Plant Biotech Denmark Annual meeting 2015 28 - 29 January

Faculty of Science University of Copenhagen

Plant Biotech Denmark Annual meeting 2015 28 - 29 January

Faculty of Science University of Copenhagen

Cover photo: Arabidopsis thaliana mutants at flowering time at DNRF Center DynaMo, Department of Plant and Environmental Sciences, University of Copenhagen. DynaMo uses the glucosinolate defense compounds within Arabidopsis thaliana as a unique model system to uncover molecular interactions that coordinate and facilitate dynamic biological processes in a multicellular organism. Head of Center: Professor Barbara Ann Halkier. Photo: Postdoc Lea Møller Jensen.

Plant Biotech Denmark Annual meeting 2015 28 - 29 January

Programme

WEDNESDAY - 28 January 2015

09.00 - 09.30 Registration, coffee/tea and croissant

09.30 - 09.35 Welcome by Henrik Brinch-Pedersen, Head of the Steering Committee, Plant Biotech Denmark

Session 1: Plant Products/Plants for Food and Feed Chair: Andreas Blennow

09.35 - 10.20 Keynote talk within the research area 'Plant Products' Advancing our understanding of starch biosynthesis in plants through work in model species and through pathway reconstruction in heterologous systems by Professor Samuel C Zeeman, ETH Zürich, Switzerland

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10.20 - 10.40 The elucidation of vanillin biosynthetic pathway in the vanilla orchid, Vanilla planifolia, by Postdoc Nethaji Gallage, University of Copenhagen, Dept. of Plant and Environmental Sciences

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10.40 - 11.00 Enzymatic degradation of soybean fiber components demonstrated by viscosity and microscopy techniques by Associate Professor Helle Juel Martens, University of Copenhagen, Dept. of Plant and Environmental Sciences

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11.00 - 11.30 Break, Coffee/tea and fruit

Session 2: Synthetic and Systems Biology/Plant Signalling and Cellular Trafficking Chair: Meike Burow

11.30 - 12.15 Keynote talk within the research area 'Synthetic and Systems Biology' The ultimate way to 'go green' by Professor Birger Lindberg Møller, University of Copenhagen, Dept. of Plant and Environmental Sciences

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12.15 - 12.35 Exploiting the Diterpenoid toolbox for the biosynthesis of high value diterpenoid compounds, by Postdoc Johan Andersen-Ranberg, University of Copenhagen, Dept. of Plant and Environmental Sciences

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12.35 - 12.55 Identification of a novel fix- mutant involved in deubiquitination pathway in Lotus japonicus using LORE1 resource by Postdoc Anna Malolepszy, Aarhus University, Dept. of Molecular Biology and Genetics, Centre for Carbohydrate Recognition and Signalling

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12.55 - 13.45 Lunch

13.45 - 13.55 Announcements

Session 3: Biomass Production and Processing/Plant Breeding - Quality, Productivity and Diseases Chair: Claus Felby

13.55 - 14.40 Keynote talk within the research area 'Biomass Production and Processing' Phenylpropanoid plasticity – opportunities and challenges for engineering for improved cell wall processing by Professor Shawn D Mansfield, University of British Columbia, Canada

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Programme

14.40 - 15.00 Comparative genomics and phylogenetical analysis in the Lolium-Festuca complex of species, by PhD student Adrian Czaban, Aarhus University, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg

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15.00 - 15.30 Coffee/tea, cake and fruit

Session 4: Elevator talk and Poster session Chair: Kåre Lehmann Nielsen

15.30 - 16.15 Elevator talks (3 minutes talks based on selected abstracts) 14

16.15 - 17.45 Poster session in the Marble Hall – Wine/beer and snacks are served

17.45 - 18.00 Walking to Gimle canteen, Dyrlægevej 9

18.00 Dinner at Gimle canteen, Dyrlægevej 9

THURSDAY - 29 January 2015

Session 5: New Perspectives on Orphan Crops Chair: Jens Stougaard

09.00 - 09.45 Development of lentil and other pulse crops in Canada by Professor Albert Vandenberg, University of Saskatchewan, Canada

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09.45 - 10.30 Is faba bean on the threshold of a golden era of gene identification? by Professor Donal O'Sullivan, University of Reading, UK

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10.30 - 11.00 Coffee/tea 11.00 - 11.45 Genomics assisted adaptation of sorghum to central European conditions

by Dr. Wubishet Abebe Bekele, University of Giessen, Germany 17

Session 6: Technologies - Genome Editing Chair: Henrik Brinch-Pedersen

11.45 - 12.25 Understanding, generating and redesigning hybrids using reverse breeding by Dr. Erik Wijnker, University of Hamburg, Germany

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12.25 - 13.10 Lunch

13.10 - 13.50 Engineering plant genomes with sequence-specific nucleases by Professor Dan Voytas, University of Minnesota, USA

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13.50 - 14.25 Developing genome editing technologies for crop improvement by Professor Caixia Gao, Chinese Academy of Sciences, Beijing, China

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14.25 - 14.40 Towards precisely glyco engineered plants, by Associated Professor Bent L. Petersen, University of Copenhagen, Dept. of Plant and Environmental Sciences

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14.40 - 14.55 Targeted mutagenesis in barley, by Senior Researcher Inger B. Holme, Aarhus University, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg

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14.55 - 15.00 Thank you for this year

15.00 Coffee and cookies

15.00 - 18.00 Master class for registered students

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Session 1: Plant Products/Plants for Food and Feed

Keynote talk within the research area 'Plant Products' Advancing our understanding of starch biosynthesis in plants through work in model species and through pathway reconstruction in heterologous systems Samuel C Zeeman

