Abstract Book

Moss 2004

The 7th annual moss international conference

Freiburg / Germany Sept 12th-15th

wwwwww..ppllaanntt--bbiiootteecchh..nneett//mmoossss22000044

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Sponsors pages 3, 4 Talk abstracts pages 5 – 31 Poster abstracts pages 32 – 73 List of participants pages 74 – 78

moss 2004 has been organized by Plant Biotechnology, University of Freiburg, headed by Ralf Reski

general organization, sponsoring and giveaways: Birgit Hoch and Stefan Rensing information, catering, excursions, accommodation: Birgit Hoch webpages, conference material, computers : Stefan Rensing

committee: Eva Decker, Wolfgang Frank, Christina Reinhard, Stefan Rensing, Ralf Reski, Eric Sarnighausen, Gabriele Schween,

Mark von Stackelberg

helper crew: Isabelle Chassignet, Anika Erxleben, Louis Gremillon, Johannes Heckmann, Verena Horstmann, Marc Kaminski, Charlotte Kleitsch, Daniel Lang, Otmar Lienhart, Anja Martin, Manuel Mildner, Juliana Parsons, Sandra Richardt, Andreas Schaaf,

Eik Schumann, Stefanie Tintelnot, Gabriele Tischlik, Getrud Wiedemann

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We would like to gratefully acknowledge our sponsors:

Silver sponsors

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Bronze sponsors

Madora

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Talk abstracts Monday, Sept 13th Page No. 09:00 am: Session I, Evolution and Development 09:00 Mitsuyasu Hasebe, National Institute for Basic Biology 6 09:40 Thomas Münster, Max Planck Institute for plant breeding research 7 10:00 Takako Tanahashi, University of Tokyo 8 10:20 Tomoaki Nishiyama, National Institute for Basic Biology 9 11:20 am: Session II, Phylogeny and Evolution 11:20 Mary Skotnicki, Australian National University 10 11:40 Mark von Stackelberg, University of Freiburg 11 12:00 Stuart McDaniel, Duke University 12 12:20 Hiroyoshi Takano, Kumamoto University 13 12:40 Nancy Hofmann, University of California at Davis 14 Tuesday, Sept 14th 09:00 am: Session I, Recombination, Transformation and Genomics 09:00 David Cove, University of Leeds/Washington University 15 09:40 Bénédicte Trouiller, INRA Versailles 16 10:00 Mamoru Sugita, Nagoya University 17 10:20 Stefan Rensing, University of Freiburg 18 11:20 am: Session II, Molecular Structures, Metabolites and Pharming 11:20 Aldwin Anterola, Washington State University 19 11:40 Gertrud Wiedemann, University of Freiburg 20 12:00 Eva Decker, University of Freiburg 21 12:20 Kieran Lee, University of Leeds 22 12:40 Yoshikatsu Sato, National Institute for Basic Biology 23 Wednesday, Sept 15th 09:00 am: Session I, Gene Regulation 09:00 Ralph Quatrano, Washington University 24 09:40 Setsuyuki Aoki, Nagoya University 25 10:00 Michael Lawton, Rutgers University 26 10:20 Magdalena Bezanilla, Washington University 27 11:20 am: Session II, Abiotic Stress 11:20 Melvin Oliver, USDA-ARS 28 12:00 Andrew Wood, Southern Illinois University 29 12:20 Wolfgang Frank, University of Freiburg 30 12:40 Andrew Cuming, Leeds University 31

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Physcomitrella patens as an indispensable model for EvoDevo studies Mistuyasu Hasebe * National Institute for Basic Biology, Okazaki 444-8585, Japan * mhasebe@nibb.ac.jp, fon/FAX +81-564-55-7546 Extant land plants are composed of five monophyletic groups; angiosperms, gymnosperms, monilophytes, lycopods, and bryophytes. These plants are all multicellular organisms, and evolved from the unicellular common ancestor more than a billion years ago. Developmental and morphological diversities of these groups are well described in the last centuries, but their molecular bases governing such diversities are mostly unknown. To perform such studies, at least several models in which forward and reverse molecular genetics are applicable are necessary for each monophyletic group. The moss Physcomitrella patens is a good model in bryophytes, and its usefulness in EvoDevo studies will be discussed in this talk. P. patens is a good model to study the evolution from unicellular to multicellular organisms. The first evolutionary step from unicellular to multicellular organisms is to form two different cells from a single cell via asymmetric cell division. The first cell division of a protoplast isolated from the protonemata of the moss Physcomitrella patens is asymmetric and gives rise to an apical meristematic cell and a differentiated non-meristematic cell. We screened genes involved in asymmetric cell division by large-scale overexpression screening of 15000 Physcomitrella full-length cDNAs. Approximately 100 candidate genes that caused defects in asymmetric cell division by overexpression were isolated. Based on characterizations of these genes, differences and similarities of molecular mechanisms in asymmetric cell divisions between plants, metazoans, and fungi will be discussed. Patterns of cell divisions and growths are varied in land plants. Protonemal cells of Physcomitrella and green algae cells do not form preprophase band, but gametophores and most cells in land plants form. Apical growth is specific in protonemata of bryophytes and pteridophytes and some types of cells in other land plants. To investigate the differences and evolution of cell division patterns, three genes specifically expressed in dividing cells were screened from the Physcomitrella gene trap lines. These genes encode kinesin-, ubiquitin-like proteins and a small molecular protein. Characterization of these genes and the comparison of their functions between Arabidopsis and Physcomitrella will be presented. The mosses and flowering plants diverged more than 400 million years ago. The mosses have haploid-dominant life cycles, while the flowering plants are diploid-dominant. The common ancestors of land plants are inferred to have been haploid-dominant, suggesting that genes used in the diploid body of flowering plants were recruited from the genes used in the haploid body of their ancestors during the evolution of land plants. To assess this evolutionary hypothesis, we constructed an expressed sequence tag (EST) library of the moss Physcomitrella patens, and compared the moss transcriptome to the genome of Arabidopsis thaliana. The functions of some candidate genes involved in diploid plant body organization in angiosperms, such as the KNOX, HD-Zip, and FLO/LEAFY genes, are being compared with those in the haploid shoot of the moss Physcomitrella. Polar auxin transport is indispensable for regular development of the diploid shoot, and investigations of transport in the haploid shoot of Physcomitrella were also performed. Implications for the evolution of diploid multicellular body plants will be discussed. The flower is the reproductive organ in angiosperms, and floral homeotic genes, such as MADS-box genes and FLO/LEAFY genes, regulate floral organ identity. To investigate the origin of floral homeotic genes, the functions of MADS-box genes and the FLO/LEAFY genes in Physcomitrella were analyzed.

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MADS-box gene networking in Physcomitrella: analysis of the protein interactions. Wolfram Faigl, Ralf Hallinger, Vanessa Quodt, Heinz Saedler and Thomas Münster Max-Planck-Institut für Züchtungsforschung, Abteilung Molekulare Pflanzengenetik, Carl-von-Linné-Weg 10, 50829 Köln, Germany Contact email: muenster@mpiz-koeln.mpg.de Plant MADS-box genes belong to a family of transcription factors present in all eukaryotic organisms. Their roles as molecular architects of the angiosperm flower and their involvement in many other developmental processes are intensively studied. On the other hand, the knowledge about MADS-box genes in non-flowering plants is limited. Therefore we choose Physcomitrella as our model system to analyse the functional evolution of this gene family in plants. The plant specific MIKC-type MADS-domain proteins (about 45 in Arabidopsis, only 12 to 15 in Physcomitrella) share a common highly conserved domain structure. They bind as protein dimers to different types of short DNA sequence motifs, the so-called CArG-boxes. In Physcomitrella 11 different MADS-box genes had been identified based on complete cDNA and partial EST sequences, respectively. We tested the DNA-binding and dimerization properties of seven different Physcomitrella MADS-domain proteins using in vitro and in vivo experimental approaches. All proteins were analysed for their protein-protein interactions in the yeast-two-hybrid system. The DNA-binding specificity of selected MADS-domain protein homo- and heterodimers was examined using electrophoretic mobility shift assays (EMSA). Finally, binding of the Physcomitrella MADS-domain proteins to a putative target promoter will be reported and first steps of a reconstruction of the MADS-box gene transcription factor network in Physcomitrella will be discussed. Henschel, K., Kofuji, R., Hasebe, M., Saedler, H., Münster, T., Theißen, G. (2002): Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens. Mol. Biol. Evol. 19: 801-814.

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Physcomitrella FLO/LFY homologues, PpLFY1 and PpLFY2 regulate the sporophyte development from the zygote stage Takako Tanahashi1*, Naomi Sumikawa2, Masahiro Kato1 and Mitsuyasu Hasebe2 1 University of Tokyo, Tokyo 113-0033, JAPAN 2 National Institute for Basic Biology, Okazaki 444-8585, JAPAN * e-mail: takakot@biol.s.u-tokyo.ac.jp, phone & fax: +81 3 5841 4047 FLORICAULA/LEAFY (FLO/LFY) genes are isolated only from land plants. Arabidopsis LFY encodes a plant specific transcription factors that positively regulates the expression of floral homeotic genes. In angiosperms, FLO/LFY genes have largely conserved function in flower development among distantly related species. The transcriptional activation of floral homeotic genes by FLO/LFY genes is conserved even in gymnosperms, conifer and Gnetum; however this is not likely the case with ferns. The function of FLO/LFY genes in bryophytes has not been identified so far. To asses the original function of FLO/LFY genes and to understand land plant evolution from the viewpoint of the regulation of flower development, we characterized FLO/LFY genes in the moss Physcomitrella patens. We isolated two FLO/LFY homologues from P. patens and designated them as PpLFY1 and PpLFY2. They were so similar to each other as to have nearly 90% identical nucleotide sequence in their coding regions. Their expressions were detected mainly in gametophore shoot apices, young leaves, archegonia and egg cells in them and developing sporophytes. Functional analyses by gene disruption showed that double disruption of PpLFY1 and PpLFY2 caused severe defect in the sporophyte formation. Only 0.8% of gametophores of PpLFY double disruptants formed a sporophyte, while more than 95% of wild type gametophores did. What caused such a critical defect in PpLFY disruptants? Sperms were likely to have no defects in PpLFY disruptants because no GUS activity was detected in antheridia in PpLFY-GUS lines and sperm entry into archegonial cavity was often observed in disruptant lines. Therefore the defect of PpLFY disruptants was likely to be derived from an egg cell or an early embryo and we observed them in detail using confocal laser scanning microscope (CLSM). When more than 70% of wild type archegonia had a developing embryo within them, no embryo nor mature zygote was observed in archegonia of PpLFY double disruptants (N=93) and which egg cells were arrested at single cells. These single egg cells were further divided into “before fertilization”, “after fertilization (=early zygote)” and “aberrant” based on our morphological criteria. These CLSM observations suggest that PpLFY disruptants are arrested at an early zygote stage after fertilization. We further investigated if PpLFY disruptants have no defect before fertilization, by crossing their egg cells with wild type sperms. Some normal sporophytes resulted from cross-fertilization were obtained and this clearly showed PpLFY disruptants having defect after fertilization. In wild type early zygote is round and small, then it expands and fills the cavity of an archegonium at maturity. The defect of PpLFY disruptants was interpreted as in this zygote expansion process. Moreover, a very little number of gametophores of PpLFY disruptants produced sporophytes. These sporophytes exhibited various abnormal morphology (Fig.) and they often failed in differentiating spores. When spores were yielded, however, they seldom germinated (only 0-60 spores germinated per a sporangium). These indicate that PpLFY1 and PpLFY2 also regulate the sporophyte development and spore formation after zygote stage. Our results demonstrate that FLO/LFY genes in Physcomitrella consistently regulate the sporophyte development from the zygote-maturating stage. We suggest that FLO/LFY genes, which generally regulate the sporophyte development in bryophytes, evolved to specifically regulate the flower development in sporophytic generations in angiosperms during land plant evolution.

Fig. Examples of abnormal sporophytes formed on PpLFY disruptants. Some sprophytes withered on their development (A) and a few had multiple sporangia (B).

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Analysis of class 1 KNOX genes in Physcomitrella patens Tomoaki Nishiyama*, Keiko Sakakibara, and Mitsuyasu Hasebe National Institute for Basic Biology, Okazaki, 444-8585 Japan * tomoaki@nibb.ac.jp, phone +81-564-55-7549 In vascular plant sporophytes, the shoot apical meristem repeatedly forms stem and lateral organs, such as leaves. However, the evolutionary origin of shoot apical meristem was unclear. The sporophyte of bryophytes forms a single sporangium without lateral organs. A part of bryophytes, mosses and leafy liverworts, form leafy shoots in the gametophyte generation. We aimed to reveal whether homologues of vascular plant genes involved in shoot apical meristem formation are involved in the sporophyte development of bryophytes and whether homologous genes are involved in the gametophytic and sporophytic shoots. Molecular genetic studies in angiosperms revealed several genes involved in the formation and maintenance of the shoot apical meristem. SHOOT MERISTEMLESS is one of such genes and is a member of KNOX class 1 group of homeobox gene superfamily. Three class 1 KNOX genes, PpKNOX1/MKN2 (PpKN1, Sakakibara et al. MOSS1998; Champagne and Ashton 2001), MKN4 (Champagne and Ashton 2001), and MKN5 (Champagne and Ashton 2001; Nishiyama et al. MOSS2003), have been isolated in Physcomitrella patens. Arabidopsis thaliana expressing PpKN1 cDNA under the control of cauliflower mosaic virus 35S promoter showed lobed leaves, which is similar to those in A. thaliana expressing other class 1 KNOX genes (Nishiyama et al. MOSS2003). To investigate the expression patterns of the three class 1 KNOX genes, we inserted the GUS reporter gene at the end of every one of the three genes (PpKN1 in Sakakibara et al. MOSS2001; MKN4 and MKN5 in this study). The transgenic mosses showed GUS activity specifically in egg cells and young sporophytes but not in gametophores (leafy shoots). We constructed triple disruptants of PpKN1, MKN5, and MKN4. The phenotypic analysis is under way, but they produced normal gametophores. These data suggest that the three class 1 KNOX genes function in the sporophyte development, but not in gametophytic shoot. The role of these genes in sporophyte development will be investigated by the phenotypic analysis of the triple disruptants. The analysis of 51 protein-coding chloroplast genes indicated that bryophytes are monophyletic with hornworts basal within bryophytes (Nishiyama et al. 2004). This implies that class 1 KNOX genes were involved in sporophyte development in the last common ancestor of extant land plants. The difference in the regulation of KNOX genes might account why vascular plants form shoots in the sporophyte generation but bryophytes do not. Champagne, CEM and Ashton, NW. (2001). Ancestry of KNOX genes revealed by bryophyte (Physcomitrella patens) homologs. New Phytologist 150: 23-36 Nishiyama, T, Wol, PG, Kugita, M, Sinclair, RB, Sugita, M, Sugiura, C, Wakasugi, T, Yamada, K, Yoshinaga, K, Yamaguchi, K, Ueda, K, and Hasebe M. Chloroplast Phylogeny Indicates that Bryophytes are Monophyletic. Molecular Biology and Evolution: in press. (2004)

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The coldest mosses in the world – evolution and survival in Antarctica Mary Skotnicki*, Anne Mackenzie and Patricia Selkirk Research School of Biological Sciences, Australian National University, Canberra, Australia and Dept. of Biological Sciences, Macquarie University, Sydney, Australia * skotnicki@rsbs.anu.edu.au Mosses at around 780S in Antarctica exist at the absolute limits for any plant growth, surviving not only extremes of temperature, but also drought, strong winds, and long months of continuous darkness in winter (and continuous light in the summer months). We are using genetic techniques to investigate their patterns of dispersal, colonisation of new areas after glacial retreat, and response to increased UV exposure due to annual expansion of the ozone hole over Antarctica. We are also using molecular taxonomy to characterise mosses from extremely remote and isolated ice-free areas of Antarctica, and to identify their origins. Mutation appears to be the main cause of the high levels of genetic diversity observed in these plants, which can only reproduce vegetatively due to the harsh environment of Antarctica. We are combining an investigation of mutagenesis in these mosses with a long-term study of their extremely slow growth rates, to better understand how these plants are evolving for survival in Antarctica.

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First Steps Towards a Genetic Map of Physcomitrella patens: High Genetic Variability in the Genus Based on Molecular Analyses Mark von Stackelberg*, Stefan A. Rensing¸ Ralf Reski, Gabriele Schween Plant Biotechnology, University of Freiburg, Schaenzlestr. 5, D-79104 Freiburg, Germany *mark.stackelberg@biologie.uni-freiburg.de With the main aim to develop a genetic map of Physcomitrella patens, we are setting up an axenic in vitro collection of worldwide Physcomitrella ecotypes. Various european, japanese and australian accessions have been collected so far (1). We investigated their genome size using flow cytometric measurement of DAPI stained nuclei and their genetic variability using microsatellites or simple sequence repeats (SSR) and internal transcribed spacer (ITS) regions. No significant differences in genome size were detected. For identification of SSR loci in silico from our EST database we used a PERL script called MISA (2) with slight modifications. Visualizing more than 70 SSR loci via PCR, we detected a high degree of intraspecific DNA polymorphism. Japanese sspec. ‘californica’ accessions reveal more than 90 % SSR polymorphism to european sspec. ‘patens’ accessions. Comparison of ITS regions supports the large genetic distance between japanese and european ecotypes. Moreover, at the intrapopulation level partially high variability has been detected. In recent years molecular biological methods have been widely used to complete classical systematic analyses. For bryophytes this had major impact on recent taxonomic concepts (3). Our initial molecular results raise the question, if the common subdivision of the genus Physcomitrella patens into four subspecies patens, readeri, californica and magdalenae (4) can be sustained, or if the subspecies have to be classified as single species. Currently we are setting up sexual crossings to develop segregating mapping populations. Acknowledgements: We would like to thank all the people, who contributed to the Physcomitrella ecotypes collection. 1. For details: http://www.cosmoss.org/ecomap.html 2. Thiel T. et al., 2003, Theor Appl Genet, 106: 411-422 3. Stenoien H. K., 2002, Lindbergia, 27: 134-140 4. Tan B., 1979, Journ Hattori Bot. Lab., 46: 327-336

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The genetic basis of protonemal growth rate variation in Ceratodon purpureus (Hedw.) Brid. Stuart F. McDaniel* and A. Jonathan Shaw Biology Department, Box 90338, Duke University, Durham NC 27708, USA *stumcd@duke.edu, fon +1 919 660 7370 Population divergence is often thought to be underlain by genetic changes at multiple interacting loci. Crosses between divergent populations can disrupt co-adapted gene networks, leading to less fit hybrid progeny. Wide crosses therefore provide a powerful means to dissect components of gene networks that underlie complex phenotypes. To better understand the genetic network underlying protonemal development in the moss model system Ceratodon purpueus, we crossed a male from Ithaca, New York, USA, and a female from Otavalo, Ecuador, and cultivated 300 recombinant progeny. Approximately half of the hybrid progeny exhibited a reduced protonemal growth rate. To understand the genetic basis of the hybrid-breakdown phenotype, we constructed a linkage map using 121 polymorphic AFLP loci and two gene-based markers. We resolved 16 linkage groups resulting in a map length of 853.9 cM. The average marker density was 11.3 cM (range 0 – 33 cM). Markers on linkage groups (LG) 10 and 14 were significantly distorted toward the New York parent, while markers on LG 10 were distorted toward the Ecuador parent. Using single-marker analysis and ANOVA we identified four loci that interact epistatically to reduce the growth rate. Our methods are conservative in estimating epistatic effects among loci. The four-locus model explains 30% of the between-class variation, although this value may increase with increasing marker saturation on our linkage map. Preliminary investigation suggests the hybrid breakdown phenotype may result from a delay in the transition from choro- to caulonema cell types. We are currently mapping candidate genes and ESTs to provide a foundation for further genomic studies.

