomimetic viewpoint new macrocyclization reactions. and in terms of selectivity. 29

Scheme 1: Ugi-type MiB combining a steroid and biarylether moiety (MiB = multiple multicomponent macrocyclization including bifunctional building blocks)

Target-oriented synthesis of macrocycles Epothilones have pronounced biological profiles, which make them interesting drugs for cancer treatment now explored in phase II/III clinical tri- als. All four diastereomers of the new epothilone D -series, e.g. isomer 1, were synthesized, and 5 2 compared to the natural isolate. In this context, the chromium-mediated Reformatsky reaction developed by us proved to be the method of The compounds synthesized proved useful in the choice for the construction of the southern part. study of plant cell cycles in cooperation with Bettina Hause in the Department of Secondary Metabolism.

Diversity-oriented synthesis of macrocycles For the synthesis of macrocycle libraries we devised three strategies. Strategy I utilizes commercially available ma- 1 crocycles like rifamycin, tylosin or erythromycin. These are modified by selective reactions, including biocatalytic ones in solution or on solid In order to produce third generation epothilones phase, to produce libraries of rim-modified deri- with enhanced accessibility and pharmacological vatives, usually without any protective group che- properties, thio-derived congeners of epothilone mistry. This allows a fast chemical screen for ac - D lacking one stereocenter, like in compound 2, cessibility and biologically importance of rapidly have been synthesized. They showed low nano- modifiable positions. A new enzyme penetrable molar activity against some cancer cell lines and resin-linker combination was designed for this patent applications have been filed. purpose. 30

Research Group: Synthesis and Method Development Heads: Ludger Wessjohann & Bernhard Westermann

Collaborators Strategy II utilizes multicomponent reactions (MCRs) like the Ugi- Uwe Bornscheuer University of Greifswald, Germany reaction (Scheme 1), which is extremely atom-economic and Antonio Luiz Braga Federal University of Santa Maria, Brazil provides ra pid access to highly di - Alexander Dömling, Wolfgang verse peptoid compounds. Utili - Richter zing specially designed bifunctional R&D Biopharmaceuticals GmbH Munich, Germany starting ma terials, the synthesis of Sabine Flitsch macro cycles can be achieved in a 4 University of Edinburgh, UK one step multi reaction procedure Lucia Gardossi University of Trieste, Italy (MiBs = Multiple Multicomponent Macrocyclization including Bifunc - Strategy III is a combination of a linear precursor Bettina Hause IPB Halle, Germany tional Building Blocks). Initially, we synthesis and a cyclizing reaction like the Grubbs- Udo Kragl have concentrated on the synthe- olefin metathesis and, e.g., the azide/al kyne- University of Rostock, Germany sis of steroid- and (vancomycin in - "click"-approach to achieve the synthesis of car- Rob Leurs, Martine Smit Free University of Amsterdam, spired) biarylether containing pro- bohydrate moiety containing macrocycles as in 5. Netherlands ducts like compounds 3 and 4. E.g., Such compounds are of particular interest due to Francisco Coll Manchado some MiBs in a one-pot re action their resemblance of aminoglycosides, a class of University of Havanna, Cuba combine twelfe building blocks as - natural compounds exhibiting antibiotic activity. Karoly Micskei University of Debrecen, Hungary sembled in 16 reactions to give a Method development MorphoChem AG Munich constitutionally defined 60-mem- Munich, Germany bered macrocycle with water as the Tamas Patonay only formal byproduct. For the first University of Debrecen, Hungary time, complex macrocycles with Romano Orru Free University of Amsterdam, non-identical subunits (cf. the repe- Netherlands titive calixarenes and cyclodex - Polymer Laboratories Ltd trines) become available in an ex - Shropshire, UK 5 tremely efficient combinatorial fashion in infinite number and quantity with almost In the report period, several selective methods unlimited size, shape, and functionality, thus ope- utilizing, e.g., biocatalysis towards asymmetric ning the door to a much improved host-guest che- acyloins and novel hetero-Diels-Alder reactions mistry, organocatalysis and artificial enzymes, cy - to form polyketide fragments, Ugi-MCRs, transi - clopept(o)id mimics, and protein-protein interaction metals and transition metal catalysts [Cr(II), tion studies. Co, Pd] have been developed and utilized in the syntheses discussed above. Selenium-based and organocatalytic processes are a more current de - velopment.

Selenium chemistry Selenium compounds are little studied in chemistry, especially in combination with their catalytic and biological significance. We set out to study the properties of selenium derivatives in comparison to the usually well-studied sulfur analogues, with a special focus on selenocystein (Sec) and Sec-containing peptides (cf. also the Research Group of Computational Chemistry). A reason - able study should be performed under (close to) 3 physiological conditions and not be limited to a single compound. This required a synthetic route 31

to libraries of (water soluble) Sec-peptide deriva- leading to the appropriate Mannich type bases, tives. In principle, the Ugi-MCR can be utilized for like 8, in high yields and selectivity. While using this purpose again, but a difficult adaptation to in - protected dihydroxy acetone derivatives, this re - corporate the initially problematic selenium con- action can be seen as a surrogate of aldolase ca - taining building blocks had to be made. With the talyzed reactions found in nature. Or gano cata - right combination of solvent, catalyst, and building lytic processes not only provide the products in blocks, we are now able to produce libraries of high stereoselectivity, they are also "green pro- selenopept(o)ids in water, including, e.g., com- cesses" due to the absence of any me tal ion. In pound 6 with the biologically important Cys-Sec- addition, a couple of these reactions can be car- moiety. Furthermore, new syntheses of seleno- ried out in aqueous solutions decreasing the and tellurocystein and related sulfur, selenium amount of organic solvents. and tellurium compounds were devised and uti - lized, e.g., for catalysis.

6 7 8

Organocatalysis Organocatalysis with proline 7, offers the possibility to carry out asymmetric Mannich reactions 32

Publications and Other Scientific Activities 2003

Publications estrogen b-selective activity of Vitex Bethke, J., Karaghiosoff, K. & Wessjohann, agnus Nguyen Thi Hoang Anh, Tran Van Sung & L. A. Synthesis of N,N-disubstituted sele- castus and isolated constituents. Planta Wessjohann, L. A. Steroidal glycosides from noamides by O/Se-exchange with sele - Med. 69, 947-950. Ophiopogon japonicus. Tap chí Hóa hoc nium-Lawesson's reagent. Tetrahedron (Vietnam. Journal of Chemistry) 41, 135- Lett. 44, 6911-6913. Kamperdick, C., Nguyen Minh Phuong, 141. Adam, G. & Tran Van Sung Guaiane dimers Braga, L. A., Paixao, M. W., Lüdtke, D. S., from Xylopia vielana. Phytochemistry 64, Ounaroon, A., Decker, G., Schmidt, J., Lott - Silveira, C. C. & Rodrigues, O. E. D. Synthe - 811-816. speich, F. & Kutchan, T. M. (R,S)-Reticuline sis of new chiral aliphatic amino disele - 7-O-methyltransferase and (R,S)-norco- nides and their application as catalysts for Kolbe, A., Porzel, A., Schmidt, J. & Adam, G. claurine 6-O-methyltransferase of Papaver 2 the enantioselective addition of diethylzinc A new synthesis of [26,28- H6] brassino - somniferum - cDNA cloning and characte- 2 to aldehydes. Org. Lett. 5 (15), 2635-2638. lide and [26,28- H6] castasterone via an rization of methyl transfer enzymes of al - un usual methyl migration. J. Label Compd. kaloid biosynthesis in opium poppy. Plant Braga, A. L., Rubim, R. M., Schrekker, H. S., Radiopharm. 46, 231-242. J., 36, 808-819. Wessjohann, L. A., de Bolster, M. W. , Zeni, G. & Sehnem, J. A. The facile synthesis of Kuhn-Wache, K., Hoffmann, T., Manhart, S., Raith, K., Neubert, R., Poeaknapo, C. chiral oxazoline for the diethylzinc addi- Brandt, W. & Demuth, H. U. The specificity Boettcher, C., Zenk, M. H. & Schmidt, J. tion to aldehydes. Tetrahedron Asymm. 14, of DP IV for natural substrates is peptide Electrospray tandem mass spectrometric 3291-3295. structure determined. Adv. Exp. Med. Biol. investigations of morphinans. J. Am. Mass 24, 57-63. Spectrom. 14,1262-1269. Black, S. L., Jales, A. R., Brandt, W., Lewis, J. W. & Husbands, S. M. The role of the side Lorey, S., Stöckel-Maschek, A., Faust, J., Richter W. G., Cappi M. W., Henkel B., Hess chain in determining relative d- and k-affi- Brandt, W., Stiebitz, B., Gorrell, M. D., Käh - S., Surivet J. P., Baeschlin D., Hubschwerlen nity in C5'-substituted analogues of nal tr in - ne, T., Mrestani-Klaus, C., Wrenger, S., Rein - C., Höfle G., Wessjohann L. & Eichelberger dole. J. Med. Chem. 46, 314-317. hold, D., Ansorge, S. & Neubert, K. Dif fe - U. Second generation epothilones and rent modes of dipeptidyl peptidase IV their biological properties. Clin. Canc. Res. Cacace, S., Schröder, G., Wehinger, E., (CD26) inhibition by oligopeptides de - (Part 2, Suppl. S) 9 (16), 6137S-6137S. Strack, D., Schmidt, J. & Schröder, J. A fla- rived from the N-terminus of HIV-1 Tat vonol O-methyltransferase from indicate at least two inhibitor binding sites. Samappito, S., Page, J., Schmidt, J., De-Ek - Catharan thus roseus performing two EJB 270, 2147-2156. namkul, W. & Kutchan, T. M. Aromatic and sequential me thylations. Phytochemistry pyrone polyketides synthesized by a stil - 62, 127-137. Luu Duc Huy & Porzel, A. Heptazoline - a bene synthase from Rheum tataricum. Phy - carbazol alkaloid extracted from Clausena to chemistry 62, 313-323. Degenkolb, T., Berg, A., Gams, W., Schlegel, heptaphilla (Roxb.) W. & ARN. Tap Chi B. & Gräfe, U. The occurrence of peptai - Duoc Hoc (Pharmaceutical Journal of Viet Schmutz, E., Steffensky, M., Schmidt, J., Por- bols and structurally related peptaibiotics Nam), 11, 11-12. zel, A., Shu-Ming Li & Heide, L. An unusual in fungi and their mass spectrometric iden- amide synthetase (CouL) from the cou-

tification via diagnostic fragment ions. J. Mrestani-Klaus, C., Brandt, W., Faust, J., mermycin A1 biosynthetic gene cluster Peptide Sci. 9, 666-678. Wrenger, S., Reinhold, D., Ansorge, S. & from Streptomyces rishiriensis DSM Neubert, K. New results on the conforma- 40489. EJB. 270, 4413-4419. Eckermann, C., Schröder, G., Eckermann, tions of potent DP IV (CD26) inhibitors S., Strack, D., Schmidt, J., Schneider, B. & bearing the N-terminal MWP structural Schütz, J., Brandt, W., Spetea, M., Wurst, K., Schröder, J. Stilbenecarboxylate biosynthe- motif. Adv. Exp. Med. Biol. 524, 65-68. Wunder, G. & Schmidhammer, H. Synthesis sis: A new function in the family of chal - of 6-amino acid substituted derivatives of cone synthase related proteins. Phyto che - Münzenberger, B., Hammer, E., Wray, V., the highly potent analgesic 14-O-methyl - mis try 62, 271-286. Schauer, F., Schmidt, J. & Strack, D. Deto xi - oxymorphone. Helv. Chim. Acta 86, 2142- fication of ferulic acid by ectomycorrhizal 2148. Gidda, S. K., Miersch, O., Levitin, A., Schmidt, fungi. Mycorrhiza13, 117-121. J., Wasternack, C. & Varin, L. Biochemical and Spiteller, P., Arnold, N., Spiteller, M. & Steg - molecular characterization of a hydroxyjas- Nguyen Thi Hoang Anh, Tran Van Sung, lich, W. Lilacinone, a red aminobenzoqui - monate sulfotransferase from Arabidopsis Franke, K. & Wessjohann, L. Phytochemical none pigment from Lactarius lilacinus. J. thaliana. J. Biol. Chem. 278, 17895-17900. studies of Rehmannia glutinosa rhizomes. Nat. Prod. 66, 1402-1403. Pharmazie 58, 593-595. Holzgrabe, U. & Brandt, W. Mechanism of Trinh Thi Thuy, Tran Van Sung & Wessjo - action of the diazabicyclononanone-type Nguyen Thi Hoang Anh, Tran Van Sung, hann, L. Chemical constituents of the roots k-Agonists. J. Med. Chem. 46, 1383-1389. Porzel, A., Franke, K. & Wessjohann, L. A. of Codonopsis Pilosula. Vietnam. J. Chem. Homoisoflavonoids from Ophiopogon 41, 119-123. Ibdah, M., Xing-Hai Zhang, J. Schmidt, J. & japonicus Ker-Gawler. Phytochemistry 62, Vogt, T. A novel Mg++-dependent O-me thyl - 1153 - 1158. Wessjohann, L. A., Brandt, W., Thiemann, T. transferase in the phenylpropanoid meta- Biosynthesis and metabolism of cyclopro- bolism of Mesembryanthemum crystalli- Nguyen Thi Hoang Anh, Tran Van Sung & pane-rings in natural compounds. Chem. num. J. Biol. Chem. 278, 43961- 43972. Wessjohann, L. A. Some homoisoflavonoi- Rev.103, 1625-1648. dal compounds from Ophiopogon japoni- Jarry, H., Spengler, B., Porzel, A., Schmidt, J., cus Ker-Gawler. Tap chí Hóa hoc (Vietnam. Westermann, B. Organokatalyse oder die Wuttke, W. & Christoffel, V. Evidence for Journal of Chemistry) 41, 114-118. Suche nach dem kleinsten Enzym. Nachr. 33

Chem. Tech. Lab., 802-806. 10355223.5 tential biocatalysts - substrate and inhibi- Patents Wessjohann, L. A., Scheid, G., Bornscheuer, tor studies on p-hydroxybenzoate oligo- Buske, A., Adam, G., Häring, A., Kekulé, A. U., Henke, E., Kuit, W. & Orru, R. V. A. Epo - prenyl transferase from E. coli. University Arzneimittel enthalten Derviate von Tetra - thilone-Synthesebausteine IV: Unsymme - of Halle-Wittenberg, Department of Che - chinolin. Brazilian Patent PI 0211026-1. trisch substituierte Acyloine und Acyloin - mistry, 22 / 09 / 2003. derivate und Verfahren zu deren Herstel- Feußner, I., Hornung, E., Krüger, C. & Por - lung. DE 0010134172A1 Mieke Toorneman: (-)-Myrtenol based trig- zel, A. Verfahren zur Herstellung von kon- ger system for the controlled formation of jugierten mehrfach ungesättigten Fett säu - Wessjohann, L. A., Schmitt, M., Allwohn, J., enediynes. Vrije Universiteit Amsterdam, ren mit mindestens drei Doppelbindungen Umbricht, G. & Braun, H.-J. Hair dyes and Fakulteit Exakte Wetenschappen 30/10/2003. in Pflanzen. DE 10308850.4 method for dying hair with 1,2,4-trihydr oxy- naphthalene glycosides and glycosidases. DE Wessjohann, L. A., Braun, H.-J., Oberson, S. 10205500 & Umbricht, G. Two-component hair dye compositions including triacetoxy-naph- Doctoral Theses thalene derivatives and a hydrolase. DE Marco Aurelio Dessoy: Synthesis and enzy- 10226061 matic coupling of prenyldiphosphates and benzoates. University of Halle-Witten berg, Wessjohann, L. A., Eichelberger, U. & Tran Department of Chemistry, 10/07/2003. Thi Phuong, Thao Neue Makrocyclen zur Behandlung von Krebserkrankungen. DE Michael Fulhorst: Prenyltransferases as po - 34

Publications and Other Scientific Activities 2004

Publications gicidal cyclopentenones from Hygro pho - Seipold, L., Gerlach, G. & Wessjohann, L. A Bräuer, L., Brandt, W. & Wessjohann, L. A. rus species (Basidiomycetes). new type of floral oil from Malpighia coc- Mo deling of the E. coli - 4-hydroxybenzoic Phytochemistry 65, 1061-1071. cigera (Mal pighiadeae) and chemical consi- acid oligoprenyl-transferase (ubiA-transfe- derations on the evolution of oil flowers. rase) and cha racterization of potential Micskei, K., Hajdu, C., Wessjohann, L., Mercs, Chemistry & Biodiversity 1, 1519-1528. active sites. J. Mol. Model. 10, 317-327. L., Kiss-Szikszai, A. & Patonay, T. Enantiose - lective reduction of prochiral ketones by Trinh Thi Thuy, Kamperdick, C., Pham Thy Brandt, W., Dessoy, M. A., Fulhorst, M., Gao, chromium(II) amino acid complexes. Ninh, Trinh Phuong Lien, Tran Thi Phuong W., Zenk, M. H. & Wessjohann, L. A. A pro- Tetra hedron: Asymm. 15, 1735-1744. Thao & Tran Van Sung. Immunosuppressive posed mechanism for the reductive ring auronol glycosides from Artocarpus tonki- opening of the cyclodiphosphate MEcPP, a Myint Myint Khine, Franke, K., Arnold, N., nensis. Die Pharmazie 59, 297-300. crucial transforma tion in the new DXP/ Porzel, A., Schmidt, J. & Wessjohann, L. A MEP-pathway to isoprenoids based on new cardenolide from the roots of Strep - Trinh Thi Thuy, Tran Van Sung & Adam, G. modeling studies and feeding ex periments. tocaulon to men tosum. Fi to te rapia 75, Study on chemical constituents of Vietna - ChemBioChem 5, 311-323. 779-781. mese Mi cro melum hirsutum. Vietnam. J. Chem. 42, 177-181. Franke, K., Nasher, A. K. & Schmidt, J. Con- Nguyen Thi Hoang Anh, Tran Van Sung & stituents of Jatropha unicostata. Biochem. Wess johann, L. Further constituents from von Roepenack-Lahaye, E., Degenkolb, T., Syst. Ecol. 32, 219-220. Ophiopo gon japonicus. Vietnam. J. Chem. Zerjeski, M., Franz, M., Roth, U., Wess jo - 42, 261 - 264. hann, L., Schmidt, J., Scheel, D. & Clemens, Frick, S., Chitty, J. A., Kramell, R., Schmidt, J., S. Profiling of Arabidopsis secondary meta- Allen, R. S., Larkin, P. J. & Kutchan, T. M.Trans- Page, J., Hause, G., Wenyun Gao, Raschke, bolites by capillary liquid chromatography formation of opium poppy (Papaver somni- M., Schmidt, J., Zenk, M. H. & Kutchan, T. M. coupled to electrospray ionization qua- ferum L.) with antisense berberine bridge Functional analysis of the final steps of the drupole time-of flight mass spectrometry. enzyme gene (anti-bbe) via somatic em - 1-deoxy-D-xylulose 5-phosphate (DXP) Plant Physiol. 134, 548-559. bryogenesis re sults in an altered ratio of pathway to isoprenoids in plants using vi- alkaloids in latex but not in roots. Trans - rus-induced gene si lencing. Plant Physiol. Wessjohann, L., Wild, H. & Schrekker, H. genic Research 13, 607-613. 134, 1401-1413. Chromium-mediated aldol and homoaldol reactions on solid support directed to - Galm, U., Dessoy, M. A., Schmidt, J., Shu- Poeaknapo, C., Fisinger, U., Zenk, M. H. & wards an iterative polyol strategy. Tetra he - Ming Li, Wessjohann, L. A. & Heide, L. In Schmidt, J. Evaluation of the mass spectro- dron Lett. 45, 9073-9078. vitro and in vivo production of new ami- metric fragmentation of codeine and mor- nocoumarins by a combined biochemical, phine after 13C-isotope biosynthetic labe- Westermann, B, Diedrichs, N., Krelaus, R., ge netic and synthetic ap proach. Chemistry ling. Phytoche mistry. 65, 1413-1420. Walter, A. & Gedrath, I. Diastereoselective & Biology 11, 173-183. synthesis of homologous bicyclic lactams- Poeaknapo, C., Schmidt, J., Brandsch, M., potential building blocks for peptide mi - Hans, J., Brandt, W. & Vogt, T. Site-directed Dräger, B. & Zenk, M. H. Endogenous for- mics. Tetrahedron Lett. 45, 5983-5986. mutagenesis and protein 3D-homology mation of morphine in human cells. Proc. modelling suggest a catalytic mechanism Natl. Acad. Sci. USA, 101, 14091-14096. Zakharova, S., Fulhorst, M., Luczak, L. & for UDP-glucose-dependent betanidin 5- Wessjohann, L. A. Synthesis, inhibitory and O-glucosyltransferase from Dorothean - Scheid, G., Kuit, W., Ruijter, E., Orru, R. V. A, activation properties of prenyldiphosphate thus bellidiformis. Plant J. 39, 319-333. Henke, E., Bornscheuer, U. & Wessjohann, mimics for aromatic prenylations with L. A new route to protected acyloins and ubiA-prenyl transferase. Arkivoc 13, 79- Hirata, K., Poeaknapo, C., Schmidt, J. & Zenk, their enzymatic resolution with lipases. 96. M. H. 1,2-Dehydroreticuline synthase, the Eur. J. Org. Chem. 5, 1063-1074. branch point enzyme opening the morphi- Zhu, M., Ruijter, E. & Wessjohann, L. New nan biosynthetic pathway. Phytochemistry Scheid, G., Ruijter, E., Konarzycka-Bessler, scavenger resin for the reversible linking 65, 1039-1046. M., Born scheuer, U.T. & Wessjohann, L. Syn - and mo noprotection of functionalized aro- thesis and resolution of a key building matic alde hydes. Org. Lett. 6, 3921-3924. Horvath,G., Wessjohann, L., Guissez, Y., Bie - block for epo th ilones: A comparison of baut, E., Caubergs, R. J. & Horemans, N. asymmetric synthesis, chemical and enzy- Books and Book Chapters Seeds of grapes of Vitis vinfera var. matic resolution. Tetra hedron Asymm. 15, Huneck, S., Lumbsch, H. T., Porzel, A. & Alphonse Lavallée (Royal) - a possible 2861-2869. Schmidt, J. Die Verteilung von Flechten in - model tissue for studying tocotrienol bio- haltsstoffen in Lecanora muralis und Leci - synthesis. Acta Hortic. 652, 415-424. Schneider, P. H., Schrekker, H. S., Silveira, C. dea inops und die Ab hängigkeit der C., Wessjohann, L. A. & Braga, A. L. First ge - Usninsäure-Konzentration vom Substrat Huneck, S., Feige, G. B. & Schmidt, J. Che - neration of cysteine- and methionine-de - und von den Jahreszeiten bei Lecanora mie von Cladonia furcata und Cladonia rived oxazoli dine and thiazolidine ligands muralis. In: Contri butions to Lichenology. rangiformis. He r zogia 17, 51-58. for palladium-catalyzed asymmetric allyla- Festschrift in Ho nour of Hannes Hertel tions. Eur. J. Org. Chem. 12, 2715-2722. (Döbbeler, P. & Ram bold, G., eds.) Bibl. Krelaus, R. & Westermann, B. Preparation Lichenol. 88, 211-221. of pep tide-like bicyclic lactams via a se - Schrekker, H. S., Micskei, K., Hajdu, C., Pa - quential Ugi reaction-olefin metathesis ap - tonay, T., de Bolster, M. W. G. & Wessjohann, Lendeckel, U., Bukowska, A., Lättig, J. H. & proach. Tetra he dron Lett. 45, 5987-5990. L. A. Involvement of an oxidation-reducti- Brandt, W. Alanyl-Aminopeptidase in hu - on equilibrium in chromium-mediated man T cells: Structure and function. In: Lübken, T., Schmidt, J., Porzel, A., Arnold, N. enantioselective Nozaki-Hiyama reactions. Ami no pepti da ses in Biology and Disease & Wessjohann, L. Hygrophorones A-G: Fun - Adv. Synth. Catal. 346, 731-736. (Hop per, N. M. & Len deckel, U., eds.) 35