Department of Biology, ETH Zurich, Switzerland Starch is a vital plant product for society. Learning more about its metabolism gives us options for crop improvement by altering starch structure, properties and yields. Starch is primarily composed of the branched glucan, amylopectin, which has an architecture that allows the formation of insoluble, semi-crystalline granules. To achieve this architecture requires multiple isoforms of the biosynthetic enzymes (starch synthases and starch branching enzymes) that initiate granules and elaborate amylopectin. The crystallisation process is also facilitated by a specialised sub-class of the debranching enzymes, which are thought to selectively remove misplaced branch points. Despite this knowledge, starch biosynthesis has not yet been recreated in-vitro or in a heterologous system, suggesting that other, as-yet undiscovered protein factors may also be involved. Much progress has been made by studying starch metabolism in the model plant Arabidopsis thaliana, where it is a primary product of photosynthesis, stored temporarily in chloroplasts during the day. Functional genomic and biochemical studies have advanced our understanding of the roles of known starch-metabolising enzymes and facilitated the discovery of new ones. However, both starch biosynthetic and degradative enzymes in the same compartment and that there is good evidence that the two processes are somehow regulated. We have observed that when using genetics to remove starch biosynthetic enzymes to examine the effect on starch structure, the results can often be complicated by the unanticipated involvement of degradative enzymes. Therefore, to complement our genetic studies in Arabidopsis, we have undertaken a programme of expressing of combinations of Arabidopsis enzymes in the heterologous system Saccharomyces cerevisiae, with the aim of defining a minimal set of enzymes/proteins required for starch production. This presentation will provide an overview of these research results – charting both the successes and the obstacles we have faced.

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Session 1: Plant Products/Plants for Food and Feed

Selected talk within the research area 'Plant Products' The elucidation of vanillin biosynthetic pathway in the vanilla orchid, Vanilla planifolia Nethaji J Gallage1,2,3 and Birger Lindberg Moller1,2,3

1 VILLUM Research Center for Plant Plasticity, 2 Center for Synthetic Biology “bioSYNergy” 3 Plant Biochemistry Laboratory, Dept. of Plant and Environmental Sciences, UCPH, DK Vanillin is the world’s most popular flavor compound. It is the key constituent of the natural vanilla flavor obtained from cured vanilla pods. A single hydratase/lyase type enzyme, vanillin synthase (VpVAN) - catalyzes direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is strictly specific to the substitution pattern at the aromatic ring. Transient expression of VpVAN in tobacco and stable expression in barley, in combination with the action of endogenous alcohol dehydrogenases and UGTs, result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco (Gallage et al., 2014). The isolation of vanillin from vanilla pods is a laborious and costly process. Currently, less than 1% of the global production of vanillin is derived from vanilla pods, while the majority is produced synthetically using e.g. lignin and eugenol as starting materials. Industrial application of bioengineered microorganisms for vanillin production has gained quite a lot of attention not only from the flavor and fragrance industries, but also from environmental groups, the general public and politicians. The recent identification of VpVAN from the vanilla orchid can contribute to an entirely new opportunity for biotechnology based production of natural vanillin. If high expression levels can be obtained in yeast production strains and the enzyme is stable and has proper kinetic characteristics, this may constitute an alternative to the current yeast production systems. The identification of vanillin synthase also offers important applications in Vanilla pod-based industries. Many orchids closely related to the vanilla orchid are present in nature but appear not to be able to synthesize vanillin. Classical mutation breeding to obtain vanillin production in such species may now be initiated. This would offer a more diverse production system less prone to disease and maybe the introduction of vanillin synthesizing orchids that would have natural pollinators in their growth habitats and thus not require hand-pollination by humans (Gallage et al., 2014, Gallage and Moller 2014). Gallage, N.J., Hansen, E.H., Kannangara, R., Olsen, C. E., Motawia, M. S., Jørgensen, K., Holme, I., Hebelstrup, K., Grisoni, M., Møller, B.L. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme. Nature Communications 5, doi:10.1038/ncomms5037 (2014). I.F. 10,742. Gallage, N.J. and Møller, B.L. Vanillin – Bioconversion and Bioengineering of the most popular plant flavour and its de novo biosynthesis in the vanilla orchid. Molecular Plant, special edition Synthetic Biology doi: 10.1093/mp/ssu105 (2014). Nethaji J. Gallage, Esben Halkjaer Hansen, Birger Lindberg Møller and Jørgen Hansen. Microbial organism and methods for producing vanillin, vanillyl alcohol, or vanillin glucoside, by vanillin synthase action on ferulic acid. International Patent Application PCT/DK2013/050357.

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Session 1: Plant Products/Plants for Food and Feed

Selected talk within the research area 'Plants for Food and Feed' Enzymatic degradation of soybean fiber components demonstrated by viscosity and microscopy techniques Jonas L. Ravn1, Helle J. Martens1, D. Pettersson2, N.R. Pedersen2

1 Department of Plant and Environmental Sciences, University of Copenhagen, Denmark 2 Novozymes A/S, Krogshoejsvej 36, 2880 Bagsværd, Denmark The content of high quality protein in soybean meal (SBM) makes it the most widely used vegetable protein source in monogastric diets. However, SBM also has a high concentration of indigestible non-starch polysaccharides (NSPs). These consist mainly of xyloglucans and rhamnogalacturonans (pectin) and may cause deleterious effects when fed to monogastrics which lack NSP degrading enzymes. Soluble NSPs may impede animal growth performance by dissolving in the gut and hold water, increase digesta viscosity and slow down digestion rates in the animal. The degradation of soybean and SBM non-starch polysaccharides by a multicomponent carbohydrase enzyme product, RONOZYME® VP, was studied in vitro using viscosity and microscopy techniques. Pectin degradation after incubation with the enzyme product was monitored as a decrease in color intensity of the fluorescent dye coriphosphine O. The degradation of specific cell wall components was observed using monoclonal antibodies targeting specific epitopes in the cell walls.