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Relationship between plastid biogenesis and genes of peptidoglycan synhtesis pathway in Physcomitrella patens Mariko Machida1, Katsuaki Takechi1, Susumu Takio2, Mitsuyasu Hasebe3, Hiroyoshi Takano1* 1Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan 2 Center for Marine Environment Studies, Kumamoto University, Japan 3 National Institute for Basic Biology, Japan * takano@kumamoto-u.ac.jp tel&fax +81 96 3423432 The endosymbiotic theory states that all plastids are derived from a single cyanobacterial ancestor with a cell wall. It is widely agreed that the plastids of red algae and land plants have no peptidoglycan layer. Therefore, the evolution from endocytobiont into a wall-less, photosynthetic organelle involved a reduction in and loss of the cyanobacterial cell wall, which is of Gram-negative type. On the other hand, some studies have shown that the cyanelles, which fulfill the functions of chloroplasts in Glaucophyta, are surrounded by a peptidoglycan wall. The structure of the cyanelle peptidoglycans resembles that of cyanobacteria, and ß-lactam antibiotics block the division of cyanelles1. The peptidoglycan synthesis pathway is a major target for antibiotics, since there is no peptidoglycan layer in animal cells. In the moss Physcomitrella patens, treatment with three antibiotics including ß-lactams, which inhibit peptidoglycan synthesis at different steps, results in giant chloroplasts2, 3. Moreover, ß-lactam antibiotic, ampicillin inhibits plastid division in the Pteridophyta Selaginella nipponica4. We isolated nine genes that were related to peptidoglycan synthesis from the P. patens EST database. Two genes were used with gene-targeting technique that is established in P. patens. In knock-out transformants, macrochloroplasts appeared. These findings suggest that a relic of the bacterial peptidoglycan synthesis pathway is retained in plants and is involved in plastid morphology and division. 1Aitken, A. and Stanier, R. Y. (1979) Characterization of peptidoglycan from the cyanelles of Cyanophora paradoxa. J. Gen. Microbiol. 112: 219-223 2Katayama, N., Takano, H., Sugiyama, M., Takio, S., Tanaka, K. and Ono, K. (2003) Effects of antibiotics that inhibit the bacterial peptidoglycan synthesis pathway on moss chloroplast division. Plant Cell Physiol. 44: 776-781 3Kasten, B., Reski, R. (1997) ß-lactam antibiotics inhibit chloroplast division in a moss (Physco-mitrella patens) but not in Tomato (Lycopersicon esculentum). J. Plant Physiol. 150: 137-140 4Izumi, Y., Ono, K. and Takano, H. (2003) Inhibition of plastid division by ampicillin in the pteridophyte Selaginella nipponica Fr. et Sav. Plant Cell Physiol. 44: 183-189 This study was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences.

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Chloroplast Protein Targeting in Physcomitrella patens: Toc64 Nancy Rosenbaum Hofmann* and Steven M. Theg# Section of Plant Biology, UC-Davis, Davis, CA 95616 *nrhofmann@ucdavis.edu, #smtheg@ucdavis.edu 530-752-0624 Toc64 has been suggested to be a part of the chloroplast protein translocation machinery at the chloroplast envelope. This protein includes a short hydrophobic domain followed by an amidase-like region and three tetratricopeptide repeats (TPRs). TPRs in other systems have been implicated in protein-protein interactions, leading Sohrt and Soll (2000) to suggest that this region mediates precursor protein docking at the translocon of the chloroplast envelope. We are using the moss, Physcomitrella patens, as model system for studying chloroplast protein targeting. We have cloned two genes (PpToc64-1 and PpToc64-2) from P. patens that encode Toc64 proteins. Both proteins are targeted to the chloroplast in in vitro targeting assays and this targeting requires the N-terminal hydrophobic domain. Their in vitro association with chloroplasts does not appear to require proteins that are sensitive to either trypsin or thermolysin proteolysis. It does, however, require the presence of nucleoside triphosphates. In order to determine whether PpToc64-1 and PpToc64-2 play roles in chloroplast protein transport, we have used homologous recombination to knock out the two genes in moss. A single knockout had no discernable phenotype, though there was still a large amount of Toc64 protein in the chloroplast. The double knockout is viable and does not have an obvious growth phenotype. We will present the further analysis of this double mutant. Sohrt K, Soll J (2000) Toc64, a new component of the protein translocon of chloroplasts. Journal of Cell Biology 148: 1213-1221

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Linear transforming DNA can be circularised before integration into the genome by homologous recombination. Yasuko Kamisugi, Andrew Cuming and David Cove* Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK *d.j.cove@leeds.ac.uk We have analysed stable transgenics produced by transformation with constructs having genomic DNA flanking a selection cassette. In some transgenics, we have only been able to detect the PCR product predicted to result from homologous recombination in either the 5’ or the 3’ flanking sequence. In order to test if this may result from circularisation of transforming DNA before integration by a single homologous recombination event, we have analysed a sample of such transgenics using PCR primers designed to amplify across the predicted boundary sequence generated by circularisation. Of 10 transgenics so far analysed, at least part of this predicted sequence, can be detected in 9. One has an almost perfect match to the predicted sequence, while the other 8 have either deletions or additions to the sequence, but contain unique signatures of a circularisation event. We conclude that most, if not all of such transgenics, must be generated by circularisation of the linear transforming DNA, prior to integration. We have compared the outcome of transformation using similar linear DNA sequences generated by restriction enzyme digestion of a plasmid construct or PCR amplification, and found no difference in the frequency with which transgenics targeted at only the 5’ or 3’ end occur. Circularisation does not therefore appear to be dependent on the presence of the sticky ends generated by RE digestion and is likely to occur by non-homologous end joining. This work was funded by the European Union as part of the PREGENE (Precision Engineering of Plant Genes) Project.

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The MSH2 gene in Physcomitrella patens preserves genomic integrity and prevents homeologous recombination between divergent sequences Bénédicte Trouiller*, Florence Charlot and Fabien Nogué Station de Génétique et d’Amélioration des Plantes, INRA, Route de St Cyr, 78026 Versailles, France * trouille@versailles.inra.fr, phone 33-1-30833009 The preservation of genomic integrity requires the proper functioning of multiple replication, repair and recombination processes. DNA mismatch repair (MMR) is one of several DNA repair pathways conserved from bacteria to humans. The MMR pathway targets base-base mismatches, and insertion-deletion loops (IDLs) that arise during replication and homologous recombination. In addition, mismatches can result from DNA damage. The protein MSH2 is a central component of the eukaryotic MMR system. Here we describe the study of the MSH2 gene in the moss Physcomitrella patens, where homologous recombination (HR) is exceptionally high. This property, unique in the plant kingdom makes the moss a convenient model to study the regulation of HR in plants. To determine the role of PpMSH2 in different aspects of DNA metabolism, we made Ppmsh2 mutants by gene targeting (GT). To assess different phenotypes of the Ppmsh2 mutants, we used the APT gene as a reporter gene. Mutation in the APT gene confers resistance to 2FA (2FluoroAdenine) a toxic compound for cells. Spontaneous mutation frequencies were estimated in the Ppmsh2 background, by estimating mutations in the PpAPT gene. Protoplasts of WT and Ppmsh2 mutants were produced and spread on a 2FA medium. Ppmsh2 mutants accumulate 200 times more spontaneous mutations than the WT. Mutation frequency and cell survival after chemical (cisplatin and MNNG) and physical (γ-irradiations and UV-irradiations) treatments are in progress. PpMSH2 gene involvement in HR has been studied by testing GT efficiency at the locus of the APT reporter gene with homologous and homeologous sequences. In the WT background 3% divergency in the APT sequence leads to a decrease of 91% of the targeting efficiency, in contrast to the msh2 background where this decrease is only 17%. This work provides a framework in which to discuss and compare MMR in other eukaryotes. By exploiting the powerful genetic tools available in Physcomitrella patens, we hope to complement studies in plants and shed more light on the role of mismatch repair proteins in higher eukaryotes.

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Plastid transformation in the moss Physcomitrella patens Chika Sugiura and Mamoru Sugita* Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan *sugita@gene.nagoya-u.ac.jp, Tel/Fax: +81-52-789-3080 Three distinct arginine tRNA genes, trnR-CCG, trnR-ACG, and trnR-UCU, are present in the moss Phycomitrella patens plastid DNA (Sugiura et al., 2003), whereas only the latter two trnR genes are present in the major vascular plants. trnR-CCG has been considered functional in plastids. However, no trnR-CCG transcript has been detected, and the codon usage of CGG is quite low in plastid protein-coding sequences. This raises the possibility that trnR-CCG is non-functional. To investigate this possibility, we constructed trnR-CCG knock-out mosses by plastid transformation via homologous recombination. For the targeted disruption of trnR-CCG, the gene was replaced with a selectable spectinomycin (spec)-resistance (aadA) gene cassette from pZS197 (Svab and Maliga, 1993) on a plastid gene fragment in the plasmid pCS∆trnR. The transforming plasmid pCS∆trnR was digested with appropriate restriction enzymes, and the DNA digests were introduced into protonema protoplasts in the presence of polyethylene glycol. The correct insertion of aadA into the targeted plastid genome in transformants were verified by Southern blot and PCR analyses. The trnR-CCG knock-out transformants (S23 and S26) grew normally and there were no apparent differences in phenotypic characters, such as the growth of protonema colonies, the size of leafy moss stems, the shape and number of chloroplasts in protonemal cells, or chlorophyll content, indicating that the P. patens trnR-CCG gene is not essential for plastid function. To investigate whether the spec-resistance of the S23 and S26 lines is inherited from the progenitors by the next generation, we examined the phenotypes of the progeny. All the moss colonies derived from regenerating spores from a single sporophyte of the S23 and S26 lines grew normally in the presence of spec. This confirms that the S23 and S26 transformants are homoplasmic transplastomic. Southern blot analysis confirmed that the progeny of the S23 and S26 lines (S23P and S26P, respectively) carry the aadA-cassette–integrated transplastome. This clearly indicates that the spec-resistant phenotype is inherited via the plastid genome. In this study, plastid transformation successfully achieved targeted gene-disruption in the moss plastid genome. This will allow investigation of the molecular basis of the regulation of plastid gene expression and the interactions between nuclear and plastid genomes. Sugiura, C, Kobayashi, Y, Aoki, S, Sugita, C and Sugita, M (2003) Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus. Nucleic Acids Res. 31, 5324–5331 Svab, Z and Maliga, P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc. Natl. Acad. Sci. USA, 90, 913–917

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News from the moss transcriptome Stefan A. Rensing*, Daniel Lang, Dana Fritzowsky and Ralf Reski Plant Biotechnology, University of Freiburg, Sonnenstrasse 5, D-79104 Freiburg, Germany * stefan.rensing@biologie.uni-freiburg.de, fon +49 761 203-6974 We have clustered and assembled the Physcomitrella patens EST and CDS data in order to represent the transcriptome. Clustering of the publicly available data resulted in a total of 19,081 non-redundant ORFs, which are expected to cover around 60% of the organisms protein-encoding genes. Whereas introns are larger on average than in Arabidopsis, position and amount of introns are approximately the same. Contrary to Arabidopsis, where protein-encoding CDS contain on average 44 % G/C, in Physcomitrella the G/C content is on average 50%, displaying less codon bias. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage towards the cress. Annotation of predicted coding regions reveals several thousand transcripts with no known homolog. In addition, there are transcripts which are not present in seed plants but can be found in other kingdoms. These are potential “retained genes” that have been lost in seed plants during the course of evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and interesting putative functions such as cytotoxicity and nucleic acid repair. In addition, species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 378 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. Both the transcriptome representation and splice site prediction have been made available on www.cosmoss.org.

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Lipoxygenases and their inhibitors from Physcomitrella patens Aldwin Anterola*, Howard Grimes and George Sellhorn School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234, USA * anterola@mail.wsu.edu, tel: (509)-335-3321, fax: (509)-335-1907 Twelve lipoxygenase genes from Physcomitrella patens were heterologously expressed in E. coli using a T7 promoter. Among the recombinant lipoxygenases produced, eight were found to be active, one of which preferentially uses arachidonic acid as substrate, while the other seven use linole(n)ic acid. These results suggest a potential mammalian-like C20-based lipoxygenases pathway in moss, in contrast and/or in addition to a C18-based lipoxygenases pathway found in higher plants. However, despite the numerous lipoxygenases putatively present in Physcomitrella, we were unable to detect any lipoxygenase activity in crude protein extracts from gametophytic cultures. We thus tested for lipoxygenase-inhibitory activities in methanolic extracts, and found potent inhibition exhibited by a hexane soluble fraction. The isolation and structural elucidation of the inhibitory compound will be presented, along with the properties of the arachidonate-specific lipoxygenase.

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Regulation of assimilatory sulfate reduction in Physcomitrella patens Gertrud Wiedemann1, Anna Koprivova2, Ralf Reski2 und Stanislav Kopriva1

1 Institute for Forest Botany and Tree Physiology, University of Freiburg Tel: +49 761-203-8307; e-mail: Gertrud.wiedemann@ctp.uni-freiburg.de 2 Plant Biotechnology, University of Freiburg The reduction from sulfate to sulfite in the sulfate assimilation pathway can be catalysed by two different enzymes. It is generally accepted that plants, algae and phototrophic bacteria use APS for reduction, while fungi and bacteria use PAPS. The moss Physcomitrella patens was the first plant in which both enzymes were found. To understand the function and coordination of the two enzymes in P. patens we made knockouts of both APR and PAPR by homologous recombination. Disruption of the genes did not affect the appearance of the plants and their thiol concentration at normal conditions. However, the knockouts are more sensitive to heavy metals. A database search revealed that beside P. patens also Selaginella lepidophylla, a vascular plant, possess PAPR. To learn more about the evolution of sulfate assimilation, different species of cyanobacteria, algae and lower plants were screened for the presence of APR and PAPR by measuring APS and PAPS dependent sulfite production. Our results indicate both APR and PAPR activity in Gingko and Equisetum. APR was also detected in several cyanobacteria, revealing that the APS dependent reduction is more widespread that originally believed. The distribution of APR and PAPR in photosynthetic organisms, evolution of plant sulfate assimilation, and the role of both enzymes in sulfate assimilation of P. patens will be discussed. Financial support through the Deutsche Forschungsgemeinschaft FOR383 is gratefully acknowledged.

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Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans Anna Koprivova1, Christian Stemmer2, Friedrich Altmann3, Axel Hoffmann1, Stanislav Kopriva4, Gilbert Gorr2, Ralf Reski1, and Eva L. Decker1* 1University of Freiburg, Plant Biotechnology, Schaenzlestr. 1, 79104 Freiburg, Germany, 2greenovation Biotech GmbH, Boetzinger Str. 29b, 79111 Freiburg, 3Institute of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria, 4University of Freiburg, Department of Tree Physiology, Georges-Koehler-Allee 053, 79085 Freiburg * Eva.Decker@biologie.uni-freiburg.de, +49 761 2032820 The use of plants as production factories for therapeutical proteins requires modification of their N-glycosylation pattern because of the immunogenicity of plant-specific sugar residues. In an attempt towards such humanisation we disrupted the genes for α1,3-fucosyltransferase and β1,2-xylosyltransferase in Physcomitrella patens by homologous recombination. The single fuc-t and xyl-t knockout plants as well as double knockout lacked transcripts of the corresponding genes, but did not differ from wild type moss in morphology, growth, development and ability to secrete a recombinant protein, the human vascular endothelial growth factor VEGF121 into the culture medium. N-glycan analysis, however, revealed absence of 1,3-fucosyl or/and 1,2-xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for production of plant-made glycosylated biopharmaceuticals with non-allergenic N-glycans.

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The role of arabinogalactan-proteins in the moss, Physcomitrella patens Kieran J.D. Lee1, Celia D. Knight1, Ralph Quatrano2, Yoichi Sakata3 and J. Paul Knox1

1 Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK 2 Department of Biology, Washington University, 1 Brookings Drive, Saint Louis, Missouri 63130-4899, USA 3 Dept. of Bioscience, Tokyo Univ. of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan The moss Physcomitrella patens is an established model system for the study of plant developmental responses, including the innovations that lead to the colonisation of land by plants 450 million years ago (1). Moss protonemal filaments exhibit tip growth, therefore this system is analogous to root hair or pollen tube growth in higher plants. Using a combined cell biology and reverse genetic approach, we have demonstrated the presence of arabinogalactan-proteins (AGPs) in Physcomitrella. AGPs are a large and heterogeneous family of extracellular proteoglycans implicated in plant cell extension (2, 3). Through a bioinformatic strategy, six Physcomitrella expressed sequence tags (ESTs) encoding putative AGP core-proteins have been identified by homology with Arabidopsis, Brassica and Oryza AGP core proteins. Genetic manipulation of one of these genes, PpAGP1 which encodes a classical AGP core protein, by means of gene knockout and over-expression driven by an actin promoter led to the production of stable AGP knock-out and over-expressing lines. These sets of lines display altered growth characteristics that indicate a role for PpAGP1 in cell expansion in Physcomitrella. Inhibitor studies using synthetic AGP-binding β-glucosyl Yariv reagent (βGlcY) revealed that the growth of Physcomitrella, and specifically apical cell expansion, is disrupted by the application of 1 µM βGlcY. The anti-(1→5)-α-L-arabinan monoclonal antibody LM6 (4) binds to AGPs in Physcomitrella and binds to all plasma membranes but binds to the cell wall surface only at the most apical regions of growing protonemal filaments. Growth in the presence of βGlcY abolishes LM6 labelling of cell walls at apical regions, which suggests that the localised movement of AGPs from the plasma membrane to the cell wall is a key step in the tip growth of apical cells. This study highlights the usefulness of this moss species as a model system to study plant cell walls and specifically the functions of AGPs in plant development. 1. Nishiyama, T., Fujita, T., Shin-I, T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., Kohara, Y., Hasebe, M. (2003). PNAS 100: 8007-8012. 2. Nothnagel, E. (1997). International Review of Cytology 174: 195-291. 3. Showalter, A. M. (2001). Cellular and Molecular Life Sciences 58: 1399-1417. 4. Willats, W. G. T., Marcus, S.E., Knox, J.P. (1998). Carbohydrate Research 308: 149-152.