Kluwer Academic/Plenum Pub lis hers, bioactive 4-oxo-2-alkenoic fatty acids from Nuhn, P. & Wessjohann, L. A. Natur stoff- New York, pp 210-227. Hygrophorus eburneus. Z. Naturforsch. B. chemie - mikrobielle, pflanzliche und tieri- Publications in Press Rodrigues, O. E. D., Perottoni, J., Paixao, M. sche Natur stoffe. 4th edition, S. Hirzel Abbas, M., Neuhaus, C., Krebs, B. & Wes - W., Zeni, G., Lobato, L. P., Braga, A. L., Ro - Verlag, Stuttgart (Germany). ter mann, B. Synthesis of g-amino acids via cha, J. B. T. & Emanuelli, T. Renal and hepatic catalytic asymmetric Homo-Mannich reac- ALA-D activity and selected oxidative Wessjohann, L. A. & Ruijter, E. Strategies tions. Synlett. stress parameters of rats exposed to inor- for total and diversity-oriented synthesis ganic mercury and organoselenium com- of Na tural product (-like) macrocycles. In: Braga, A. L., Lüdtke, D. S., Wessjohann, L. A., pounds. Fd. Chem. Toxic. 42, 17-28. Top. Curr. Chem. Vol. 243 (J. H. Mulzer, Paixão, M. W. & Schneider, P. H. A chiral di- ed.), Springer, Heidelberg. sulfide derived from (R)-cysteine in the Ruijter, E., Schültingkemper, H. & Wessjo - enantioselective addition of diethylzinc to hann, L. A. Highly substituted tetrahydro - Patents aldehydes: Loading effect and asymmetric pyrones from hetero-diels-alder reac tions Lübken, T., Arnold, N., Wessjohann, L., Fa. amplification. Synthesis. of 2-alkenals with stereochemical inducti- Morphochem. Hygrophorone und deren on from chiral dienes. J. Org. Chem. Derivate. German Patent 10 2004 015566.6. Braga, A. L., Alves, E. F., Silveira, C. C., Zeni, G., Appelt, H. R. & Wessjohann, L. A. A new Wessjohann, L.A. & Ruijter, E. Macrocycles Wessjohann, L., Eichelberger, U., Tran Thi cysteine derived ligand as catalyst for the rapidly produced by multiple multicompo- Phuong, T. Neue Makrocyclen zur Behand - addition of diethylzinc to aldehydes: The nent reactions including bifunctional buil- lung von Krebserkrankungen. International importance of a "free" sulfide site for ding blocks (MiBs). Molecular Diversity 9. Patent PCT/EP 2004/013451. enantioselectivity. Synthesis. Wessjohann, L. A., Ruijter, E., Garcia-Rivera, Doctoral Theses Brandt, W. & Wessjohann, L. A.The functio- D. & Brandt, W. What can a chemist learn Henri Stephan Schrekker: Selective orga - nal role of selenocysteine (Sec) in the ca - from nature s macrocycles? - A brief, con- nic reactions promoted by chromium(II) talysis mechanism of large thioredoxin re- ceptual view. Molecular Diversity 9. ions. Vrije Universiteit Amsterdam, ductases: Proposition of a swapping cataly- 20/02/2004. tic triad including a Sec-His-Glu state. Books and Book Chapters in Press ChemBioChem. 6. Mrestani-Klaus, C., Faust, J., Golbik, R., Lars Seipold: Blütenöle - Chemische Analy - Brandt, W., Wrenger, S., Reinhold, D. & se, Biosyn these und Betrachtungen zur Nguyen Thi Hong Van, Nguyen Thi Hoang Neubert, K. De tection of PPII-helix-like Ent stehung von Ölblumen. University of Anh, Tran Van Sung, Franke, K. & Wessjo - structural features in short proline-contai- Halle-Wittenberg, Department of Che - hann, L. Stil bene ferulic acide and its deri- ning peptide inhibitors of the cell-surface mistry, 24/05/2004. vatives from the roots of Angelica sinensis. protease dipeptidyl peptidase IV. In: Pep ti - Vietnam. J. Chem. 42. des 2004 - Proceedings of the 3rd Inter na - Diploma These tional and 28th European Peptid Sym po - Axel Teichert: Fungitoxische Inhaltsstoffe Teichert, A., Lübken, T., Schmidt, J., Porzel, sium, Prag. aus Fruchtkörpern der Gattung Hygro - A., Arnold, N. & Wessjohann, L. Unusual pho rus (Basidiomycetes). University of Leip-

Department of Bioorganic Chemistry 36

Department of Stress and Developmental Biology Head: Dierk Scheel, Professor Secretary: Ruth Laue until March 2004, afterwards Susanne Berlin

Department of Stress and Developmental Biology 37

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

The basis for those processes is the ability of plants to perceive environmental factors and initiate signal transduction networks that modify gene expression patterns. The investigation of the molecular mechanisms underlying this course of events is the main topic of the Department of Stress and Developmental Bio logy.

Plant pathogens play a major role in biotic stress. The work of several research groups of the department focuses on the analysis of recognition, signal transduction and gene activation processes in plant-pathogen interactions. The work on abiotic environmental factors centers around metal homeostasis in plants, using hyperaccumulating model organisms.

Plant responses to biotic and abiotic environmental factors finally result in altered patterns of proteins and metabolites. In order to be able to directly monitor such alterations, mass spectrometry-based methods have been established allowing the comprehensive profiling of proteins and secondary metabolites. Both me thods are also being employed for biochemical phenotyping of mutants. 38

Research Group: Molecular Communication in Plant-Pathogen Interactions Head: Wolfgang Knogge

Many phytopathogenic microorganisms colonize the intercellular spaces of their host plants. This region is relatively poor in nutrients and in order to optimize their life style pathogens therefore needed to develop strategies that aim at improving the nutrient supply by the host cells. To prevent just this, plants on their part evolved mechanisms that enable the efficient recognition of invaders as the prerequisite for their rejection. These communication processes are crucial for the outcome of an interaction. They are mediated by membrane-loca lized or intracellular receptors in the plant, which are the targets of pathogen-secreted mole cules, frequently of proteinaceous nature. As the consequence of these protein-protein interactions two scenarios can be envisaged. The plant metabolism is redirected in favor of the pathogen and disease ensues. Alternatively, upon recognition of the pathogen through secreted compounds the plant defense is induced leading to the expression of plant resistance. Furthermore, in particular in compatible interactions pathogen receptors may be involved that sense stimuli of plant origin and, hence, allow the adaptation of the pathogen physiology to the specific environment of the host.

To identify pathogen genes whose pro- the host plant, one of these virulence factors, NIP1, Group Members ducts are involved in the communica - becomes a specific recognition signal that serves as Barbara Degner tion with the host and, hence, play a the trigger of plant resistance reactions. A specific Technician role during pathogenesis, mutagenesis NIP1-binding site was detected on membranes Claudia Mönchmeier strategies can be applied. Loss-of-func - from resistant and susceptible barley cultivars as Diploma Student since September 2004 tion mutations in genes whose products well as from other cereals. This indicates that the Sylvia Siersleben contribute to optimal pathogenesis (vi - NIP1 receptor is not encoded by the Rrs1 gene PhD Student since April 2004 ru lence genes) or that are even essen - and that (an) additional component(s) are required Marina Wibe tial for microbial penetration and deve- for resistance-related signal transduction. Diploma Student since April 2004 lopment in planta (pathogenicity genes) can be presumed to generate a pheno- Recently, two different mutagenesis approaches, in - type that deviates more or less obviously from the sertion mutagenesis and promoter trapping, led to wild type situation. Isolation and detailed molecular the identification of several fungal pathogenicity analysis of the affected genes and their protein pro- and virulence genes. The deduced functions of the ducts will lead to the unraveling of the processes that are important for the interaction. An alternative strategy aims at identifying the proteins that are secreted by the pathogen ("secretome"). These proteins are either required for the synthesis of mi crobial extracellular structures or candidates for factors involved in the interaction with the host.

Rhynchosporium secalis, the causal agent of a leaf spot disease on several grasses, is of particular economic importance as a pathogen of barley. After pe netration of host leaves the fungus grows in the extracellular region beneath the leaf cuticle and the epidermal cells of susceptible plants. During early stages of pathogenesis epidermal cells collapse, whereas necrotic lesions only become visible du - ring later stages when the mesophyll cells are affec- Schematic depiction of the communication between R. secalis ted as well. Small proteins that are secreted by the and barley. (1) NIP1, NIP2, NIP3; (2) transcription factor; (3) permease; (4) P450 protein; (5) histidine protein kinase; (6) fungus are involved in the expression of disease intramembrane protease. (C) cuticle, (CW) cell wall, (H) fun- symptoms, as was shown using fungal knock-out gal hypha, (N) nucleus, (PC) plant cytoplasm, (PM) plasma membrane, (T) tonoplast, (V) vacuole. mutants. In the presence of resistance gene Rrs1 in 39

gene products, although biochemically not yet terpreted as indicating an impor- confirmed, suggest their role in various pathoge- tant role in microbial adaptation to Collaborators nic processes. Thus, a protein with sequence ho - their respective host plants. In this James Brown mology to a class of transcription factors is li kely context the question arises as to John Innes Centre, Norwich, UK to control fungal genes that are only ex pressed the existence and na ture of (a) Barbara Howlett University of Melbourne, Australia during fungal growth in the plant. A g-aminobuty- plant factor(s) that may affect fun- Bruce McDonald ric acid/ami no acid permease is supposed to me - gal development through these en - Swiss Federal Institute of Technology, diate fungal uptake of specific plant nutrients, zyme sys tems. Finally, a gene was Zurich, Switzerland where as a P450 protein may be involved in the identified that encodes an intra- Dirk Warnecke University of Hamburg, Germany oxidation of substrate with an as yet unknown membrane protease of the rhom- function. One of the genes encodes a histidine boid type. These enzymes ca ta lyze protein kinase. These enzymes are part of intra- the release of ex tracellular signals from trans- cellular information processing systems that link membrane precursor proteins. However, neither sig nals from the environment with specific cellu- the identity of the sig nal generated by R. secalis lar adaptive processes. Al though the im mediate nor its target or the role of "regulated intramem- function of most of these enzymes is not known, brane proteolysis" in fungus-plant interactions in their high number in phytopathogenic fungi is in - general are known to date. 40

Research Group: Cellular Signaling Heads: Dierk Scheel & Justin Lee

Group Members The sessile lifestyle of plants compels the development of a wide spectrum of defense reactions to counteract and deter potential pathogens, insects and parasites. Our research Reetta Ahlfors emphasis is on plant defense signal transduction pathways - in non-host plant- pa thogen Guest Scientist until April 2003 interactions as well as the so-called gene-for-gene type of disease resistance. In addition, Gerit Bethke Diploma Student since February 2004 infection strategies of Cuscuta sp., a parasitic plant, are also being investigated. Mandy Birschwilks Postdoctoral Position since April 2003 Jutta Elster The interaction of parsley with the as a cytoplasmic anchor for MAPK prior to elici- Technician soybean pathogen, Phytophthora tor stimulus. Phosphorylation of MAPK by MKK Anja Halbauer Diploma Student since April 2004 sojae, constitutes the main model for leads to its release from a MAPK-containing pro- Franziska Handmann studies of plant non-host disease resi- tein complex, possibly allowing the activated PhD Student since March 2003 stance in our group. This form of resi- MAPKs to interact with their substrates.The spa- Jan Heise stance is comparable to the basal or tial redistribution into the nucleus indicates that Postdoctoral Position since March 2004 innate immunity of animal systems. some of its substrates are likely to be nuclear Nora Köster-Eiserfunke Pep-13, an oligopeptide derived from a proteins, presumably factors that control gene Diploma Student since February 2004 P. sojae cell wall transglutaminase, ser- transcription. Transient expression of dominant Sylvia Krüger Technician ves as a pathogen-associated mo - inactive versions of PcMPK3a and/or PcMPK6 lecular pattern (PAMP) to trigger de - and/or PcMKK5 interfered with the expression Violetta Macioszek Postdoctoral Position until April 2004 fense reactions in parsley cells. Upon of co-transfected pathogenesis-related (PR1 or Kai Naumann recognition of the Pep-13 elicitor by a PR2) gene promoter-reporter constructs. Expe - Postdoctoral Position since November 2004 plasma membrane-localized receptor, riments with inactive mutants of other, non-Pep- Anja Nickstadt a network of cellular signaling events 13-activated, MAPK or MKK do not show this PhD Student is triggered that culminates in the ap - effect. In contrast, the inhibitor of the NADPH Jason Rudd propriate defense response. The sig - oxidase, DPI, did not affect expression of this par- Postdoctoral Position until February 2004 naling events elucidated in the parsley- ticular subset of defense genes but blocked phy- Christel Rülke Pep-13 system include ion fluxes at toalexin accumulation in parsley. Hence, the oxi- Technician the plasma membrane, activation of dative burst and the MAPK cascade are required Rita Schlichting mitogen-activated protein kinases in the induction of distinct subsets of defense PhD Student (MAPK) and the accumulation of sig - genes by Pep-13. Claudia Spielau Diploma Student until September naling molecules such as reactive oxy- 2004, afterwards PhD Student gen species, phospholipids and jasmo- A constitutive active version of PcMKK5 was suf- Christiane Unger nates. In particular, studies with the ficient to activate PcMPK3/6 and drive PR-gene Diploma Student until January 2004 calcium-sensitive reporter, aqueorin, expression in parsley. He terolo gous expression Ivy Widjaja PhD Student since April 2004 have shown that calcium influx, with a of this constitutive active PcMKK5 in transgenic characteristic signature of sustained Arabidop sis, similarly led to ac ti vation of the Ara - cytosolic Ca2+ increase, is an early and key step bi dop sis MPK3/6 that is essential for all other downstream events or thologs and the studied. Thus, molecular characterization of the mRNA ac cumu la - calcium channel involved will be pur sued. tion of many de - fense-related ge - Parsley genes encoding MAPKs and their up - nes. Binding sites stream MAPK kinases (MKK) have been isolated. for WRKY trans - The genes designated PcMPK3a/b, PcMPK6 and cription factors are PcMKK5 have been identified as being the Pep- over-represented 13-activated components in the parsley MAPK in the promoters cascade. Immunofluorescence studies indicate of these ge nes;

that the activated MAPKs are rapidly transloca- suggesting that ac - Elicitation of parsley cells leads to translocation of ted in to the nucleus while the upstream PcMKK5 tiva ted MPK3/6 the activated MAPK (PcMPK6, upper panel) into is seen to accumulate at the perinuclear region might target WRKY the nucleus while the corresponding upstream MAP kinase kinase (PcMKK5, lower panel) remains upon elicitation. It appears that MKK might serve factors directly or cytoplasmic or is localized in perinuclear region de - spite activation. Right panels are the corresponding DAPI staining (blue) to visualize nuclei position. 41

indirectly. We are currently attempting to identify ferences/similarities between non- Collaborators MAPK substrates using different biochemical, host and the ge ne -for-gene type of proteomic and molecular techniques. The com- disease resistance, the analysis will Thomas Boller pleted genome sequence and the extensive post- be extended to transgenic plants University of Basel, Switzerland genomic tools available for the model plant, Ara - that ex press a Phy toph tho ra sojae Jeff Dangl University of North Carolina, Chapel Hill, bidopsis thaliana, render it the plant of choice for Necrosis-Inducing Pro tein (NIP), USA such studies. which represents a ge neral elicitor Jerome Giraudat Institut des Sciences du Végétal, CNRS, of defense in the non-host Ara bi - Gif-sûr-Yvette, France Monospecific antibodies to AtMPK3, AtMPK6 dopsis plant. Minna Haapalainen, Suvi and AtMPK4 have been generated. As observed Taira, Martin Romantschuk for the parsley-Pep-13 system, activation of The interaction of the holoparasi- University of Helsinki, Finland Bettina Hause MAPKs, e.g. by ozone treatment, also led to their tic plant, Cuscuta sp., with Ara bi - Leibniz Institute of Plant Biochemistry, nuclear translocation. Furthermore, diverse gene- dopsis was established for study- Halle, Germany ral elicitors and abiotic stresses were shown to ing angiosperm parasites. Not unli- Heribert Hirt University of Vienna, Austria activate these kinases. The convergence of va - ke other phytopathological sys - Jonathan Jones rious stress factors at the same MAPK activation tems, successful invasion requires The Sainsbury Laboratory, Norwich, UK also introduces the question on how specificity in the transfer of nutrients and water Sophien Kamoun signaling is maintained. One possibility is the exis - from the host to the parasite; in Ohio State University, Wooster, USA tence of defined protein complexes (signalo - this case via a multicellular hausto- Birgit Kersten Max Delbrück Center for Molecular somes). Indeed, size-exclusion chromatography rial structure. Specific xylem-mo - Medicine, Berlin, Germany showed that MAPKs in protein extracts, albeit bile or phloem-mobile dyes de - Chris Lamb only a small portion, migrate as larger molecular mon stra t ed a functional connec - John Innes Centre, Norwich, UK complexes.Transgenic plants with tandem-affinity tion of the invading haustorium to John Mundy University of Copenhagen, Denmark tagged versions of the elicitor-activated MAPKs the vascular system of the host. Teun Munnik have been generated to facilitate the isolation of The high efficiency of nu trient University of Amsterdam, The Netherlands such protein complexes. transfer to the parasite can even- Karsten Niehaus tually result in death of the host. University of Bielefeld, Germany We have established high-resolution two-dimen- In com patibility between Cus cuta Thorsten Nürnberger University of Tübingen, Germany sional PAGE analysis of proteins from transgenic and its host has occasionally been Jane Parker, Imre Somssich, Arabidopsis plants with inducible expression of observed, where the functional Joachim Uhrig the bacterial avirulence gene, AvrRPM , in the haustoria is terminated at various Max Planck Institute of Plant Breeding 1 Research, Cologne, Germany presence or absence of the corresponding resi- developmental stages. A screen in Jose Sanchez-Serrano stance RPM1 vari ous ecotypes and defense-re - Autonomous University, Madrid, Spain gene. Pro tein lated mu tants has been initiated to spots that are dif- elucidate the components responsible for incom- ferentially ac cu - patibility. mulated are identified via MALDI- TOF spectrometry. In order to un - derstand the dif -

Two-dimensional polyacrylamide gel electrophore- sis analysis of proteins in transgenic Arabidopsis plants that are induced to express the bacterial AvrRPM1 gene. The ar rows point to protein spots Cuscuta twining around the stem of an Ara bidop sis inflo- that are more abundant in these plants as compa- rescence. The close-up shows an en larged area where the red to isogenic plants lackingg the RPM1 resistance haus torial region is visible. Inset is a transverse section illu- gene, that is required for percep tion/transducing strating the development of a haustorium towards the vas- the AvrRPM1 signal. cular systems of the host. 42

Research Group: Induced Pathogen Defense Heads: Dierk Scheel & Sabine Rosahl

The oomycete, Phytophthora infestans, is the causal agent of late blight disease, one of the most deva- stating diseases of potato. To characterize defense mechanisms against this pa thogen, we are studying the interaction of P. infestans with its host plant potato and with the non-host plant Arabidopsis thaliana. For potato, our major interest is the analysis of pathogen recognition, signal transduction and characterization of the defense reactions. On the other hand, we are identifying and characterizing mutants of the non-host plant Arabidopsis, which are compromised in non-host resistance to P. infestans.