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Session 2: Synthetic and Systems Biology/Plant Signalling and Cellular Trafficking

Keynote talk within the research area 'Synthetic and Systems Biology' The ultimate way to ‘go green’ Birger Lindberg Møller1,2 1 Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark 2 Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark Photosynthetic organisms are able to use solar energy and carbon dioxide for the production of organic compounds. Based on initial formation and subsequent turn-over of carbohydrates, plants channel energy flux and carbon into specific biosynthetic pathways to optimize growth and development and adapt to environmental challenges by producing bioactive defense compounds when attacked by insects and microbes. Several of these bioactive defense compounds are structurally complex and highly valuable flavor ingredients or pharmaceuticals used in the treatment of serious human diseases like cancer. Unfortunately such bioactive natural products are typically produced in small amounts by plants. Most of the pathways responsible for the formation of the compounds involve cytochrome P450 catalyzed key steps difficult to copy using organic chemical synthesis. Using the “share-your-parts” principle of synthetic biology, we have now succeeded in breaking the evolutionary compartmentalization of energy generation and production of bioactive natural products by relocating an entire P450-dependent pathway for a bioactive natural product into to the chloroplast and driving the pathway by direct use of the reducing power generated by photosystem I in a light-dependent manner. The enzymes catalysing the pathway were targeted to the chloroplast by being expressed as fusion proteins with the transit peptide sequence from ferredoxin. The long term goal is to build a supra-molecular enzyme complex catalysing light driven synthesis of food ingredients, pharmaceuticals and other interesting bioactive molecules. The production systems are being developed and optimised using transient expression in tobacco as the experimental system followed by stable transformation of cyanobacteria and moss strains grown in transparent plastic bags mounted in green houses enabling solar energy driven synthesis and environmental containment. Key target compounds are structurally complex diterpenoids of which the entire biosynthetic pathway for forskolin has now been elucidated. 1: L.M. Lassen, A.Z. Nielsen, B. Ziersen, T. Gnanasekaran, B.L. Møller, P.E. Jensen: Redirecting Photosynthetic Electron Flow into Light-Driven Synthesis of Alternative Products Including High-Value Bioactive Natural Compounds. ACS Synthetic Biol. (2014) 3:1–12. 2: I.Pateraki, J. Andersen-Ranberg, B. Hamberger, A.M. Heskes, H.J. Martens, P. Zerbe, S.S. Bach, B.L. Møller, J. Bohlmann and B. Hamberger: Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, is Synthesized in Specialized Root Cork Cells in Coleus forskohlii. Plant Physiol. (2014) 164: 1222-1236. 3: A.Z. Nielsen, B. Ziersen, K. Jensen, L. M. Lassen, C.E. Olsen, B.L. Møller, and P.E. Jensen: Redirecting Photosynthetic Reducing Power towards Bioactive Natural Product Synthesis. ACS Synthetic Biology (2013) 2:308–315. 4: B.L. Møller, R.G. Ratcliffe: Editorial overview: Synthetic plant biology: the roots of a bio-based society. Current Opinion in Biotechnology 26: ix-xvi (2014). 5: B.L. Møller: Disruptive innovation: channeling photosynthetic electron flow into light-driven synthesis of high-value products. Specialist Periodical Reports. Synthetic Biology 1: 330-359 (2014) (Eds: M. Ryadnov, L. Brunsveld, H. Suga) The Royal Society of Chemistry.

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Session 2: Synthetic and Systems Biology/Plant Signalling and Cellular Trafficking

Selected talk within the research area 'Synthetic and Systems Biology' Exploiting the Diterpenoid toolbox for the biosynthesis of high value diterpenoid compounds Johan Andersen-Ranberg1,2, Irini Pateraki1, Morten Thrane Nielsen1, Niels Bjerg Jensen3, Björn Hallström4, Sileshi Wubset5, Dan Stærk5, Carl Erik Olsen1, Birger Lindberg Møller1,2 and Björn Hamberger1,2

1 Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Denmark; 2 Center for Synthetic Biology “bioSYNergy”, Copenhagen, Denmark; 3 Evolva A/S, Copenhagen, Denmark; 4 Science for Life Laboratory, KTH - Royal Institute of Technology , Stockholm, Sweden; 5 Department of Drug Design and Pharmacology, Faculty of Health and medical Sciences, University of Copenhagen, Denmark With 12,000+ known structures, diterpenoids are a prime example of a vast repository of plant natural products. The key to this diversification may lie in the highly modular nature of the diterpenoid biosynthetic routes, where initially coupled pairs of diterpene synthases (diTPS) catalyze the formation of the diterpene core structure, which subsequently are functionalized by cytochrome P450 enzymes (CYPs). By mining transcriptome data from medicinal plant producing high value diterpenoid compounds, our group has generated the “Diterpenoid tool box” consisting of biosynthetic parts which can be used for the biosynthesis of both known and new-to-nature diterpenoid compounds. For the identification and functional characterization of diterpenoid specific biosynthetic parts, our laboratory has developed an optimized expression platform in Nicotiana Benthamiana. This platform not only enabled the high-through put screening of candidate biosynthetic parts for diterpenoid biosynthesis, it also facilitated exploitation of the Diterpenoid toolbox for the construction of new-to-nature modules of biosynthetic parts for the biosynthesis of complex diterpenoids. One of our target compounds is the high value diterpenoid forskolin, which is produced the in root cork cells of Coleus forskolhii and recently we have described two diTPS involved in initial steps of forskolin biosynthesis*. In addition we have now identified CYP76AH8 and CYP76AH11 which catalyze three out four oxidation steps in the forskolin biosynthesis, and CYP76D381 catalysing regio- and stereo- specific oxidations of diterpene core structures. All three CYPs have been added to Diterpenoid toolbox and through academic (PlantPower) and commercial (Evolva) collaborations the biosynthetic parts for forskolin biosynthesis have now been implemented in Synechocystis PCC 6803 and Saccharomyces cerevisiae for the development of biosustainable production platforms of forskolin. * Pateraki I, Andersen-Ranberg J, Hamberger B, Heskes AM, Martens HJ, Zerbe P, Bach SS, Møller BL, Bohlmann J, Hamberger B. (2014). Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii. Plant Physiology, 164, 1222-1236.