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Live cell imaging of microtubules and actin filaments in Physcomitrella patens Yoshikatsu Sato1, 2, Mitsuyasu Hasebe1, 3, Masamitsu Wada2, 4, Akeo Kadota2 1Division of Speciation Mechanism 2, National Institute for Basic Biology, Myodaiji, Okazaki Aichi 444-8585, Japan; 2Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-Osawa 1-1, Hachioji, Tokyo 192-0397, Japan; 3Department of Molecular Biomechanics, The Graduate University for Advanced Studies, Okazaki, 444-8585 Japan, 4Division of Biological Regulation and Photobiology, National Institute for Basic Biology, Myodaiji, Okazaki Aichi 444-8585, Japan E-mail; yoshi@nibb.ac.jp Cytoskeleton plays a crucial role in plant development and morphogenesis. Gametophyte cells of the moss Physcomitrella patens are an excellent model to study dynamics of cytoskeleton because gametophytes have simpler structures than sporophyte tissue in other plants. Further, we can make stable transformants with an appropriate construct in a given locus by homologous recombination. We made reporter constructs by fusing a GFP with P. patens a-tubulin or an actin binding domain of mouse talin, which are expressed under the control of rice actin promoter to visualize microtubules and actin filaments. In the MOSS meeting 2003, we showed the dynamic properties of these cytoskeletons in protonemal cells expressing GFP-talin or GFP-α-tubulin but the inserted locus and the copy number of each plasmid has not been estimated. Then, we performed DNA blots and isolated strains that each plasmid inserted low copy numbers at only the targeting locus, Pphb7 gene, which is a member of HD-Zip gene family. Since the expression of Pphb7 gene is limited to rhizoids and no phenotype was detected in any tissues except for rhizoids1), we can analyze dynamic changes of microtubules and actin filaments organization during the various types of cellular processes such as establishment of cell polarity, cell division, cell elongation, and intracellular organelle movement. Using the strains, we will report the reorganization of these filaments during protoplast regeneration and chloroplast movement in 4D microscopy which was accomplished by a laser scanning microscope (Yokogawa electric corp. Japan) equipped with Meta Morph software (Molecular Devices, Japan) and in dual irradiation microscopy which was achieved by a fluorescent microscope equipped with microbeam irradiator. 1) Sakakibara et al. (2003) Development 130, 4835-4846

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The Role of the ABI3 homolog in Physcomitrella in ABA-Regulated Gene Expression Ralph S. Quatrano Department of Biology, Washington University, St. Louis, MO 63130-4899, USA rsq@biology2.wustl.edu, Fon +1 314 935-6850 The phytohormone abscisic acid (ABA) controls a variety of processes in both reproductive and vegetative processes in Angiosperms, including embryogenesis, inhibition of germination, stomatal function, desiccation tolerance and a major signal in response to abiotic stresses. There are several Arabidopsis mutants in the ABA signaling pathway, including abscisic acid insensitive-3 (abi3). The phenotype of this mutation is characterized by premature germination of the embryo and sensitivity to water loss. The abi3 gene has been cloned and encodes a transcription factor homologous to viviparous-1 (vp1) of maize. ABA signaling through ABI3 or VP1 regulates a set of genes controlling the acquisition of desiccation tolerance during embryo maturation.This same Em-GUS fusion was activated by ABA in Physcomitrella patens cells, indicating a similar ABA signaling pathway is present in a non-seed plant (Knight, CD et al. (1995), Plant Cell 7: 499-506). More recently, homologs to the abi3 genes in moss have been characterized in our lab, allowing for a comparative approach in exploring the role of ABI3/VP1 in ABA-regulated gene expression. We will discuss recent evidence indicating the structural and functional properties of the ABI3-like gene from moss with respect to its role in ABA-regulated gene expression in moss and in higher plants.

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Circadian clock-controlled gene expression in Physcomitrella patens Setsuyuki Aoki1*, Seiji Kato1, Mamoru Sugita2 and Kazuhiro Ichikawa1 1Graduate School of Information Science, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan 2Center for Gene Research, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan *aoki@is.nagoya-u.ac.jp, phone +81-52-789-5376 The circadian clock, a self-sustained oscillator with a period of about one day, controls the expression of Lhcb genes encoding the chlorophyll a/b-binding proteins broadly in seed plants. In bryophytes, however, there have so far been no reports of any circadian rhythms, including circadian expression of any genes. We show that clock-controlled expression of Lhcb genes is also conserved, partly in a different manner, in Physcomitrella patens. Protonema cells grown in 12-hr:12-hr light-dark cycles showed a robust daily oscillation of Lhcb mRNA levels that damped rapidly in continuous darkness. In continuous light (LL), by contrast with typical profiles in seed plants, Lhcb mRNA levels only peaked during the first day and thereafter it showed constant levels. Some other clock-controlled genes also showed this property, arhythmicity in LL, suggesting a possibility that Physcomitrella has a circadian clock, the molecular mechanism of which is diverged from those of higher plants. Aoki, S., Kato, S., Ichikawa, K. and Shimizu, M (2004) Circadian expression of the PpLhcb2 gene encoding a major light-harvesting chlorophyll a/b-binding protein in the moss Physcomitrella patens. Plant and Cell Physiology, 45(1):68-76.

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Physcopathology: programmed cell death and defense responses of Physcomitrella patens Hemalatha Saidasan1, Didier G. Schaefer2, Andrija Finka2, Jean-Pierre Zrÿd2 and Michael Lawton1*, 1Biotech Center and Plant Biology and Plant Pathology Department , Cook College, Rutgers University, New Brunswick, NJ 08901. USA. 2Institute of Ecology, Laboratory of Plant Cell Genetics, Biology Building, University of Lausanne, CH-1015 Lausanne, Switzerland *lawton@aesop.rutgers.edu, fon +1 732 932-8165 Little is known about the defense responses of lower plants to challenge with pathogenic microorganisms. Recent EST sequencing projects have revealed that a many components of the defense response of higher plants are conserved in Physcomitrella patens, along with some notable differences. To establish a system for studying disease resistance, defense responses and programmed cell death (PCD) pathways in lower plants, we examined the responses of P. patens to several elicitors of defense pathways and PCD. These studies show that P. patens displays characteristic pathways for cell death, as characterized by distinct cellular morphologies, changes in nuclear architecture and the expression of biochemical and molecular markers. Evidence will be presented for the conservation of cell death pathways as measured by the effects of gene knock-out and transgenic over-expression of anti-apoptotic genes. The effects of manipulating cell death pathways on development will also be discussed.

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RNAi Silencing in Physcomitrella patens: An effective Method for Dissecting Plant Gene Function Magdalena Bezanilla*, Aihong Pan, Paul Klueh, Pierre-Francois Perroud and Ralph S. Quatrano Department of Biology, Washington Unviersity, St. Louis, MO 63130 * manena@biology.wustl.edu We developed a method to rapidly screen for phenotypes induced by RNAi in Physcomitrella patens. We designed a vector to express a hairpin loop of mRNA under the control of a strong constitutive promoter. The stalk of the hairpin consists of inverted repeats of GFP fused to a cDNA of interest. This RNAi fusion construct is transformed into a reporter line, which expresses a nuclearly localized GFP. Active silencing of target genes is thus monitored by loss of GFP expression. Transformation of a control vector containing only the GFP inverted repeat shows that approximately 50% of transformants have active silencing, as indicated by absence of GFP fluorescence. To screen for RNAi-induced phenotypes due to loss of cytoskeletal genes, we have transformed moss with constructs targeting silencing of subunits of the Arp2/3 complex, class VIII myosins, components involved in actin dynamics (ADF/cofilin, profilin, capping protein), formins, expansins, Rho of plants, and calmodulin. We found that loss of function of genes involved in actin dynamics led to dramatic reduction in cell size and abnormal cell morphology. Loss of myosin-VIII function was lethal and showed defects in cell shape and/or division. Most phenotypes appear as early as six days after transformation and are stable for at least 21 days. Using RNAi, we are able to determine for the first time phenotypes associated with loss of function of genes within large gene families. RNAi, a method based on cDNA sequence information, provides a powerful tool for studying plant gene function in moss. On the evolution of bryophytes

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The Rehydration Transcriptome:Translatome of Tortula ruralis: A combined bioinformatics and microarray approach to investigate the desiccation response of tolerant mosses. Melvin J. Oliver, Paxton Payton, Scot Dowd and Steve Mauget. USDA-ARS Plant Stress and Water Conservation Laboratory. 3810 4th St Lubbock TX moliver@lbk.ars.usda.gov Gene expression studies of desiccation-tolerant plants are limited to only a few species one of which is the desiccation-tolerant moss Tortula ruralis. From an evolutionary standpoint T. ruralis represents the primitive mechanism for tolerance of dehydration and thus an understanding of its genome level response has important implications for plant biology and agriculture. We have demonstrated that the alteration in gene expression in response to desiccation in this plant occurs following rehydration, in large part as the result of a change in translational controls. In addition, during drying certain transcripts, encoding rehydrins, are sequestered in messenger ribonucleoprotein particles (mRNPs) for storage in the dried state. To characterize the extent and importance of translational versus transcriptional events we have taken a combined genomics and bioinformatics approach to analyze gene expression in response to desiccation. A Tortula EST collection, 10,368 primary ESTs (9,159 passing quality controls), has been established and a total of 5.93 million nucleotides have been submitted to Genbank. The EST collection was established from a non-normalized 2h rehydrated gametophyte cDNA library, the nonnormalized library was used in order to gain an appreciation of individual transcript abundance within the transcriptome. The 9,159 ESTs formed 7,272 contigs which in turn reduced to 5,563 clusters (a cluster is an assembly of ESTs that could represent transcripts from the same gene but either from separate alleles or alternate splice forms). Even though the ESTs derive from a non-normalized library 96.5% of the clusters still have 5 or fewer EST members. Consensus cluster sequences were subjected to a Blastx search and annotated according to the degree of similarity to known genes. Of he 5,563 clusters, 3,321 (59.7 %) could be matched to known sequences and 2,242 (40.3%) were categorized as unknowns. Functional classification of the Tortula rehydration transcripts were achieved using Gene Ontology (GO) and KEGG pathway mapping strategies. The results of these analyses will be discussed in context and are also available at our website, http://199.133.147.108/bryobase/. The 5,563 clusters formed the basis of a Tortula “uni-cluster” cDNA microarray that we have used to complete a comprehensive expression profile for a desiccation:rehydration cycle. Using both total and polysomal mRNA transcripts for the source of cDNA probes we have evaluated the relative importance of transcript abundance and recruitment in the response for each represented gene. The data we will present, coupled with the bioinformatics, offers a unique picture of the transcriptome and translatome level response of T. ruralis to severe stress.

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Characterization of TrDr3 from Tortula ruralis—a Membrane Protein of Unknown Function That is Unique to Mosses. Congyue A. Peng and Andrew J. Wood* Department of Plant Biology, Southern Illinois University, Carbondale, IL 62901, USA *wood@plant.siu.edu The resurrection moss Tortula ruralis is an important experimental system for examining the molecular and biochemical mechanisms of vegetative desiccation tolerance in plants. We have employed EST analysis to discover genes that control vegetative desiccation-tolerance (Wood et al. 1999), and describe the characterization of the EST-derived cDNA TrDr3 (Tortula ruralis Desiccation-stress Related) (1, 2). The deduced polypeptide TRDR3 has a predicted molecular mass of 25.5 kDa, and a predicted pI of 6.7. SOSUI, SMART & TMHMM predict 6 trans-membane helical domains, each 23 amino acid residues in length, with no predicted transit sequence(s). BlastX, BlastN, BlastP & PSI-Blast searches of the electronic databases reveal that Tortula TRDR3 shares significant similarity to the hdeD (HNS-dependent expression) from Eschericia coli. With the exception of several ESTs from Physcomitrella patens, TRDR3 is not found in any other eukryotic organism. The function of HdeD in E. coli is unknown, but it is postulated to be involved in a mechanism of acid-stress defense, and expression of the gene is negatively regulated in response to decreased pH (3). To establish the role of E. coli HdeD in abiotic stress-tolerance, we determined viable cell counts from shaking cultures of wild-type bacteria and the hdeD mutant in the presence of sorbitol (10% (w/v) and 20% (w/v)), NaCl (5% (w/v)), low pH (4.5), low temperature (28˚ C) or elevated temperature (42˚ C). In response to elevated temperature, the hdeD mutant was more sensitive. Suprisingly, the hdeD mutant was less senestive, as comapared to wild-type E. coli, in response to cold, decreased pH and 5% NaCl; no differences were observed between the genotypes for sorbitol at either 10% or 20%. On-going RNA gel blot experiments will determine the expression of TrDr3 within Tortula gametophytes in response to desiccation, salinity, altered pH & altered temperature. (1) Wood, AJ, Duff RJ and Oliver MJ (1999) Plant & Cell Physiology 40: 361-368 (2) Wood AJ and Oliver MJ (2004) (pp) 71-90, In, New Frontiers in Bryology: Physiology, Molecular Biology and Applied Genomic AJ Wood, MJ Oliver, DJ Cove (eds.), Kluwer Academic Press (3) Masuda, N and Church GM (2003) Molecular Microbiology 48: 699-712

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Investigation of the abiotic stress response in Physcomitrella patens Sandra Richardt, Jan Hoffbauer, Ralf Reski and Wolfgang Frank Lehrstuhl für Pflanzenbiotechnologie, Albert-Ludwigs-Universität Freiburg Tel.: +49-(0)761-2032820; e-mail: wolfgang.frank@biologie.uni-freiburg.de In order to establish the moss Physcomitrella patens as a model system to analyse molecular mechanisms underlying the adaptation to abiotic stress we have examined its tolerance against salt, osmotic and dehydration stress. Plants that have been treated with NaCl tolerated concentrations up to 350 mM. Treatments with sorbitol revealed that plants are able to survive concentrations up to 500 mM. Furthermore, plants which have lost 92% water on a fresh weight basis were able to recover successfully. To identify genes involved in the abiotic stress response of Physcomitrella we have initiated different experimental approaches. We have analysed a comprehensive Physcomitrella EST database to search either for genes showing homology to stress-associated genes from other plants or to identify sequences which do not show any homology to sequence entries in public databases. The expression pattern of these genes has been studied by cDNA macroarrays. We were able to identify homologues of stress-associated genes as well as novel genes that show an enhanced expression level upon stress treatments. Furthermore, we intend to perform expression profiling analyses using a cDNA microarray covering all putative transcription factors of Physcomitrella. The results of these experiments and the putative function of the identified genes will be discussed.

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Molecular responses to ABA and desiccation stress in the moss Physcomitrella patens Yasuko Kamisugi1, Ralph Quatrano2 and Andrew Cuming1

1Centre for Plant Sciences, Leeds University 2Department of Biology, Washington University at St. Louis In higher plants, adaptive responses to desiccation are largely restricted to the production of highly desiccation-tolerant seeds. By contrast, the bryophytes - which represent one of the earliest groups of plants to colonise terrestrial habitats - demonstrate high levels of desiccation tolerance in their vegetative tissues. The ability to withstand extreme fluctuations in the availability of water must have been a key element to the success of plants in their conquest of the land, and molecular and biochemical adaptations to water stress must consequently be of ancient evolutionary origin. Building on our earlier demonstration that the regulation of the expression of the seed-specific desiccation-stress associated “Em” gene of wheat (encoding a Group 1 Late Embryogenesis Abundant (LEA) protein) was regulated in Physcomitrella by ABA and osmotic stress using a molecular mechanism apparently identical to that used in cereals, we have set out to characterise the nature of the ABA and stress-response in Physcomitrella. We have used three approaches. (i) Candidate genes have been identified among the EST collections that show homology with ABA- and stress-regulated genes in higher plants. These include members of the LEA gene families, and like their higher plant counterparts, are also ABA- and stress-regulated in Physcomitrella. (ii) Bioinformatic analysis has identified ESTs that are relatively overrepresented among those cDNAs isolated from a library corresponding to ABA-treated tissue: closer analysis demonstrates that a significant number are also homologous with higher plant stress-regulated genes. (iii) The development of a Physcomitrella oligonucleotide microarray has enabled us to take a transcriptomic approach to identify genes up-regulated in response to ABA and stress treatments. Again, a significant number of the strongly up-regulated genes are similar to those identified as late embryogenesis- and stress-related in seed plants. We are currently analysing the regulatory mechanisms that control the expression of a number of Physcomitrella genes involved in the stress response, and will present evidence relating both to the transcriptional control of gene activity by ABA and osmotic stress, and to the contribution made by mRNA stability to the accumulation of Physcomitrella transcripts.