The Phytophthora sojae-derived oli- oxophytodienoic acid levels were achieved in Group Members gopeptide elicitor, Pep-13, originally plants with reduced expression of genes enco- identified as an inducer of plant de- ding allene oxide cyclase and 12-oxophytodienoic Simone Altmann Diploma Student since October 2004 fense in parsley and shown to act as a acid re ductase. Lennart Eschen-Lippold pathogen-associated molecular pat- Diploma Student since January 2004 tern (PAMP) in evoking innate im- Oxylipins synthesized via the lipoxygenase path- Vincentius A. Halim PhD Student mune responses, triggers defense re - way possess antimicrobial activity and can act as sponses in potato, such as the oxida- signaling molecules. We tested 49 different oxyli- Jörn Landtag PhD Student tive burst, defense gene activation and pins for their inhibitory effects against P. infestans Grit Rothe the accumulation of the signaling com- in an in vitro assay. 18 were able to significantly Postdoctoral Position pound salicylic acid. Interestingly, deri- reduce mycelial growth of the oomycete or inhi- Angelika Weinel Technician vatives of Pep-13 show similar elicitor bit spore germination. Among those were pro- activity in parsley and potato, sugge- ducts of the 9-lipoxygenase pathway, which also Lore Westphal Postdoctoral Position sting a receptor-mediated induction accumulate in potato leaves in response to Pep- Dorothea Wolf of defense responses in potato similar 13, as well as after pathogen infection. To analyze Diploma Student since June 2004 to that observed in parsley. However, the role of 9-lipoxygenase-derived oxylipins for unlike in parsley, infiltration of Pep-13 the response to pathogens, transgenic potato into leaves leads to hypersensitive-like lesions in plants expressing RNA interference constructs potato, which show characteristics of pro gram - targeted at the pathogen-induced 9-lipoxygenase med cell death. Analyses of transgenic potato and the 9-divinyl ether synthase were generated. plants, which are unable to accumulate salicylic Significantly reduced levels of 9-lipoxygenase pro- acid, due to the expression of a salicylate hy - ducts and of the divinyl ethers colneleic and col- droxylase gene, suggest that a subset of the Pep- nelenic acids were observed in several transgenic 13-induced de fense responses in potato leaves is plants. Lipid peroxidation during the hypersensi - salicylate-de pendent. tive cell death was not significantly altered in the absence of 9-lipoxygenase activity. Real time PCR In addition to the local accumulation of salicylic assays to determine pathogen biomass also did acid, infiltration of Pep-13 or the phytopathoge- not reveal significant differences in the growth of nic bacteria, Pseudomonas syringae pv. maculico- P. infestans on a susceptible host plant with re - la, induces increases in the levels of the signaling duced levels of 9-lipoxygenase activity. Whether compounds, jasmonic acid, as well as its biosyn- oxylipins from solanaceous plants like potato can thetic precursor, 12-oxophytodienoic acid. Interestingly, 12-oxophytodienoic acid, but not jasmonic acid, accumulates also systemically, which correlates with the expression of systemic acquired resistance. To analyze the role of jasmonic and 12-oxophytodienoic acid for both local and syste mic defense responses, transgenic plants were ge nerated, which express RNA interfe- Oxylipins inhibit germination of Phytophthora infestans in vi tro. When added to a zoospore suspension of P. infestans, rence constructs targeted at several genes enco- oxygenated polyunsaturated fatty acids are able to efficient- ding en zymes of the jasmonic acid biosynthetic ly inhibit germination (right panel) compared to untreated controls (left panel). pathway. Significant reduction in jasmonic and 12- 43

accumulation of autofluorescent Collaborators material and hypersensitive cell Rod Casey death. Analysis of more than 40 John Innes Institute Norwich, UK Arabidopsis ecotypes revealed Ivo Feussner that, despite differences in their University of Göttingen, Germany hypersensitive response, none al - Markus Frank BASF Plant Science Ludwigshafen, lows significant growth or multi- Germany plication of the oomycete. The Joëlle Fournier, Marie-Thé Arabidopsis mutant, pen2, which rèse Esquerré-Tugayé Université Paul Sabatier Toulouse, France was isolated by Volker Lipka and Christiane Gebhardt, Paul Elicitation of the oxidative burst by Pep-13 is dependent on Paul Schulze-Lefert (MPI Köln) as Schulze-Lefert salicylic acid. The oligopeptide elicitor Pep-13 induces the allowing enhanced penetration of Max Planck Institute for Breeding Research Köln, Germany accumulation of hydrogen peroxide in potato plants as vi - the non-host pathogen barley sualized by staining with diaminobenzidine (left). However, Mats Hamberg in plants unable to accumulate the signaling compound sali- pow dery mildew (Blumeria gram- Karolinska Institute Stockholm, Sweden cylic acid, accumulation of reactive oxygen species is im - nis f. sp. hordei), shows a higher paired (right). Volker Lipka number of penetrated cells and University of Tübingen, Germany in creased cell death in response William Albert Rensink The Institute for Genomic Research also be effective against pathogens in other plants to infection by P. infestans. Seeds (TIGR), Rockville, USA is being tested by transferring the respective of the pen2 mutant were mutage- Jose Sanchez-Serrano genes from potato into A. thaliana. nized and approximately 70,000 Universidad Autonoma de Madrid, Spain lines were screened for altera - Claus Wasternack Leibniz Institute of Plant Biochemistry, Arabidopsis is not a host plant for P. infestans. tions in their response to infec - Halle, Germany Therefore, the identification of genes involved in tion by P. infestans. Several mu - this non-host resistance should elucidate mecha- tants were isolated, which show increased hyper- nisms of defense against the infectious agent of sensitive cell death, or which allow enhanced late blight disease. P. infestans spores germinate penetration of epidermal cells as well as en - on Arabidopsis leaves and attempt to penetrate hanced growth of P. infestans. Mapping of the cells which react with the deposition of callose, position of the affected genes is in progress.

Arabidopsis mutants impaired in non-host resistance to Phytophthora infestans. In contrast to wild type Arabidopsis (left leaf), two mutants react to infection with P. infes tans with en - hanced cell death, indicated by stronger staining with trypan blue (middle and right leaves). 44

Research Group: Metal Homeostasis Head: Stephan Clemens

Plants - like all other organisms - are able to tightly regulate the intracellular concentration and the distribution of essential metals such as zinc and copper. Also, the cytosolic concentrations of non- essential toxic metals (e.g. cadmium, lead) have to be minimized. Some plant species (so-called metallophytes) can tolerate otherwise toxic concentra tions and grow on metal-contaminated soil. The projects of our group are aiming at elucidating the molecular mechanisms underlying plant metal homeostasis, metal tole rance and metal hyperaccumulation. Plants under investigation are Arabidopsis thaliana and its close relative Arabidopsis halleri, which grows, for instance, on medieval mining sites in the Harz mountains. In addition, we are working with Schizosaccharomyces pombe as a cellular model for metal homeostasis.

Group Members Comparative transcript profiling with The identified genes are still responsive to eleva- A. thaliana and A. halleri has led to ted metal levels in A. halleri albeit at concentra- Annegret Bährecke Diploma Student until March 2004, after- im portant insights into the molecular tions orders of magnitude higher than in A. thalia- wards PhD Student mechanisms of metal hyperaccumula- na. This led us to hypothesize that one molecular Frank Bretschneider tion. A. halleri is a hyperaccumulator mechanism behind metal hyperaccumulation might Diploma Student until September 2004 growing on metal-contaminated sites be a de-regulation of metal deficiency re sponses. Sophie Bundtzen Diploma Student until November 2004 in Central Europe. We found (pre- Additional microarray experiments with the "full- Sandra Franz vious cDNA-AFLP re sults) that the genome" ATH1 chip confirmed this pattern. It is Diploma Student until September 2004 similarity of coding se quences of A. an emerging picture that plant adaptation to ex - Thomas Fritsche thaliana and A. halleri is high enough treme environments is mostly a regulatory pheno- Diploma Student until September 2004, afterwards PhD Student to use A. thaliana GeneChips also for menon and our microarray data lend further sup- Marina Häußler A. halleri.We performed a number of port to this. It is now of paramount importance to Technician microarray experiments to find con- elucidate the molecular ba sis for this altered regu- Emiko Harada stitutive differences in the root tran- lation. We therefore cloned the promoters of se - Postdoctoral Position until January 2004, Guest Scientist since September 2004 scriptomes of the two species and to veral metal homeostasis genes from both Ara - Claudia Simm identify transcriptional changes in re - bidopsis species and generated various reporter PhD Student until March 2004 sponse to various doses of both es - lines. Preliminary results indicate that the A. halleri Pierre Tennstedt sential and non-essential metal ions. promoters retain most of their activity in A. tha- PhD Student The constitutive comparison revealed liana. The immediate goal of these experiments is Aleksandra Trampczynska Postdoctoral Position since December se veral genes that are expressed at a the identification of cis elements responsible for 2003 much higher level in A. halleri. In te - the de-regulated ex pres sion in A. halleri. Tino Unthan res tingly, some of these encode Diploma Student until September 2004 known metal homeostasis factors. The results of our GeneChip studies are also of Christoph Veß PhD Student until June 2003 The two genes showing highest ex - general importance.They demonstrate the feasibi- Susan Wassersleben pression in A. halleri roots relative to lity of comparative microarray analysis of clo sely PhD Student until August 2004 A. thaliana roots encode a nicotian- related species and their potential to become an Michael Weber amine synthase (NAS) and a putative extremely valuable tool for the molecular elucida- PhD student Zn2+ uptake system (ZIP). The signifi- tion of biodiversity. cantly high er activity of these and other genes in - volved in metal homeostasis could be confirmed. The involvement of nicotianamine in plant metal A. halleri roots also show higher NAS protein le - homeostasis and Zn hyperaccumulation is studied vels. Fur ther more, we developed a CapLC-ESI- in more detail. All known isoforms from A. halleri QTOF-MS-based nicotianamine (NA) analysis pro- were cloned and functionally characte rized. Re gu - cedure and found higher NA levels in roots of A. lation of the genes in response to varying supplies halleri. Ex pression of a NAS in a Zn2+-hypersensi - of essential micronutrients is being analyzed by tive Schi zo saccharomyces pombe mutant demon- real-time PCR. In addition, field samples from sites strated that formation of NA can confer Zn2+ tole- in the Harz mountains that differ in degree of me - rance. Taken together, these observations impli - tal contamination are under investigation. Trans - cate NA in plant Zn homeostasis and in the genic approaches are aiming at modulating NA hyperaccumula tion of Zn by A. halleri. levels in both Arabidopsis species. 45

Microarray data on the metal responses provided cad1-3 mutant with AtPCS2-HA. Collaborators clues as to the modes of toxicity and helped iden- A homozygous insertion line for tifying common as well as species-specific tran- AtPCS2 is being studied. Compa - Sebastian Fettig University of Bayreuth, Germany scriptional changes that will guide functional stu- ra tive studies both in vitro and in Rüdiger Hell dies. In particular, "core" response genes, genes re - vivo on the activities of PCS1 and 2 University of Heidelberg, Germany sponsive specifically to excess of one particular from A. thaliana and A. halleri were Ute Krämer Max Planck Insitute metal and putative signal transduction compo- initiated. of Molecular Plant Physiology, Golm, nents were identified. A collection of T-DNA in - Germany sertion lines is being established for putative sig nal A second major question we are Youngsook Lee POSTECH, Pohang, South Korea transduction components that, based on their me - addressing concerning PC syntha- Andreas Meyer tal responsiveness, are hypothesized to be in - ses is their mode of activation. University of Heidelberg, Germany volved in the transcriptional regulation of genes They are constitutively expressed Dietrich H. Nies upon changes in external metal concentrations. yet PC synthesis occurs only in the University of Halle, Germany Homozygous lines are being characterized with presence of elevated metal ion le - Julian I. Schroeder University of California San Diego, La Jolla, respect to tolerance, accumulation and transcrip- vels. Several metal ions are known USA tome changes. to elicit PC synthesis both in vivo Sébastien Thomine and in vitro. Peptide scanning Institute des Sciences du Végétal, CNRS Gif-sûr-Yvette, France The synthesis of phytochelatins (PCs), small me tal- experiments helped to identify pu - Nathalie Verbruggen binding peptides derived from glutathione, is es - tative metal binding sites. Also, we Université de Bruxelles, Brussels, Belgium sential for the detoxification of Cd and As ions in have identified differences in the Kuo-Chen Yeh Institute of BioAgricultural Sciences, plants, some fungi and certain animals. A number re sponse range among various PC Academia Sinica, Taipeh, Taiwan of questions concerning the function of the ubiqui- synthases and are now trying to tously present PC synthase encoding genes (PCS establish the structure-function relationships genes) remain unanswered. Foremost, it is not underlying these differences. This might help to clear, how the sporadic need to detoxify non- gain insight into the interaction of metal ions and essential metal ions could have provided the se - metal-ligand complexes with proteins - a research lective pressure to account for the presence of topic that has come into focus with the realization PCS genes throughout the plant kingdom (with that aberrant metal-binding might be involved in the exception of some mosses). We found two the pathogenesis of various human disorders, lines of evidence that might help to solve this my - including Alzheimer s and prion diseases. stery: (i) studies on two PC-deficient A. thaliana mutants clearly showed a key role of PC forma tion S. pombe cells are being used as tools for the func - also in buffering excess es sential metal ions; (ii) tional characterization of plant proteins and in or - characterization of one of the bacterial proteins der to learn more about the mechanisms of cellu- with similarity to the PCS Pfam domain, alr0975 lar metal homeostasis in an easily workable sys - from Nostoc spec., revealed that this protein cata- tem, that can serve as a model for PC-forming lyzes only the first formal step in PC synthesis, the cells. The analysis of putative metal homeostasis cleavage of the C-terminal glycine from glutathione. factors in S. pombe was extended. We construc- This finding provides a clue as to the evolutionary ted and characterized additional S. pombe knock- origin of PC synthases and points to possible addi- out mutants - especially for the transporters of tional roles in glutathione metabolism, e.g. the cata- the ZIP family. In order to help understand the ho - bolism of GS-conjugates. meo static function of the sole metallothionein in S. pombe and to develop an additional tool for the With respect to additional PCS functions, we are analysis of the "metal status" of S. pombe cells, a also interested in the physiological role of the se - "metal array" was established that covers known cond PCS gene in Arabidopsis, AtPCS2. We show- metal homeostasis genes as well as general stress ed partial complementation of the PC-deficient response and sulphur metabolism genes. 46

Interdepartmental Research Group: Metabolite Profiling in Arabidopsis and Crop Plants Heads: Stephan Clemens, Jürgen Schmidt, Ludger Wessjohann & Dierk Scheel

Our project is aiming at contributing to the "post-genomic" analysis of the model organism Arabidopsis thaliana and of crop plants by establishing an extensive profiling of metabolites and, in addition, of proteins and peptides. These profiles are used for the detection and identification of early stress responses. Also, they are valuable tools for the analysis of various developmental and stress-induced changes as well as for the biochemical phenotyping of mutants and the exploration of natural diversity. Exemplary biotic and abiotic stresses un der investigation are pathogen attack and toxic metal exposure, respectively. The metabo lite profiling is to be used also for crop plant biotechnology.

The main emphasis of the group has near relationship between compound abundance Group Members been on developing a novel and com- and signal strength of the CapLC-ESI-Qq-TOF-MS Christoph Boettcher petitive metabolite profiling platform analysis was found for most of the signals tested. Postdoctoral Position since August 2004 exploiting the potential of hybrid mass Thomas Degenkolb Postdoctoral Position until June 2003 spectrometers developed in the past The raw data generated by CapLC-ESI-Qq-TOF-MS Mathias Franz few years. The chemical diversity of are very complex and require deconvolution. Major Student the metabolome necessitates the use efforts therefore had to be devoted to developing Kerstin Körber-Ferl of different analytical techniques to co - and optimizing tools for the automatic data decon- Technician until March 2004 ver the wide range of polarities found volution and data processing. These achievements Edda v. Roepenack-Lahaye Postdoctoral Position among the metabolites occurring in a greatly increased the depth of analysis and the Udo Roth cell. Thus, we decided to complement throughput. Reliability of data was found to be Postdoctoral Position until September existing GC-MS approa ches with a comparable to manual data processing. On average 2003 profiling scheme using li quid chroma- about 1,400 signals are detected in leaf extracts Michaela Winkler Technician since August 2004 tography (LC). We established an ex - and about 800 signals in root extracts. Given the Michael Zerjeski perimental system based on ca pillary limited overlap between root and leaf metabolites Student LC coupled to electrospray io nization a total of about 2,000 different signals can be de - quadrupole time-of-flight mass spec- tected in extracts from Arabidopsis plants grown trometry (CapLC-ESI-Qq-TOF). Major question under control conditions. Most likely the numbers marks concerning this approach were the robust- will be significantly higher once the analysis is ex - ness of the analysis, its reproducibility, and its suita- tended to other tissues such as flowers, to different bility for high-throughput and quantitative analysis. developmental stages and to plants exposed to en - We performed an extensive evaluation of the pro- vironmental stimuli. cedures we had developed earlier. Our results de - monstrated, that CapLC-ESI-Qq-TOF-MS princi - Thus, this technique is suitable for detecting subtle pally meets all the necessary criteria for powerful metabolic changes in response to environmental metabolic analysis. Retention times of compounds stimuli or metabolic differences dependent on ge - eluting from the CapLC column were found to netic background. This ability was proven already in show only little variation. Evaluation of the mass ac - pilot studies on Arabidopsis mutants affected in curacy showed that the target value in the range of metabolic enzymes and on Arabidopsis stress re - five ppm error can be reached for many signals sponses. Furthermore, identification of metabolites even in a complex mixture of compounds allowing, is feasible. Electrospray as a soft ionization method as discussed below, the calculation of elemental combined with the enhanced resolution and mass compositions and thereby the tentative identifica - accuracy of the TOF instrument and tandem MS al - tion of compounds. Robustness of the analysis was low to determine the elemental composition and il lustrated by the fact that the average deviation for to detect characteristic in-source fragments. Struc - a range of mass signals was no higher than eleven tural information can also be obtained in a targeted percent when the same extract was run several way by performing tandem MS analysis of mass sig - times. Reproducibility of retention time and mass nals showing interesting changes in abundance. In accuracy provided a sufficient basis for the unequi- conclusion, the principles of separation and mass vocal detection and identification of mass signals. analysis of this technique together with its sensiti- Quantification is possible because a satisfactory li - vity and resolution greatly expand the range of me - 47

tabolic profiling.The development of tools for the the establishment of data bases Collaborators automatic data extraction and processing make it and the ease and speed of data Jeff Dangl an invaluable tool for addressing a myriad of bio- analysis. Also, the fo cus of the me - University of North Carolina, Chapel Hill, logical questions. tabolite profiling has re cently been USA extended to rapeseed biotechno- Gunhild Leck band Norddeutsche Pflanzenzucht To date, the profiling in its current form has been logy within the frame of the Hans Georg Lembke KG, Hohenlieth, Germany used mostly for studying metabolic changes in re - GABI 2 program. Volker Lipka sponse to excess metals and for elucidating pa - University of Tübingen Germany thogen resistance by analyzing an Arabidopsis The profiling of Arabidopsis pro- Paul Schulze-Lefert mutant with partial loss of non-host resistance. teins is based on large-format high- Max Planck Institute for Breeding Research Köln, Germany Extensive analysis of different alleles has allowed resolution two-dimensional gel elec- to identify metabolic changes caused by loss-of- trophoresis (2D-PAGE). Following the improve- function of the gene in question. ment of image analysis this technique has mostly been ap plied to identifying proteins that are sho- Besides trying to answer biological questions con- wing changes in abundance upon expression of siderable effort is still invested in further im pro - bacterial avirulence genes in Arabidopsis leaves. ving the profiling platform with regard to scope, 48

Publications and Other Scientific Activities 2003

Publications plant steroid carrier. J. Mol. Biol. 325, 123- ringae pathovars and related pathogens. In: Clemens, S. & Simm, C. Schizo sac cha ro my - 133. De ve lopments in Plant Pathology 10 -(Ia ces pombe as a model for metal ho me os - Steiner-Lange, S., Fischer, A., Böttcher, A., cobellis, N.S. et al., eds.) Kluwer Aca de mic ta sis in plant cells: The phytochelatin- Rouhara, I., Liedgens, H., Schmelzer, E. & Pub lishers Dordrecht, pp. 165-173. dependent pathway is the main Cd detoxi- Knogge, W. Differential defense reactions fication mechanism. New Phytol. 159, 323- in leaf tissue of barley in response to infec- Neumann, D. Silicon in plants. In: Progress 330. tion by Rhynchosporium secalis and to in Molecular and Subcellular Biology 33 treatment with a fungal avirulence gene (Müller, W. E. G., ed.) Springer-Verlag Ber lin Genger, R. K., Brown, A. H. D., Knogge, W., product. Mol. Plant-Microbe Interact. 16, Hei del berg, pp. 149-160. Nesbitt, K. & Burdon, J. J. Develop ment of 893-902. SCAR markers linked to a scald resistance Patents gene derived from wild barley. Euphytica van't Slot, K. A. E, van den Burg, H. A., Lipka, V., Schulze-Lefert, P., Scheel, D., Ro - 134, 149-159. Kloks, C. P. A. M., Hilbers, C. W., Knogge, sahl, S. & Landtag, J. Polynucleotides enco- W., & Papavoine, C. H. M. Solution struc - ding a beta glucosidase. Göbel, C., Feussner, I. & Rosahl, S. Lipid ture of the fungal disease resistance-trig- European Patent EP 2003000743388 peroxidation during the hypersensitive re - gering protein NIP1 from the fungus Rhyn - International Patent PCT/EP03/02315 sponse in potato in the absence of 9-lip- chosporium secalis shows a novel b-sheet Canadian Patent 2,475,572 oxygenases. J. Biol. Chem. 278, 52834- fold. J. Biol. Chem. 278, 45730-45736. Australian Patent 3227037. 52840. Varet, A., Hause, B., Hause, G., Scheel, D. & Diploma These Kroj, T., Rudd, J. J., Nürnberger, T., Gäbler, Y., Lee, J. The Arabidopsis NHL3 gene encodes Engelhardt, Stefan: Struktur- und Funk - Lee, J. & Scheel, D. Mitogen-activated pro- a plasma membrane protein and its overex- tionsanalyse des bakteriellen Effektor - tein kinases play an essential role in oxida- pression correlates with increased resis - proteins HrpZ aus Pseudomonas syringae tive burst-independent expression of pa - tance to Pseudomonas sy ringae pv. tomato pv. phaseolicola. University of Halle-Wit - thogenesis-related genes in parsley. J. Biol. DC3000. Plant Physiol. 132, 2023-2033. tenberg, Department of Biochemistry/ Chem. 278, 2256-2264. Biotechnology, 17/07/2003. Wysocki, R., Clemens, S., Augustyniak, D., Legatzki, A., Franke, S., Lucke, S., Hoffmann, Golik, P., Maciaszczyk, E., Tamás, M. J. & Habilitations T., Anton, A., Neumann, D. & Nies, D. H. Dziadkowiec, D. Metalloid tolerance based Clemens, Stephan: Molekulare Mechanis - First step towards a quantitative model on phytochelatins is not functionally equi- men der Aufnahme, Detoxifizierung und describing Czc-mediated heavy metal resi- valent to the arsenite transporter Acr3p. Akkumulation von Metallen. Univer sity of stance in Ralstonia metallidurans. Biochem. Biophys. Res. Commun. 304, 293- Halle-Wittenberg, Faculty of Ma thematics, Biodegra dation 14, 153-168. 300. Natural Sciences and Techno logy, Depar - tment of Biology, 22/10/2003. Maier, T., Yu, C., Küllertz, G. & Clemens, S. Books and Book chapters Localization and functional characteriza - Clemens, S., Simm, C. & Maier, T. Heavy Nürnberger, Thorsten: Signalerkennung- tion of metal-binding sites in phytochelatin metal binding proteins and peptides. In: und Signalumwandlungsprozesse bei der synthases. Planta 218, 300-308. Bio polymers, Vol. 8 Polyamides and com- Auslösung nichtkultivar-spezifischer Pa - plex pro teinaceous materials II (Stein bü - thogenabwehrmechanismen in Pflanzen. Markovic-Housley, Z., Degano, M., Lamba, D., chel, A. & Fahnestock, S. R., eds.) Wiley- University of Halle-Wittenberg, Faculty of von Roepenack-Lahaye, E., Cle mens, S., Su - VCH, Wein heim, pp. 255-288. Mathematics, Natural Sciences and Tech - sani, M., Ferreira, F., Scheiner, O. & Brei ten - nology, Department of Bioche mistry/Bio - eder, H. Crystal structure of a hypoallergenic Lee, J. & Nürnberger, T. Is pore formation technology, 16/09/2003. isoform of the major birch pollen allergen activity of HrpZ required for defence acti- Bet v1 and its likely biological function as a vation in plant cells? Pseudomonas sy -