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Session 2: Synthetic and Systems Biology/Plant Signalling and Cellular Trafficking

Selected talk within the research area 'Plant Signalling and Cellular Trafficking' Identification of a novel fix- mutant involved in deubiquitination pathway in Lotus japonicus using LORE1 resource Anna Malolepszy1, Niels Sandal1, Dorian Urbanski1, Euan James2, Erika Isono3, Jens Stougaard1, Stig Uggerhøj Andersen1

1 Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark 2 The James Hutton Institute, Invergowrie, Dundee DD2 5DA, Scotland UK 3 Department of Plant Systems Biology, Technische Universität München, 85354 Freising, Germany E-mail: ann@mbg.au.dk

In the last 4 years we developed a new reverse and forward genetics tool based on insertional mutagenesis – the Lotus retrotransposon 1 (LORE1) collection – to facilitate investigation of gene function in the model legume Lotus japonicus. LORE1 is a 5kb long Gypsy-type retrotransposon that can be derepressed during tissue culture (Fukai et al., 2010) and has an exonic insertion preference (Urbanski et al., 2012). At present, the LORE1 resource consists of ~85.000 individual lines where 65% of Lotus genes have exonic LORE1 insertions. Importantly, 81% of genes with exonic length longer that 1kb, were tagged by LORE1. The insertion information, software-designed genotyping primer sequences and seeds are freely available to the community on the website: http://carb.au.dk/lore1.

To take full advantage of the LORE1 population we initiated forward screening. Initially we identified two alleles of a novel fix- mutant. Thanks to the LORE1 collection we obtained six additional independent alleles showing similar phenotype. The mutant plants are smaller, and are able to form only nodule primordia. Microscopic characterization revealed that the mutant forms significantly lower numbers of infection threads. The mutated gene is involved in deubiquitination and the mutant accumulates ubiquitin conjugates. Detailed microscopic and biochemical analysis will be presented. These results highlight the importance of deubiquitination in legume-rhizobia symbiosis establishment in Lotus japonicus.

Fukai et al., 2010, PLOS Genetics 6, e1000868. Urbanski et al., 2012, Plant J. 69, 731-741.

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Session 3: Biomass Production and Processing/Plant Breeding - Quality, Productivity and Diseases

Keynote talk within the research area 'Biomass Production and Processing' Phenylpropanoid plasticity – opportunities and challenges for engineering for improved cell wall processing Shawn D. Mansfield Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver BC, Canada, V6T 1Z4 Plants possess the innate ability to adapt their resource allocation to match resource acquisition, acquire resources more effectively, or avoid deleterious conditions. This plasticity can have a profound effect not only on the development and physiology of plants, but on the industrial utility of the plant matter, which is largely dependent on the ultrastructure and chemistry of the secondary cell wall. This is particularly true when considering lignocellulosics bioenergy crops, crops for ruminant digestibility, or woody material as a feedstock for processes such as pulping and papermaking. For example, the effective removal of lignin is expensive and often the limiting factor to many applications, and is therefore a key target in plant breeding or genetic engineering efforts. Perturbing plants by mis-regulating genes integral to major cell wall pathways, such as the monolignol biosynthetic pathway, can produce at times striking alterations in lignin composition and structure, and is well authenticated in various plants. Moreover, these efforts have clearly shown that previously “non-traditional monolignols” can be incorporated into, and accumulate to significant levels, in lignins. Given the inherent plastic nature of monomer substitution, we now can actually design lignins to improve the ease with which they can be removed from the cell wall. Here we highlight recent progress in the use of promising monolignol conjugates for in planta lignification to produce plant materials that are better optimized for processing.

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Session 3: Biomass Production and Processing/Plant Breeding - Quality, Productivity and Diseases

Selected talk within the research area 'Plant Breeding – Quality, Productivity and Diseases' Comparative genomics and phylogenetical analysis in the Lolium-Festuca complex of species Adrian Czaban, Stephen Byrne, Sapna Sharma, Manuel Spannagl, Istvan Nagy, Matthias Pfeifer, Heidrun Gundlach, Klaus Mayer, Torben Asp Department of Molecular Biology and Genetics, Aarhus University, Research Centre Flakkebjerg, Forsøgsvej 1, DK-4200 Slagelse The Lolium-Festuca complex incorporates species from the Lolium genera and the broad leaf Fescues. Plants belonging to this complex exhibit significant phenotypic plasticity for agriculturally important traits, such as annuality/perenniality, establishment potential, growth speed, nutritional value, winter hardiness, drought tolerance and resistance to grazing. In this study we have sequenced and assembled the low copy fraction of the genomes of Lolium westerwoldicum, Lolium multiflorum, Festuca pratensis and Lolium temulentum. We have also generated de-novo transcriptome assemblies for each species, and these have aided in the annotation of the genomic sequence. Using this data we were able to generate annotated assemblies of the gene rich regions of the four species to complement the already sequenced Lolium perenne genome. Using these gene models we have identified orthologous genes between the species, and analyzed their phylogentic relationship using protein families. Our dataset enabled us to perform comparative gene family analysis for LEA (Late Embryogensis Abundant) proteins, which are responsible for dehydration stress response in plants.

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Session 4: Elevator talks

As appetizer for the poster session short talks have been selected based on submitted abstracts to the respective research areas. Research Area: Plant Products Adam Takos Repeated evolution of cyanogenic glucoside biosynthetic gene clusters

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Giuseppe Dionisio Barley (Hordeum vulgare L.) endoprotease B2 controlled by the D-Hordein promoter is expressed and active in developing barley grains. Differential proteomics and lipidomics analysis

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Research Area: Plants for Food and Feed Fariha Tanwir Biosynthesis of benzoxazinoids during germination in wheat and rye as well as the functional characterization of a BX6-like ortholog in rye

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Research Area: Synthetic and Systems Biology Alice Jara De Porcellinis Bifunctional sedoheptulose/fructose bisphospahtase activity acts as a metabolic switch enhancing the calvin cycle and photosynthesis in cyanobacteria

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Claus Krogh Madsen Secreted expression libraries of plant proteomes

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Research Area: Signalling and Cellular Trafficking Marie Pireyre Specific activation of MYB transcription factors is regulated by subcellular protein-protein interactions in Arabidopsis