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Poster abstracts Session I: Monday, Sept 13th, 2:00-3:30 pm odd numbers Session II: Monday, Sept 13th, 4:00-5:30 pm even numbers Poster

# Pres.

author Title Page #

1 Kubešová Bryophyte species composition on cliffs of south-western Moravia (Czech Republic) 34

2 Potters Physcomitrella ascorbate metabolism – a story written in the sand 35

3 Lunde Characterising Monodehydroascorbate Reductase (MDHAR) in Physcomitrella patens 36

4 Lienard Aquaporin and transpiration in Physcomitrella patens 37

5 Yevdakova Studies of cytokinin biosynthetic genes from Physcomitrella 38

6 Olsson Evidence that the balance between chloronemal and caulonemal growth is regulated by the energy supply in the moss Physcomitrella patens 39

7 Parsons Optimization of culture conditions for the production of recombinant pharmaceuticals 40

8 Lucumi Production of recombinant proteins in photoreactors by means of submers moss cells 41

9 Baur Genetic stability of transgenic Physcomitrella patens plants 42

10 Jost Production of monoclonal antibodies in Physcomitrella patens 43

11 Kaufmann Heterologous expression of a human protein in transiently transformed protoplasts of Physcomitrella patens 44

12 Launhardt In vivo reconstitution of a human gene from two non-functional fragments by intermolecular recombination in Physcomitrella patens 45

13 Stemmer Marker-free transformation of Physcomitrella patens 46

14 Kamisugi Analysis of the outcome of transformation of Physcomitrella patens with constructs containing genomic DNA. 47

15 Younousse High conditional expression of recombinant protein in Physcomitrella patens using a soybean heat-shock promoter 48

16 Verelst Identification and phylogeny of a new MADS-box gene in Physcomitrella patens 49

17 Bushoven Physcomitrella patens: a model system for investigating the evolution and organization of cellulose-synthesizing terminal complexes. 50

18 Finka Heat-shock inducible promoter allows efficient GFP-talin labeling of the actin cytoskeleton in Physcomitrella patens 51

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19 Grémillon Functional proteomics of plastid division in the moss Physcomitrella patens 52

20 Tintelnot Transcriptional regulation of genes encoding extracellular proteins by phytohormones in Physcomitrella patens 53

21 Erxleben Proteome analysis of mutants in the moss Physcomitrella patens 54

22 Hartmann Phospholipase C From Physcomitrella patens: Biochemistry and Knockout 55

23 Trouiller Cytokinin signalling in Physcomitrella patens is mediated by histidine kinase receptors with partially overlapping functions 56

24 Mosquna From Physcomitrella to Arabidopsis: the role of chromatin remodeling by polycomb proteins in controlling plant development. 57

25 Lienhart Analysis of extracellular differentiation signals in Physcomitrella patens 58

26 Arazi Deciphering RNA silencing in moss 59

27 Minami Abscisic acid-induced freezing tolerance in Physcomitrella patens 60

28 Itouga Tolerance of the moss Physcomitrella patens subsp. patens (Funariales) to inorganic arsenate, As (V) 61

29 Carballo A knockout mutant of the dehydrin DHNA is impaired in recovery from osmotic stress in Physcomitrella patens 62

30 Schween Gene-ID in Physcomitrella patens 63

31 Stenøien Adaptive basis of codon usage in Physcomitrella patens 64

32 Lang Annotation and representation of the Physcomitrella patens transcriptome: an integrated approach 65

33 Schumann Alternative splicing in Physcomitrella patens 66

34 Riese Molecular and functional characterization of SBP-box genes in the moss Physcomitrella patens 67

35 Horstmann Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude 68

36 Richardt Expression profiling of transcription factors during phytohormone action in Physcomitrella patens 69

37 Lindsay Genomics-driven comparative physiology of GATA transcription factor function in Physcomitrella patens 70

38 Menand RHD6-like transcription factors: from rhizoids to root hairs? 71

39 Kobayashi Isolation and characterization of a nuclear rpoA gene knockout moss reveal the existence of two different plastid RNA polymerases 72

40 Miyata RNA editing in the moss Physcomitrella patens chloroplast 73

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Bryophyte species composition on cliffs of south-western Moravia (Czech Republic) Svatava Kubešová Botanical department, Moravian Museum, Hviezdoslavova 29a, Brno, CZ 627 00 Czech Republic skubesova@mzm.cz Bryophytes of rock habitats was studied in south-western Moravia (Czech Republic). The total number of species was 140 (23 liverworts and 117 mosses). The most frequent species were Bryum argenteum, B. capillare, Cephaloziella divaricata, Ceratodon purpureus, Dicranella heteromalla, Dicranum scoparium, Grimmia pulvinata, Hedwigia ciliata, Hypnum cupressiforme, Pleurozium schreberi, Pohlia nutans, Polytrichum formosum and P. piliferum. Temperate and subboreal chorological types were the most frequent. Among the growth-forms, rough mat and short turf types were the most often. Most frequent life strategies were perennials and colonists. Life strategies and distribution range type spectra do not differ significantly between the rock habitats and the surrounding landscape. Variability of species data of rocks, revealed by detrended correspondence analysis, was related to potential direct irradiation and position on slopes.

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Physcomitrella ascorbate metabolism – a story written in the sand Geert Potters1,*, Annick Van de Wiel1, Mario C. De Tullio2 and Nele Horemans1 1Dept. of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium 2Dept. Plant Biology and Pathology, University of Bari, Italy * geert.potters@ua.ac.be , phone +32 3 265 3421, fax +32 3 265 3417 Ascorbate and glutathione are two major components in the antioxidative defensive system of higher plant cells (Noctor and Foyer, 1998). In addition, they are involved in cell growth and division (Potters et al., 2002), and even differentiation (Potters et al., in preparation). Beside its antioxidant role, ascorbate is also known to be involved in the synthesis of hydroxyproline-containing proteins and plant hormones (Arrigoni and De Tullio, 2000). A first step in the analysis of the role of ascorbate and glutathione used the rich in silico resources for Physcomitrella. Different genes, related to ascorbate or glutathione metabolism, were selected and the Arabidopsis sequence was blasted against the Physcomitrella databases. Several homologues were picked up: - ascorbate peroxidase, coding for a protein which detoxifies H2O2 using ascorbate as its substrate - ascorbate oxidase, an enzyme of still uncertain function (De Tullio et al., 2004) - monodehydroascorbate reductase, involved in the reduction of the free radical form, monodehydroascorbate ; - glutathione reductase, assisting in the reduction of oxidised glutathione. Activities of some enzymes of the ascorbate-glutathione cycle have already been detected in other Bryophyta (Paciolla and Tommasi, 2004). The presence of these genes suggests the existence of the ascorbate-glutathione cycle in Physcomitrella. Their functionality should still be proven, for example with native polyacrylamide gel assays. Arrigoni O, De Tullio MC (2000) The role of ascorbic acid in cell metabolism: between gene-directed functions and unpredictable chemical reactions, J Plant Physiol, 157: 481-488 De Tullio MC, Liso R, Arrigoni O (2004) Ascorbate oxidase: an enzyme in search of a role. Biol Plant 48: 161-166 Horemans N, Foyer CH, Potters G, Asard H (2000) Ascorbate function and associated transport systems, Plant Physiology and Biochemistry 38: 531-540 Kerk NM, Feldman LJ (1995) A biochemical model for the initiation and maintenance of the quiescent center: implications for organization of root meristems, Development 121, 2825-2833 Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control, Annu Rev Plant Physiol Plant Mol Biol 49: 249-279 Paciolla C, Tommasi F. (2004) The ascorbate system in two bryophytes: Brachythecium velutinum and Marchantia polymorpha. Biol Plant 47: 387-393 Potters G, De Gara L, Asard H, Horemans N (2002) Ascorbate and glutathione: guardians of the cell cycle, partners in crime ?, Plant Physiology and Biochemistry 40: 537-548

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Characterising Monodehydroascorbate Reductase (MDHAR) in Physcomitrella patens Christina Lunde*¶, Damian Drew and Geoffrey B. Fincher Australian Centre for Plant Functional Genomics, University of Adelaide, SA 5064, South Australia * christina.lunde@acpfg.com.au / Tel.: +61 8 8303 7174 Exposure of plants to biotic or abiotic stress often leads to the generation of Reactive Oxygen Species (ROS). Since ROS in high concentrations are toxic to the cell, it is important to have an efficient scavenging system. In plants, the majority of ROS is scavenged by the oxidation of ascorbate. This process is catalysed by an ascorbate peroxidase. In order to maintain the scavenging potential of the ascorbate pool, oxidised ascorbate has to be re-reduced. This process is in part catalysed by MDHAR using NAD(P)H as an electron donor. In addition, MDHAR has been shown to catalyse the reduction (detoxification) of phenolic radicals. MDHAR activity is found in many cellular compartments and in different tissues, but only in plants and algae. Based on homology the most likely ancestor is a bacterial flavo-oxidoreductase. One objective of our current research at the Australian Centre for Plant Functional Genomics (ACPFG) is to characterise the different isoforms of MDHAR found in Physcomitrella patens, with respect to their physiological roles under stress conditions, as well as their biochemical properties. A BLAST search of EST databases has so far revealed three different isoforms of MDHAR in P. patens. However, EST analysis has revealed that each isoform is found with 3-5 different 5’UTR. This variation is mainly due to alternative splicing. Quantitative PCR has revealed that MDHAR expression does not appear to change under oxidative stress and we speculate that the variations in the 5’UTR's may play a regulatory role. We are currently generating MDHAR knock-outs in Physcomitrella patens. Furthermore, we are expressing the three different isoforms of MDHAR in heterologous systems for protein isolation and characterisation. ¶ Dr. Christina Lunde is supported by a two-year post-doc grant from the Danish Agricultural and Veterinary Research Council, Copenhagen, Denmark.

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Aquaporin and transpiration in Physcomitrella patens David Liénard, Gaëlle Durambur, Véronique Gomord, Jean-paul Lassalles Glycosylation et Transport chez les Végétaux CNRS- UMR 6037 IFRMP 23 Université de Rouen, 76821 Mont-Saint-Aignan Cedex France Contact E-mail: david.lienard@univ-rouen.fr Aquaporins (AQPs) are membrane-spanning channel proteins highly specific for water. This channel family is ubiquitous from bacteria to animal (Agre et al., 2001) and plant cells. In plants, many aquaporins have been detected in all kinds of tissue (Chrispeels et al., 2001) but their physiological role in water transport may be questionned. The water permeability of the lipid part of the membranes already allows important water exchanges between a cell and its surroundings and for some authors, the main function of AQPs could be to detect osmotic changes rather than increasing the membrane permeability (Hill et al., 2004). We decided to focus our attention on Physcomitrella patens: as a poikilohydric plant it has a relationship with water different than that of higher plants. It does not regulate its water content so it could be interesting to study the evaporation of P. patens and the involvement of aquaporins in this process. Our studies have demonstrated that the limiting step for the evaporation was dependent on the size of the leaves. The magnitude of the evaporation rate (~ 1 µm s-1) that we measured on small leaves implied values > 100 µm s-1 for the osmotic water permeability (Pos) of the cellular membranes. Such large Pos values were also measured on isolated protoplasts from the gametophyte (median: 159 µm s-1), while the values obtained from protoplasts isolated from protonema were much lower (median: 2.7 µm s-1). These results were confirmed by the cloning of two putative aquaporin genes, PpPIP2,1 and PpPIP2,2, showing strong homologies with the PIP2 (Plasma membrane Intrinsic Protein class II) of A. thaliana. Their expression was detected in the gametophyte but not in the protonema. We think that the large Pos values and the corresponding PIP genes were required to facilitate water transfer between the two sides of a leaf. For the small leaves partly in contact with the air on one side and with liquid water on the other side, an increase in the transcellular water flux could delay the loss of cellular water for a given RH. Our work tends to prove that more than 400x106 years before vascular plants, P. patens was already using aquaporins to increase the permeability of its cellular membranes to water in order to cope with the high evaporation rate from the small leaves of gametophytes. David Liénard was supported by a grant from the "Conseil Régional de Haute-Normandie" Agre, P., M.J. Borgnia, M. Yasui, J.D. Neely, J. Carbrey, D. Kozono, E. Beitz, J. Hoffert, V. Leitch, and L.S. King. 2001. Discovery of the aquaporins and their impact on basic and clinical physiology, p. 1-38 Aquaporins, Vol. 51. Chrispeels, M.J., R. Morillon, C. Maurel, P. Gerbeau, P. Kjellbom, and I. Johansson. 2001. Aquaporins of plants: Structure, function, regulation, and role in plant water relations, p. 277-334 Aquaporins, Vol. 51. Hill, A.E., H.B. Shachar, and H.Y. Shachar. 2004. What are aquaporins for? Journal of Membrane Biology 197:1-32.

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Studies of cytokinin biosynthetic genes from Physcomitrella Natalya Yevdakova, Klaus von Schwartzenberg Biocentre Klein Flottbek and Botanical Garden, University of Hamburg, Germany The moss Physcomitrella patens provides valuable tools such as cytokinin overproducing mutants (ove mutants) and large genomic resources to investigate the cytokinin biosynthesis. Isopentenyltransferases (IPT) catalyse a rate limiting step in cytokinin biosynthesis. There are two routes; the tRNA pathway and the AMP/ADP/ATP pathway, which have been proposed for cytokinin biosynthesis in plants. Isopentenylation of tRNA is catalysed by tRNA- IPT, and the resulting t-RNA containing cytokinins as hypermodified bases can be a source of cytokinins. Larger quantities of cytokinins are deriving from de novo biosynthesis of free isopentenyladenosine-5’-nucleotides by adenylate- IPTs with dimethylallyl-pyrophosphate (DMAPP) and AMP/ADP or ATP as substrates. To pick up putative ipt genes of Physcomitrella we screened EST databases and carried out in silico analyses using the nine A. thaliana ipts as queries. By PCR analysis we identified two Physcomitrella ipt candidates named PpiptN1 and PpiptN2. Expression of the identified genes has been verified and quantified by real time RT-PCR, comparatively in wild type (WT) and in two ove mutants (oveSt25 and oveB300). Preliminary results indicate a difference in expression level of these genes with a clear tendency of higher level in cytokinin overproducing mutants. Functional studies will clarify whether these genes belong to the group of tRNA- ipts or to adenylate- ipts.

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Evidence that the balance between chloronemal and caulonemal growth is regulated by the energy supply in the moss Physcomitrella patens Tina Olsson1,2*, Mattias Thelander1,2, Anders Nilsson1,2 and Hans Ronne1,2

1Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, 75123 Uppsala, Sweden 2Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, Box 7080, 75007 Uppsala, Sweden * e-mail: Tina.Olsson@imbim.uu.se The filamentous gametophyte of the moss Physcomitrella patens consists of two filament types called chloronemata and caulonemata. Chloronemal cells are photosynthetically active with numerous chloroplasts, while caulonemal cells have fewer and smaller chloroplasts. Caulonemata instead help to spread the colony by radial growth and provide the cells from which buds are formed that develop into leafy shoots. The balance between chloronemal and caulonemal filaments is affected by external factors such as light and plant hormones. We have monitored caulonema formation and chloronemal branching during high and low light conditions, and in the presence of glucose, auxin or cytokinin. These experiments were performed both in a wild type strain and in a hxk1 knockout mutant (1) which lacks the major hexokinase of Physcomitrella. We found that caulonema formation is induced by high energy conditions such as high light and external glucose, while chloronemal branching instead is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant. We are also investigating how the expression of genes involved in the carbon metabolism is affected both in the hxk1 knockout mutant, and in a snf1a snf1b double knockout mutant that lacks the two genes encoding the SnRK1 protein kinase (2). The latter kinase is the closest plant homologue of Snf1 or AMPK, a key metabolic regulator in animals and fungi. Our previous studies have shown that the snf1a snf1b double knockout has a number of pronounced phenotypes. This includes an inability to grow in a normal day-night light cycle, which suggests that the carbon and energy metabolism in the snf1a snf1b knockout mutant is disturbed (2). 1. Olsson, T., Thelander, M., and Ronne, H. (2003) A novel type of chloroplast stromal hexokinase is the major glucose phosphorylating enzyme in the moss Physcomitrella patens. J. biol. Chem. 278, 44439-44447. 2. Thelander, M., Olsson, T., and Ronne, H. (2004) Snf1-related protein kinase 1 is needed for growth in a normal day-night light cycle. EMBO J. 23, 1900–1910 .

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Optimization of culture conditions for the production of recombinant pharmaceuticals Juliana Parsons*,Marc Kaminski, Ralf Reski and Eva Decker Plant Biotechnology, University of Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany * juliana.parsons@biologie.uni-freiburg.de, fon +49 761 - 203 - 2820 In order to satisfy the increasing demand of new and higher amounts of pharmaceutically valuable proteins, it is necessary to develop and optimize new expression systems. Physcomitrella patens as an expression system offers the advantages of eukaryotic posttranslational modifications (such as glycosylation, the formation of disulphide bonds and assembly of multimeric proteins), low risk of product contamination (with oncogenes, bacterial toxins, mammalian viruses and other pathogens), photoautotrophical growth in a strict containment with low susceptibility of suspension cultures to shear stress.Finally as it grows fully differentiated, somaclonal variation, typical for cell cultures of higher plants, is avoided (Decker and Reski 2004). In order to use P. patens as a protein production platform not only optimal conditions for the moss growth should be achieved, but also those for the maximum protein yield. In this context we tested the growth and protein production of moss cultures expressing the human vascular endothelial growth factor (hVEGF121) under different conditions in transient expression and stable plants. The transient expression system allows to test different genetic constructs for new proteins in a fast and easy way. On the other hand stable transgenic plants offer the advantage of constant protein yield and the ability for upscaling and cultivation in mossbioreactors. The use of the auxin α-Naphthaleneacetic acid (NAA) produces at least a threefold increase of hVEGF121 concentration in the culture medium and in the cells, both in the stable plant culture and the transient expression. This effect seems to be independent from the genetic background, as similar results were achieved with the expression of varying constructs containg different promoters and secretory signal sequences. Also higher pH values associated with lower salt concentration produces an increase in the VEGF detected in the culture medium in the stable plant without affecting the growth. Physcomitrella was already shown to be a valuable expression system for the production of recombinant proteins. Here we show an increase of at least three times in the heterologous protein concentration by manipulating some of the culture conditions. We expect that further optimization of the system will lead us to even higher yields. Decker, E.L. and Reski R. (2004) The moss bioreactor. Curr. Opinion Plant Biol. 7: 166-170.