Department of Stress and Developmental Biology 49

Publications and Other Scientific Activities 2004

Publications tabolites by capillary liquid chromatogra- University of Halle-Wittenberg, Depart - Ahlfors, R., Macioszek, V., Rudd, J., Brosché, phy coupled to electrospray io nization ment of Bio che mistry/Biotech nology, M., Schlichting, R., Scheel, D., & Kan gasa jär- quadrupole time-of-flight mass spec tro - 20/09/2004. vi, J. Stress hormone-independent activa - metry. Plant Physiol. 134, 548-559. tion and nuclear translocation of mitogen- Fritsche, Thomas: Funktionelle Cha rak teri - activated protein ki nases (MAPKs) in Ara - Weber, M., Harada, E., Vess, C., von Roe - sierung der MAP-Kinase MPK11 aus Ara - bidopsis thaliana during ozone exposure. penack-Lahaye, E. & Clemens, S. Compara - bi dopsis thaliana. University of Halle-Wit - Plant J. 40, 512-522. tive microarray analysis of Ara bi dop sis tenberg, Department of Bioche mi stry/ thaliana and Arabidopsis halleri roots Biotechnology, 15/09/2004. Gao, L. L., Knogge, W., Delp, G., Smith, F. A. identifies nicotianamine synthase, a ZIP & Smith, S. E. Expression patterns of de - transporter and other genes as potential Köster-Eiserfunke, Nora: Analyse von pu - fense-related genes in different types of metal hyperaccumulation factors. Plant J. tativen Phosphoproteinen in transgenen arbuscular mycorrhizal (AM) development 37, 269-281. Arabidopsis-Pflanzen mit erhöhter MAP - in wild-type and mycorrhiza-defective mu - Kinase-3 und-6 Aktivität. University of Hal - tant of tomato. Mol. Plant-Microbe Publications in Press le-Wittenberg, Department of Bioche mis - Interact. 17, 1114-1125. Li, C.-M., Haapalainen, M., Lee, J., Nürn ber - try/Biotechnology, 09/12/2004. ger, T., Romantschuk, M. & Taira, S. Harpin Halim V., Hunger A., Macioszek V., Landgraf of Pseudomonas syringae pv. phaseolicola Müller, Claudia: Struktur- und Funk tions a - P., Nürnberger T., Scheel D., & Rosahl S. The harbors a protein binding site. Mol. Plant- nalyse einer mikrobiellen Transglutami nase oligopeptide elicitor Pep-13 induces salicy- Microbe Interact. aus Phytophthora sojae. University of lic acid-dependent and -independent de - Halle-Wittenberg, Department of Bio che - fense reactions in potato. Physiol. Mol. Books and Book chapters mis try/ Biotechnology, 22/01/2004. Plant Pathol. 64, 311-318. Scheel, D. & Nürnberger, T. Signal transduction in plant defense responses. In: Fungal Schwedt, Anne: Molekulare Charak terisie - Harada, E., von Roepenack-Lahaye, E. & Disease Resistance in Plants. Bio che mistry, rung einer Rezeptorkinase aus Ara - Clemens, S. A cyanobacterial protein with Molecular Biology, and Genetic Engi - bidopsis thaliana. University of Halle- similarity to phytochelatin synthases cata- neering. (Punja, Z. K., ed.) The Haworth Wittenberg, Department of Bioche - lyzes the conversion of glutathione to g- Press, Inc., Binghamton, U.S.A., pp. 1-30. mistry/Biotech no lo gy, 09/07/2004. glutamylcysteine and lacks phytochelatin synthase activity. Phy to chemistry 65, 3179- Rosahl, S. & Feussner, I. Oxylipins. In: Plant Spielau, Claudia: Grundlagen der Analyse 3185. Lipids: Biology, Utilisation and Mani pu - von Proteinkinase-Komplexen mittels Tan - lation. (Murphy, D., ed) Blackwell Pub - dem Affinity Purification (TAP) aus trans- Lee, J., Rudd, J. J., Macioszek, V. K. & Scheel, lishing, Ox ford, pp. 329-354. genen Arabidopsis thaliana. Univer sity of D. Dynamic changes in the localization of Halle-Wittenberg, Department of Bio che - MAP kinase cascade components con trol - Diploma Theses mistry/Biotechnology, 15/09/2004. ling pathogenesis-related (PR) gene ex - Bährecke, Annegret: Funktionelle Cha rak - pression during innate immunity in parsley. terisierung von ZIP-Transportern aus Ara - Unger, Christiane: Beteiligung von Lipi den J. Biol. Chem. 279, 22440-22448. bidopsis thaliana und Arabidopsis halleri. an der Pep-13-initiierten Signal trans duk - Uni versity of Halle-Wittenberg, Depart - tion in Petersilie. University of Halle-Wit - Nickstadt, A., Thomma, B. P. H. J., Feuss ner, ment of Biology, 19/05/2004. tenberg, Department of Bio logy, 12/01/2004. I., Kangasjärvi, J., Zeier, J., Loeffler, C., Scheel, D. & Berger, S. The jasmonate-in - Bethke, Gerit: Analyse der Protein-Pro - Unthan, Tino: Untersuchungen zur Rolle der Nicotianamin-Synthese für die Me tall - sensitive mutant jin1 shows increased re - tein-Interaktionen zwischen Kompo nen - sistance to biotrophic as well as ne cro - ten aus Arabidopsis MAP-Kinase-Kas ka - hyperakkumulation. University of Halle- trophic pathogens. Mol. Plant Pathol. 5, den mittels Fluoreszenz-Reso nanz-Ener - Wittenberg, Department of Bio chemis try/ 425-434. gietransfer (FRET). University of Halle- Biotechnology, 20/09/2004. Wittenberg, Department of Biology, Schürch, S., Linde, C. C., Knogge, W., Jack - 13/12/2004. Doctoral Theses son, L. F. & McDonald, B. A. Molecular po - Hofmann, Ingo: Erzeugung, Isolation und pulation genetic analysis differentiates two Bretschneider, Frank: Zellbiochemie der Charakterisierung von Suppressor mu - virulence mechanisms of the fungal aviru- Phytochelatinsynthase aus Schizosac cha ro - tanten für Transcriptional Gene Silen cing myces pombe. University of Halle-Wit ten - in Arabidopsis thaliana. University of lence gene NIP1. Mol. Plant-Microbe Interact. 17, 1103-1113. berg, Department of Biochemistry/Bio - Halle-Wittenberg, Department of Bio - technology, 15/09/2004. chemistry/ Biotechnology, 03/11/2004. Takeda, S., Tadele, Z., Hofmann, I., Probst A. V.,Angelis, K. J., Kaya, H., Araki, T., Mengiste, Eschen-Lippold, Lennart: Entwicklung ei - Hunger, Astrid: Die Rolle von Jasmonat, T., Mittelsten-Scheid, O., Shibahara, K., ner Realtime-PCR basierten Methode zur 12-Oxophytodiensäure (OPDA) und Sali - Scheel D. & Paszkowski, J. BRU1, a novel Bestimmung des Wachstums von Phy toph - cylat bei der induzierten Resistenz in Kar - link between responses to DNA damage thora infestans auf Kartoffel pflan zen mit toffel. University of Halle-Witten berg, De - and epigenetic gene silencing in Arabi - unterschiedlicher Resistenz. University of part ment of Biochemistry/Biotechnology, dopsis. Genes & Development 18, 782- Halle-Wittenberg, Depart ment of Biology, 20/12/2004. 793. 06/12/2004. Simm, Claudia: Das Metallothionein Zym von Roepenack-Lahaye, E., Degenkolb, T., 1 Franz, Sandra: Charakterisierung von Ge - aus Schizosaccharamyces pombe - Unter - Zerjeski, M., Franz, M., Roth, U., Wess jo - nen, die in Microarray-Experimenten als pu - su chungen zur Funktion des Me - hann, L., Schmidt, J., Scheel, D. & Cle mens, tative Metallakkumulations fakto ren in tallothineins in einem phytochelatin-bil- S. Profiling of Arabidopsis secondary me - Arabi dopsis halleri identifiziert wurden. denden Orga nis mus. University of Hal le- 50

Department of Secondary Metabolism Head: Dieter Strack, Professor Secretary: Heidemarie Stolz until October 2004, afterwards Ildikó Birkás

Department of Secondary Metabolism 51

ork of the department is concerned with the molecular regulation Wof plant secondary metabolism, evolution of the enzymes involved and the role of secondary products in interactions of plants with their en - vironment.

The work on metabolic regulation includes isolation and characterization of the enzymes and the encoding genes, focusing on transferases. We currently investigate malate and choline hydroxycinnamoyltransferases as well as several hydroxycinnamate glucosyltransferases from Arabidopsis thaliana and oilseed rape (Brassica napus) as well as flavonoid and betanidin glucosyltransferases from betacyanin-accumulating plants or flavonoid methyltransferases from the ice plant (Mesem bryan themum crystallinum).

The aim of the work on glucosyl- and hydroxycinnamoyltransferases is to elucidate their evolutionary origin and structure-function relations to pre- dict substrate specificity. Glucosyltransferases involved in betacyanin biosynthesis are considered to be oligophyletic and originate from different clusters of flavonoid glucosyltransferases. A new class of methyltransferases was identified, whose substrate specificities can be modulated by metal cations and N-terminal deletions. Hydro xy cinna moyl transfe ra ses, which are dependent on b-acetal esters as acyldonors, are vacuolar serine carb - oxypeptidase-like (SCPL) proteins as found for the enzyme involved in the formation of si napoylmalate in Arabidopsis.This plant harbors a gene family encoding 53 SCPL proteins, of which 21 cluster to a distinct group of SCPL acyltransferases.The existence of such acyltransferases proves a new concept in the regulation of plant secondary metabolism.

Special emphasis is also placed on programs focusing on the molecular interactions of plants with arbuscular mycorrhizal fungi. The work of two groups is concerned with fungus-induced alterations in plant isoprenoid metabolism, in particular carotenoid biosynthesis and degradation, accompanied by a dramatic reorganization of plastid population in arbuscule-harboring root cells. Another main objective is the analysis of the role of phy- tohormones, in particular jasmonates, in development and functional main- tenance of mycorrhizal symbiosis. It was found that jasmonates obviously play a crucial role in the establishment of arbuscular my - corrhizas. These studies are supported by comprehensive analysis of primary and secondary metabolites (metabolite profiling) in wild type and transgenic mycorrhizal plants. 52

Research Group: Molecular Physiology of Mycorrhiza Head: Michael H. Walter

Arbuscular mycorrhizas are mutualistic symbiotic associations between a small number of specialized fungi and the roots of most terrestrial plants. The fungi deliver mineral nutrients to the plant and recei- ve carbohydrates in return. The focus of the group is plant isoprenoid biosynthesis and its molecular regulation in mycorrhizal roots. Starting from an analysis of mycorrhiza-specific apocarotenoids specific gene targets in the plastidial non-mevalonate methylerythritol phosphate (MEP) pathway and in carotenoid cleavage reactions have been selected. In the first step of the MEP pathway, catalyzed by -deoxy-D-xylulose 5-phosphate synthase (DXS), an ancient gene duplication has evolved into specia- 1 lized DXS genes for the generation of primary and secondary isoprenoids. The my corrhiza-regulated DXS2 gene has duplicated again recently and occurs as a tandem repeat in the genome of the model legume Medicago truncatula.

Two classes of apocarotenoids (ca - and by isolating a spruce DXS1 cDNA clone. Group Members ro tenoid cleavage products) accu- Future work will try to trace back this gene Daniela Floß mulate in the roots of various plants duplication even further in evolution. PhD Student since April 2004 upon colonization by mycorrhizal fun -

Joachim Hans gi. These are linear C14 mycorradicin Current experiments on the angiosperm Me di ca - Postdoctoral Position until October 2004 and glycosylated C13 cyclohexenone go truncatula have identified an additional gene Kerstin Manke derivatives, which are predicted to duplication of DXS2 in this system. The duplicate Technician originate from a common C40-ca - paralogous genes MtDXS2-1 and MtDXS2-2 are rotenoid or xanthophyll precursor. almost identical in coding regions, similar in in - Collaborators The first step of plastidial carote- tron and proximal promoter regions and occur as Harro Bouwmeester noid precursor biosynthesis in the a tandem repeat.These characteristics argue for a Plant Research International, Wageningen, The Netherlands MEP pathway is catalyzed by 1-de - recent duplication event. Both genes are ac ti va - Jörg Degenhardt oxy-D-xylulose 5-phosphate syn tha se ted during mycorrhization. Possible early sub- Max Planck Institute for Chemical Ecology, Jena, Germany (DXS). We have discovered dupli - functionalization features (alterations in ex pres - Thomas Fester, Bettina cate genes for DXS in Medi cago sion) of MtDXS2-2 are currently being investiga- Hause, Willibald Schliemann, truncatula, which are distantly related. An RNAi strategy has been devised to Jürgen Schmidt Leibniz Institute of Plant Biochemistry, ted and differentially regulated. The down-regulate both genes concomi tantly and Halle, Germany general concept of dedica- Franziska Krajinski ted roles of DXS in the University of Hannover, Germany 1 biosynthesis of primary iso prenoids (e.g. leaf ca ro - te noids) and of DXS2 in secondary isoprenoid formation holds for all plants investigated so far. The only exception is Arabidopsis thaliana, which has two DXS1 co pies but no DXS2 copy. Ex amples for DXS2-correlated biosynthesis of se condary isoprenoids are the mo no terpenes of pep - per mint leaf trichomes, the steviol diterpenes of Ste via rebaudiana leaves and the terpenoid indole alkaloids of Catharanthus roseus cells. The gene duplication and subfunctionalization leading to this diversification must be very old, since we have shown that duplicate DXS genes occur in gym - Changes in root color of Medicago truncatula plants upon colonization by mycorrhizal fungi nosperms by data base mi ning of ESTs due to the accumulation of apocarotenoids, one of them being the "yellow pigment" (mycorradicin derivatives). The inset shows an RNA blot indicating elevated transcript levels of the biosynthetic enzyme MtDXS2 exclusively in mycorrhizal roots (R, M). 53

cre ate loss-of-function mutants devoid of apoca- DXS2 copy. rotenoid accumulation without interfering with The second step of the MEP pathway cata - biosynthesis of primary carotenoids in leaves.The lyzed by 1-deoxy-D-xylulose 5-phosphate re- RNAi-constructs are expressed in roots of Me - duc toisomerase (DXR) is or ga nized in a dicago truncatula after transformation via Agro - more simple fashion than DXS. At least in bacterium rhizogenes. Transformed roots sprea- maize, rice, tomato, peppermint and Catha - ding from the injection site of bacteria are recog- ranthus roseus only single genes have been nized by the fluorescent marker DsRed and can identified for DXR. A single maize DXR gene thus be fur ther propagated, while non-transgenic ex hibits elevated transcript levels in leaves as root parts are discarded. Preliminary re sults indi- well as in mycorrhizal roots. The generation cate a significantly lower concentra tion of mycor- of antibodies created from recombinant radicin de rivatives in mycorrhizal roots transge- maize DXR has allowed for detailed immuno- nic for the RNAi construct as compared to con- localization studies on maize DXR in arbus- trol mycorrhizal roots. In addition, MtDXS2-1 cule-containing mycorrhizal roots. The results promoter-GUS fusions and promoter deletion- demonstrate an extensive proliferation of GUS fusions are being created to eventually iden- DXR-containing plastids during mycorrhiza - tify promoter elements relevant to my corrhizal tion, which form a network of interconnec- regulation of plant genes. ted plastids (stromules) around the arbus - M. truncatula plant lets injected with Agro bac te rium rhizogenes cules in co lonized maize root cells. The DXR below the hy po cotyl develop new A recent gene duplication of DXS2 has occurred ac cumulation reaches its maximum at or trans genic hairy roots at the injection si te (a bo ve, plant to the right). also in the genome of African marigold (Tagetes slightly af ter the peak of arbuscule matura - Trans genic roots of M. truncatula erecta). Amplification of genomic fragments from tion and declines thereafter indicating a close flu o resce in red due to ex pres sing the fluorescent mar ker DsRed T. erecta DXS2 genes indicates very strong simi- correlation of (apo)carotenoid formation with larity of two TeDXS2 genes similar to the the arbuscular life cycle. MtDXS2 duplicates. Comparative analyses have identified the DXS2 duplicates in six out of seven Carotenoid cleavage constitutes a late step in apo - Tagetes species and cultivars. On ly T. tenuifolia carotenoid biosynthesis requiring regiospecific di - appears to lack the second gene copy. By lowe- oxygenases. A carotenoid cleavage enzyme (CCD) ring the stringency in PCR amplification experi- clone with specificity for the 9,10 (9',10') double ments a dis tantly related DXS1 fragment could bond creating C13 and C14 products has been isola- be amplified from this species but never a second ted from maize. Again, a single gene appears to be responsible for elevated transcript levels in leaves and in mycorrhizal roots. Ex pression of the maize CCD in E. coli strains engineered to produce carotenoids leads to a discoloration of bacterial colo- nies. However, product identification after incuba - tion of recombinant CCD with carotenoid substrates has not yet been accomplished.

Highly branched fungal arbuscule in a mycorrhizal maize root cell visualized by WGA-TRITC staining (red). Im - munolocalization of DXR (green) reveals strong prolifera - tion of plastids around the arbuscule. DNA in plant nucleus and fungal hyphae is shown in blue by DAPI staining. 54

Research Group: Cell Biology of Mycorrhiza Head: Bettina Hause

Plant hormones are believed to play a role in the establishment and development of sym- Group Members biotic interactions between plants and arbuscular mycorrhizal (AM) fungi. Jasmonates, Sabine Both known as regulators in plant response to biotic or abiotic stress, are good candidates for Trainee until November 2004 such a role. Therefore, the functional ana lysis of jasmonates during the interaction bet- Ana Maria Cencano ween Glomus intraradices and barrel medic (Medicago truncatula) is the main objective DAAD Fellow until November 2004 of our group. In a second project, the massive proliferation of plastids in AM colonized Thomas Fester Leader Junior Group root cortical cells is studied. Metabolic and cytological changes as well as respective Ulrike Hintsche/Huth molecular mechanisms will be elucidated. In a third project, we are analyzing the influen- Technician ce of epothilones on the structure of plant microtubules. Stanislav Isayenkov Postdoctoral Position until October 2004 Nina Kreißig A possible role of jasmonates in the control of the CaMV35S promoter leading to Student until June 2004 mycorrhizal interaction is indicated chimeric plants with wild-type shoots and trans- Sandra Lischewski by the following data: Jasmonic acid formed roots. In comparison to samples transfor- Trainee until March 2004 (JA), applied exogenously, promotes med with a control vector (35S::uidA), roots with Swanhild Lohse PhD Student colonization and development of my - suppressed expression of MtAOC1 ex hibited Cornelia Mrosk corrhizal structures, and the en do - lower JA levels and a remarkable delay in the pro- PhD Student since July 2004 genous JA level of mycorrhizal roots cess of colonization with G. intraradices. Both Daniel Peisker is remarkably higher than that of the mycorrhization rate, quantified by fungal Student until February 2004 non-mycorrhizal roots.The in crease rRNA, and the formation of arbuscules, analyzed Silke Pienkny Student until May 2003 of JA content in barley roots upon by the expression level of the AM-specific gene Sara Schaarschmidt mycorrhization is accompanied by MtPT4, were affected. In the future work, analy- PhD Student the expression of genes coding for ses of transcript and metabolite patterns by Rostand Tonleu Tonfack enzymes of JA biosynthesis and for cDNA microarrays (collaboration with H. Küster, Diploma Student since November 2004 jasmonate-induced proteins. Uni versity of Bielefeld) and metabolite profiling Carola Tretner Research Scientist (collaboration with W. Schliemann, IPB Halle), Gerlinde Waiblinger To analyze the function of JA during both in wild-type and transgenic roots, will provi- Technician until July 2004 the development of the AM symbio- de answers to the question, which processes of sis, we intend to perform functional mycorrhization are mediated by JA. analyses by modulating the JA content in mycorrhizal roots of M. Because higher levels of JA within mycorrhizal truncatula. Two cDNAs coding for roots could be caused by the higher sink func tion the allene oxide cyclase (AOC), the of such roots, transgenic tobacco (Nicotiana taba- en zyme performing the crucial step cum) plants specifically modulated in root su gar in JA biosynthesis, were isolated metabolism were studied as well. For this, tobacco from M. truncatula. The AOC pro- plants expressing a yeast-derived apoplast-di rec ted tein was localized to plastids and invertase under the control of an alcohol-inducible found to occur constitutively in all promoter (alc) were used. Soil-drenching with aqu - vascular tissues of M. truncatula. In e ous acetaldehyde solutions resulted in a rapid and leaves and roots, MtAOC1 is temporary induction of the alc gene ex pression expressed upon JA application. system exclusively in roots. In ad dition, the split- Enhanced expression accompanied root system allowed an activation restricted to the by elevated JA levels was also treated part of the root. In duc tion of the invertase observed during mycorrhization upon treatment with acetaldehyde led to a change with G. intraradices. A partial sup - in the ratio of suc rose to hexoses specifically wit- pression of MtAOC1 expression hin the roots. To include in vertases targeted to the Chimeric M. truncatula plant after root was achieved in roots following cytoplasm and vacuole in our analyses, suitable transformation with 35S::uidA. The plant transformation with Agrobacterium vectors were used for root transformation of to - was processed for GUS staining showing rhizogenes harboring the MtAOC bacco. All the change described, how ever, did not the transgenic part of the root in blue. 1 cDNA in antisense direction under lead to alterations in AM colonization according to 55

Invertase in situ staining of cross sec tions of alc::cwINV- to bacco root tips indicates a high invertase activity in all root cells after induction of the yeast-derived invertase by soil-drenching with 0.05 percent acetaldehyde (right side).Wa ter-treated control plants show only a weak purpur staining (left side). Bars represent 25 µm.