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Research Area: Biomass production and processing Daniele Silvestro Modified brassinosteroids accumulation affects saccharification in Brachypodium distachyon

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Eric van der Graaff A systems biology analysis of early-stage sugar beet storage root development

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Lorenzo Fimognari Cell wall acetylation and plant fitness: suppressor screening of the REDUCED WALL ACETYLATION 2 reveals mutants with wild type surface permeability

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Research Area: Plant Breeding - Quality, Productivity and Diseases Agnieszka Siwoszek Peptide aptamer-mediated resistance to barley powdery mildew

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Session 5: New Perspectives on Orphan Crops

Development of lentil and other pulse crops in Canada Albert Vandenberg and Kirstin Bett Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK Canada S7K 2S9 www.knowpulse.usask.ca In 1970 there was no grain legume industry in western Canada. Today we produce more than three million ha of grain legumes in a rotational system that includes common bean, chickpea, faba bean, soybean, field pea and lentil. A completely new sector of the agricultural economy now exists as result of the replacement of the practice of fallowing land. The lentil crop is an example of development of an unknown crop in an agricultural region which now profitably produces almost 50% of the world supply and the majority of world exports. This was accomplished primarily by small but steady investment in agronomy and plant genetics initially, with contributive partnerships from farmer organizations, a university, and a provincial government. As the lentil industry grew, so did multi-faceted research needs at the applied and basic level. The short to medium applied research spectrum targets the needs of consumers, producers and processors. The medium to long term research spectrum includes application of genomics to lentil genetic improvement, and the development of genetic resources for the future of the lentil crop. Research targeted at consumer needs deals with how to elevate the global desire to eat more lentils through knowledge of the nutritional and culinary profile. For the production and processing sectors, research is aimed at ways of increasing productivity, profitability, and value of pulse crops like lentil through improvements in yield to minimize economic stress, and resistance to ever-changing biotic and abiotic stresses. Investments in the development of genetic, and more recently, genomic resources for lentil, are now leading to development of useful knowledge and strategies for leveraging the potential of downstream and upstream research. This will increase our ability to respond quickly to environmental and economic changes.

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Session 5: New Perspectives on Orphan Crops

Is faba bean on the threshold of a golden era of gene identification? Donal O’Sullivan School of Agriculture, Policy and Development, University of Reading d.m.osullivan@reading.ac.uk Vicia faba L. (faba bean) is a globally significant grain legume, with 4-5 million tonnes annual production worldwide in recent years (FAO, 2014), providing the predominant affordable dietary source of protein to subsistence farmers and urban populations across North Africa, Horn of Africa (including Ethiopia) and the Middle and Near East as well as parts of China.

Faba bean varieties that are nutritionally optimised, that resist fungal pathogens, viral vectors and parasitic weed attack and that retain high yields under temperature and drought stress are required to meet future demand. The identification of DNA polymorphisms that are either very tightly linked to or cause variation in such traits opens up a new pallet of tools for marker-assisted breeding, targeted mutagenesis and GM approaches that have yet to be seriously deployed in faba bean.

In my talk, I will describe progress on two fronts. First, in the creation of an entirely SNP-based medium density consensus map for faba bean which provides a straightforward means to predict gene order and content across most of the faba bean genome. The benefits of this new-found navigational ability are illustrated by two examples – 1. the cloning of a putative causative deletion in the WD40 transcription factor underlying the ZeroTannin1 (ZT1) gene, seed coat tannins being an important anti-nutritional factor in feed uses of faba bean, and 2. the mapping of the VC locus controlling the levels of another important anti-nutritional factor, and subsequent isolation of a suite of marker polymorphisms for ongoing fine-mapping of the interval. Second, in the development of a community mutant population resource, which will facilitate reverse genetics approaches to gene function assignment as well as providing the raw materials for isolation of novel mutations by ‘forward’ phenotypic screens e.g. herbicide resistance.

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Session 5: New Perspectives on Orphan Crops

Genomics assisted adaptation of sorghum to central European conditions Wubishet A. Bekele1,2, and Rod Snowdon1 1 Department of Plant Breeding, Justus Liebig University, Giessen, Germany 2 Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-food Canada, Ottawa, Canada The hardy C4 plant sorghum has considerable potential as an alternative bioenergy feedstock to maize, particularly for production under reduced-input or stress conditions where maize yields are suboptimal. The small, sequenced genome of Sorghum bicolor and the vast phenotypic diversity for energy-related and adaptation traits make sorghum an excellent model for application of genomics assisted breeding strategies that can increase selection gains and accelerate breeding progress.

The first part of the talk will focus on the development of genomics tools in sorghum. Whole genome resequencing and bulk segregant analysis methods used during the array development will be discussed. The second part will focus on the genetic analysis of sorghum adaptation and yield. This will be demonstrated by showing the phenotypic dissection strategies followed during the initial parts of the project. Examples from the genetic analysis that combine the genomic tools and comprehensive phenotypes will be presented.

The final part will deal with the potential of genomic selection in sorghum adaptation breeding. Genomic selection using genome-wide single-nucleotide polymorphism (SNP) markers has been highly successful in livestock breeding and is becoming increasingly applicable to crops as genome resources become available. We applied statistical models for genomic prediction of important adaptation (temperature-dependent emergence) and energy-related traits (stem sugar content, biomass yield), based on genome-wide SNP profiles, in energy sorghum breeding populations. Especially for traits that are difficult to phenotype and select, this opens the possibility to pre-select breeding lines which are more likely to be the best performers, thus improving the selection intensity and selective gains. Our results suggest that breeding with the help of genomic prediction can lead to rapid adaptation of sorghum as an alternative bioenergy crop for methane production under central European conditions. The presenting author will also show additional examples from his current work on oat (Avena sativa).