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Production of recombinant proteins in photoreactors by means of submers moss cells

Alexander Lucumi*, Clemens Posten Institute of mechanical process engineering and mechanics, Department of bioprocess engineering, University of Karlsruhe, Karlsruhe, Germany * alexander.lucumi@mvm.uka.de

The moss Physcomitrella patens has been recently recognized as an ideal producer of recombinant proteins with respect to glycosylation. Due to the elaborated post-translational capabilities of moss cells, glycosylation patterns can be manipulated to obtain proteins similar to those found in animal cells. This production system using moss in suspension offers important advantages in comparison to the cultures of animal cells. Moss cells grow photoautotrophic, requiring only a simple mineral medium, carbon dioxide and light[1]. Therefore, the contamination risk is minimized and the production cost is low. In addition, there are no known diseases of plants which can be transfered to humans and due to the fact that, recombinant proteins can be targeted extracellular, the down stream processing is simplified. Moss cells are cultivated in a filamentous stage called protonema. Phototrophic growth in bioreactors is investigated where light quantity and quality, stress, concentration of growth factors and morphology of the organisms influence cell differentiation . An original 30 liter, 40 mm diameter, tubular photoreactor, externally illuminated with metal halide lamps is used to characterize the response of moss cells to different flow conditions, light/dark cycles and CO2 concentrations. A size reduction system based on the rotor-stator principle is used parallel to the reactor to keep the average length of the moss threads at a desired level and then, delay the formation of leaflets (gametophores). In order to reduce the effect of phytohormones produced by the cells, separate the product, exchange medium and feed the productive cells back, a perfusion system using cross flow filtration is introduced. Cultivations are effectuated in periods of time ranging between two and four weeks. Online measurements of CO2 consumption and O2 production and offline data of moss cells concentration, total and recombinant protein, fatty acids and chlorophyll content along the time are described. [1] Hohe, A. and Reski, R. 2002. Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts. Plant Sci. 163: 69-74.

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Genetic stability of transgenic Physcomitrella patens plants Armin Baur, Barbara Timm, Andreas Weise, Marta Rodriguez-Franco and Gilbert Gorr* greenovation Biotech GmbH, Boetzingerstr. 29b, 79111 Freiburg, Germany *ggorr@greenovation.com, fon: ++49761-47099130 Transgenic moss plants offer a unique production system for biopharmaceutical proteins. Economic photoautotrophic growth, advanced safety aspects, easy downstream processing of the target protein from a simple mineral medium and unique genetical manipulation are some of the advantages of mosses. One important criteria for successful expression of a therapeutic protein from a recombinant cell is to obtain a transgenic plant that maintains stability of production and in addition stability at the molecular level. Here we focused on stable transformed plants to verify the stability of expression during prolonged culture with and without selection pressure. Several plants aging 2 and 7 years were examined concerning expression of the target protein human vascular endothelial growth factor (hVEGF) and antibiotic resistance (nptII). Human VEGF is a homodimeric, glycosylated protein of 26KDa that induces angiogenesis and endothelial cell proliferation. Protein levels of rhVEGF were measured by ELISA and found to be unchanged. Furthermore 100% of the transgenic plant material showed resistance to the antiobiotic G418 even after several years of cultivation without selection pressure. In addition analysis at the molecular level were performed. We can conclude that transgenic Physcomitrella patens plants show genetic stability of the transgene over a long period of time even under subcultivation without selection pressure. This makes the moss an ideal production system for biopharmaceuticals under strict regulatory requirements.

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Production of monoclonal antibodies in Physcomitrella patens Wolfgang Jost, Eija Schulze, Sandra Link, Michael Schledz, Andreas Weise, Marta Rodriguez-Franco and Gilbert Gorr* greenovation Biotech GmbH, Boetzingerstr. 29b, 79111 Freiburg, Germany * ggorr@greenovation.com fon +49 (761) 47099 130 Physcomitrella patens allows cost-effective production of therapeutic proteins and humanisation of the glycosylation pattern through homologous recombination (see also abstract Koprivova et al.). In this study we investigated the expression of a humanized IgG4 Kappa monoclonal antibody (ABC-48 / AERES Biomedical) in Physcomitrella. This antibody targets P-selectin on activated platelets and is currently in pre-clinical development at AERES Biomedical for the prevention of deep vein thrombosis. ABC-48 transiently secreted by moss protoplasts is correctly assembled as tetramere (HC2/LC2), glycosylated and fully functional, as measured by binding to its ligand. Here we report that proper antibody assembly is strongly influenced by the stoichiometry of expressed heavy (HC) and light chains (LC) using a) the same or different signalpeptides for either chain and b) unequal amounts of HC and LC constructs in transient transformations. Interestingly, not an equimolar stoichiometry but an excess of HC constructs gave highest yields of fully assembled and functional ABC-48.

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Heterologous expression of a human protein in transiently transformed protoplasts of Physcomitrella patens Franz Kaufmann, Helene Rolli, Andreas Weise, Marta Rodriguez-Franco and Gilbert Gorr* greenovation Biotech GmbH, Boetzingerstr. 29b, 79111 Freiburg, Germany *ggorr@greenovation.com fon +49 (761) 47099 130 Transiently transformed protoplasts of Physcomitrella patens can be employed for the heterologous expression of proteins into the culture medium. Here we will present data on the optimization of the transient expression systems with respect to protein yield and proper processing of the product. Human vascular endothelial growth factor (VEGF) was used as the model protein. As could be expected the promotor sequence and the signal peptide strongly influenced the expression level with moss promotors (as compared to the 35S promoter) and plant signal peptides (as compared to the native VEGF signal peptide) showing the highest expression. The conditions during transformation (protoplasts number, PEG concentration, osmolarity) and during culturing of the transformed protoplasts were optimized. Under optimized conditions the expression rate remained constant over several weeks and up to 1 µg VEGF per transformation per week were produced. VEGF was secreted as a homodimer with the subunits being linked by disulfide bonds. These data show that Physcomitrella protoplasts can be used to express recombinant proteins with complex modifications in quantities sufficient for structural and functional characterization.

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In vivo reconstitution of a human gene from two non-functional fragments by intermolecular recombination in Physcomitrella patens Heike Launhardt, Carla Hehn and Gilbert Gorr* greenovation Biotech GmbH, Boetzingerstr. 29 b, 79111 Freiburg, Germany * ggorr@greenovation.com, fon +49 (761) 47099 130 The moss Physcomitrella patens is a widely-used plant model organism. The clearly defined but simple differentiation pattern together with its high rate of homologous recombination events are the main reasons for the use of Physcomitrella as a powerful tool for reverse genetics by targeted knock-outs. However, the reconstitution of genes is another interesting aspect of homologous recombination. To analyse the reconstitution of genes in Physcomitrella we cloned the gene coding for the human vascular endothelial growth factor (VEGF) in two nonfunctional and nonoverlapping fragments. As an overlapping region we introduced a moss intron sequence on both constructs resulting in construct a) comprising a promoter, the cDNA coding for the N-terminus of VEGF and the moss intron and b) comprising the moss intron, the cDNA of the C-terminus of the VEGF and a termination sequence. Transformation of Physcomitrella with either construct a) or b) did not result in any measurable VEGF amounts. In one approach both constructs were introduced into the genome one after the other. In 39 out of 46 (> 80%) analysed plants the VEGF gene was reconstitued. In an additional approach co-transformation with both constructs in parallel was performed and again stably transformed plants were generated. Comparable reconstitution rates were achieved with this method. The plants were analysed by PCR and VEGF specific ELISA. Furthermore , from several plants the reconstituted gene and as well the corresponding cDNA were isolated and sequenced. Sequencing of the cloned DNA and cDNA confirmed the base pair correct recombination event and that the RNA was spliced accurately.

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Marker-free transformation of Physcomitrella patens Christian Stemmer, Anja Koch and Gilbert Gorr* greenovation Biotech GmbH, Boetzingerstr. 29 b, 79111 Freiburg, Germany * ggorr@greenovation.com, fon +49 (761) 47099 130 Despite the large number of marker genes that exist for plants, only a few marker genes are used for bryophytes. In some cases the integration of the marker gene can be a major disadvantage. Investigations at the physiological level in transgenic plants can be influenced by the co-expression of the marker gene. To generate marker-free higher plants some developments were performed e.g. cotransformation approaches followed by segregation of the marker genes. Nevertheless, this procedures are generally time consuming and not useful for plants that grow vegetatively. Especially for model organisms like the moss Physcomitrella patens a marker-free transformation method would be a major improvement with regard to many topics. Therefore we established a PEG-mediated-marker-free transformation method by improving the transformation efficiency. This was achieved by down-scaling the transformation conditions and by changing some parameters which significantly influenced the transformation rate. Depending on the construct the analysis of randomly taken plants from different transformation experiments revealed a transformation efficiency between 5% and 20% of the regenerated protoplasts. Such high transformation rates allowed us to isolate transgenic plants without the use of any selection marker. This new PEG-mediated-marker-free transformation procedure is the method of choice for generating transgenic plants not only for physiological studies but also for the recombinant expression of biopharmaceuticals and many other applications.

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Analysis of the outcome of transformation of Physcomitrella patens with constructs containing genomic DNA. Yasuko Kamisugi, Celia Knight, Andrew Cuming and David Cove Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK y.kamisugi@leeds.ac.uk Constructs with varying lengths of genomic DNA flanking an antibiotic-resistance cassette, have been used for transformation, using both PEG-mediated DNA uptake by protoplasts and microprojectile bombardment. The outcome of transformation has been determined using PCR amplification, Southern hybridisation and DNA sequencing. Key findings are: • PEG-mediated DNA uptake by protoplasts generates about twice the frequency of gene targeting compared to microprojectile bombardment. • Targeting may result in homologous recombination occurring in only one of the sequences flanking the selection cassette. • There is no difference in the length dependency of flanking sequences 5’ or 3’ to the selection cassette. • There are no differences in targeting frequencies for the four loci analysed. • The rate of allele replacement in asymmetric constructs, is dependent on the length of the shorter homologous flanking sequence. • Comparisons of the frequency of targeting and allele replacement, of copy number and of the number of integration sites for two loci, have not revealed significant differences. • Circularisation of linear transforming DNA may occur before integration by a single homologous recombination event. This work was funded by the European Union as part of the PREGENE (Precision Engineering of Plant Genes) Project.

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High conditional expression of recombinant protein in Physcomitrella patens using a soybean heat-shock promoter Younousse Saidi*, Pierre Goloubinoff, Jean-Pierre Zryd Department of Plant molecular Biology, UNIL 1015 Lausanne, SWITZERLAND *Younousse.Saidi@ie-pc.unil.ch In order to develop an inducible gene expression system in moss, we constructed a stably transformed Physcomitrella Patens with the uidA reporter gene -coding for ß-Gluruconidase (GUS)- under the control of a soybean promoter for the heat shock protein Gmhsp17.3. The optimal temperature for 1h heat-shock induction of the Gmhsp17.3 promoter in P. Patens was determined. Whereas at 25°C, there was a constant minimal GUS expression, following 1h heat-shock at 38°C and 15h at 25°C, the GUS activity was over 300 fold higher, with no apparent cell damage. As a control, we created another stably transformed clone carrying the GUS gene under the maize ubiquitin-1 promoter. The GUS activity in this strain was constitutive, about 30-40 fold higher than that of the Gmhps17.3-GUS at 25°C, and did not significantly change following heat-shock treatment. We also analyzed various times of induction at 38°C for the Gmhsp17.3-GUS construct. Already after 5 min heat-shock, the level of GUS protein –detected 15h later- was as high as the level of ubiquitin1p-GUS. Maximum GUS activity was observed following 60 min induction, yet induction times as long as 3h did not significantly affect the high level of GUS (detected after 15h recovery)(see Figure). This system is a powerful tool to modulate gene expression in Physcomitrella Patens and generate controlled amounts of recombinant proteins using a relatively mild and non-invasive mode of induction.

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Identification and phylogeny of a new MADS-box gene in Physcomitrella patens Wim Verelst*, Annemarie Matthes, Katrin Münster, Heinz Saedler, Thomas Münster Max-Planck-Institute for Plant Breeding Research, D-50829 Cologne, Germany * verelst@mpiz-koeln.mpg.de, Phone: +49-221-5062-123 MADS-box genes encode transcription factors with crucial roles in plant development, such as floral organ identity determination in higher plants (e.g. Schwarz-Sommer et al, 1990). MIKC-type MADS-domain proteins have a modular structure, consisting of a DNA-binding M-domain, an intervening I-domain and keratin-like K-domain, both involved in protein dimerisation, and a C-terminal C-domain, which is required for the formation of higher complexes. While the MADS-box gene-family in Arabidopsis thaliana consists of 104 genes, it seems to be much smaller in the Physcomitrella genome. Nevertheless, the two main MADS-box classes that can be distinguished in higher plants - MIKCc and MIKC* - are both also represented in moss (Henschel et al, 2002). MIKC*-type proteins are characterized by a prolonged I-domain, which is encoded by 4 or 5 exons, instead of only one exon in MIKCc genes (Henschel et al, 2002). Here we report the identification of a new MIKC* gene from Physcomitrella, bringing the total number of known MADS-box genes in moss to 11, from which 6 are MIKC*-type. The phylogenetic relation between all these genes will be discussed, and in addition preliminary expression data will be shown for some of them. Henschel K, Kofuji R, Hasebe M, Saedler H, Münster T, Theißen G (2002) Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens. Mol Biol Evol 19: 801-814 Schwarz-Sommer Z, Huijser P, Nacken W, Saedler H, Sommer H (1990) Genetic control of flower development by homeotic genes in Anthirrhinum majus. Science 250: 931-936

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Physcomitrella patens: a model system for investigating the evolution and organization of cellulose-synthesizing terminal complexes from algae to land plants. John T. Bushoven1, Chessa Goss and Alison W. Roberts2 Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881 USA 1jbus7623@mail.uri.edu, 2aroberts@uri.edu, 401-874-4098 Terminal complexes (TCs) synthesize cellulose at the plasma membrane as microfibrils that vary in diameter from about 3 nm in land plants to 25 nm in some algae. This variation in microfibril structure is determined largely by TC organization. The TCs are composed of cellulose synthase catalytic subunits, which are encoded by CesA genes. Bacteria and land plants share conserved regions of CesA gene products; however, extensive differences do exist. We are investigating whether such differences contribute to the variation in TC organization and microfibril structure by comparing the structure and function of CesA genes of algae to those of bacteria and land plants. Physcomitrella patens provides a model for this investigation because the predominance of the haploid generation, in addition to gene integration by homologous recombination, enables analysis of gene function through allelic exchange. Probes derived from P. patens CesA gDNA fragments were used to screen available P. patens gDNA, cDNA, and BAC libraries. To date, we have identified 8 CesA and 6 CesA-like (CslD) genes expressed in P. patens, as well a one CesA pseudogene. A phylogenetic analysis will be presented. These data provide insight into the evolutionary history of diversification and specialization of CesA genes from algae to land plants. As a test of the feasibility of conducting allelic exchange experiments, we have designed a transformation vector, in which a 2347 bp genomic fragment of expressed gene PpCesA5 was disrupted by insertion of the nptII selection cassette excised from pMBL5. Primers for PCR-based testing of gene integration have been designed to distinguish between targeting of PpCesA5 and pseudogene PpCesA2. Data from analysis of potential P. patens CesA knockout transformants will be presented and the relation to TC organization in P. patens discussed. We gratefully acknowledge gifts of cDNA, genomic, and BAC libraries, and pMBL5 from the University of Leeds, and EST clones from the University of Leeds and RIKEN Biological Resources Center. This work was funded by the U.S. Department of Agriculture NRI-GCP and the University of Rhode Island Foundation.

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Heat-shock inducible promoter allows efficient GFP-talin labeling of the actin cytoskeleton in Physcomitrella patens Andrija Finka, Didier Schaefer and Jean-Pierre Zrÿd Department of Plant Molecular Biology, Faculty of Biology and Medicine, University of Lausanne, Switzerland Inducible reporter gene systems are required to study gene expression in a developmental context where it is necessary to minimize potential deleterious effect caused by constitutive overexpression. Among inducible promoters, the small heat-shock promoters react rapidly to stress caused by an increase in temperature. Although heat-shock causes pleiotropic effect, the heat inducible promoter of soybean protein GmHsp17.3 proved to be very effective in plants [1]. In our work on actin cytoskeleton dynamics during the development of Physcomitrellapatens we have encountered difficulties with the use of the constitutive 35S promoter. Inconsistencies in the labeling of microfilaments using 35S-GFP-mTn [2] have prompted us to use heat stress inducible promoter. We demonstrate here that this promoter is behaving perfectly well in the moss P. patens. We constructed a HSP-GFP-mTn vector, which can be used either for transient or stable transformations. Upon thermal induction for one hour at 37°C, cells of P. patens plants were all homogenously labeled, this in contrast with the patchy labeling obtained with 35S-GFP-mTn. It is documented that permanent overproduction of GFP-mTn impaired cell growth [2]. The temporary overexpression of GFPmTn induced by heat shock response had no effect on cell morphology through the life cycle of P. patens. The labeling disappears progressively during the following 72 hours after induction. The detailed spatiotemporal organization of labeled F-actin in moss cells was revealed by confocal imaging. The network of actin bundles, apical cap structures, cortical star-like structures and small patches were present in regenerating protoplasts, protonemal cells and caulinary organs. We propose the heat induced GFP-mTn system as a powerful tool in temporary flashing of plant F-actin network without interfering with cell functions.

Organization of GFP-mTn labeled F-actin network in moss cells fifteen hours upon heat induction: Left : two days old single cell protoplasts Middle: four days old regenerating protonemal colony Right: part of the labeled juvenile leaflet REFERENCES 1. Prandl, R. and F. Schoffl, Plant Molecular Biology, 1996. 31(1): p. 157-162. 2. Kost, B., P. Spielhofer, and N.H. Chua, Plant Journal, 1998. 16(3): p. 393-401.

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Functional proteomics of plastid division in the moss Physcomitrella patens Grémillon, Louis 1; Martin, Anja 1; Suppanz, Ida 1; Reski, Ralf 1; Sarnighausen, Eric 1

1Freiburg University, Plant Biotechnology, Schänzlestr. 1, 79104 Freiburg, Germany; Email : louis.gremillon@biologie.uni-freiburg.de FtsZ is a filament-forming protein that resembles tubulin in structure and thus is considered to be the evolutionary ancestor of this cytoskeletal protein. In bacteria, FtsZ is essential for cell division whereas plant FtsZ homologues have been reported to be involved in plastid division. In bacteria, it was shown that the formation of ring-like FtsZ structures is followed by the recruitment of other interacting proteins to build the divisome. However, in plants, such proteins are not known. Our main focus of work is the identification of FtsZ interaction partners in the moss Physcomitrella patens whose genome encodes at least four different FtsZ proteins. Since cultivation and handling of the plant are easily feasible and gene targeting by homologous recombination is highly efficient, Physcomitrella patens is a unique model plant. We successfully used the FRET technique to investigate interactions between FtsZ isoforms. In order to purify FtsZ protein complexes, we are applying the TAP method and co-immunoprecipitation. After being separated on SDS-gels the subunits are identified by mass spectrometry. Results are confirmed by FRET analyses and targeted gene knock outs. Financial support by the Deutsche Forschungsgemeinschaft (SFB 388) is gratefully acknowledged.