microscopical, metabolite and transcript analy- root cortical cells. The extent of ses. network formation, however, see- Collaborators Plastids in colonized root cortical cells are cha- med to be less pronounced when Frank van Breusegem University of Gent, Belgium racterized by an active carotenoid biosynthesis compared to tobacco. Fur ther Ivo Feussner, Katharina Pawlowski and by a strong proliferation close to fungal analyses re gard the characteriza- University of Göttingen, Germany structures. In the current project we have been tion, lo cali za tion and functional Philipp Franken looking for further changes in plastid metabo- analysis of molecular markers in- Institute of Vegetables and Ornamental Crops, Großbeeren, Germany lism, for a functional reason for the activation volved in processes like plastid Gerd Hause of carotenoid biosynthesis and for mechanisms division or plastid deformation. In University of Halle, Germany involved in changing plastid number and shape. the case of plastid division, we Helge Küster Transcript levels of the genes referring to the have chosen the plastid division University of Bielefeld, Germany main plastid biosynthetic pathways have been protein FtsZ for such an analysis. Thomas Roitsch University of Würzburg, Germany analyzed by "Electronic Northern" and real - First results from immunolocali- Uwe Sonnewald time RT-PCR. Increased transcript levels have zation of this protein suggest that Institute of Plant Genetics and Crop Plant been observed in particular for genes involved the division of plastids is connec- Research, Gatersleben, Germany Claus Wasternack, Otto Miersch, in fatty acid and asparagine biosynthesis. These ted to a specific phase of arbuscu- Willibald Schliemann, Ludger findings correlate with data from the research lar development. An antisense ap - Wessjohann Leibniz Institute of Plant Biochemistry, Halle, group Biochemistry of Mycorrhiza, indicating proach re gar ding this protein will Germany in creased levels of metabolites of the two res - reveal the functional significance Victor Wray pec -tive pathways. Regarding carotenoid bio- of plastid division for the AM German Research Centre for Biotechnology, Braunschweig, Germany synthesis, we are currently studying a possible symbiosis in general and for pla- connection of the activation of this pathway to stid biosynthetic metabolism in the accumulation of reactive oxygen species particular. Re garding a similar analysis of plastid (ROS) in AM roots. AM roots from transgenic deformation, we are currently searching for tobacco plants containing reduced levels of suitable proteins in volved in this process. catalase (cooperation with Frank van Breusegem, Uni versity of Gent) accumulate Our group is further interested in studying the increased amounts of apocarotenoids. A link influence of epothilones, macrocyclic lactones between ca rotenoid biosynthesis and the pre- from culture filtrates of the myxobacterium So - sence of ROS is fur ther suggested by the fin- rangium cellulosum, on the structure of plant ding that both processes occur predominantly mi crotubules (MTs). Stability and dynamics of in the phase of arbuscule degradation. MTs are influenced by natural compounds like Regarding the analysis of plas tid number and taxol, the most prominent example for inhibi- shape, we have analyzed M. truncatula after A. tors of MT depolymerization. Epothilone and rhizogenes-mediated root transformation using its derivatives are known as taxol-like microtu- a construct targeting the green fluorescent bular drugs in human medicine, but their effects protein to plastids. A significant proliferation of on plants are still unknown. We have analyzed this compartment was ob served in colonized the effect of Epo thilone D on the plant cell- cycle in cultivated cells.The treatment of toma- to cell suspension cultures resulted in a conti- nuous in crease of the mitotic index, visible al - ready after two hours. As shown by immuno-

cytological methods, abnormal spindles are for- Immunofluorescence images of med during metaphase leading to a random dis- tomato cells in metaphase showing the microtubular pattern tribution of chromosomes in the whole cell labelled with anti-a-tubulin an ti - body (green) and the distribu - without formation of a metaphase plate. The tion of chromosomes stained effects of Epo thilone D seem to be irreversible, with DAPI (blue) in non-treated cells (a) and cells treated with Confocal laser scanning mi cro graph of an arbuscule (Ni co tia na taba- because cells with an abnormal spindle could 1.5 µM Epothilone D for 8 h (b). cum colonized by Glomus in tra radices) after staining for reactive Bar represents 5 µm for both oxygen spe cies using dihydrorhodamine-123. Bar represents 20 µm. not be recovered after removal of the drug. micrographs. 56

Research Group: Biochemistry of Mycorrhiza Head: Willibald Schliemann

Besides transcriptomics and proteomics, metabolomics as a further functional genomic approach cha- racterizes a biological system at a given time. Our research focus is arbuscu lar mycorrhiza originating from the interaction of fungi (Glomeromycota) with the roots of the majority of plants. In a project of the DFG focus program 1084 Molecular Basics of Mycorrhizal Symbioses the pattern of primary and secondary metabolites of developing ar buscular mycorrhizal roots of Medicago truncatula colonized with Glomus intraradices are analyzed by metabolite profiling. The ultimate aim is the characterization of causal relationships between mycorrhiza-specific gene expression and metabolite profiles to assign processes relevant for mycorrhiza development and symbiotic functioning.

Group Members On the basis of metabolite profiling t ween the metabolites at different inoculation results of preliminary mycorrhiza - stages during mycorrhization (Pearson correla - Christian Ammer Research Scientist tion kinetics an extended mycor- tion and network analysis, cluster, principal com- rhization experiment was per - ponent and regression analysis). The re sults ob - Barbara Kolbe Technician formed including additional contained up to now refer to a general activation of trols with plants fully provided with mi tochondrial (tricarboxylic acid cycle) as well as Collaborators phos phate.This should allow to dif- plastid metabolism (lipid biosynthesis, N-assimila- Hermann Bothe, Ulrich Hildebrandt ferentiate between metabolite alte- tion) during mycorrhization, which correlates University of Cologne, Germany rations induced by phosphate only with corresponding increased transcript levels (in Bettina Hause, Thomas Fester, from those caused by mycorrhizal silico and real-time RT-PCR analyses-collabora - Alfred Baumert, Jürgen Schmidt, in teraction. Furthermore, additio- tion with Thomas Fester, IPB Halle). Furthermore, Martina Lerbs Leibniz Institute of Plant Biochemistry, nal harvest time points and paral- higher Pearson correlation coefficients of meta- Halle, Germany lels enable a more reliable statisti- bolite pairs in mycorrhizal root extracts (M) indi- Joachim Kopka Max Planck Institute of Molecular Plant cal evaluation of the metabolite da - cate a closer metabolic relationship among them- Physiology, Golm, Germany ta obtained by GC/MS, LC/ Pål Axel Olsson ESI-MS and DAD-RP-HPLC. University of Lund, Sweden For a more substantial me - tabolite identification, the Lloyd W. Sumner Samuel Roberts Noble Foundation, NIST MS-database was sup- Ardmore, USA plemented by the plant-specific MSRI database (collaboration with Joachim Kopka, MPI of Mole cular Plant Physiology, Golm). The large data sets are subjected to a detailed quantitative analysis requiring self-programmed tools for automated data processing and visualiza - tion.The utilized analytical techniques and bioinformational tools are the key elements of our metabolite profiling platform. In the course of preprocessing the raw data are a ligned, smoothed, averaged and graphically summarized for re cog ni - zing general trends. Qualitative and quantitative differences in the chromatograms are revealed in calculated differential plots. The quantitative (integrated) me ta bolite data are similarly preprocessed as the raw data. In subsequent steps the data are subjected to various statistical me thods Metabolite profiling platform: analytical methods and bioinformational tools to reveal correlations and interactions be - 57

selves than in non-mycorrhizal controls (NM). components. The accumulation of the fungus-specific palmit- The levels of sa - vaccenic acid (C16:1D11) can be detected as early ponins and es pe - as 14 days after inoculation (dai), whereas the cially isoflavo noids fungal trehalose and the mycorrhiza-induced apo- (malonylononin carotenoids (cyclohexenones, mycorradicin-de - as the main com - rived compounds and the complex "yellow pig- po nent) are slight- ment") are accumulating continuously during the ly higher in my - complete analysis period. The developed ratio cor rhizal than in plot for comparison of metabolite data from ave- non-my corrhi zal raged HPLC runs of different sample sets allows roots. The me ta - to detect wavelength-dependent differences be - bolite profiling ap - tween them immediately. The controls supplied proach is comple- Retention time-wa velength ratio plot of averaged root HPLC data with increased concentration of phosphate did mented by pre- of my cor rhi zal plants (M)/100 % phosphate controls (100 % P) not show any of these mycorrhiza-characteristic parative isola tion metabolite altera tions. How ever, they showed of secondary compounds and their structural elu- strong reduction or dis ap pearan ce of at least two cidation by MS and NMR.

HPLC profiles and accumulation of mycorrhiza-induced apocarotenoids MtCH1,2,3 - M. truncatula cyclohexenone derivative 1,2,3; MtApo1,2 - M. truncatula mycorradicin-derived compound 1,2 58

Research Group: Glycosyl- and Methyltransferases Head: Thomas Vogt

Modifications of plant natural products are performed by a variety of transferases with such prominent families like glucosyltransferases (UGTs) and methyltransferases (OMTs), which can share the same substrates and lead to the observed diversity of plant secondary metabolites. These enzymes may both solubilize and deactivate bioactive low-molecular weight compounds. Sequence identities cluster the UGTs as well as the OMTs according to regiospecificities rather than strict substrate preference. Predictions imply that both superfamilies of proteins have evolved oligophyletically as one of the primary adaptive mechanisms of plants to meet the changing environmental conditions in a timely and developmentally controlled manner. Recent developments have focused to elucidate the structural properties of both classes of enzymes with specific emphasis on substrate and position specificities as well as the reaction mechanisms.

Based on earlier work, our group Our second model system, the ice plant (Mes - Group Members started on the molecular physiology embryanthemum crystallinum), adapts to extre- Judith Hans of betalains, but lately as a result of me light intensities by the rapid accumulation of PhD Student until March 2004 the ongoing work, the major focus glycosylated and methylated flavonol and beta- Dagmar Knöfel has shifted towards two classes of cyanin conjugates in leaf epidermal layers. The fla- Technician modifying enzymes involved in the vonoid-methylating activity (PFOMT) is in volved Collaborators modification of the two classes of in the accumulation of a variety of conjugated Wolfgang Brandt, Jürgen plant natural products under inves- quercetagetin 6-3 -diOMe derivatives upon light Schmidt tigation, the betacyanins and the fla- induc tion. Based on sequence identities and sub- Leibniz Institute of Plant Biochemistry, vonoids. By detailed sequence analy- strate specificities, it defines a new subgroup of sis and substrate specificity studies cation-dependent OMTs, characterized by a of two UGTs, the betanidin broad substrate specificity, previously as sumed to UGT73A5 and UGT71F2 from Do - be involved only in the methylation of the lignin rotheanthus bellidiformis (Dbs), we precursor caffeoyl coenzyme A. Light-inducible were able to demonstrate an oligophyletic origin of the corresponding glucosyltransferase genes from different clusters of flavo - noid UGTs. Several lines of evidence, including cloning of highly homologous UGT73A5 and UGT71F1 sequences from red beet (Beta vulgaris), suggest that these position specific enzymes involved in betanidin glucosylation have derived from common ancestors, flavonoid and other hy - droxylated phenylpropanoid-modifying enzymes. Site-directed mutagenesis, performed with the heterologously expressed UGT73A5 indicated the importance of a highly conserved N-terminal histidine and a glutamate of the conserved UDP- glucose binding site to be catalytically essential. Ba sed on a crystal structure of the microbial UGT from Amycolatopsis orientalis, the first 3D- structure of UGT73A5 was modelled, and a new SN-1-like reaction mechanism proposed, which confirms the essential role of the N-terminal his - Predicted tertiary structure of UDP-glucose: be tanidin 5-O- tidine as the catalytic base. Docking studies also glucosyltransferase (UGT73A5) from Dorothe an thus belli- demonstrated the substrate preference of diformis with the docked ligands betanidin and UDP-gluco- UGT73A5 for betanidin. se. transcript accumulation and localization of the 59

en zyme in epidermal tissues correlates with its im portant role in phenylpropanoid accumulation.

Optimized heterologous expression resulted in high yields of recombinant PFOMT from E. coli, after purification used for subsequent crystalliza- 1 2 tion. Crystals were obtained under various preci- pitant conditions and several data sets re corded with resolutions down to 1.4 Å, in case of the recombinant PFOMT and 1.8 Å for its seleno- methionine-derivative, respectively. Both datasets were sufficient to result in a preliminary 3D-crys - 3 4 5 tal structure of this cation-dependent enzyme. Like in case of the UGT, the N-terminal part of Potential substrates of cation-dependent OMTs. 1, caffeoyl CoA; 2, quercetin; 3, caffeic acid; the enzyme is important for position specificity. 4, caffeoylglucose; 5, 3,4-dihydroxybenzoic acid; Compared to the (N-terminally trun cated) native plant protein, with eleven ami no acids missing, additional fluorescent flavonol 5-OMe derivati- the full length sequence showed an altered proves, never observed in the plant. Tests with rela- duct profile, when assayed with the endogenous ted cation-dependent OMTs revealed that not substrate quercetagetin. The profile of the re - only the ami no acid sequence but also that the combinant PFOMT-isoform, lacking tho se eleven nature of bivalent cation within the active site is amino acids is consistent with the observed pro- crucial for the position and substrate specificity duct profile in vivo, whereas the full length of this class of enzymes. recombinant enzyme catalyzes the formation of

Typical crystals of PFOMT obtained with polyethylene glycol-based precipitants. 60

Research Group: Hydroxycinnamic Acids Head: Dieter Strack

The work of our group is focused on biochemistry, genetics and molecular regulation of sinapate ester biosynthesis in oilseed rape (Brassica napus) and the model plant Ara bi dopsis with the aim to improve rapeseed quality by metabolic engineering.The sinapate esters sinapoylcholine (sinapine) and sinapoylglucose (SinGlc) are abundant compounds, known to accumulate in seeds of Brassicaceae plants. To produce the highly diverse pattern of metabolites, plants have evolved a wide array of enzymes to fulfill the need for driving plant secondary metabolism. SinGlc-dependent acyltransferases like SCT belong to this group of enzymes. They are homologous to hydrolases of the serine carboxypeptidase type. Our research interest is aimed at elucidating the molecular changes necessary for conversion of pep- tidases to acyltransferases by analysis of structure-function relationships. Sinapate esters are regarded as antinutritive factors. In B. napus they prevent the valuable seed protein from being used as human food supplement, a significant reduction of sinapine content is therefore a key requirement for establishing rape as a protein crop.

Based on the isolation of cDNAs en - expressed in E. coli, one of these enzymes dis- coding the enzymes for sinapine bio- plays HCA-GT activity. From seeds various synthesis, UDPglucose:sinapate gluco- secondary compounds were isolated and identi- syltransferase (BnSGT1) and sinapoyl- fied by MS and NMR as sinapic acid esters of glu- glucose:choline sinapoyltransferase cose, gentiobiose and kaempferol glycosides, (BnSCT) we analyzed the regulation of besides sinapine, sinapoylmalate and an unusual sinapate ester metabolism in B. napus. cyclic spermidine amide. BnSGT1 and BnSCT activities were found to be subject to a pronounced We started a transgenic approach to suppress transcriptional regula tion. BnSGT1 sinapine accumulation. A dsRNAi construct de - transcript level in creases throughout signed to silence the BnSGT1 gene was used to early stages of seed development until transform B. napus (cv. Drakkar).Transgenic plants the early cotelydonary stage and stays homozygous for the insertion of the BnSGT1 constant in later stages. In two-day suppression cassette showed a severe reduction old seedlings the BnSGT1 transcripts of sinapine content to about 20 percent of the reach their high est abundance follo- un transformed plants accompanied by a strong wed by a dramatic decrease. Ex pres - de crease in total sinapate ester content. sion of BnSCT is restricted to seeds. Enzyme activities in crude extracts and metabo - HPLC analysis of seed extracts of the trans - lite concentrations reflect the RNA abundance of formed plants revealed that the concentration of BnSGT1 and BnSCT indi cat ing that transcriptio- si napoylglucose and most of the the minor sina- nal regulation of these genes is of major im - pate esters was portance for regulation of sinapine biosynthesis. under the detec- Southern-blot analyses revealed that the amphi- tion level. This in - diploid ge nome of B. na pus contains at least two dicates that 1-O- genes for SGT and SCT, each derived from its sinapoylglucose is progenitors B. rapa and B. oleracea. As the rela- a central precur- ted genome of Arabidopsis was shown to encode sor not on ly for besides SGT three less-specific ester-forming sinapine biosyn- hydroxycinnamic acid glucosyltransferases thesis but also for (HCA-GTs) we assume for B. napus the presence transacyla tion of HCA-GT homologues po tentially in volved in reac tions lea ding sinapine biosynthesis. to all the other si - Transcript levels of BnSGT1 and BnSCT genes, enzyme activities and metabolite accumulation in napate es ters in developing rape seeds. Maximal enzyme activity was By homology-based cloning techniques two full- B. napus seeds. set to 100% (SGT=127 pkat/mg protein; SCT= 185 length cDNAs with strong sequence identity to As the si napoyl - pkat/mg Protein); metabolite concentration is given in nmol/seed. A-G = stages of seed development HCA-GTs could be isolated from B. napus. When trans ferases 61

known so far, such as SCT and SMT, have been ate amounts of func tional en zyme shown to dis play high accep tor spe ci ficity we hypo- for crystallization experiments. Collaborators thesize that each of the sinapate es ters should be Since SMT is subject to several post- Clint Chapple pro du ced by a distinguished acyltransferase of the translational mo di fications like gly- Purdue University, West Lafayette, USA SCPL-type protein giving rise to a lar ge protein cosylation or formation of disul fide Martin Frauen, Gunhild Leck - band family of SCPL acyltransferases in B. napus as it has brid ges functional expression re - Norddeutsche Pflanzenzucht, Hans Georg been shown for Arabidopsis. To optimize suppres- quires an eu ka ryotic system. By op - Lembke KG, Hohenlieth, Germany sion of sina pine synthesis in rape, additional vec- timiza tion of in duction conditions Christian Möllers University of Göttingen, Germany tors have been constructed making use of the and construct se quences we were José Orsini CaMV 35S promoter for constitutive silencing of able to produce en zy matically active Saaten-Union Resistenzlabor GmbH, SCT sequences for BnSCT to be suppressed or of SMT in Saccharomy ces cerevisiae. Leopoldshöhe, Germany a double in verted repeat including BnSGT and However, further work is ne cessary Jürgen Schmidt, Sabine Rosahl 1 Leibniz Institute of Plant Biochemistry, BnSCT se quences for simultaneous silencing of to in crease the yield. As the homo- Halle, Germany both genes. logous serine carboxypeptidases be - Milton T. Stubbs long to the best characterized pro- University of Halle, Germany Energy-rich -acetal esters like -O-sinapoylglu - teins, a structure modelling ap proach Victor Wray b 1 German Research Centre for cose have been characterized as acyl donors in has been started. Model structures Biotechnology, Braunschweig, Germany transacylation reactions of plant secondary meta- ob tained give first evidence for ami - bolism. In Arabidopsis, four related genes (AtSGT no acid residues that are critical for and AtHCAGT1-3) were found to encode gluco- transacylation function and sub strate recogni - syltransferases catalyzing the formation of hydro- tion. Site-directed mu tagenesis will be performed xycinnamate glucose esters in vitro. We have star - to prove the functional relevance of these amino ted a sys tematic approach including modulation acid positions. Sequence analyses reveal that of gene ex pression, mutant analysis and crossing SCPL acyltransferases form a distinct group with - as well as promoter testing to elucidate the functi- in the large family of SCPL proteins. They are spe- on of these genes in planta. In wild type plants, it cific for higher plants obviously reflecting the di - could be shown by real-time RT-PCR that all four versity of secondary metabolism. genes are expressed during seed development. However, AtSGT-mRNA is the most abundant one and ex pression of AtSGT1 follows roughly that of BnSGT1 in B. napus with highest expression in two-day-old seedlings. To modulate gene expression in Ara bi dopsis, overexpression vectors as well as specific dsRNAi constructs designed to silence each of the four genes have been constructed and trans formed into wild type plants. Several Arabidopsis in sertion lines are se gregating for isolation of ho mozygous mutants. For each of the genes promoter-GUS fusions have been constructed and transformed. To elucidate potential links to other metabolic pathways we have included several mutants in our analyses.