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Session 6: Technologies - Genome Editing

Understanding, generating and redesigning hybrids using reverse breeding Erik Wijnker

University of Hamburg, Biozentrum Klein Flottbek, Department of Developmental Biology, Ohnhorststr. 18 - 22609 Hamburg, Germany The highest producing crop varieties are F1 hybrids: the offspring of a cross between two parental lines. The observation that heterozygotes grow more vigorously than homozygous lines (an effect known as heterosis) lies at the base of modern day plant breeding. It is commonly understood that the genome of a heterozygous plant cannot be maintained, as its traits segregate in offspring. Hybrid seeds are therefore produced anew each year by crossing parental lines. To speed up the development of new hybrid crops, it was hypothesized that new hybrid varieties might best be chosen from segregating populations (because in segregating populations all plants have are unique genotypes). But therefore one needs a method that allows the propagation of that single hybrid. Reverse breeding allows one to do just this: to generate parental lines from a heterozygous plant. It will be shown that by suppressing crossover recombination during meiosis, it is indeed possible to produce parental lines for any heterozygote of choice.

Apart from this, reverse breeding allows one to systematically generate chromosome substitution lines: plants in which one or more chromosomes of one accession have been replaced by chromosomes of another accession. It will be explained how such lines can be used for the systematic dissection of complex traits, and perhaps equally important: on how these lines, that are a by-product in reverse breeding, can quickly provide a lot of information in the hybrid that was subjected to reverse breeding. Reverse breeding is not only a tool to generate parental lines for an uncharacterized heterozygote, but generates lines that may well hold the key to dissecting heterosis.

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Session 6: Technologies - Genome Editing

Engineering plant genomes with sequence-specific nucleases Dan Voytas

Dept. of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455 USA The ability to precisely alter DNA sequences in living cells makes possible detailed functional analysis of genes and genetic pathways. In plants, targeted genome modification has applications ranging from understanding plant gene function to developing crops with new traits of value.

We have enabled efficient methods for targeted modification of plant genomes using sequence-specific nucleases. With zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR/Cas9 reagents, we have achieved targeted gene knockouts, replacements and insertions in a variety of plant species. Current work is focused on optimizing delivery of nucleases and donor DNA molecules to plant cells to more efficiently achieve targeted genetic alterations.

19

Session 6: Technologies - Genome Editing

Developing genome editing technologies for crop improvement Caixia Gao State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Genome editing using sequence-specific nucleases (SSNs) provides good opportunities for crop improvement. The precise genome modifications involved are generated by SSNs, namely zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas). SSNs enable precise genome engineering by introducing DNA double-strand breaks (DSBs) that subsequently trigger DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR). By exploiting NHEJ and HR, SSNs can be used to generate targeted genome modifications including mutations, insertions, replacements and chromosome rearrangements.

To date, SSNs have been used to create gene knock-out plants in a variety of important crops, such as rice, maize, wheat, barley and soybean. However examples of real improvement of agronomic traits or the creation of excellent novel genotypes using SSNs are very limited. A successful example occurred in bread wheat. By knocking out all six alleles encoding the MILDEW-RESISTANCE LOCUS (MLO) protein with one pair of TALENs, we generated a mutant line with broad-spectrum resistance to powdery mildew, a devastating fungal disease.

I give another example of using genome editing methods to develop new traits with value. Fragrance is an important quality trait of rice, which is mainly controlled by loss-function of the betaine aldehyde dehydrogenase gene (badh2) on rice chromosome 8. The presence of a defective badh2 allele results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. We used TALEN technology to knockout OsBADH2 and obtain mutant plants carrying only the desired DNA sequence change but not the TALEN transgene by segregation in the T1 and T2 generations. Homozygous mutants with significantly increased content of the fragrant chemical 2AP; the level produced is similar to or even higher than in the fragrant rice cultivar Daohuaxiang. Our work provides plant breeders good example of the use of SSNs for molecular breeding of crop plants.

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Session 6: Technologies - Genome Editing

Towards precisely glyco engineered plants Bent Larsen Petersen

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Plants hold a huge potential as green biofactories for sustainable, affordable and safe production of therapeutic proteins and natural products. The precise genetic editing tools, the Zinc Finger- & TALE Nucleases (ZFNs, TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, allow for full freedom of precise genome editing in in principle any organism without adding additional changes than those intended. Although these technologies are now operational in e.g. mammalian cell lines and rodents, implementation in plants has been slow due to inefficient delivery of nucleases and inefficient hereditary transmission of the generated genome changes. Our first attempt to glyco engineer Arabidopsis by means of TALENs appeared to be futile. The CRISPR/Cas9 system, however, was shown to functionally restore a reporter gene disrupted with sgRNA glycogene targets by HR when transiently expressed in N. benthamiana thus providing a means of testing CRISPR sgRNA targets and ultimately generating precisely glyco engineered plants.

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Session 6: Technologies - Genome Editing

Targeted mutagenesis in barley Inger B Holme1, Toni Wendt1, Javier Gil2, Colby C Starker2, Daniel F Voytas2, Henrik Brinch-Pedersen1 1 Aarhus University, Department of Molecular Biology and Genetics, Research Centre Flakkebjerg, Slagelse, Denmark 2 Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA Sequence specific nucleases are molecules with nuclease activity able to bind and induce double strand breaks (DSBs) at specific sites in a genome. These DSBs are repaired by the cells own DNA repair machinery. The most common type of repair is non-homologous end-joining which frequently generates mutations in the form of short deletion and/or insertions at the break site. Thus the DSBs often result in mutations at the targeted sites. Currently, the most widely used sequence specific nucleases in plants are TALENs and the CRISPR/Cas9-system. Molecular constructs for both enzymes are easy to assemble and can be designed to target almost any location in a genome. We have recently used TALENs to induce mutations in barley and shown that TALENs controlled by the 35S-promoter can be used to induce mutations at a specific site in the barley genome (Wendt et al. 2013). However, for efficient inheritance of TALEN-induced mutations to the next generation, high activity levels of the TALENs are needed (Christian et al., 2013). We are presently testing various TALEN constructs and a CRISPR/Cas9-construct for improved activity in barley. Current results show that it is possible to increase the activity of the TALENs through this approach. Wendt T., Holm P.B., Starker C.C., Christian M., Voytas D.F., Brinch-Pedersen H., Holme I.B. (2013). TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants. Plant Mol Biol. 83, 279-285. Christian, M., Qi Y., Zhang Y.. and Voytas D.F. (2013) Targeted Mutagenesis of Arabidopsis thaliana Using Engineered TAL Effector Nucleases. G3: Genes|Genomes|Genetics. 3: 1697-1705.