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Transcriptional regulation of genes encoding extracellular proteins by phytohormones in Physcomitrella patens Stefanie Tintelnot*, D. Heintz, E. Sarnighausen, E. Decker, R. Reski University of Freiburg, Plant Biotechnology, Schänzlestraße 1, 79104 Freiburg, Germany *Stefanie.tintelnot@biologie.uni-freiburg.de The extracellular environment plays an important role in the life of animals and plants. It is necessary for the control of the development and the communication between cells. In plants, the extracellular compartment consists of the apoplastic space and the cell wall. Besides physiological processes, the cell wall fulfills physical functions like the stability of the whole organism and regulation of the growth. In Physcomitrella patens protonema, the phytohormones ABA, auxin, and cytokinin are known to induce specifically the few and well known developmental steps, i. e. the transition from chloronema to caulonema (auxin), the initiation of buds (cytokinin), and the formation of brachycytes (ABA). As phytohormones regulate moss development and extracellular proteins are involved in the developmental processes, the genes encoding extracellular proteins might be regulated by those hormones. Extracellular proteins from the medium of Physcomitrella protonema cultures were isolated and identified by peptide sequencing. The transcriptional regulation of ten of the corresponding genes by the phytohormones ABA, auxin, and cytokinin was studied by northern blot analyses as well as real time PCRs. These analyses showed that the transcription of most of the investigated genes is regulated by abscisic acid, mainly of those genes coding for proteins putatively involved in cell wall modification and signal transduction. The extracellular localisation of the proteins was determined by computational prediction and confirmed by confocal laserscanning microscopy with gfp fusions as well as ELISA demonstrating that the analysed signal peptides could efficiently target a recombinant protein to the supernatant of transformed Physcomitrella cultures.

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Proteome analysis of mutants in the moss Physcomitrella patens Anika Erxleben*, Ralf Reski, Eric Sarnighausen

Freiburg University, Plant Biotechnology, Schänzlestr. 1, 79104 Freiburg, Germany * Anika.Erxleben@biologie.uni-freiburg.de, fon +49 761 2032814 The moss Physcomitrella patens is the only land plant known with highly efficient homologous recombination in its nuclear DNA . This feature renders gene targeting feasible and - in conjunction with advantages - makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. The reverse genetics approach is a basic tool of functional genomics and allows deriving clues to the function of a gene by characterising the influence of its manipulation on the phenotype of the mutants. Out of 75,814 mutant plants analysed to date, 26.7% display a clearly deviating phenotype compared to the wild type. This number of obvious phenotypical aberrations is rather high as compared to rates reported for other plants species. To further extend the potentialities of this lower plant we are currently exploring the Physcomitrella patens proteome. Here, we present a protocol optimised to analyse differences expressed at the proteome level of the Physcomitrella mutants. Our special focus is on those silent mutants which display the same visible phenotype as the wild type but show variations in the protein patterns. The advantages of proteome analysis for further studies of gene functions in Physcomitrella patens are discussed.

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Phospholipase C from Physcomitrella patens: biochemistry and knockout Alexander Repp1, Chantal Brüggemann1, Franz Mittmann2, Koji Mikami3 and Elmar Hartmann1

1Freie Universität Berlin, Institut für Biologie – Pflanzenphysiologie, Königin-Luise-Str. 12-16, 14195 Berlin 2Justus-Liebig Universität Gießen, Institut für Pflanzenphysiologie, Senkenbergstr. 3, 35390 Gießen 3National Institute for Basic Biology, 38 Nishigonaka Myodaiji-cho, Okazaki 444-8585, Japan As sessile organisms plants have to respond effectively to environmental change. Different sensory and signal transduction pathways serve to detect and collate signals as diverse as light, gravity and nutrient levels. A key enzyme in numerous signaling pathways in animals is the phosphoinositide-specific phospholipase C (PI-PLC). This generates the two second messengers - inositol-1,4,5-trisphosphate (IP3) and diacylglycerol. In plants several physiological responses are thought to be associated with the phosphoinositide (PI) pathway, including ABA-mediated stomatal movement and the perception of gravitropic stimuli. However, our understanding of PI-signaling in plants is still poor. To elucidate the physiological functions of PI-PLC we have cloned two PLC from Physcomitrella, characterized the biochemical properties and generated targeted knockout mutants via homologous recombination. At micromolar Ca2+ concentrations, PpPLC1 preferentially hydrolyzed phosphatidylinositol-4,5-bisphosphate (PIP2), while PpPLC2 showed no specificity. Furthermore, at millimolar Ca2+, phosphatidylinositol was hydrolyzed by PpPLC2 but not by PpPLC1. Thus, PpPLC1 and PpPLC2 belongs to type II and type I of plant PI-PLC, respectively. The plc1 knockouts show several striking phenotypic differences compared to wild type. The most prominent are an altered gravitropic response of protonema filaments and a dramatically reduced gametophore formation which was accompanied by a loss of sensitivity to cytokinin. Further molecularbiological and physiological characterisation of plc2 knockouts are in progress. The current results of plc1 imply a hitherto unknown role for PLC in plant signaling.

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Cytokinin signalling in Physcomitrella patens is mediated by histidine kinase receptors with partially overlapping functions Bénédicte Trouiller2*, Fabien Nogué2, Florent Brun1, Kenjiro Fujiwara3, Didier Schaefer4, Akio Toh-e3, Michel Laloue1 and Martine Gonneau1

1Laboratoire de Biologie Cellulaire, INRA, Route de St Cyr, 78026 Versailles, France 2Station de Génétique et amélioration des plantes, INRA, Route de St Cyr, 78026 Versailles, France 3Laboratory of Genetics, Department of Biological Sciences,Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan 4Laboratoire de Phytogénétique Cellulaire, Institut d'Écologie, Université de Lausanne, Bâtiment de Biologie, CH-1015 Lausanne-Dorigny, Switzerland. * trouille@versailles.inra.fr The cytokinin bud induction in moss is a quantitative response and has been used as a cytokinin bioassay. Cytokinin receptors identified in Arabidopsis thaliana are two components hybrid histidine kinases (Inoue et al., 2001). In Arabidopsis triple mutants for histidine kinases (AHK4/CRE1/WOL, AHK2, AHK3) that function as cytokinin receptors, are small and infertile but they still possess basics organs: roots, stems and leaves. Using degenerate oligonucleotides designed from AHK4 and other AHK4 plant homologs, we identified two P. patens AHK4 homologs (PpCRE1 and PpCRE2). Both cDNA are able to complement the sln1 histidine kinase deficient mutant of yeast in a cytokinin dependant manner. Single knockout transformants for PpCre1 & PpCre2 didn’t show marked phenotypical alterations. Cell proliferation from the newly formed buds was examined in the various knockout-background. PpCRE1 knockout lines show partial resistance to high levels of cytokinin regarding gametophore development, whereas PpCRE2 knockout lines do not. Double KO transformants for PpCRE1 and PpCRE2 have been obtained. Negative gravitropism in the dark is not affected in single or double KO. The cytokinin bud induction on dark grown caulonema as well as polarotropic responses and senescence process in the various backgrounds will be discussed. This study provides the first molecular evidence that: 1) the higher plant two components system of cytokinin perception is conserved in P. patens, 2) the two P. patens hybrid kinases have partially distinct roles in the cytokinin signalling pathway, 3) PpCre1 and PpCre2 are functionnaly redundant for cytokinin-induced bud formation. Inoue, T; Higuchi, M; Hashimoto, Y; Seki, M; Kobayashi, M; Kato, T; Tabata, S; Shinozaki, K and Kakimoto, T (2001) Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409 : 1060-1063.

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From Physcomitrella to Arabidopsis: the role of chromatin remodeling by polycomb proteins in controlling plant development. Assaf Mosquna, Aviva Katz and Nir Ohad Department of Plant Sciences, Tel-Aviv University, Tel Aviv, 69978 Israel Plant development requires the establishment and maintenance of particular gene expression programs along the entire plant life cycle. In Arabidopsis, a protein complex containing FIE and MEA, homologues of the Drosophila chromatin remodeling Polycomb-group proteins (PcG), were shown to regulate the development of the gametophyte. Mutation in either protein triggered the central cell female gametophyte within the embryo sac, to develop autonomous endosperm with out fertilization. Further more, silencing of the FIE gene in the sporophyte resulted in pleiotropic phenotypes. Based on our results we propose that FIE (a WD40 protein) and various members of the PcG SET domain protein family may form different PcG complexes serving as central regulators of particular developmental programs along the entire plant life cycle. The evolution of plants depended upon the establishment of mechanisms controlling cell division and differentiation. To understand the contribution of PcG proteins to these processes in plant evolution, we propose to study the role of PcG proteins during the development of Physcomitrella as a model system representing an early stage in terrestrial plant evolution. Through a search of the public data base (http://www.cosmoss.org/bm/BLAST) we have identified putative candidates of Physcomitrella PcG proteins. Homologues members of the WD40 and SET domain PcG proteins were identified displaying high similarity at the protein level within the above domains. Our long term goal is to elucidate the function of these proteins in Physcomitrella. To this end, we are generating knockout plants to test the Physcomitrella PcG protein function in vivo as well as transgenic plants bearing a GFP fusion to monitor their pattern of protein localization and accumulation. The comparison of PcG functions between lower and higher plants will shade light on the underling mechanisms controlling plant development.

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Analysis of extracellular differentiation signals in Physcomitrella patens by RNAi Otmar Lienhart*, Ralf Reski and Eva L. Decker Plant Biotechnology, University of Freibug, Schänzlestraße 1, D-79104 Freiburg, Germany * otmar.lienhart@biologie.uni-freiburg.de, fon +49 761 2036944 Developmental processes in multicellular organisms are based on the integration of signals, which single cells receive from their environment. In plants extracellular polypeptides and their respective receptors fill an important position besides the classical phytohormones in regulating differentiation (Ryan et al. 2002). Most of the known receptors of polypeptides belong to the leucine-rich repeat receptor kinase (LRR-RLK) family. There are 216 predicted LRR-RLKs existing in the Arabidopsis thaliana genome, but for only a few of these there is evidence for ligands and the vast majority of the LRR-RLKs are orphan receptors. In contrast to the rather complex seed plants the simple and well characterized protonema development of Physcomitrella patens is a critical advantage to study the role of polypeptides and LRR-RLKs during cellular differentiation. Homologs to the polypeptide families of Rapid Alkalinization Factors (RALF) and CLV3/ESR-related proteins (CLE) were identified from a clustered EST database covering more than 95% of the Physcomitrella transcriptome (Rensing et al. 2002). Furthermore 57 EST-clones showed to be closely related to LRR-RLKs. Current experiments carry out the functional analysis of RALF and CLE homologs with a loss of function strategy by RNAi and with overexpression studies with respect to their role in developmental processes. For this purpose a transient expression system was established which allows the monitoring of regenerating protoplasts in different growth media over three weeks. Ryan, C. A., G. Pearce, J. Scheer und D. S. Moura (2002). Polypeptide hormones. Plant Cell 14: S251-64. Rensing, S. A., S. Rombauts, Y. Van de Peer und R. Reski (2002). Moss transcriptome and beyond. Trends Plant Sci 7: 535-8.

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Deciphering RNA silencing in moss Tzahi Arazi*1, Ran Stav1 and David Baulcombe2 1Department of Ornamental Horticulture, Volcani Center, P.O.B. 6, Bet-Dagan, 50250, Israel 2The Sainsbury Laboratory, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK *tarazi@agri.gov.il, Tel: +972-3-9683498 Recently, it was demonstrated that RNA interference and miRNA target sequences occurs in Physcomitrella patens (P. patens) suggesting that RNA silencing and miRNA regulation mechanisms are conserved in moss. Our study is aimed to identify moss genes which are involved in PTGS and miRNA regulation. To do so, we have screened P. patens EST database with amino acid sequences of Arabidopsis SDE1, AGO1, DCL1 and HEN1 genes, demonstrated to be involved in PTGS or miRNA metabolism. Several ESTs showing significant homology to SDE1and AGO1 were identified. Firstly, we have used rapid amplification of DNA ends protocol to clone the full length P. patens SDE1 homologue (PpRdRp). PpRdRp gene spans 4.5 Kb and contains 3 introns. To characterize the ORF of PpRdRp gene, its cDNA was cloned by RT-PCR. Sequencing of the amplified fragment revealed a 3459 bp cDNA that is predicted to encode an 1153 aa protein. PpRdRp protein is highly similar to AtSDE1 (46%), which was shown to be required for PTGS mediated by transgenes. SMART Analysis of PpRdRp protein sequence indicates that it contains an RNA recognition domain in its N-terminus and a highly conserved RNA dependent RNA polymerase domain, both characteristic of an RdRp enzyme. Moss genes showing homology to Arabidopsis AGO1 and HEN1 were then cloned and were successfully utilized together with the PpRdRp gene to generate knock out moss plants. Preliminary phenotypic analysis of different KO lines shows that under normal autotrophic conditions all plants developed similarly to wild-type plants. In depth analysis of various KO plants and characterization of PpRdRp is ongoing.

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Abscisic acid-induced freezing tolerance in Physcomitrella patens Anzu Minami*, Manabu Nagao, Keita Arakawa, Seizo Fujikawa and Daisuke Takezawa Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan Graduate School of Agriculture, Hokkaido University, Sapporo 060-0819, Japan * anzu@pop.lowtem.hokudai.ac.jp, fon +81-11-706-7356 Many plants growing on the regions exposed to freezing temperature have tolerance to cellular dehydration caused by extracellular ice formation. We are investigating mechanisms of freezing tolerance in the moss Physcomitrella patens. P. patens protonema cells had low levels of freezing tolerance with LT50 (lethal temperature for 50% mortality) of -2˚C. Abscisic acid (ABA) plays an important role in tolerance to drought, osmotic, cold and salinity stress in higher plants. In P. patens, exogenous application of 10 µM ABA increased freezing tolerance changing LT50 to -10˚C (Minami et al. 2003). In P. patens, cell growth is inhibited by short-term ABA treatment and formation of 'brood cell' is accelerated by long-term ABA treatment. Our results indicated that 10 µM ABA treatment for one day, which dramatically induced freezing tolerance, did not induce brood cell formation but induced morphological alterations in organelles such as chloroplast flattening, vascular segmentation and cell wall thickening (Nagao et al. in press). The development of freezing tolerance by ABA treatment was inhibited by a protein synthesis inhibitor cycloheximide, suggesting critical roles of synthesis of nuclear encoded proteins for freezing tolerance. ABA also induced physiological changes such as expression of various genes, accumulation of boiling stable proteins and increase in soluble sugar contents. Recently, we isolated mutants with reduced ABA sensitivity by UV mutagenesis. These mutants grew normally in a medium containing ABA, whereas the wild type showed inhibited growth. In these mutants, long-term ABA treatment did not induce brood cell formation. Freezing tolerance in all mutants after ABA treatment was lower than that of wild type, but the levels of freezing tolerance varied among different mutants. We present gene, protein and sugar analyses of these mutants to dissect ABA signaling pathways leading to development of freezing tolerance. Minami, A., Nagao, M., Arakawa, K., Fujikawa, S. and Takezawa, D. (2003) Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. Journal of Plant Physiology 160, 475-483 Nagao, M., Minami, A., Arakawa, K., Fujikawa, S. and Takezawa, D. Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens. Journal of Plant Physiology, in press

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Tolerance of the moss Physcomitrella patens subsp. patens (Funariales) to inorganic arsenate, As (V) Misao Itouga1*, Makoto Kimura1, Yoshiro Ono2, and Isamu Yamaguchi1 1RIKEN Plant Science Center, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, JAPAN 2Okayama University, 1-1-1 Tsushima-naka, Okayama, 700-8530, JAPAN * itouga@psc.riken.jp, Phone:+81-45-503-9490 We investigated toxicity of arsenate, As (V), to the moss Physcomitrella patens (Hedw.) Bruch & Schimp. subsp. patens Tan for a cultivation period of six months. Metal nutrients in gametophytes were quantified by X-ray analytical microscope (Horiba XGT-5000W) and inductively coupled plasma mass spectrometry (ICP-MS; Perkin Elmer Elan6100DRC). Plants treated with Na2HAsO4�7H2O did not accumulate detectable amount of As: they showed similar growth compared to the control plants. However, the leaf tended to become slightly smaller with increasing concentration of As (Fig.1). Na2HAsO4�7H2O caused the highest Na gametophytes concentrations and simultaneously the lowest gametophytes K levels (antagonism K-Na), with a significant decrease in gametophytes Ca concentration. Indeed, chloronema of this moss dominantly grew in the presence of 10 mM Na2HAsO4. P. patens subsp. patens shows tolerance to the inorganic arsenate, As (V), without actively absorbing As (V) on its surface, while As (V) significantly affected macro- and micro-nutrient concentrations and development of leaf. Control 10μ Ｍ

As: 0.75ppm100μ Ｍ

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A knockout mutant of the dehydrin DHNA is impaired in recovery from osmotic stress in Physcomitrella patens. Valentina Carballo*, Laura Saavedra, Jan Svensson, Darwin Izmendi, Björn Welin and Sabina Vidal. Laboratorio de Biología Molecular Vegetal, Facultad de Ciencias, Universidad de la República, Iguá 4225, CP 11400, Montevideo, Uruguay. *vcarballo@fcien.edu.uy phone +598 2 5258618 ext. 213 Considerable research in the last decade has focused on the analysis of stress-induced gene expression in order to find answers to the genetic and molecular mechanisms underlying plant cellular dehydration tolerance. One of the most studied protein family that accumulates in response to water stress in higher plants are the dehydrins (DHNs). Despite extensive studies clear evidence showing a physiological role in abiotic stress tolerance for these plant proteins is still lacking. In this study we have employed a reverse genetics approach, using the moss Physcomitrella patens, in order to contribute to the understanding of the function of DHNs in plants. We have isolated a dehydrin-like gene fragment, PpDHNA, from P. patens by PCR amplification, using degenerate primers directed against conserved amino acid segments of DHNs of higher plants. The gene encodes a 59.9 kDa glycine-rich protein, DHNA, showing typical characteristics of DHNs from higher plants. Expression analysis of PpDHNA reveals both mRNA and protein accumulation in response to abscisic acid, salinity or osmotic stress treatment. Interestingly, transcript and protein levels rapidly decrease when plants are transferred from stress conditions to optimal growth conditions. To analyze the contribution of DHNA to osmotic stress tolerance, a knockout mutant (dhnA) was generated by interrupting the coding sequence of PpDHNA with the nptII gene. Growth and stress response studies of the mutant showed that dhnA was severly impaired in its capacity to resume growth after a severe osmotic stress treatment. This is the first direct evidence in any plant species for a physiological role of a dehydrin, protecting the plant during cellular dehydration, allowing plant recovery when optimal growth conditions are resumed. These findings suggest that dehydrins constitute part of a general molecular mechanism used by all land plants to protect them from injury during cellular dehydration. The mechanism of action of DHNA in stress tolerance is still unclear. The deduced amino acid sequence of DHNA reveals the presence of 11 repeated segments that theoretically can form a class A amphipathic α-helix. This type of structure has been suggested to have the capacity to interact with the plasma membrane. Based on this, we postulate a membrane preservation mode of action for this protein, by interacting with phospholipids or/and membrane proteins during stress conditions. Immunohistochemical studies showing the DHNA sub-cellular localization during stress will be presented together with stress response studies of the null-mutant dhnA.