Another main interest in our research is to eluci - date molecular factors that govern the functional adaptation of hydrolases to perform transacyla - tion reactions. As model protein we investigate the sinapoylglucose:L-malate sinapoyltransferase (SMT) from Arabidopsis. A key requirement for this research aimed at investigation of structure- Similarity tree of SCPL proteins from Arabidopsis and functionally proven enzymes, the carbox- function relation ships is the heterologous over- ypeptidases CPDY from yeast and CPDWII from wheat as well as the acyltransferases from Arabidopsis (SCT, SMT), rape (BnSCT) and wild tomato (GAC-Lp). expression of SMT-cDNA to produce appropri - 62

Publications and Other Scientific Activities 2003

Publications transformation on barley (Hordeum vulga- Varet, A., Hause, B., Hause, G., Scheel, D. & Cacace, S., Schröder, G., Wehinger, E., re L.) with the JIP23 cDNA in sense and Lee, J. The Arabidopsis NHL3 gene en - Strack, D., Schmidt, J. & Schröder, J. A fla- antisense orientation. Cereal Res. codes a plasma membrane protein and its vonol O-methyltransferase from Catha - Commun. 31, 17-24. overexpression correlates with in creased ran thus roseus performing two sequen tial resis tance to Pseudomonas syringae pv. me thylations. Phytochemistry 62, 127-137. Münzenberger, B., Hammer, E., Wray, V., tomato DC3000. Plant Physiol. 132, 2023- Schauer, F., Schmidt, J. & Strack, D. De - 2033. Doll, J., Hause, B., Demchenko, K., Paw - toxification of ferulic acid by ectomycor- lowski, K. & Krajinski, F. A member of the rhizal fungi. Mycorrhiza 13, 117-121. Wang, W., Hause, B., Peumans, W. J., germin-like protein family is a high ly con- Smagghe, G., Mackie, A., Fraser, R. & Van served mycorrrhiza-specific induced gene. Opitz, S., Schnitzler, J.-P., Hause, B. & Damme, E. J. M. The Tn-antigen specific lec- Plant Cell Physiol. 44, 1208-1214. Schneider, B. Histochemical analysis of tin from Glechoma hederacea is an in sec - phenylphenalenone-related compounds in ticidal protein with an unusual physiology. Eckermann, C., Schröder, G., Eckermann, Xiphidium caeruleum (Haemodo raceae). Plant Physiol. 132, 1322-1334. S., Strack, D., Schmidt, J., Schneider, B. & Planta 216, 881-889. Schröder, J. Stilbenecarboxylate biosynthe- Books and Book Chapters sis: A new function in the family of chal - Peng, Z. F., Strack, D., Baumert, A., Sub - Fester, T. Plastiden bei der arbuskulären My - cone synthase-related proteins. Phy toche - ramaniam, R., Goh, N. K., Chia, T. F., Tan, S. korrhizasymbiose. In: Wurzelindu zier te Bo - mis try 62, 271-286. N. & Chia, L. S. Antioxidant flavonoids from denvorgänge, 14. Borkheider Se mi nar zur leaves of Polygonum hydropiper L. Phyto - Ökophysiologie des Wurzelraums Hause, B., Hause, G., Kutter, C., Miersch, O. che mistry 62, 219-228. (Merbach, W., Egle, K. & Augustin, J., eds.) & Wasternack, C. Enzymes of jasmonate B.G. Teubner, Stuttgart, pp. 39-42. biosynthesis occur in tomato sieve ele- Proels, R. K., Hause, B., Berger, S. & Roitsch, T. ments. Plant Cell Physiol. 44, 643-648. Novel mode of hormone in duction of tan- Stenzel, I., Hause, B., Feussner, I. & Was ter- dem tomato invertase genes in floral tissues. nack, C. Transcriptional activation of jas- Hause, B., Stenzel, I., Miersch, O. & Was- Plant Mol. Biol. 52, 191-201. monate biosynthesis enzymes is not re - ternack, C. Occurrence of the allene ox ide flected at protein level. In: Advanced Re - cyclase in different organs and tissues of Stenzel, I., Hause, B., Maucher, H., Pitzschke, search on Plant Lipids (Murata, N., Ya mada, Arabidopsis thaliana. Phytoche mistry 64, A., Miersch, O., Ziegler, J., Ry an, C. A. & Was- M., Nishida, I., Okuyama, H., Sekija, J. & Ha - 971-980. ternack, C. Allene oxide cyclase de pendence jime, W., eds.) Kluwer Academic Publi - of the wound re sponse and vascular bundle- shers, Dordrecht, pp. 267-270. Ibdah, M., Zhang, X.-H., Schmidt, J. & Vogt, T. specific ge neration of jasmonates in tomato A novel Mg++-dependent O-me thyl trans fe - - amp lification in wound signalling. Plant J. 33, Strack, D. & Milkowski, C. Recruitment of rase in the phenylpropanoid metabolism 577-589. se rine carboxypeptidase-related proteins of Mesembryanthemum crystallinum. J. in to phenylpropanoid metabolism. In: Poly - Biol. Chem. 278, 43961-43972. Stenzel, I., Hause, B., Miersch, O., Kurz, T., phenols 2002: Recent Advances in Poly - Maucher, H., Weichert, H., Ziegler, J., Feuss - phenols Research. Vol. 1 (El Hadrami, I. & Liu, S., Chen, K., Schliemann, W. & Strack, D. ner, I. & Wasternack, C. Jasmonate biosynthe- Daayf, F., eds.) Elwatanya Faculté des Scien - Isolation and identification of two flavone sis and the allene oxide cyclase family of Ara - ces, Sem lalia, Marrakech (Morocco), pp. 50- glycosides in burdock (Arctium lap pa L.) bidopsis thaliana. Plant Mol. Biol. 51, 895-911. 61. leaves by polyamide column chromatography and high performance liquid chroma- Strack, D., Fester, T., Hause, B., Schlie mann, Stumpe, M., Stenzel, I., Weichert, H., Hau se, tography in combination with e lec trospray W. & Walter, M. H. Arbuscular my corrhiza: B. & Feussner, I. The lipoxygen ase pathway in ionization-mass spectrometry. Chinese J. Biological, chemical, and molecular as - mycorrhizal roots of Me dicago trun catula. In: Anal. Chem. 31, 1023. pects. J. Chem. Ecol. 29, 1955-1979. Advanced Research on Plant Lipids (Murata, N., Yamada, M., Nishida, I., Oku ya ma, H., Monostori, T., Schulze, J., Sharma, V. K., Strack, D., Vogt, T. & Schliemann, W. Recent Sekiya, J. & Hajime, W., eds.) Kluwer Maucher, H., Wasternack, C. & Hause, B. advances in betalain research. Phy toche - Academic Publi shers, Dordrecht, pp. 287- Novel plasmid vectors for homologous mis try 62, 247-269. 290.

Thorson, J. S. & Vogt, T. Glycosylated na -

Department of Secondary Metabolism 63

Publications and Other Scientific Activities 2004

Publications Maucher, H., Stenzel, I., Miersch, O., Stein, Publications in Press Bücking, H., Förster, H., Stenzel, I., Miersch, N., Prasa, M., Zierold, U., Schwei zer, P., Do - Günther, C., Hause, B., Heinz, D., Krauze - O. & Hause, B. Applied jasmonates accumu- rer, C., Hause B. & Waster nack, C. The al - wicz, N., Rudolph, R. & Lilie, H. Com bi na - late extracellularly in to ma to, but in tra - lene oxide cyclase of barley (Hordeum tion of listeriolysin O and a tumor-specific cellularly in barley. FEBS Lett. 562, 45-50. vulgare L.) - Cloning and organ-specific ex - antibody facilitate efficient cell-type speci- pression. Phytochemis try 65, 801-811. fic gene delivery of conjugated DNA. Can - Camacho-Cristóbal, J. J., Lunar, L., Lafont, F., cer Gene Ther. Baumert, A. & González-Fontes, A. Bo ron Miersch, O., Weichert, H., Stenzel, I., Hau - deficiency causes accumulation of chloro- se, B., Maucher, H., Feussner, I. & Waster - Hause, B. & Fester, T. Molecular and cell bio- genic acid and caffeoyl polyamine conju- nack, C. Constitutive overexpression of al - logy of arbuscular mycorrhizal symbiosis. gates in tobacco leaves. J. Plant Physiol. lene oxide cyclase in tomato Planta. 161, 879-881. (Lycopersicon esculentum cv. Lukullus) elevates levels of some jasmonates and Doctoral Theses Hans, J., Brandt, W. & Vogt, T. Site-directed octadecanoids in flower organs but not in Hans, Joachim: Isoprenoidbiosynthese in mutagenesis and protein 3D-homology leaves. Phyto che mistry 65, 847-856. my korrhizierten Maiswurzeln: Klonie rung, modelling suggest a catalytic mech anism Charakterisierung und Lokalisie rung einer for UDP-glucose dependent be ta nidin 5- Milkowski, C., Baumert, A., Schmidt, D., Neh - 1-Desoxy-D-xylulose-5-phosphat-Reduk - O-glucosyltransferase from Doro - lin, L. & Strack, D. Molecular regula tion of toisomerase (DXR). Uni versity of Halle- theanthus bellidiformis. Plant J. 39, 319- sinapate ester metabolism in Bras si ca napus: Wittenberg, Depart ment of Biology, 333. Expression of genes, properties of the enco- 24/02/2004. ded proteins and correlation of enzyme Hans, J., Hause, B., Strack, D. & Walter, M. H. activities with metabolite accumulation. Hans, Judith: Klonierung und Charakte ri - Cloning, characterization, and immuno lo ca - Plant J. 38, 80-92. sierung von Flavonoidglucosyl transfe ra sen lization of a mycorrhiza-indu cible 1-deoxy- aus Beta vulgaris L. University of Halle- D-xylulose 5-phosphate re duc toisomerase Milkowski, C. & Strack, D. Serine carbo xy - Wit tenberg, Department of Bio chemis try/ in arbuscule-contain ing cells of maize. Plant peptidase-like acyltransferases. Phyto che - Biotechnology, 23/06/2004. Physiol. 134, 614-624. mis try 65, 517-524. Habilitation Isayenkov, S., Fester, T. & Hause, B. Rapid Schaarschmidt, S., Qu, N., Strack, D., Son - Hause, Bettina: Biosynthese und Funk tio - determination of fungal colonization and newald, U. & Hause, B. Local induction of nen von Jasmonaten - eine zellbiologische arbuscule formation in roots of Me di cago the alc gene switch in transgenic tobacco Analyse. Univer sity of Halle-Wit tenberg, truncatula using real-time (RT) PCR. J. plants by acetaldehyde. Plant Cell Physiol. Faculty of Mathematics, Natural Sciences Plant Physiol. 161, 1379-1384. 45, 1566-1577. and Techno logy 02/12/2004.

Vogt, T. Regiospecificity and kinetic proper- Lukacin, R., Matern, U., Specker, S. & Vogt, T. Cations modulate the substrate specifi- ties of a plant natural product O-me thyl - city of bifunctional class I O-me thyl trans - transferase are determined by its N-termi- fe rase from Ammi majus. FEBS Lett. 577, nal domain. FEBS Lett. 561, 159-162. 3 6 7 - 370.

Professor Dieter Strack organized a DFG supported symposium in Japan entitled "Germany-Japan Seminar on Mole cular Regulation of Plant Secondary Metabolism". The Japanese organizer was Kazuki Saito of the Chiba University. The symposium took place from September 20-23, 2004 at the Kazusa Akademia Center, Kisarazu, Chiba, Japan and was meant to serve as a starting point for more cooperation and exchange between Japanese and German scientists in the area of plant natural product research. Ten German and 19 Japanese groups presented their work and discussed future plants for possible collaborations. 64

Administration and Technical Services Head: Lothar Franzen Secretary: Heide Pietsch until April 2004, afterwards Cindy Maksimo

Administration and Technical Services 65

uring the past two years, two construction projects have dominated Dthe activities of the IPB administration. The construction of a new sha red-use building started in January 2003 and was completed in March 2004. In June 2004, construction of additional fully climate-controlled greenhouses was initiated.

With a total area of 500 square meters, the shared-use building provides space for 15 researchers and technicians from the Department of Bio - organic Chemistry. In addition to the two wide-span laboratories, there are many rooms for special uses such as fermentation, distillation, liquid chromatography and robotics for combinatorial syntheses. With an inert gas extinguishing system, experiments can also be carried out in a special "night lab" without constant supervision. Laboratories for gas chromatography, mass spectrometry, and DNA sequencing are used by scientists from all departments. The total cost of about three million Euros was provided by the Federal Ministry of Education and Research and by the Mi - nistry of Education and Cultural Affairs of the State of Saxony Anhalt. Nearly half the building outlay is required for the technical facilities for ventilation, special gases, heating and a closed-cycle cooling system.

The new greenhouses, with an additional 350 square meters of floor space, will meet the growing demand of the departmental research programs in- volving transgenic plants. The nine air-conditioned chambers will be ope- ned in the near future for cultivation of tomato, poppy, tobacco, rape and plants from the mycorrhization projects. In addition, there will be two tro- pical chambers and incubators for Arabidopsis. The technical equipment for the fine control of climatic conditions is state-of-the-art and costs about 1.1 million Euros. The total construction cost of 2.5 million Euros was also provided by the Federal Ministry of Education and Research and by the Ministry of Education and Cultural Affairs of the State of Saxony Anhalt. These new plant growth facilities will be ready for occupancy in May 2005.

The building that currently houses employees of facilities and construction management, graphic arts and horticulture will be replaced by two new buildings in the near future. Within this new complex, an additional 200 square meters of laboratory space for biological and chemical re - search for guest profes- sors is planned as well as new offices for the In - formation Technology spe- cialists from the De part - ment of Stress and Deve - lopmental Biology. 66

Administration Employees

Head of the Administration Oliver Prudyus, since August 2003 Lothar Franzen Mandy Schatkowski, since August 2003 Clemens Schinke, until July 2003 Accounting Head: Barbara Wolf Graphic Arts Gudrun Schildberg Head: Christine Kaufmann Burgunde Seidl, until September 2004 Annett Kohlberg Kerstin Wittenberg Horticulture Chauffeur Head: Iris Rudisch Jürgen Gaul Martina Allstädt Annett Grün, Trainee since August 2004 Computer Support Nicole Mühlwald, Trainee until September Head: Hans-Günter König 2003 Holger Bartz Christian Müller Kevin Begrow, trainee since August 2003 Philipp Plato, Trainee since August 2004 Ronald Scheller Kristina Rejall Steffen Rudisch Katja Scheming, Trainee Construction Management Andrea Voigt, Trainee Head: Heike Böhm Catrin Timpel Human Resources Head: Kerstin Balkenhohl Facilities Management Alexandra Burwig Head: Michael Kräge Cindy Maksimo, until April 2004 Detlef Dieckmeyer, until February 2004 Antje Olschewski, until September 2004 Carsten Koth Rita Stelzer, until August 2004 Michael Kräge Kathleen Weckerle Jörg Lemnitzer Klaus-Peter Schneider Library Services Eberhard Warkus Head: Andrea Piskol Jessica Ackermann, Trainee until February General Administration 2003 Head: Rosemarie Straßner Anja Gärtner, Trainee since August 2004 Christel Düfer, until June 2004 Antje Werner, Trainee until July 2004 Heide Pietsch, since April 2004 Elviera Schotte Press and Public Relations Trainees Head: Sylvia Pieplow Maike Hildebrandt, since August 2003 Jana Krupik, until September 2004 Antje Olschewski, until July 2003 67

Employee Statistics

2003 2004

Average number of employees 172 172

Full-time employees in % 73 67

Part-time employees in % 27 33

Permanent positions 92 92

Temporary positions 26 23

Employees externally funded 41 31

Employees remunerated by “University Science Funds Program” (Hochschulwissenschaftsprogramm/HWP) 5 6

Proportion of female employees in % 61 59

Personnel turnover in % 20 16

Averade age of employees in years 39 37

Scholarship/fellowship holders 25 26

Vocational training general administration 3 3 horticulture 2 4 library sciences 1 1 computer support 1 1 research technical assistant - 2

Succesfully completed vocational training 3 1

Average number of apprentices 7 11 68

Involvement of the IPB in National and International Scientific Networks

CERC 3 many, Spain Chairmen of the European Research Coun cils COMPARATIVE GENOMICS Chemistry Committees Comparative genomics between Arabi dopsis German Research Foundation and Brassica for genes directing seed-specific flavonoid biosynthesis COMBIOCAT GABI-Génoplante-bilateral Cooperation, Evolutionary Discovery of Novel Drugs by Ger many, France Orchestration of Polymer-Supported Com bi - natorial Bio-/Chemistry HEATOS Fifth Framwork Programme of the European A Vietnamese opiate detoxification symp tom Com munitiy medication German Federal Ministry for Education, Re - EPILA search and Technology (BMBF) Opioid Treatment of Chronic Pain and In flam - mation of the Locomotor System MOLECULAR ANALYSIS Fifth Framwork Programme of the European OF PHYTOHORMONE ACTION Com munitiy Priority Program SPP 1067 of the German Re - search Foundation (DFG) EVOMET Evolution of Metabolic Diversity MOLECULAR Priority Program 1152 of the German Re - CELL BIOLOGY OF PLANT SYSTEMS search Foundation Collaborative Research Center SFB 363 of the German Research Foundation GABI Genome Analysis MOLMYK of the Plant Biological Sys tem Molecular Basics of Mycorrhizal Sym bio ses German Federal Ministry for Education, Re - Priority Program SPP 1084 of the German Re - search and Technology (BMBF) and Business search Foundation Companies NAPUS 2000 GABI-NONHOST Rapeseed breeding A consortium-based functional geno mics ini - for improved human nutrition tiative on plant non-host disease resistance German Federal Ministry for Education, Re - GABI 1b search and Technology (BMBF)

METABOLOMICS PLATFORM SELF-ORGANIZATION THROUGH COORDINA TION Metabolite Profiling AND NON- COVALENT INTERACTIONS in Arabidopsis and Crop Plants Graduate Program of the German Re search GABI 2 Foundation

SARA SELENOPROTEINS Functional genomics of local and systematic Priority Programm SPP 1087 of the German acquired resistance in Arabidopsis Research Foundation GABI-trilateral Cooperation, France, Ger - 69

Resources and Investments

Research grants listed on this and the in Mio. Euro in % following pages were given by Basic financing funds AFGN Arabidopsis Functional Genomics Network (DFG) Personnel 9.7 37.9

BPS BASF Plant Science Consumables 4.4 17.2

BMBF Bundesministerium für Grants/Subsidies 0.2 0.8 Bildung und Forschung - Federal Minsitry for Education and Research Investments 7.4 28.8 “University Science Funds Program” DAAD Deutscher Akademischer (HWP) 0.3 1.2 Aus tauschdienst - German Aca demic Exchange Service Subtotal 22.0 85.9

D-B Gottlieb Daimler and Karl Funds from external sources Foundation Benz Foundation

DBU Deutsche Bundesstiftung BMBF 0.8 3.1 Umwelt - German Environ - mental Foundation MK-LSA 0.1 0.4

DFG Deutsche Forschungsgemein - DFG 1.5 5.9 schaft - German Research Foun dation Industry 0.5 2.0

Elsevier Elsevier Science Publisher EU 0.5 2.0 EU European Union other 0.2 0.7 Firmenich Firmenich Company Subtotal 3.6 14.1 Hopsteiner Hopsteiner Company Total 25.6 100 HWP Hochschulsonderprogramm - University Science Funds Program Investments in Million Euro Icon Icon Genetics AG Genetics Equipment 2.3

MK-LSA Kultusministerium des Landes Construction 5.1 Sachsen-Anhalt - Ministry of Education and Cultural Affairs of the State of Saxony Anhalt Total 7.4

MLU Martin Luther Universität Halle-Wittenberg - University of Halle Funds from HWP Investments external sources 1.2 % 28.8 % 14.1 % PPP Hungary Projektbezogener Personenaustausch (DAAD)- Project Based Personnel Ex - change Program Probiodrug Probiodrug AG

VW Volkswagen Foundation Foundation Consumables & Personnel Grants 37.9 % Wella Wella AG 18.0 % 70

Funds from External Sources

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2003-2004) Funded

Department of Natural Product Biotechnology

Jasmonate biosynthesis regulation 02/04 DFG 49,300 1 Prof. C. Wasternack & O. Miersch 04/05 DFG 16,000 1

12-Hydroxyjasmonic acid 03/05 DFG 30,200 1 Prof. C. Wasternack & O. Miersch

Glutamat cyclase 01/05 Probiodrug 24,400 0 Prof. C. Wasternack

Allenoxidcyclase 01/03 Firmenich 6,500 0 Prof. C. Wasternack

Analysis of genes 00/04 Icon Genetics 57,200 1 Prof. T. Kutchan

Molecular genetics of isoquinoline alk. biosynth. 01/04 DFG 93,700 2 Prof. T. Kutchan 04/05 DFG 41,000 2

Molecular genetics in Liana Triphyoph. Pellatum 03/05 DFG 82,800 1 Prof. T. Kutchan

Cellular signaling 02/04 DFG/MLU 46,400 1 Prof. T. Kutchan

Transformation and regeneration of P. somniferum 02/04 DFG 115,300 2 S. Frick

Metabolic engeneering 04/06 DFG 15,000 2 S. Frick

Modulation of jasmonates by transgenic plants 02/04 DFG/SFB 363 126,100 1 Prof. C. Wasternack & O. Miersch

Salvia fragrances 02/03 DBU 31,200 0 Prof. T. Kutchan

HUM-NEU 03/05 Hopsteiner 54,200 1 J. Page

Subtotal: 789,300 16

Department of Bioorganic Chemistry

HEATOS 00/03 BMBF 90,300 2 Prof. L. Wessjohann

COMBIOCAT 01/04 EU 185,600 2 Prof. L. Wessjohann

EPILA 01/04 EU 3,700 2 W. Brandt 71

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2003-2004) Funded

MCR ligand synthesis 02/04 DAAD/Probral 11,900 0 Prof. L. Wessjohann

Chrom-(II)-mediated reactions DAAD/ 02/03 5,200 0 Prof. L. Wessjohann PPP Hungary

Daimler Benz fellowship 02/03 D-B Foundation -1,100 1 Prof. L. Wessjohann

Reactivity of selenopeptids 03/05 DFG 100,400 1 Prof. L. Wessjohann & W. Brandt 04/06 DFG 15,800 1

CERC-3 04/06 DFG 0 Prof. L. Wessjohann 23,000

MUSKARIN 03/04 MLU 31,900 1 W. Brandt

HUMULUS 03/04 Hopsteiner 20,000 0 Prof. L. Wessjohann

Fungi metabolites 04/06 DFG 21,000 1 N. Arnold & J. Schmidt

Screening 04/06 Wella 42,000 1 Prof. L. Wessjohann

Subtotal: 549,700 13

Department of Stress and Developmental Biology

Heavy metal tolerance 02/04 DFG/SFB 363 70,600 1 D. Neumann & S. Clemens

CRISP 01/04 EU 39,000 1 Prof. D. Scheel

Heavy metal tolerance and silicon 00/04 MK-LSA 28,500 1 U. zur Nieden & S. Clemens

Resistence in potatoes 04/05 DFG 34,500 1 Prof. D. Scheel & S. Rosahl

The role of jasmonates in pathogene defense 01/03 DFG 22,900 1 Prof. D. Scheel

Jasmonate-insensitive mutant 99/03 MK-LSA 13,400 1 Prof. D. Scheel

Arabidopsis halleri 00/03 DFG 7,600 1 S. Clemens

Metallophytes 01/04 EU 101,800 1 S. Clemens 72

Funds from External Sources

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2003-2004) Funded

Metalhome 03/06 EU 64,700 1 S. Clemens

Biomineralisation 01/04 DFG 23,700 1 D. Neumann

Cooperation with South Africa 01/03 VW Foundation 28,100 0 Prof. T. Nürnberger

NODO 02/04 EU 147,800 1 S. Rosahl

Receptor kinases 02/04 DFG/AFGN 95,000 2 Prof. T. Nürnberger

Bioinformatics and mass spectronomy 02/03 BMBF 152,100 6 Prof. D. Scheel

GABI-NONHOST BPS 02/06 304,700 4 Prof. D. Scheel BMBF

Metal homeostasis 04/06 DFG 20,200 1 S. Clemens

GABI-GENOPLANTE 04/06 BMBF 10,000 1 S. Clemens

SARA 04/07 BMBF 11,200 1 Prof. D. Scheel

Subtotal: 1,175,800 26

Department of Secondary Metabolism

Betanidin-Glucosyltransferases 01/03 DFG 22,800 2 T. Vogt & Prof. D. Strack

Metabolism of isoprenoids 00/03 DFG 6,800 1 M.H. Walter & T. Fester 03/04 DFG 49,700 1 M.H. Walter 04/06 DFG 20,000 1