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Research Areas

Abstracts that are submitted in the oral presentation category undergo a selection process and participants with selected abstracts are invited to give a 20 minute talk or a short elevator talk (see programme). The Selection committees for the six different research areas are: Plant Products: starches, fibres, pharmaceuticals, specialized metabolites, biopesticides, proteins and enzymes, oil, phytochemicals, fuels, natural colors, bioplastics

Barbara Halkier, University of Copenhagen Eva Vincze, Aarhus University Søren Bak, University of Copenhagen Plants for Food and Feed: healthy food and feed, fatty acids, fibres, vitamins, minerals, proteins, biofortification, flour, malt, allergens, anti-nutritional factors

Henrik Brinch-Pedersen, Aarhus University Birte Svensson, Technical University of Denmark Søren K. Rasmussen, University of Copenhagen

Synthetic- and Systems Biology: metabolic engineering, molecular farming, omics technologies, bioinformatics, meta genomics, epigenetics

Kåre Lehmann, Aalborg University Mathias Pribil, University of Copenhagen Per Gregersen, Aarhus University

Plant Signalling and Cellular Trafficking: cell-cell signaling, bioimaging, biomembranes, signal transduction, organism wide and community wide signaling, membrane trafficking, cellular trafficking

Jens Stougaard, Aarhus University Anja Thoe Fuglsang, University of Copenhagen Hans Thordal-Christensen, University of Copenhagen

Biomass Production and Processing: micro and macro nutrients, plant uptake, resource use efficiency, bioenergy, carbohydrate polymers, biorefinery

Yumiko Sakuragi, University of Copenhagen Jan K. Schjørring, University of Copenhagen William Willats, University of Copenhagen

Plant Breeding – Quality, Productivity and Diseases: yield, modern breeding techniques, genomics, adaptation of crops to climatic change, pathology, plant/microbe interactions, pest managements, epidemiology, disease resistance, phenomics

Torben Asp, Aarhus University Niels Sandal, Aarhus University Svend Christensen, University of Copenhagen

23

Poster

Research Area: Plant Products Page

Nethaji Gallage The elucidation of vanillin biosynthetic pathway in the vanilla orchid, Vanilla planifolia

7

Adam Takos Repeated evolution of cyanogenic glucoside biosynthetic gene clusters 27 Aldo Ricardo Almeida Robles

α-Onocerin is an unusual triterpene in Ononis spinosa 28

Daniel Vik Integrated analysis of metabolites, transcripts and proteins gives insight into regulation of plant defense chemistry

29

Daniela Lai Alternative biosynthetic gene clusters for oxime-derived compounds in the genus Phaseoulus show the evolutionary dynamics of plant chemical defence

30

Giuseppe Dionisio Barley (Hordeum vulgare L.) endoprotease B2 controlled by the D-Hordein promoter is expressed and active in developing barley grains. Differential proteomics and lipidomics analysis

31

Lea Møller Jensen Investigation of regulatory networks controlling glucosinolate profiles 32 Lemeng Dong Bioprospecting of natural and aynthetic cucurbitane related compounds 33 Pernille Ø Erthmann What determines product specificity of oxidosqualene cyclases in

triterpenoid biosynthesis? 34

Qing Liu Unlocking the code of saponin structure-activity relationships for use in pest management

35

Sebastian Nintemann How to visualize the orchestration of glucosinolate biosynthesis? 36 Stefan Pentzold Resistance in insect herbivores to cyanogenic glucosides by avoidance,

metabolism and excretion 37

Svend Roesen Madsen Elucidating the role of transport processes in leaf glucosinolate distribution 38 Yunjia Tang Natural variation among Arabidopsis thaliana accessions in expression of

miR826 and corresponding alkenyl glucosinolate synthesis 39

Research Area: Plants for Food and Feed

Helle J Martens Enzymatic degradation of soybean fiber components demonstrated by viscosity and microscopy

8

Fariha Tanwir Biosynthesis of benzoxazinoids during germination in wheat and rye as well as the functional characterization of a BX6-like ortholog in rye

40

Nicola Zagari Protein quality control regulating carotenoid biogenesis 41 Zelalem Eshetu Bekalu Hydrolase inhibitors in barley and wheat grains: significance in feed 42

Research Area: Synthetic and Systems Biology

Johan Andersen-Ranberg Exploiting the Diterpenoid toolbox for the biosynthesis of high value diterpenoid compounds

10

Alice Jara De Porcellinis Bifunctional sedoheptulose/fructose bisphospahtase activity acts as a metabolic switch enhancing the calvin cycle and photosynthesis in cyanobacteria

43

Artur Wlodarczyk Engineering cyanobacteria to produce dhurrin 44 Bekzod Khakimov Novel unbiased derivatization methodology for broad-spectrum GC-MS

metabolomic analysis of complex mixtures 45

Brian King In planta assembly of multiple functional DNA fragments via homologous recombination

46

24

Poster

Claus Krogh Madsen Secreted expression libraries of plant proteomes 47 Érika de Castro Evolutionary aspects of cyanogenic glucoside biosynthesis in butterflies and

moths and their food plants 48

Konstantinos Vavitsas Engineering of isopimaric acid pathway in Nicotiana benthamian; a step towards light-driven terpenoid biosynthesis

49

Mikael Kryger Jensen Reincorporation of heme into apo-CYP79A1 50 Niels Sandal Forward screening of Lotus japonicus LORE1 insertion mutant lines 51 Rosanna C Hennessy Microbial biosynthesis of secondary metabolites involved in biocontrol 52 Silas Mellor Improving the efficiency of electron transfer for light-driven cytochrome

P450 reactions 53

Svenning Rune Møller Towards glyco engineered plants – using precise genetic engineering 54