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Gene-ID in Physcomitrella patens Gabriele Schween*, Mark von Stackelberg, Christina Reinhard, Ralf Reski Plant Biotechnology, University of Freiburg, Sonnenstrasse 5, 79104 Freiburg * gabriele.schween@biologie.uni-freiburg.de, fon +49 761 203-6972 The moss Physcomitrella patens (Hedw.) B.S.G. is a novel tool in plant functional genomics as the rate of homologous recombination in its nuclear DNA is several orders of magnitudes higher, compared with seed plants. By using large scale mutagenenesis approaches, about 75,000 Physcomitrella knock-out plants were generated (Egener et al. 2002). HoMi mutants were produced by protoplast transformation of a limited pool of 20 distinct mutagenized cDNAs. Afterwards transformants were regenerated, selected, documented, and cryoconserved. After metabolic profiling of about 10,000 transformants, 54 plants were analysed so far to establish gene/function-relationships. Knockouts were detected by a simple PCR-screen. After sequencing the cDNAs used for transformation, primers were derived, which will amplify a fragment of about 300 bp in wildtype plants. This product will disappear or shift up in KO-plants, because of the integration of the npt-II-cassette in the locus in case of homologous recombination. 54.7 % of the 1174 cDNAs used for transformation could be tested via this easy and fast PCR-approach. In 45.3 % of the cases, either the sequencing, primer derivation or PCR failed. A comparison of results obtained via genomic PCR and RT-PCR was performed (n=23 plants). In 28.7 % the PCR-band in the control was bigger than calculated. When comparing results of PCR and RT-PCR, the RT-PCR-band had the calculated product size. Thus the shift of the PCR band was due to introns in the genomic locus. Using RT-PCR, more knockouts could be identified. For one homologous knockout, the result of the RT-PCR was consistent with genomic PCR. In this case, homologous recombination has taken place with exchange of the original genomic locus with the cDNA-construct. In three other cases, the genomic PCR amplified the wildtype band in the transformed plant, whereas the RT-PCR did not give the expected product, thereby revealing the knockout. The original wildtype locus seems to be somewhere present, but its transcription is clearly knocked out. Although the rearrangements at the genomic level is not fully understood, the targeted locus is non-functional, nevertheless. To identify all functional knock-outs the analysis at RNA-level is therefore necessary. In 10 transformants gene knock-outs were detected via RT-PCR. In one plant four cDNAs were targeted, in one plant three, in two plants two different cDNAs and in six plants a single cDNA. We tested, how many of the the 20 cDNAs used for the transformation had integrated in the transformants (n=9). 98 out of 115 cDNAs (85.2 %) were detected in the plants, indicating, that the cDNAs form large heteroconcatemers. Southern and Northern-analyses confirmed the knockouts and showed, that the heteroconcatemers integrated at only a few loci in the genome. BLAST-searches at the amino acid level yielded no significant hit for 11 of a total of 17 targeted cDNAs. Previous analyses of the Physcomtrella transcriptom already indicated, that Physcomitrella patens is a valuable source for novel genes (Rensing et al. 2002). This work has been performed in a joint project with BASF Plant Science GmbH. Metabolic profiles of the transformants were generated at Metanomics GmbH. Egener, T., J. Granado, M.-C. Guitton, A. Hohe, H. Holtorf, J.M. Lucht, S. Rensing, K. Schlink, J. Schulte, G. Schween, S. Zimmermann, E. Duwenig, B. Rak, R. Reski (2002): High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. BMC Plant Biology 2, 6. Rensing, S.A., S. Rombauts, Y. Van de Peer, R. Reski (2002): Moss transcriptome and beyond. Trends Plant Sci. 7, 535-538.

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Adaptive basis of codon usage in Physcomitrella patens Hans K. Stenøien*

Department of Ecology and Evolution, Evolutionary Biology Centre, Uppsala University, Villav. 14, SE-756 32 Uppsala, Sweden *hans.stenoien@ebc.uu.se, phone: +46 73 708 1148 Patterns of codon usage bias have been studied in Physcomitrella patens. 92 nuclear, protein coding genes have been employed and estimated levels of gene expression has been tested for association with two measures of codon usage bias and other variables hypothesized to be associated with gene expression. Codon bias is found to be positively associated both with estimated levels of gene expression, as well as GC contents in the coding parts of studied genes. However, GC contents in non-coding parts, i.e. introns and 5’ and 3’ UTRs, are not associated with estimated levels of gene expression. It is argued that codon bias is not shaped by mutational bias but rather by weak natural selection for translational efficiency in P. patens. The ecology and overall biology of this species suggest that random genetic drift could have a significant impact on molecular evolution. Contrary to this expectation, the present results indicate that genetic drift cannot prevent the action of weak selection in studied genes. Stenøien, HK (2004) Adaptive basis of codon usage in the haploid moss Physcomitrella patens. Heredity in press.

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Annotation and representation of the Physcomitrella patens transcriptome: an integrated approach D. Lang*, R. Reski, S.A. Rensing University of Freiburg, Plant-Biotechnology, Sonnenstr. 5, D-79104 Freiburg * Daniel.Lang@biologie.uni-freiburg.de, Phone +49761-2036988, Fax +49761-2036990

The general management and representation of the vast amount of data generated during and in the aftermath of large-scale sequencing projects in a user-friendly way is crucial in order to extract biologically meaningful conclusions and hypotheses. For most projects, especially in an academic context, an all-in-one solution for the production, storage, annotation and representation of the information is either not available or too expensive. Thus, a variety of programs is being used in the different steps of e.g. sequencing, clustering, annotation of the sequences, each with a different input/output format and information content. Additionally, scientists working with the data generate their own results and conclusions. The variability in terms of format of these contents is even higher. Therefore the accessibility and usability for third parties is hardly ensured. Redundancy of work carried out and data generated/stored as well as loss of information are the consequences. Hence, the goal is to create a central storage, that allows distinct views and representations of the collected data and the creation of so-called data warehouses, e.g. by using a database management system. Here we present an integrated approach to generate a knowledge resource for the transcriptome of Physcomitrella patens (i.e. the representation of an organisms protein encoding transcripts) using mainly open source software. The underlying dataset consists of the public ESTs and full-length cDNAs of Physcomitrella patens. The sequences were clustered using the Paracel Transcript Assembler (6) using a species-specific parameter set (1). The main view of the database will represent each transcript as an entity containing all the annotation that can be gathered automatically as well as optional manual additions (added by the researcher). The automatic annotation was done using homology searching against protein sequence databases as well as motif and profile collections. Through a set of decision trees we inferred annotations and gene ontology associations (2) for the transcriptome. We link the information gathered during clustering of the ESTs and the ORF prediction as well as other sequence features to every single transcript. The implementation is object oriented using the bioperl (3) sequence object framework. The main database backend is an instantiation of the biosql/OBDA database schema standard (4) developed by the Open Bioinformatics Foundation. Biosql is meant to be a generic unifying schema for the storage of sequences from different sources (e.g. Genbank, EMBL or Swissprot) and functions as rich sequence object persistence framework for all Bio* toolkits (5). Additionally we developed several other database schemas to support the mapping of the different filtering and annotation steps to the sequence objects. E.g. to provide efficient retrieval and non-redundant storage, the data-oriented XML format (6) representing the filtering and the clustering of the sequences was mapped to these relational databases. The whole clustering process is modeled, so that not only the respective contig or singleton is represented in the database, but also the clusters they belong to. This results in 5 different sequence datasets representing the annotated Physcomitrella transcriptome with different granularity: the raw unfiltered input sequences, the filtered input sequences, the assembled transcriptome, the assembled non-redundant transcriptome and the predicted ORFs. These datasets are searchable and accessible in different formats through web interfaces at http://www.cosmoss.org. (1)Rensing, S.A., S. Rombauts, A. Hohe, D. Lang, E. Duwenig, P. Rouze, Y. Van de Peer, R. Reski (2002): The transcriptome of the moss Physcomitrella patens: Comparative analysis reveals a rich source of new genes. http://www.plant-biotech.net/Rensing_et_al_transcriptome2002.pdf (2)Gene Ontology: tool for the unification of biology. The Gene Ontology Consortium (2000) Nature Genet. 25: 25-29 http://www.geneontology.org (3)Stajich JE, Block D, Boulez K, Brenner SE, Chervitz SA, Dagdigian C, Fuellen G, Gilbert JGR, Korf I, Lapp H, Lehvaslaiho H, Matsalla C, Mungall CJ, Osborne BI, Pocock MR, Schattner P, Senger M, Stein LD, Stupka ED, Wilkinson M, Birney E. The Bioperl Toolkit: Perl modules for the life sciences. Genome Research. 2002 Oct;12(10):1161-8. http://www.bioperl.org (4)http://obda.open-bio.org/ (5)O|B|F - Open Bioinformatics Foundation http://open-bio.org (6)Paracel Transcript Assembler (PTA) and Paracel Clustering and Assembly Markup Language (CAML) http://www.paracel.com

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Alternative Splicing in Physcomitrella patens E. Schumann, R. Reski, S.A. Rensing University of Freiburg, Plant Biotechnology, Sonnenstr. 5, 79104 Freiburg, Germany. Eik.Schumann@biologie.uni-freiburg.de; fon: +49 761 2036988. Alternative Splicing increases gene expression complexity at the transcript level. Different ways of processing the pre-mRNA can lead to many transcripts which differ in sequence composition and length, and thus may yield altered protein functionality. A lot is known about alternative splicing in animals, whereas knowledge about the mechanism in plants is scarce. It is estimated that about 40% of human genes are alternatively spliced (Modrek et al. 2001). In plants, the frequency seems to be lower, but so far the study of this phenomenon is mostly restricted to individual genes. In Arabidopsis, for example, 100 cases of alternative splicing were detected in 5000 screened genes (Haas et al. 2003). We are looking for alternative splicing in Physcomitrella patens by using both computational approaches and checking promising transcripts in the wet lab. In silico prediction of putative splice forms is based on the clustering of an extensive Physcomitrella EST dataset (Rensing et al. 2002). Multiple contigs per cluster can be caused by alternative splicing. Combining knowledge about alternatively spliced genes in other plants with the clustering data increases the chance to find alternative splice forms in Physcomitrella. Finally, alternative splicing must be proven for each single transcript by sequencing and comparing cDNA and genomic DNA. In addition, tissue specificity of the transcripts can be proven using RT-PCR. Modrek B., Resch A., Grasso C., Lee C.(2001) Nucleic Acids Res. 29:2850-9. Genome-wide detection of alternative splicing in expressed sequences of human genes. Haas B., Delcher A., Mount S., Wortman J., Smith Jr R., Hannick L., Maiti R., Ronning C., Rusch D., Town C., Salzberg S. and White O.(2003) Nucleic Acids Res. 31: 5654-5666. Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Rensing S., Rombauts S., Van de Peer Y., Reski R.(2002) Trends Plant Sci. 7: 535-538. Moss transcriptome and beyond. Trends Plant Sci. 7: 535-538.

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Molecular and functional characterization of SBP-box genes in the moss Physcomitrella patens Maike Riese, Peter Huijser, Thomas Münster, Vanessa Quodt, Susanne Höhmann, Wolfram Faigl and Heinz Saedler Max-Planck Institute for plant breeding research, Carl-von-Linné-Weg 10, 50829 Köln contact: riese@mpiz-koeln.mpg.de SBP-box genes encode a family of plant specific putative transcription factors sharing a conserved DNA-binding domain, i.e. the SBP-domain. SBP-domain proteins were discovered through their interaction in vitro with a sequence element found in the promoter region of the Antirrhinum floral meristem identity gene SQUAMOSA. Hence the name SBP for SQUAMOSA-promoter-Binding-Protein. In Arabidopsis the SBP-box genes are known as SPL-genes and form a family with sixteen structurally heterogeneous members showing characteristic temporal and spatial expression patterns suggesting different roles in development. However, to date for only one of these a role in anther development could be elucidated. Another SBP-box gene in maize, i.e. LG1, has previously been found to affect ligule development. Thus, despite their evolutionary conservation from green algae to higher plants, the role of most of the SBP-box genes remains unknown due to a general lack of well-defined mutants. Therefore, and to contribute to the elucidation of these possible roles in plant development, we choose the moss Physcomitrella patens as a model system to exploit reverse genetics. In contrast to other land plants, Physcomitrella has extraordinary high gene targeting rates, which should allows us to make some real SBP-box gene knock-outs. As a first step towards this goal, we isolated four SBP-box genes, called PpSBP1 to 4, by cDNA library screening. From the deduced amino acid sequences we identified short conserved motifs in addition to the SBP-domain on the basis of which we can divide the Physcomitrella SBP-proteins in at least two different groups. Detailed genomic and cDNA sequence analysis revealed the presence of a positionally conserved intron in the SBP-box, as has been found in all Arabidopsis SPL genes. Furthermore, a putative miRNA target site predicted for several of the Arabidopsis SPL gene transcripts could also be recognized in the PpSBP3 cDNA sequence. In the light of these evolutionary conserved features of SBP-box genes and their encoded proteins, it will be interesting to find out if the SBP-box genes also conserved some function in the development of lower plants like Physcomitrella and higher plants like Arabidopsis.

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Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude Verena Horstmann1, Claudia M. Huether1, Wolfgang Jost2, Ralf Reski1, Eva L. Decker1

1 University of Freiburg, Plant Biotechnology, Schaenzlestr. 1, 79104, Germany, 2 greenovation Biotech GmbH, Boetzinger Str. 29b, 79111 Freiburg, Germany Background In addition to gene function and developmental analyses, biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors equipped with heterologous and endogenous promoters respectively were constructed. Results Promoter activity was quantified using the dual-luciferase reporter assay system. The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude. Of these, the complete rice actin 1 gene promoter showed the highest activity in Physcomitrella, followed by a truncated version and three different versions of the cauliflower mosaic virus 35S promoter. In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly. Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella. In addition, the 5′-regions of two Physcomitrella glycosyltransferases (i.e. α1,3-fucosyltransferase and β1,2-xylosyltransferase) were identified and functionally characterised in comparison to the heterologous promoters. Furthermore, motifs responsible for enhancement of translation efficiency - such as the TMV omega element and a modified sequence directly prior the start codon - were tested in this model. Conclusion We developed an established vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research

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Expression profiling of transcription factors during phytohormone action in Physcomitrella patens Sandra Richardt*, Ralf Reski and Wolfgang Frank Plant Biotechnology, University of Freiburg, Schänzlestraße 1, D-79104 Freiburg, Germany * sandra.richardt@biologie.uni-freiburg.de, fon +49 761 2036944 In moss protonema cytokinin, auxin and abscisic acid (ABA) control distinct developmental changes. Together with the advantages in morphology, cell lineage and reverse genetics, this facilitates the study of phytohormone action on a molecular basis in this phylogenetically old land plant . Nevertheless, the genome structure of Physcomitrella, its regulation and expression are very similar to higher plants (Reski 1999, Rensing et al. 2002). The rising amount of sequence data among the plant kingdom enables comparative studies and identification of homologues. The basis of our functional analysis is a clustered EST database covering more than 95% of the transcriptome (Rensing et al. 2002). Following different approaches around 900 ESTs coding for putative transcription factors (TFs) in Physcomitrella have been identified. As a basis annotated TF genes of Viridiplantae and Algae have been used for extensive BLAST searches. These results were completed by additional searches with existing PFAM and PROSITE profiles of all kingdoms. In this step a remarkable amount of ESTs without relation to known plant TF genes was identified. The expression profile of these 900 putative TF genes upon hormone treatment will be studied using a cDNA microarray. The microarray system has been tested and evaluated successfully for this purpose. In this preliminary study for the purpose of normalization housekeeping genes of Physcomitrella have been identified. Additionally two common reference designs were compared. An universal oligo nucleotide reference (Dudley et al. 2002) showed excellent performance and will be used for further expression profiling experiments with the transcription factor cDNA microarray. As results of the preliminary experiment, the evaluation of two common references and the identification of Physcomitrella housekeeping genes will be discussed. A second part will focus on details of the homology searches for Physcomitrella transcription factors. Dudley, AM, Aach, J, Steffen, MA and Church, GM (2002): Measuring absolute transcript abundance with microarrays with calibrated reference sample and an extended signal intensity range. PNAS 99(11):7554-7559 Rensing, SA, Rombauts, S, Van de Peer, Y and Reski, R (2002): Moss transcriptome and beyond. Trends in Plant Science 7:535-538. Reski, R (1999): Molecular genetics of Physcomitrella. Planta 208:301-309

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Genomics-driven comparative physiology of GATA transcription factor function in Physcomitrella patens Scott Lindsay*, Celia Knight, Phil Gilmartin and Iain Manfield Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK * bgysal@leeds.ac.uk, +44 113 3432863 GATA factors are a class of transcriptional regulators whose members are characterised by the presence of either one or two highly conserved type IV zinc finger DNA-binding domains. These zinc fingers proteins bind specifically to promoter elements containing the DNA sequence motif (A/T)GATA(A/G). In plants GATA transcription factors are implicated in the control of light-responsive transcription. Indeed, many plant light-regulated genes have been shown to contain a consensus GATA motif within their promoter sequence. A small sub-group of GATA factors in Arabidopsis thaliana contain a motif which has been observed in CONSTANS (CO) and TIMING OF CAB 1 (TOC1), further implicating these genes in light-regulated transcription. In Arabidopsis thaliana a family of 29 GATA factors have been characterised. I have identified 8 GATA factor homologues in Physcomitrella patens. These genes have been cloned, sequenced and, for many, extensive amounts of promoter sequence identified. Phylogenetic analysis indicates significant homology between GATA factors in both plants. The ‘CONSTANS/TOC1’ motif is also conserved in moss. I have made constructs to target several moss GATA factors and, using the power of homologous recombination in this model organism, I intend to functionally characterise these genes by analysing the knockout mutants. In addition, by comparing results with data from Arabidopsis GATA mutants, it should be possible to ascertain whether functionality is conserved between similar genes in both plants. This comparative approach could also reveal important evolutionary clues as to how this family has evolved in the transition from primitive to higher plants.