NAPUS 2000 99/04 BMBF 186,200 2 Prof. D. Strack

The role of jasmonates 00/04 DFG 62,400 1 during the establishment of mycorrhiza 04/06 DFG 16,700 1 B. Hause & Prof. D. Strack Carotenoid biosynthesis 00/04 DFG 42,800 1 in arbuscular mycorrhizal roots 04/06 DFG 24,000 1 T. Fester

Metabolite profiling 02/04 DFG 115,200 1 W. Schliemann 04/06 DFG 23,000 1

Phytochemistry 02/04 Elsevier 53,200 1 Prof. D. Strack 73

Amount in Euro Personnel Posts Title & Head of Project Duration Financial Source (2003-2004) Funded

HCA-Glucosyltransferases 03/05 DFG 58,200 1 C. Milkowski & A. Baumert

SCPL-Ayltransferases 03/05 DFG 48,500 1 C. Milkowski & Prof. D. Strack

PFOMT 04/05 DFG 3,200 0 T. Vogt

Subtotal: 732,700 16

Joint projects

Metabolite profiling in Arabidopsis and crop plants, GABI 00/04 BMBF 228,300 4 Department of Stress and Developmental Biology Department of Bioorganic Chemistry S. Clemens

Metabolite profiling in Arabidopsis and crop plants, GABI-2 Department of Stress and Developmental Biology 04/07 BMBF 87,800 3 Department of Bioorganic Chemistry Prof. D. Scheel, S. Clemens, Prof. L. Wessjohann, J. Schmidt

Subtotal: 316,100 7

Total projects granted: 3,563,600 78

General overview

DFG 1,497,400 38

BMBF 765,900 19

EU 542,600 8

Industry 534,400 8

Other sources 181,400 3

MK-LSA 41,900 2

Total: 3,563,600 78

HWP 350,900 4

Total: 3,914,500 82 74

Seminars, Workshops and Colloquia 2003

January 14 Molecular Plant Physiology, Golm, Germany Luc Varin, Professor, Multidimensionale LC-MS für die Pflanzen - Franziska Krajinski, Dr. Concordia University Montreal, Canada pro teomanalyse. University of Hannover, Germany Functional analysis of sulfotransferases in Ara - Transcriptional changes in the model plant bi dopsis thaliana. March 12 Me di cago truncatula in response to symbiotic Christine Schimek, Dr. and pa thogenic microorganisms. January 23 University of Jena, Germany Ekkehard Neuhaus, Professor Trisporoids, apocarotenoid signal molecules June 5 University of Kaiserslautern, Germany in zy gomycetes. Burkhard Schulz, Dr. Physiology and evolution of nucleoside trans- University of Tübingen, Germany porters in plastids and bacteria. March 25 The Arabidopsis immunophilin AtFKBP42 Mike Toorneman, Dr. (TWIS TED DWARF) integrates hormonal Yves Marco, Dr. University of Amsterdam, The Netherlands signaling pathways in plants. Laboratoire de Biologie Moleculaire des Re - A (-)- Myrtenol based trigger system for the lations Plantes-Mi cro-Organism CNRS, France controlled formation of enediynes. June 6 Resistance and disease development in Arabi - Elisabeth Fuss, Dr. dop sis thaliana in response to the bacterial April 3 University of Düsseldorf, Germany pathogen Ralstonia solanacearum. Jörg Hacker, Professor Biosynthesis of lignans in Linum spp. University of Würzburg, Germany January 30-31 Escherichia coli as a model to study microbial June 12 First colloquium of Priority Program 1152, pa thogenicity. Bruno Lemaitre, Dr. Evolution of Metabolic Diversity Centre de Genetique Moleculaire, April 10 CNRS, Gif-sur-Yvette, France February 4 Patrick Covello, Dr. Genomic and genetic analysis of the Athanassios Giannis, Professor NRC Plant Biotechnology Institute Drosophila immune response. University of Leipzig, Germany Saskatoon, Kanada Synthese und biologische Untersuchungen Genomics approaches to bioactive lignan bio- Károly Micskei, Dr. von In hibitoren der Angiogenese. synthesis in Podophyllum. University of Debrecen, Hungary Assymmetric synthesis induced by natural February 6 April 15 amino acids in aqueous medium. Zsuzsanna Schwarz-Sommer, Dr. Ralph Panstruga, Dr. Max Planck Institute for Max Planck Institute for Tamás Patonay, Professor Plant Breeding Research, Cologne, Germany Plant Breeding Research,Cologne, Germany University of Debrecen, Hungary Homeotic control of floral organ identity in Corruption of host seven-transmembrane Alpha-azido ketones: Useful tools in the syn- snapdragon: The problem of restricting gene proteins by pathogenic microbes: A common thesis of polyfunctionalized synthons and expression domains. theme in animals and plants? heterocycles.

February 18 April 30 June 19 Guy R. Cornelis, Professor Bernhard Westermann, Dr. Regine Kahmann, Professor Biocenter of the University of Paderborn, Germany Max Planck Institute for University of Basel, Switzerland Synthesen zu Peptid- und Glycopeptidmimetika. Terrestrial Micro biology, Marburg, Germany How to switch off the innate immunity: The The Ustilago maydis/maize pathosystem: Yer sinia lesson. May 20 Mating and beyond. Helmut Duddeck, Professor February 20 University of Hannover, Germany June 24 Mugio Nishizawa, Professor Chirale Erkennung von Phosphorderivaten mit Naohiro Kato, Professor Bunri University, Tokushima, Japan ei nem Dirhodiumkomplex als NMR-Auxiliar. University of New Brunswick,USA Synthetic approach into sterol biosynthesis by Live from plant cells: Analysis of plant nuclear de veloping mercuric triflate. May 21 functions using fluorescent proteins. Gernot Frenking, Professor February 25 University of Marburg, Germany June 25 Klaus Naumann, Dr. Die Natur der chemischen Bindung-alte Peter Bäuerle, Professor Bayer AG, Germany Fragen, neue Antworten. University of Ulm, Germany Chlor - ein wichtiger Substituent in biologisch Selbstorganisierende Oligo-, Poly- und aktiven Substanzen aus Natur und Synthese. May 26 Cyclothio phene. Bernadette Lippok, Dr. March 3 University of Freiburg, Germany June 26 Yang Ye, Professor Pythium and Arabidopsis - an interaction that Peter Bäuerle, Professor Institute of Materia Medica, Shanghai is complex and full of surprises. University of Ulm, Germany Chemical studies on traditional chinese medi - Combinatorial Organic Materials Research cine. June 4 (COMR) A faster route to organic materials. Robert A. Dietrich, Dr. March 5 Syngenta Biotechnology, North Carolina, USA Ralf Oelmüller, Professor Wolfram Weckwerth, Dr. The Arabidopsis Bos mutants affect tolerance University of Jena, Germany Max Planck Institute of to both biotic and abiotic stresses. Regulatory proteins and novel processes in 75

photosynthesis. and plants: Virulence functions and resistance A medley of ribonucleases: How plastids responses. adjust mRNA levels during plant develop- June 30 ment. Volkmar Vill, Professor October 15 University of Hamburg, Germany Harald Schmidt, Professor November 19 Naturstoff-Datenbank Phytobase-aktueller University of Linz, Austria Lutz H. Gade Stand der Arbeiten. Neues vom Holz. University of Strasbourg, France Strategien in der Homogenkatalyse: Von der July 2 October 16 Ko ordinationssphäre in Einkernkomplexen zu R. Madsen, Professor Hugo Kubinyi, Professor ma kromolekularen Dendrimerkatalysatoren. Technical University of Denmark, Lyngby BASF AG Ludwigshafen, Germany Synthesis of natural products from carbohy- Wirkstoffe:Vom Zufall zum gezielten Entwurf. November 20 drates by the use of ring-closing olefin me ta - Georg Frank, Professor thesis. Bayer Bitterfeld GmbH, Germany Murray Grant, Dr. Phasen der Arzneimittelforschung am Beispiel July 3 Imperial College London, UK As pirin. Axel Mithöfer, Dr. Local and systemic signalling networks me - University of Munich, Germany diated by the plant disease resistance protein November 27 Aspects of friend and foe discrimination in RPMI. Klaus Apel, Professor legume-microbe interactions. Swiss Federal Institute October 21 of Technology, Zurich, Switzerland July 8 Reinhard Agerer, Professor Singlet oxygen-mediated stress responses of Thomas Weimar, Dr. University of Munich, Germany Ara bidopsis. University of Lübeck, Germany Ökologische und systematische Aspekte der Untersuchung biomolekularer Wechsel- Ekto mykkorrhiza. December 1 wirkungen mit Hilfe der NMR-Spek troskopie Karin Groten, Dr. und der Ober flächen-Plasmonen-Resonanz. October 22 Rothamsted Research, Harpenden, UK Teun Nunnik, Dr. Mechanisms contributing to the senescence Ahlert Schmidt, Professor University of Amsterdam, The Netherlands of pea nodules. University of Hannover Phosphatidic acid - an emerging plant lipid se - Thioredoxine steuern die assimilatorische cond messenger. December 4 Sulfat reduktion und die assimilatorische Andreas Freialdenhoven, Dr. Nitrat re duk tion. October 23-24 Max Planck Institute for Second colloquium of Priority Program 1152, Plant Breeding Research, Cologne, Germany July 15 Evolution of Metabolic Diversity Control of race-specific resistance and cell- Franz Nussbaum, Dr. death in barley. Bayer AG, Germany October 23 Warum-und vor allem wie-sollte man bioakti- Birgit Piechulla, Professor December 10 ve Naturstoffe derivatisieren? Anregungen aus University of Rostock, Germany Thomas Schrader, Professor der Chemie des -(-)Galiellalactons. Control of time-specific scent emission in University of Marburg plants. Self-organization and molecular recognition July 16 of biomolecules by artificial receptors. Werner Engewald, Professor October 24 University of Leipzig, Germany Shin-Ichi Aizawa, Dr. December 11 Die Kopplung GC-MS/MS-wieviel Core Research for Evolutional Science Guido van den Ackerveken, Dr. Chromatogra phie brauchen wir noch? and Technology, Tokyo, Japan University of Utecht, The Netherlands Flagella as the type III secretion system. Susceptibility to downy mildew in August 2003 Arabidopsis: Mo lecular aspects of hosting an Wolfgang Knogge, Dr. October 28 uninvited pathogenic guest. University of Adelaide, Australia Günter Gerlach, Dr. NIPping grass leaves - a favorite fungal pasti- Botanical Garden Munich, Germany me and its consequences. Blütenöle als Belohnung - Über die Biologie Workshops des Ölblumensyndroms. October 2 Februrary 2003 Ulrich Zähringer, Professor November 6 Collaborative Research Center Molecular Leibniz Center for Thomas Martin, Dr. Cell Biology of Plant Systems (SFB 363) of the Medicine and Biosciences, Borstel, Germany University of Cambridge, UK Ger man Research Foundation Peptidoglycan - Toll or Nod? Pathogenic pat- Are Arabidopsis T-DNA mutants suitable for tern recognition in innate immunity. the analysis of 14-3-3 specificity? June 2003, GABI Nonhost October 6 November 13 David Mackey, Dr. David Stern, Dr. The Ohio State University, Columbus, USA Boyce Thompson Institute Meetings The interface between bacterial pathogens for Plant Research, New York, USA January 2003 76

Seminars, Workshops and Colloquia 2004

January 8 trans cription factors involved in plant defense Andreas Schaller, Professor and leaf senescence. Randolph J. Alonso-Herrera, Dr. University of Hohenheim, Germany March 25 Venezuelan Institute Jasmonate signaling in plant defense and pol- Bonnie Carolyn McCaig, Dr. for Scientific Research Caracas, Venzuela len development. Michigan State University, USA Synthetic derivatives of natural products with The role of jasmonate perception in tomato po tential chemotherapeutic applicability. January 12 re production and development. Ian W. M. Smith, Professor May 12 University of Birmingham, UK March 31 Burghard König, Professor Chemistry amongst the stars: Reaction kine- Ute Wittstock, Dr. University of Regensburg, Germany tics at a new frontier. Max Planck Institute for Molecular recognition with coordination Chemical Ecology, Jena, Germany compounds. January 14 Special weapons - exceptional countermeasu- Joachim Stöckigt, Professor res: How insects cope with an activated plant May 18 University of Mainz, Germany defense system. Jens Rohloff, Dr. Molekulare Analyse der Vinorin Synthase-ein Norwegian University of zentrales Enzym der Alkaloidbiosynthese in April 7 Science and Technology, Trondheim, Norway Rau volfia. Jürgen O. Metzger, Professor Approaches in Arabidopsis research: Space University of Oldenburg , Germany biology and differences of smell. Manfred Psiorz, Professor Massenspektrometrische Untersuchung von Boehringer Ingelheim Re aktionen in Lösung oder wie kann man May 26 Pharma GmbH & Co KG, Germany Carbe niumionen bei SN1-Reaktionen und Jakob Ley, Dr. Tropan-Stukturen in Natur- und Wirkstoffen. Radikale bei Radikalkettenreaktionen sehen. Symrise GmbH & Co KG, Holzminden, Germany January 15 Ruth Niemetz, Dr. Geschmack-Physiologie, Moleküle, Iwona Adamska, Professor University of Ulm, Germany Modifizierung. University of Konstanz, Germany Zur Biosynthese komplexer Gallotannine in Protective mechanisms against light stress in Rhus typhina und Ellagitannine in Tellima gran- June 3 the diflora. Herman Spaink, Professor chloroplast of higher plants. University of Leiden, The Netherlands April 22 Similarities of microbial recognition by plants February 12 Martin Parniske, Dr. and animals. Georg Coupland, Professor John Innes Centre, Norwich, UK Max Planck Institute for Plant genetics of root symbiosis with fungi June 9 Plant Breeding Research, Cologne, Germany and bacteria. Birgit Kersten, Dr. The regulation of plant development by sea- Max Planck Institute sonal cues. April 29 for Molecular Genetics, Berlin, Germany Widmar Tanner, Professor Towards plant proteomic studies using pro- February 18 University of Regensburg tein mi croarrays. A. Llobet, Professor Plant membrane transport: From physiology University of Girona, Spain to molecular biology and back. June 10 Azomacrocyclic complexes and their applica- Markus Pauly, Dr. tion in bioorganic and coordination chemi- May 3 Max Planck Institute of stry. Laurent Zimmerli, Dr. Molecular Plant Physiology, Golm, Germany University of Fribourg, Switzerland Life on the outside: Why do plant cell walls February 19 Early events in Arabidopsis nonhost resi- have to be so complex? Steffen Backert, Dr. stance. University of Magdeburg, Germany June 18 Function of two type IV secretion systems in May 6 Cornelia Mrosk, Dr. He liobacter pylori: Protein translocation and Hanjo Hellmann, Professor University of Jena, Germany conjugative chromosomal DNA transfer. Freie Universität Berlin, Germany Die ß-Amylase in Turionen von Spirodela Cullins as regulators in the ubiquitin protea- polyrhiza: Regulation durch Licht und Nitrat. February 26 som pathway. Helle Ulrich, Dr. June 22 Max Planck Institute for May 10 Harro J. Bouwmeester, Dr. Terrestrial Micro biology, Marburg, Germany György Horvath Plant Research International, Control of genome stability by ubiquitin and University of Antwerp, The Netherlands Wageningen, The Netherlands SUMO. Biosynthesis of tocotrienols. Role of terpenoids in signaling between plants and other organisms. March 18 May 11 Imre Somssich, Dr. Koop Lammertsma, Professor June 24 Max Planck Institute for University of Amsterdam, The Netherlands Olivier Voinnet, Dr. Plant Breeding Research, Cologne, Germany Generating and applying new organophorus Institut de Biologie Moleculaire des Plantes Search for in vivo target genes of WRKY rea gents. CNRS, Strasbourg, France 77

Mechanisms and roles of RNA silencing in Interplay of gene-specific resistance to barley- G. Ungar, Professor plants and animals. powdery mildew and the suppression of host- University of Sheffield, UK re sponses. Periodic and quasiperiodic patterns in dendri- mer nanostructures. June 29 September 8 November 16 Pierre Potier, Professor Virinder Parma, Professor Christa Kamperdick, Dr. Institut de Chimie des Substances Natu - University of Massachusetts, Lowell, USA Institute of relles, CNRS, Giv sur Yvette, France Biocatalytic generation of novel materials of Molecular Biotechnology Jena, Germany Research and discoveries of new antitumor im portance in drug and gene delivery. Detection of protein-ligand interactiuons by drugs: NAVELBINE ® & TAXOTERE ®. saturation transfer difference NMR October 4-5 Spectroscopy (STD). July 5 Third colloquium of Priority Program 1152, Vincenzo de Luca, Professor Evolution of Metabolic Diversity November 18 University of Ontario, Kanada Beat Keller, Professor New tools for understanding metabolic path- October 10 University of Zurich, Switzerland ways in single cells: The case for Catharanthus Ko Shimamoto, Dr. Diversity and evolution of resistance genes in roseus indole alkaloid biosynthesis. Nara Institute of Science and Technology, Japan cultivated and wild wheat: Exploring the Rac GTPase is a key regulator of defense resources of a crop plant. July 8 signaling in rice. Steffen Abel, Professor November 24 University of California, Davis, USA October 18 Klaus Müllen, Professor Glucosinolates: From biosynthesis to pathway Bertil Helgee, Professor Max Planck Institute for Polymer Research re gulation. University of Technology of Göteborg, Sweden Graphitmoleküle. N-Vinylpyrrolidone co-polymers, possible July 15 support materials for chemical reactions. November 26 Jeff Dangl, Professor Gopalan Selvaraj, Dr. University of North Carolina, Chapel Hill, USA October 21 National Research Council of Canada P. syringae type III effector proteins manipula- Joachim Uhrig, Dr. Molecular aspects of reproductive develop- te host cell biology, and plant disease resi- Max Planck Institute ment in cereals and oilseeds. stance gene products stop them. for Plant Breeding Research, Köln, Germany Protein interaction networks: Functional ana- December 8 July 23 lysis of plant protein families and plant-virus Albrecht Berkessel, Professor Gynheung An, Professor interac tions. University of Cologne, Germany Pohang University Biomimetik und Organokatalyse für die of Science and Technology, South Korea October 27 Synthese enantiomerenreiner Epoxide, Aldole T-DNA insertional mutagenesis for reproduc- Gerd Jürgens, Professor und Amino säuren. tive development in rice. University of Tübingen Apical-basal pattern formation in Arabidopsis December 9 September 2 em bryogenesis. Anne Osbourn, Dr. Stefanie Ranf John Innes Centre Norwich, UK University of Souht Carolina, Columbia, USA November 4 Secondary metabolism and plant defense. Virus induced gene silencing of MAP kinases Raoul Bino, Professor in tomato. University of Wageningen, The Netherlands Plant Metabolomics for Plant Breeding. Roger Wise, Professor Workshop Iowa State University, USA November 11 78

Guest Researchers and Fellows

Department of Bioorganic Chemistry

Susanne Aust, Dr., Germany Dick, Germany DAAD Fellow 01.03. 2003 - 31.12. 2004 Fellow Stifterverband für die Deutsche 16.04. 2003 - 15.10. 2003 Wissenschaft Lilechi Danstone Baraza, Tanzania 01.02. 2004 - 31.12. 2004 Paulo Henrique Schneider, Brazil NAPRECA-DAAD Fellow DAAD Fellow 01.06. 2004 - 30.11. 2004 Simon Dörner, Germany 16.04. 2003 - 15.10. 2003 Fellow Studienstiftung des Deutschen Alessandra Basso, Dr., Italy Volkes Jasqer Alonso Sehnem, Brazil EU Fellow 01.03. 2004 - 31.12. 2004 03.10. 2004 - 31.12. 2004 12.01. 2004 - 29.01. 2004 Kanchana Dumri, Thailand Jana Selent, Germany Claudia Bobach, Germany DAAD-Leibniz Fellow 12.01. 2004 - 30.04. 2004 01.10. 2004 - 30.11. 2004 01.03. 2004 - 31.12. 2004 Katalin Sepreny, Hungary Christiano Rodrigo Bohn Rhoden, Othilie Vercillo Eichler, Brazil DAAD Fellow Brazil DAAD Fellow 01.11. 2003 - 30.11. 2003 DAAD Fellow 01.08. 2004 - 31.12. 2004 01.10. 2004 - 31.12. 2004 Tran Van Sung, Prof., Vietnam Gergely Gulyás, Hungary February, June and September 2004 Antonio Luiz Braga, Prof., Brazil 01.08. 2004 - 30.09. 2004 DAAD Fellow Mieke Toorneman, 16. 09. 2004 - 31.10. 2004 Zsuzsa Juhasz, Hungary The Netherlands DAAD Fellow 01.01. 2003 - 30.10. 2003 Tran Van Chien, Vietnam 01.10. 2003 - 31.10. 2003 07.10. 2002 - 30.09. 2003 Larissa Vasilets, Dr., Russia Christine Kamperdick, Dr., Germany 01.01. 2003 - 31.05.2004 Nguyen Manh Cuong, Vietnam Humboldt Fellow DAAD Fellow 22.04. 2003 - 21.10. 2003 Nguyen Hong Thi Van, Vietnam 01.06. 2004 - 31.08. 2004 01.01. 2003 - 16.04. 2003 Myint Myint Khine, Myanmar Katalin Czifrak, Hungary Daimler Benz Fellow Heike Wilhelm, Dr., Germany DAAD Fellow 01.01. 2003 - 31.12. 2004 Fellow BioService GmbH, EU and the 04.08. 2003 - 30.09. 2003 State of Saxony Anhalt Christine Neuhaus, Germany 01.01. 2004 - 31.12. 2004 01.03. 2004 - 30.09. 2004 Hasliza Yusof, Malaysia Luay Rashan, Prof., Jordan 06.10. - 20.10.2004 Humboldt Fellow 12.07. 2004 - 30.07. 2004

Daniel Garcia Rivera, Cuba Graduiertenkolleg Victor 01.10. 2003 - 31.12. 2003 Juliana Schneider, Brazil

Myint Myint Khine, Tran Thi Phuong Thao and Kanchana Dumri (from left to right).