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RHD6-like transcription factors: from rhizoids to root hairs? Benoît Menand1, Paul Linstead1, Didier Schaefer2 and Liam Dolan1. 1Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR47UH, United Kingdom. 2Laboratoire de Phytogénétique Cellulaire, Institut d’Ecologie, Université de Lausanne, CH 1015, Lausanne, Switzerland. Bryophytes rhizoids and the root hairs of vascular plants are epidermal tip growing cells involved in the anchorage of land plants to their growth substrate. In this regard, rhizoids have been essential structures for the colonisation of land by plants. Similarities between rhizoids and root hairs might suggest an evolutionary link between these two cellular structures. Our work on Arabidopsis thaliana has identified members of the RHD6 subfamily of basic-Helix-Loop-Helix transcriptions factors that are essential regulators of root hair development. Because the only role of the Arabidopsis RHD6-related genes is in root hair development, characterising the function of these genes in more ancestral species will shed light on the evolutionary relationship between rhizoids and root hairs. We identified RHD6-related genes in Physcomitrella from the Riken EST database. We are currently using gene targeting to investigate the function of these Physcomitrella RHD6-like genes. Furthermore, we are using degenerate primers to amplify others putative members of this subfamily in Physcomitrella. If the Physcomitrella genes control rhizoid development it suggests that the same regulatory genes control the development of rhizoids and root hairs and that this function has been conserved over the past 450 million years.

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Isolation and characterization of a nuclear rpoA gene knockout moss reveal the existence of two different plastid RNA polymerases Yuki Kobayashi* and Mamoru Sugita Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan * kyuki@gene.nagoya-u.ac.jp, fon +8152 789 3080 Chloroplasts have their own transcriptional apparatus and most chloroplast genes are transcribed by a plastid-encoded plastid RNA polymerase (PEP), although some chloroplast genes are transcribed by a nucleus-encoded plastid RNA polymerase (NEP) in higher plants (Kobayashi et al. 2002). PEP is a prokaryotic multi-subunit RNA polymerase, the core subunits are encoded by rpoA, rpoB, rpoC1, and rpoC2 on the chloroplast genome. In contrast, NEP is a phage-type single-subunit RNA polymerase. We have recently determined the complete chloroplast DNA sequence (122, 890 bp) of Physcomitrella patens and found that rpoA gene encoding the α subunit of PEP is completely absent from the chloroplast genome (Sugiura et al. 2003). Interestingly, rpoA was identified as a nuclear gene. This suggests that the P. patens PEP is distinct from the PEP of higher plants. In this study, we generated and characterized the nuclear rpoA knockout moss to examine whether either PEP or NEP or both occur in transcription of chloroplast genes in P. patens. Chloroplast DNA macroarray analysis revealed that the transcripts of photosynthesis genes accumulated in rpoA knockout moss, at the same levels of wild-type moss. Run-on transcription assay using chloroplasts isolated from 3-day old protonemata showed that the transcription activity was totally accomplished by PEP in wild-type. For this assay we used both a PEP-specific inhibitor, tagetitoxin, and a global transcriptional inhibitor, actinomycin D. The rpoA-knockout moss chloroplasts retained 60% of transcription activity in the presence of tagetitoxin. These results indicate that PEP and NEP occur in transcription of chloroplast genes in P. patens. Both RNA polymerases are known to recognize specific promoters of chloroplast genes in higher plants. In contrast, the moss PEP and NEP may recognize the promoters, in a different manner from higher plant enzymes. We discuss on transcription machinery unique to the moss chloroplasts. Kobayashi, Y, Dokiya, Y, Kumazawa, Y, and Sugita, M (2002) Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris. Biochemical and Biophysical Research Communications 299:57-61 Sugiura, C, Kobayashi, Y, Aoki, S, Sugita, C, and Sugita, M. (2003) Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast the nucleus. Nucleic Acids Research 31:5324-5331

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RNA editing in the moss Physcomitrella patens chloroplasts Yuki Miyata* and Mamoru Sugita Center for Gene Research, Nagoya University, Nagoya, Japan *miyata@gene.nagoya-u.ac.jp, tel/fax: +81-52-789-3080 RNA editing is a posttranscriptional process changing individual nucleotides in transcripts and usually occurs in chloroplasts of land plants. Approximately 30 RNA editing sites are identified in chloroplast genomes of the major vascular plants. In contrast, the number of editing sites is significantly divergent among the bryophytes. For instance, RNA editing is absent in hloroplasts of the liverwort Marchantia polymorpha, whereas extensive RNA editing has been found in the hornwort Anthoceros formosae. We have previously identified a new RNA editing site, a cytidine (C)-to-uridine (U) conversion, in the rps14 transcripts of the moss Physcomitrella patens (Miyata et al. 2002). The editing occurs at an ACG codon to create an AUG translation initiation codon. This editing site is unique to the P. patens rps14 transcript and has not been found in chloroplasts of other plants. In addition, we have identified a novel C to U RNA editing site has been identified at the –1 position relative to the edited AUG translation initiation codon (Miyata and Sugita, 2004). We have recently determined the complete chloroplast DNA sequence of the moss P. patens (Sugiura et al. 2003). In this study, we have systematically investigated identification of RNA editing sites in P. patens chloroplasts. By comparison of the putative amino acid sequences of chloroplast-encoded proteins of P. patens to those of other plant species, we predicted possible 100 editing sites. To investigate the possibility of RNA editing for 49 sites out of 100 possible sites, we amplified and sequenced cDNAs using appropriate primers. No RNA editing was identified so far. This suggests that RNA editing rarely occurs in the chloroplasts of the moss P. patens. Miyata, Y, Sugiura, C, Kobayashi, Y, Hagiwara, M and Sugita, M (2002) Chloroplast ribosomal S14 protein transcript is edited to create a translation initiation codon in the moss Physcomitrella patens. Biochim. Biophys. Acta, 1576:346-349 Miyata, Y and Sugita, M (2004) Tissue- and stage-specific RNA editing of rps14 transcripts in moss (Physcomitrella patens) chloroplasts. J. Plant Physiol., 161:113-115 Sugiura, C, Kobayashi, Y, Aoki, S, Sugita, C and Sugita, M (2003) Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus. Nucleic Acids Res., 31:5324-5331

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Moss 2004 participant list, alphabetically sorted name e-mail affiliation, country

Abel, Wolfgang - Hamburg, Germany

Anterola, Aldwin anterola@mail.wsu.edu Washington State University, USA

Aoki, Setsuyuki aoki@is.nagoya-u.ac.jp Nagoya University, Japan

Arazi, Tzahi tarazi@volcani.agri.gov.il The Volcani Center, Israel

Baur, Armin abaur@greenovation.com greenovation Biotech GmbH, Germany

Bezanilla, Magdalena manena@biology.wustl.edu Washington University, USA

Bopp, Martin no email address University of Heidelberg, Germany

Brüggemann, Chantal chantal.brueggemann@gmx.de FU Berlin, Germany

Bushoven, John jbus7623@mail.uri.edu University of Rhode Island, USA

Cadoret, Jean-Paul jpcadore@ifremer.fr IFREMER, France

Carballo, Valentina vcarballo@fcien.edu.uy University of the Republic; Faculty of Sciences, Uruguay

Chassignet, Isabelle isabelle.chassignet@biologie.uni-freiburg.de University of Freiburg, Germany

Cove, David d.j.cove@leeds.ac.uk University of Leeds/Washington University, UK/USA

Cuming, Andy a.c.cuming@leeds.ac.uk Leeds University, UK

Decker, Eva eva.decker@biologie.uni-freiburg.de University of Freiburg, Germany

Dröge, Markus bberemski@mwgdna.com MWG Biotech AG, Germany

Enríquez Schäfer, Ramón

renriquez-schaefer@beckman.com Beckman Coulter, Germany

Erxleben, Anika anika.erxleben@biologie.uni-freiburg.de University of Freiburg, Germany

Fernández Núnez, Marta mfernandez@iangbot.uni-hamburg.de University of Hamburg, Germany

Finka, Andrija afinka@ie-pc.unil.ch University of Lausanne, Switzerland

Frank, Wolfgang wolfgang.frank@biologie.uni-freiburg.de University of Freiburg, Germany

Gorr, Gilbert ggorr@greenovation.com greenovation Biotech GmbH, Germany

Gremillon, Louis louis.gremillon@biologie.uni-freiburg.de University of Freiburg, Germany

Hartmann, Elmar hartmann@zedat.fu-berlin.de FU Berlin, Germany

Hasebe, Mitsuyasu mhasebe@nibb.ac.jp National Institute for Basic Biology, Japan

Heckmann, Johannes johannes.heckmann@biologie.uni-freiburg.de University of Freiburg, Germany

75

name e-mail affiliation, country

Hedman, Harald harald.hedman@ebc.uu.se Swedish University of Agricultural Sciences, Uppsala, Sweden

Herbst, Roswitha rherbst@beckman.com Beckman Coulter, Germany

Himmelbauer, Heinz himmelbauer@molgen.mpg.de Max-Planck-Institute of Molecular Genetics, Germany

Hoch, Birgit birgit.hoch@biologie.uni-freiburg.de University of Freiburg, Germany

Hofmann, Nancy nrhofmann@ucdavis.edu University of California at Davis, USA

Horlemann, Christoph Horlemann@PlantClimatics.de CLF Plant Climatics, Germany

Horstmann, Verena Verena.Horstmann@biologie.uni-freiburg.de University of Freiburg, Germany

Hughes, Jon jon.hughes@uni-giessen.de Justus Liebig University Giessen, Germany

Itoga, Misao itouga@psc.riken.go.jp RIKEN Plant Science Center, Japan

Jost, Wolfgang wjost@greenovation.com greenovation Biotech GmbH, Germany

Kaminski, Marc marc.kaminski@biologie.uni-freiburg.de University of Freiburg, Germany

Kamisugi, Yasuko bmbyk@leeds.ac.uk University of Leeds, UK

Kaufmann, Franz fkaufmann@greenovation.com greenovation Biotech GmbH, Germany

Kleitsch, Charlotte charlotte.kleitsch@biologie.uni-freiburg.de University of Freiburg, Germany

Knight, Celia c.d.knight@leeds.ac.uk University of Leeds, UK

Kobayashi, Yuki kyuki@gene.nagoya-u.ac.jp Nagoya University, Japan

Kumar, G.V. fivestar_23in@yahoo.com Bharathidasan University, India

Lang, Daniel daniel.lang@biologie.uni-freiburg.de University of Freiburg, Germany

Launhardt, Heike hlaunhardt@greenovation.com greenovation Biotech GmbH, Germany

Lawton, Michael lawton@aesop.rutgers.edu Rutgers University, USA

Lee, Kieran bgykjdl@leeds.ac.uk University of Leeds, UK

Lienard, David david.lienard@univ-rouen.fr University of Rouen, France

Lienhart, Otmar otmar.lienhart@biologie.uni-freiburg.de University of Freiburg, Germany

Lindsay, Scott bgysal@leeds.ac.uk University of Leeds, UK

Lucumi, Alexander Alexander.Lucumi@mvm.uka.de Universität Karlsruhe (TH), Germany

Lunde, Christina christina.lunde@adelaide.edu.au University of Adelaide, Australia

Martin, Anja anja.martin@biologie.uni-freiburg.de University of Freiburg, Germany

McDaniel, Stuart stumcd@duke.edu Duke University, USA

76

name e-mail affiliation, country

Menand, Benoit benoit.menand@bbsrc.ac.uk John Innes Centre, UK

Mildner, Manuel manuel.mildner@biologie.uni-freiburg.de University of Freiburg, Germany

Minami, Anzu anzu@pop.lowtem.hokudai.ac.jp Hokkaido University, Japan

Mishler, Brent bmishler@socrates.Berkeley.EDU University of California, USA

Miyata, Yuki miyata@gene.nagoya-u.ac.jp Nagoya University, Japan

Münster, Thomas muenster@mpiz-koeln.mpg.de Max-Planck Institute for plant breeding research, Germany

Nilsson, Anders anders.nilsson@imbim.uu.se Uppsala University, Sweden

Nishiyama, Tomoaki tomoaki@nibb.ac.jp National Institute for Basic Biology, Japan

Nogué, Fabien nogue@versailles.inra.fr INRA Versailles, France

Ohad, Nir NirO@tauex.tau.ac.il Tel-Aviv University, Israel

Oliver, Mel moliver@lbk.ars.usda.gov USDA-ARS, USA

Olsson, Tina Tina.Olsson@imbim.uu.se Uppsala University, Sweden

Özlem, Yayýntaþ yayintasozlem@hotmail.com University of Çanakkale Onsekiz Mart, Turkey

Pakrasi, Himadri Pakrasi@wustl.edu Washington University, USA

Parsons, Juliana Juliana.Parsons@biologie.uni-freiburg.de University of Freiburg, Germany

Potters, Geert geert.potters@ua.ac.be University of Antwerp, Belgium

Pulz, Otto pulz@igv-gmbh.de IGV Institut für Getreideverarbeitung GmbH, Germany

Quatrano, Ralph rsq@wustl.edu Washington University, USA

Quodt, Vanessa quodt@mpiz-koeln.mpg.de Max-Planck Institute for plant breeding research, Germany

Reinhard, Christina christina.reinhard@biologie.uni-freiburg.de University of Freiburg, Germany

Reiss, Bernd reiss@mpiz-koeln.mpg.de Max-Planck Institute for plant breeding research, Germany

Rensing, Stefan stefan.rensing@biologie.uni-freiburg.de University of Freiburg, Germany

Reski, Ralf ralf.reski@biologie.uni-freiburg.de University of Freiburg, Germany

Richardt, Sandra Sandra.Richardt@biologie.uni-freiburg.de University of Freiburg, Germany

Riese, Maike riese@mpiz-koeln.mpg.de Max-Planck Institute for plant breeding research, Germany

Roberts, Alison aroberts@uri.edu University of Rhode Island, USA

Rombauts, Stephane strom@gengenp.rug.ac.be University of Gent, Belgium

Ronne, Hans Hans.Ronne@imbim.uu.se Uppsala University and SLU, Sweden

77

name e-mail affiliation, country

Saavedra, Laura laura.saavedra@plantbio.lu.se Lund University, Sweden

Sarnighausen, Eric Eric.Sarnighausen@biologie.uni-freiburg.de University of Freiburg, Germany

Sato, Yoshikatsu yoshi@nibb.ac.jp National Institute for Basic Biology, Japan

Schaaf, Andreas andreas.schaaf@biologie.uni-freiburg.de University of Freiburg, Germany

Schumann, Eik Eik.Schumann@biologie.uni-freiburg.de University of Freiburg, Germany

Schween, Gabriele Gabriele.Schween@biologie.uni-freiburg.de University of Freiburg, Germany

Shaw, Blanka Blanka@duke.edu Duke University , USA

Shaw, Jon shaw@duke.edu Duke University, USA

Skotnicki, Mary skotnicki@actewagl.net.au Australian National University, Australia

Stenøien, Hans K. hans.stenoien@ebc.uu.se Uppsala University, Sweden

Strack, Volker bberemski@mwgdna.com MWG Biotech AG, Germany

Sugita, Mamoru sugita@gene.nagoya-u.ac.jp Nagoya University, Japan

Takano, Hiroyoshi takano@kumamoto-u.ac.jp Kumamoto Univ., Japan

Tanahashi, Takako takakot@biol.s.u-tokyo.ac.jp University of Tokyo, Japan

Tashpulatov, Alisher alisher.tashpulatov@univie.ac.at University of Vienna, Austria

Thelander, Mattias mattias.thelander@imbim.uu.se Uppsala University / Swedish University of Agricultural Sciences, Sweden

Tintelnot, Stefanie stefanie.tintelnot@biologie.uni-freiburg.de University of Freiburg, Germany

Trouiller, Benedicte trouille@versailles.inra.fr INRA Versailles, France

Van de Wiel, Annick geert.potters@ua.ac.be University of Antwerp, Belgium

Verelst, Wim verelst@mpiz-koeln.mpg.de Max-Planck Institute for plant breeding research, Germany

von Stackelberg, Mark mark.stackelberg@biologie.uni-freiburg.de University of Freiburg, Germany

Wambutt, Rolf wambutt@agowa.de AGOWA GmbH, Germany

Weise, Andreas aweise@greenovation.com greenovation Biotech GmbH, Germany

Wickett, Norm norman.wickett@uconn.edu University of Connecticut, USA

Wiedemann, Gertrud Gertrud.Wiedemann@ctp.uni-freiburg.de University of Freiburg, Germany

Winterhalter, Franz Franz.Winterhalter@binder-world.com Binder GmbH, Germany

Wood, Andrew wood@plant.siu.edu Southern Illinois University, USA

Wood, Jeanne wood@plant.siu.edu Southern Illinois University, USA

78

name e-mail affiliation, country

Yayintas, Özlem yayintasozlem@hotmail.com University of Çanakkale Onsekiz Mart, Turkey

Yevdakova, Natalya NYevdakova@iangbot.uni-hamburg.de University of Hamburg, Germany

Younousse, Saidi younousse.saidi@ie-pc.unil.ch Lausanne University, Switzerland

Zobell, Oliver zobell@mpiz-koeln.mpg.de Max Planck Institute for Plant Breeding Research, Germany

Zoriniants, Svetlana svetlana.zoriniants@chello.at University of Vienna, Austria

Zrÿd, Jean-Pierre jzryd@ie-pc.unil.ch University of Lausanne, Switzerland