Department of Stress and Developmental Biology

Reetta Ahlfors, Finland Ingo Hofmann, Germany Claudia Simm, Germany DAAD Fellow 01.01. 2003 - 31.12. 2003 Graduiertenkolleg 08.07. 2002 - 30.04. 2003 since 01.10. 2003

Emiko Harada, Dr., Japan Thorsten Nürnberger, Prof., Aleksandra Trampczynska, Dr., Humboldt Fellow Germany Poland since 22.02. 2002 01.08. 2003 - 31.12. 2003 10.11. 2003 - 30.11. 2003 79

Department of Natural Product Biotechnology

Andrea Andrade, Argentina DAAD Fellow DAAD Fellow 01.10. 2003 - 30.09. 2004 Alfonso Lara Quesada, Costa Rica 05.08. 2003 - 30.09.2003 DAAD Fellow Aphacha Jindaprasert, Thailand since 01.04. 2003 Andrea Borgogni, Italy DAAD Fellow 09.04. 2004 - 16.06. 2004 since 18.10. 2004 Luc Varin, Prof., Canada 01.10. 2002 - 31.01. 2003 Kum-Boo Choi, Dr., Korea Kristin Krukenberg, USA Humboldt Fellow Fulbright Fellow 07.10. 2002 - 30.09. 2003 23.09. 2002 - 15.07. 2003

Bonnie Carolyn McCaig, Dr., USA Natsajee Nualkaew, Thailand 06.01. 2004 - 02.04.2004 DAAD Fellow Maria Luisa Diaz Chavez, Mexiko 01.11. 2004 - 31.12.2004

Department of Secondary Metabolism

Ma Hnin Hnin Aung, Singapore 13.01. 2003 - 28.02. 2003

Ana Cenzano, Dr., Argentina 01.09. - 30.11. 2004

Diana Schmidt, Germany Fellow, Bio Service GmbH, EU and the State of Saxony Anhalt 01.08. 2001 - 31.07. 2004 Rostand Tonleu Tonfack

Administration and Technical Services 80

Press and Public Relations Head: Sylvia Pieplow Assistant: Jana Krupik

Press and Public Relations 81

National Year of Chemistry 2003 panies and private ci tizens in The public relations activities in 2003 were effec- Germany, Aus tria and Switzer - ted by the national Year of Chemistry. This the- land. The response to the game matic year, initiated by the Donors Association was consistently positive and re - for the Promotion of Sciences in Germany, has sulted both in numerous press been enriched by our chemists with many pro- articles and in an increase in ge - jects and ideas. neral name recognition of the IPB in Ger many. Special Lectures In the context of special lectures for the Year of Public Events The Phytolator crowned with a doctoral hat. Chemistry, Professor Ludger Wessjohann spoke 2003 and 2004 in March 2003 about flavors and pharmaceutically Germany’s Day of Re uni fica tion important ingredients from plants. The lecture, On October 3, 2003, the IPB participated toge - named Pizza Taste and Jungle Drugs-Che mical ther with other scientific institutes of the state of Substances in Nature, was addressed to primary Saxony Anhalt in the celebration of Germany's school students, high school students and inter- Day of Reunification in Magde burg.Together with ested laymen. the university of Halle, the IPB exhibited the differences between human and plant cell cultures. Very interesting as well was the lecture about the biology of oil flowers in October, given by the cu - Long Night rator of the botanical garden in Munich, Günter of Science 2003 and 2004 Gerlach. The talk was organized by the IPB re - The second and the third Long searchers who are investigating the biological Night of Science were both great role of the poorly understood flower oils in co - successes for our institute. On this operation with the botanical garden. first Friday in July, the institute's doors were open from 7 pm to The Ship of Chemistry 2003 midnight to welcome more then In summer 2003 the, so-called Ship of Che mistry 300 members of the public. Visi - started as a floating exhibition its tour on the tors enthusiasti cally participated Carsten Milkowski showed electrophoretic Rhine. The exhibits about chemistry in every day in guided tours through the labs experiments in the Long Night of Science 2004. life could be visited by a large number of people and greenhouses. In addition, our during the stops of the ship in every major har- scientists displayed experiments and ex hibits in bor.The IPB participated in this exhibition with a the foyer. Special lectures were fa vo red by the pu - multimedia project about the scientific work on blic. Stephan Cle mens spoke about the chances the institute. The presentation contained a virtual for and risks of green gene technology, whereas tour through the IPB and a game, designed to Professor Ludger Wess jo hann gave an overview inform people about bioorganic chemist’s work. about plant natural products and their applica tion The player has to isolate and analyze active sub- as pharmaceuticals. stances from medical plants. At the end of the game, the player knows the structure of the com- Guided Tours for Scholars pounds and is crowned with a doctoral hat. The As in the previous years, the IPB organized many game was planned mainly by our scientists Lars guided tours through the institute for school Bräuer, Wolfgang Brandt, Andrea Porzel and Pro - classes. Teachers and foreign students were also fessor Ludger Wessjohann from the De partment very interested in this possibility to learn more of Bio organic Chemistry. about the institute. In addition, all of the four scientific departments sponsored several periods The game and the virtual tour were burned on of practical training, which allowed many high CD and distributed in August 2004 to many se - school students to gain insight into lab work and condary schools, universities, firms, publishing com- to try out experiments on the bench. 82

Participation in Trade Shows Also in April, Christel Rieman-Hahnewinckel, Ul - Biotechnika in 2003 rich Kasparick, and Dr. Wolfgang Eichler visited In October 2003, the IPB partici- the IPB to gain information on the major re - pated in Biotechnika, Germany's search projects on the institute. The members of most important international bio- the German Lower House of Parliament were technology exhibition. Together impressed by the excellent research conditions at with universities and many scien- the IPB. With this visit, the politicians were re - tific institutes and firms, our re - sponding to an initiative of the government to searchers presented their work at provide more support to the best scientific insti- In Biotechnika 2003 in Hannover, the IPB, re - the Saxony Anhalt booth in Han - tutes in the eastern part of Germany. Together presented by Dr. Michael H. Walter (left), exhibi- nover. In this trade show, Mi chael with the directors, they discussed the necessary ted topics on mycorrhiza. Here Dr. Walter is dis- cussing with Dr. Horst Rehberger (right), Saxony- H. Walter from the Depart ment steps of economic fortification by a better pre- Anhalt's Minister for Work and Eco no my. of Secondary Metabolism display- sentation of the whole scientific landscape in this ed topics on mycorrhiza - a sym- region. "In spite of the fact that many of East Ger - biosis between plant roots and many's research institutes work on an excellent fungi. professional level, they have image problems in the western part of the country", Ulrich Kas pa - ABIC in 2004 rick said. These problems could be resolved by In a similar Saxony Anhalt booth the, IPB partici- visiting the corresponding institutes and by more pated in September 2004 in ABIC in Cologne. personal contact between scientists and politi- For this Agricultural Biotechnology International cians. Conference, resear chers of the Department of Stress and Developmental Biology presented me - President of the Leibniz Society chanisms of metal hyperaccumulation in plants. tours our Labs and Greenhouses With pleasure, members of the IPB welcomed Projects in 2004 the president of the Leibniz Society Hans Olaf In 2004, our institute was highly present in the Henkel in June 2004. Henkel, who is visiting at re - regional and national press. Some of our scientific gular intervals all of the 84 Leibniz Institutes, was work also resulted in interest by public broadca- impressed by the scientific projects of the insti - sters. In addition, several high-ranking dignitaries tute. After the tour, he discussed with the direc- visited the IPB to get an impression about our tors some general questions about the future scientific projects and the conditions of work. role of the Leibniz Society within national and in - With the organization of two art exhibitions, we ternational science politics. could revitalize an old custom of the past, which gave many artists the possibility to show their European Commission Of ficer own perspectives about science and other im - visits important scientific insti- portant aspects of life. tutes of Saxony Anhalt In context of the recently star - Visits of Dignitaries ted biotechnology offensive by Members of the German Lower House of the State of Saxony Anhalt, in Parliament visit the IPB Octo ber 2004, Dr. Christian In April 2004, Dr. Christoph Bergner, member of Pater mann from the Eu ropean the German Lower House of Parliament, visited Di rectorate-General for Re - the IPB not only for nostalgic reasons, but also to search viewed the IPB and discuss further promotion of the Leibniz Society other re gional scientific esta- Hans Olaf Henkel (right), Presi - institutes. One central point in discussions with blishments with similar orien- dent of the Leibniz So ciety, visited the former scientist of this institute was the re - tation in plant biochemistry. In the IPB in June 2004. Here, he speaks with Prof. Dierk Scheel, Ma - solution of the new gene technology law in the addition to green biotechnolo- na ging Director of the institute. near future. gy, the im ple mentation strate- 83

gy of this of fensive aims to support the pharma- ked for many years as scientific co-wor- ceutical industry, neu rotechnology and red bio- ker at the IPB. His paintings displayed a technology as well. Af ter the circuit, Dr. wide range of themes from landscapes Patermann informed interested persons about and scientific motifs to dreams and current and planned scientific projects of the EU. human relationships. All pictures were made as monotype, a spe cial printing New information about Mycorrhiza technology, which was modified and Our multimedia presentation about mycorrhiza improved by the painter. was in this year completely updated by its crea- tor Thomas Fester. The scientist of the De part - Oil on Canvas Our CD ment of Secondary Metabolism completed the from Giacomo Piccoli about mycorrhiza in a new layout. presentation with the newest scientific re sults The Italian painter and sculptor Giaco - and with references from the literature. The first mo Piccoli abducted us into the border- version of the interactive course about this fasci- land between science and philosophy. nating biological interaction was produced in His exhibition named Perpetuum Mobile 2001 for students and interested nonscientists - Magic of Biology in Imaginary Worlds alike. In September 2004, the renewed program was shown from October 2004 until was again burned on CD and sent to many January 2005 at the IPB. Inspired by schools, firms, publishing companies and private plants, fruits and microscopic structu- citizens throughout the German-speaking region res, Giacomo Piccoli displayed amazing in Europe. spaces of surrealistic perspectives. Giacomo Piccoli, born in Catania Art Exhibitions 2004 (Sicilia) in 1949, he was elected in 1983 Monotypes from Hanno Lehman National President of the ANTITESI cul- The exhibition Farbansichten in July and August tural circuits in Rom. Currently he is 2004 showed pictures of the painter Hanno Leh - chancellor of the Italian Bibliographical Temptation man. Hanno Lehmann studied chemistry and wor- Center. Monotype from Hanno Lehmann.

Hair Cell Oil in canvas, Giacomo Piccoli 84

Publications and Press Releases 2003

Articles and Press Releases king, www.berlinews.de STETE BÖDEN, PRESS PELEASE. JANUARY 2003 25.09.2003 Stäudner, F. Pflanzen als Schadstoff schluk - Daur, V. Biotechnologie im Land gewinnt Book Chapter ker. Leibniz, p. 5. Profil. Mitteldeutsche Zeitung. February 2003 Pieplow, S. Pflanzenforschung im interna- Pieplow,S. Pflanzliche "Staubsauger" ziehen 25.09.2003 tionalen Rampenlicht. In Bauen für die Schwermetalle aus dem Boden. Wirt - Daur, V. Bund setzt Tradition in Qued lin - Wissenschaft Sachsen-Anhalt,GEHRIG schaftsspiegel, p. 40. burg fort. Mitteldeutsche Zeitung. Verlagsgesellschaft mbH, p. 80-81. This article was also published by: 08.10.2003 - www.analytik-news.de WALTER, M. H., NATÜRLICHE HELFER FÜR DIE - www.chemie.de PFLAN ZENERNÄHRUNG: BIO DÜN GUNG DURCH Radio Transmission SYMBIONTISCHE MY KOR RHIZA PIL ZE, PRESS - ww.e2nn.de RELEASE. January 2003 - www.innovations-report.de Schilling, J. Pflanzenstaubsauger. NDR4. - www.uni-protokolle.de 23.10. 2003 PIEPLOW, S., BLÜTENÖLE UND DAS ÖL - 17.03.2003 - www.unitools.de BLUMENSYNDROM, PRESS PELEASE. Smiljanic, M. Grüne Staubsauger - wie - www.vln-saar.de Pflanzen vergiftete Böden sanieren. - www.zalf.de 04.12. 2003 Leonardo - Wissenschaft und mehr, MAX-PLANCK-GESELLSCHAFT, WDR5. 08.01.2003 NEUE HOFFNUNG FÜR SCHWERMETALL-BELA- Jäckel, E. Land strebt in die Weltklasse - Netzwerk Innoplanta sorgt für Aufsehen. Mitteldeutsche Zeitung, p. 17. 28.01.2003 Crodel, C. Junge Gärtner lösen manch knifflige Frage. Mitteldeutsche Zeitung. 15.02.2003 Pieplow, S. Staubsauger und Schnellrei ni - ger. Laborjournal, p. 34-35. 21.03.2003 PIEPLOW, S., PIZZAGESCHMACK UND UR - WALDDROGE, PRESS RELEASE. 24.03.2003 PIEPLOW, S., SUBTILE STRATEGIEN DER PFLANZEN GEGEN KRANKHEITSERREGER, PRESS RELEASE. Based on this press release articles were published by - www.innovations-report.de - www.uni-protokolle.de - www.zalf.de

MARCH 2003 Niemann,T. Nachgefragt Stephan Cle mens. Bild der Wissenschaft. 16.03.2003 Krause, I. Biologe mit Schwäche für die höchsten Gipfel der Welt. Mitteldeutsche Zeitung. 13.05.2003 Pieplow, S. Wenn das Grünzeug einen Schnupfen hat. Mitteldeutsche Zeitung, p. 17. 13.05.2003 Krause, I. Entscheidung für Halle als positi- ves Signal. Mitteldeutsche Zeitung, p. 17. 01.08.2003 PIEPLOW, S., EINLADUNG ZUM RICHT FEST, PRESS RELEASE 08.08.2003 Krause, I. Richtfest auf dem Campus - Institut erhält neues Labor. Mitteldeutsche Zeitung. 11.08.2003 Ronzheimer, M., Die WGL im DFG-Ran- 85

Publications and Press Releases 2004

Articles and Press Releases PFLANZENBIOCHEMIE, PRESS RELEASE. January 2004 29.06.04 30.09.04 Winter, S., Schlachten in der Furche, Der Grafiker zeigt Ansichten von der Farbe, PIEPLOW, S., AUSSTELLUNG PERPETUUM MOBILE SPIEGEL 4/2004, p. 80. Mitteldeutsche Zeitung, p.17. VON GIACOMO PICCOLI AM LEIBNIZ-INSTITUT FÜR PFLANZEN BIO CHEMIE, PRESS RELEASE 28.01.2004 01.07.04 Witthuhn, B., Die grünen Sanierungshelfer, Gabriele Herrmann vom Institut für Pflan - Berliner Zeitung, p. 13. zenbiochemie setzt frische Zellkultu ren für This press release was also published by: - www.interconnections.de 19.03.04 die Lange Nacht der Wissen schaf ten an, Mitteldeutsche Zeitung, Photography, p. 15. PIEPLOW, S., LEIBNIZ-INSTITUT ERÖFFNET WEITE- - www.prowissenschaft.de RES FORSCHUNGSGEBÄUDE AUF DEM WEINBERG 09.07.04 - www.qinx.de CAMPUS, PRESS RE LEASE. Krause, I., Forscher entdecken Ursachen - www.uniprotokolle.de 26.03.04 für den bitteren Geschmack, - www.webinfo1.de Mitteldeutsche Zeitung, p.19. Neubau für Biochemiker, Mitteldeutsche 09.10.04 Zeitung, p. 9. 31.07.04 Italiener zeigt die “Magie der Biologie”, 27.03.04 Lehmanns Farbansichten im Institut, Mit - Mitteldeutsche Zeitung, Photography. tel deutsche Zei tung, Photography, p.12. Krause, I., Viel Licht und ein Nachtlabor, 11.10.04 Mitteldeutsche Zeitung, p. 12. 31.07.04 Ausstellung am IPB, Mitteldeutsche 27.03.04 Schmundt, H., Dschungel unter den Füßen, Zeitung, p.9. DER SPIEGEL 31/2004, p. 116-118. Richter, S., Erst Kellerkind - jetzt im Licht, 12.10.04 Wochenspiegel. 24.08.04 Hoffman, P., Das “Stille-Post-Spiel” um die 29.03.04 PIEPLOW, S., MIT DEM PHYTOLATOR IN DEN Leibniz-Gemeinschaft, Magdeburger Volks - DSCHUNGEL DER ERKENN TNIS, PRESS RELEASE. stimme. Neues Forschungsgebäude seiner Be stim - mung übergeben, Based on this press release articles 14.10.04 were published by: www.weinbergcampus.de. KULTUSMINSITERIUM SACHSEN-ANHALT, - www.analytik.de HOHER EU-BEAMTER INFORMIERT SICH ÜBER 24.05.04 - www.bildung-branden burg.de BIOTECHNOLOGIE-OFFENSIVE DES LANDES SACHSEN-ANHALT, PRESS RE LEASE. WINGERT, N., HALLES BIOLOGEN ENTTARNEN DIE - www.bildungsklick.de GE HEIM NISSE VON PFLANZEN UND PILZEN, PRESS - www.chemie.de 22.10.04 RE LEA SE. - www.chemikalien.de Im Dschungel der Erkenntnis, Chemie in unserer Zeit 38 /2004, p. 367. 07.06.2004 - www.chemlin.de PIEPLOW, S., HANS OLAF HENKEL BE SUCHT - www.lehrer-online.de 22.10.04 DAS LEIBNIZ-INSTITUT FÜR - www.studieren-im-netz.de Wein, M., Lange Nacht war viel zu kurz, PFLANZENBIOCHEMIE, PRESS RELEASE. - www.uniprotokolle.de Scienta hallensis 10/2004, p. 6-7. 09.06. 2004 - www.wgl.de Krause, I., Henkel besucht Institut für - www.wifoe.halle.de Pflanzenbiochemie, Mitteldeutsche Zeitung, p.12. 01.09.04 Rapssamen ohne Bitterstoffe, Leibniz 3, p. 09.06.04 3. PIEPLOW, S., TRANSGENER RAPS PRODUZIERT SAMEN OHNE BITTERSTOFFE, 03.09.04 PRESS RELEASE. Gauselmann, K., Verunsicherung in "Exzel - 11.06.04 lenz-Zentren", Mitteldeutsche Zeitung, p.6. Transgener Raps produziert Samen ohne Bitterstoffe, www.innovations-report.de. 04.09.04 Gauselmann, K., Zusage an Institute, 18.06.04 Mittel deutsche Zeitung, p.1. Präsident der Leibniz-Gemeinschaft am 8. Juni zu Besuch in Halle, www.weinbwerg- 10.9.04 campus.de. Chemiespiel kostenlos, Mitteldeutsche Zei tung, p. 13. 20.06.2004 Pieplow, S., Bitte weniger bitter, Labor - 29.09.04 journal 06/2004, p. 34-35. Schwägerl, C., Eine Pflanze ändert ihr Ima - ge - der Raps als Edelgewächs, Frank furter 21.06.04 Allgemeine Zeitung 227. PIEPLOW, S., AUSSTELLUNG FARBAN SICH TEN VON HANNO LEH MANN AM LEIBNIZ-INSTITUT 30.9.04 FÜR PFLAN ZEN BIO CHEMIE, PIEPLOW, S., MYKORRHIZA IM NEUEN GE WAND, PRESS RELEASE. PRESS RELEASE. 23.06.04 This press release was also published by: Lehmann zeigt Farbansichten, Mitteldeut - - www.innovationsreport.de sche Zeitung. - www.interconnections.de 25.06.04 - www.mygeo.de Krause, I., Einblicke auf dem Campus, Mit - - www.vdbiol.de teldeutsche Zeitung, p.16. - www.wgl.de 28.06.04 - www.wissensschule.de PIEPLOW, S., LANGE NACHT DER WIS - - www.zeus.zeit.de SENSCHAFTEN AM LEIBNIZ-INSTITUT FÜR 86

Publications and Press Releases 2004

25.10.04 This press release are also published by: 20.12.2004 PIEPLOW, S., NEUE DIREKTORIN AM LEIB NIZ- - www.chemie.de Stäudner, F., Gehaltvolle Pilze, Leibniz 4, p. INSTITUT FÜR PFLANZENBIO CHEMIE, PRESS - www.chemlin.de 4. RELEASE. - www.forstverein.de 20.12.2004 This press release was also published by: - www.geoscience-online.de Bank, M. Morphium in Zellen produziert, - www.chemlin.de - www.journalmed.de Mitteldeutsche Zeitung, p. 24. - www.innovationsreport.de - www.medicineworldwide.de - www.interconnections.de - www.physik-forum.de Radio Transmission - www.uniprotokolle.de - www.shortnews.stern.de 05.06.04 - www.weinbergcampus.de - www.tk-online.de Jacobs, D., Schnecklinge als Antibiotika, 12/2004 - www.vdbiol.de Mitteldeutscher Rundfunk, Radio - Laborpraxis , p. 14. Sachsen-Anhalt - BIOforum 12/2004, p. 8 04.11.04 03.11.04 26.10.04 Schilling, T., Mit dem Trick-”Phytolator” eine neue Medizin entdecken, Mittel deut - Kienzlen, G., Medizin aus dem Wald, Bank, M., Neue CD über Symbiosen, Deutschlandfunk, Forschung aktuell Mitteldeutsche Zeitung, p.20. sche Zeitung, p. 22. 30.10.04 26.11.2004 Television Transmission Bank, M., Frau aus Amerika an der Spitze, Pieplow, S., Pilze kontra Eiter und Bak - terien, Mitteldeutsche Zeitung, p. 22. 26.03.04 Mitteldeutsche Zeitung, p. 20. Neues Gebäude für Leibniz-Institut, Mitteldeutscher Rundfunk, Sachsen- 01.11.04 10.12.04 Spilok, K., Neue Bioprodukte haben Zu- Anhalt heute PIEPLOW, S., NEUE WIRKSTOFFE AUS HEIMI- satznutzen, VDI Nachrichten. SCHEN WÄLDERN, PRESS RELEASE 16.06.04

Press and Public Relations 87

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Publisher: SCIENTIFIC REPORT 2003 - 2004 Leibniz Institute of Plant Biochemistry May 2005

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Editor: Sylvia Pieplow Press and Public Relations Phone: +49 (0) 3 45 - 55 82 11 10 Fax: +49 (0) 3 45 - 55 82 11 19 email: [email protected]

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Copyright © 2005, all rights reserved Leibniz Institute of Plant Bio chemistry (IPB), Halle, Germany. No parts of this publication may be reproduced by any mechanical, photographic, electronic process, or in form of a photographic recording, nor may be stored in a retrieval system, transmitted or otherwise copied for public or private use, without written permission from the publisher.