Research Area: Signalling and Cellular Trafficking

Anna Malolepszy Identification of a novel fix- mutant involved in deubiquitination pathway in Lotus japonicus using LORE1 resource

11

Anna Holzwarth Identifying novel regulators of photosynthetic acclimation to light changes 55 Christoph Crocoll Cross-talk between Methionine Biosynthesis and Aliphatic Glucosinolates in

Arabidopsis thaliana 56

Mads Eggert Nielsen VPS9a mediates pre- and post-invasive disease resistance against powdery mildew infection

57

Marie Pireyre Specific activation of MYB transcription factors is regulated by subcellular protein-protein interactions in Arabidopsis

58

Marijn Knip Discovering the mechanisms behind cross-kingdom RNA transfer: Host-Induced Gene Silencing (HIGS) in the Arabidopsis powdery mildew Golovinomyces orontii

59

Morten Egevang Jørgensen

AtGTR3 is an indole-specific glucosinolate transporter responsible for controlling root/shoot indole glucosinolate distribution

60

Pablo Pulido Chloroplastic isoprenoid biosynthesis is specifically regulated by the protein quality control formed by chaperones and proteases

61

Raquel Sánchez-Pérez Secondary metabolites in almond flowers 62 Seong Wook Yang COP1 E3 ligase protects HYL1 to retain microRNA biogenesis 63 Sisse Gjetting In vivo calcium signaling induced by secreted RALF peptide hormones 64 Sophie K Lambertz Monitoring of Hormone Dynamics in the Plant Body of Arabidopsis thaliana 65

Wenjun Xie Idenfication of novel plant defence components using pen1 syp122 as a tool 66

Research Area: Biomass production and processing

Anna Segečová Cultivation of photoautotrophic plant suspension cultures in photobioreactors

67

Daniele Silvestro Modified brassinosteroids accumulation affects saccharification in Brachypodium

68

Domenico Sagnelli Plant Factory Hybrid Biopolymers for Future Materials-Related Applications 69

Eric van der Graaff A systems biology analysis of early-stage sugar beet storage root development

70

Jan Van Hecke Structural cell wall proteins of winter wheat (Triticum aestivum) affected by nitrogen status

71

25

Poster

Ken Suszkiewicz Krogholm Impact of Manganese stress on Photosystem I and II in Arabidopsis thaliana 72 Lorenzo Fimognari Cell wall acetylation and plant fitness: suppressor screening of the REDUCED

WALL ACETYLATION 2 reveals mutants with wild type surface permeability 73

Pernille Louise Munke Hansen

GWAS of winter wheat cultivars – identification of genes relevant for biomass and grain yield

74

Sylwia Glazowska Low silicon Brachypodium mutant as a tool for investigation of silicon effects on cell wall composition

75

Research Area: Plant Breeding - Quality, Productivity and Diseases

Agnieszka Siwoszek Peptide aptamer-mediated resistance to barley powdery mildew 76 Carsten Pedersen Functional analyses of barley powdery mildew effector candidates 77 Changhai Liu Puccinia effector candidate PEC6 suppresses PAMP triggered immunity and

targets adenosine kinase 78

Chris Sørensen The effect of the hemoglobin level in barley (Hordeum vulgare) on the interaction with both compatible and incompatible races of Blumeria graminis

79

Cristiana Paina Public Private Partnership for pre-breeding in perennial ryegrass: developing cultivars with suitable adaptation to future climates in the Nordic countries

80

Dagmara Podzimska-Sroka

Barley plants over-expressing the NAC transcription factor gene HvNAC005 shows an early senescence and drought-mimicking phenotype

81

Dew Kumari Sharma Hunting QTLs for heat tolerance from exotic wheat cultivars 82 Dominik Grosskinsky Physiological phenotyping by determination of phytohormone and enzyme

activity signatures 83

Istvan Nagy Genome-wide analysis of pollen allergen gene families in perennial ryegrass (Lolium perenne L.)

84

Kacper Piotr Kaminski QTL Analysis by Sequencing of Total Glycoalkaloid (TGA) Content in Potato 85 Louise de Bang An essential model plant for food and bioenergy crops – Brachypodium

distachyon 86

Maria-Ermioni Constantin Two Argonaute genes are necessary for the control of cellular functions during the vegetative stage in Blumeria graminis f. sp. hordei

87

Massimiliano Carciofi Regulation of the hemoglobin/NO cycle in barley infected with powdery mildew or yellow (stripe) rust

88

Nele Gjendal Light-dependent lesion formation might rather correlate with resistance than with susceptibility in the pathosystem barley <-> B. sorokiniana

89

Qiongxian Lu Characterization of erectoides barley mutants ert-c and ert-d 90 Remy Kronbak Resistance gene specificity in cereals 91 Stephen Byrne Partitioning SNPs identified by GBS into genome annotation classes and

calculating variance components for heading date and disease resistance from the resulting genomic relationship matrices

92

26

Posters: Plant Products

Repeated evolution of cyanogenic glucoside biosynthetic gene clusters Adam Takos, Daniela Lai, Camilla Knudsen, Birger Lindberg Møller, Fred Rook

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Cyanogenic glucosides (CNGs) are plant chemical defense compounds that occur in a wide variety of plant families, ranging from ferns to angiosperms. CNG biosynthesis is catalyzed by the sequential reactions of two cytochrome P450s and a UDP-dependent glucosyl transferase. We elucidated the biosynthetic pathway for CNGs in Lotus japonicus due to the observation that the biosynthetic genes were organized in a short chromosome region forming a biosynthetic gene cluster. Gene clusters for cyanogenic glucoside biosynthesis were also found in crops species such as cassava and sorghum. The gene identity and the diverse genomic structure of these clusters suggested that they evolved independently. Although the first enzyme in each of the three pathways is encoded by a member of the CYP79 gene family, they are not orthologous genes but a member of this family is repeatedly recruited. Genes encoding the second cytochrome P450 of the pathway either belong to the CYP71 family or the CYP736 family. The independent clustering of the biosynthetic pathways for CNGs in three plant lineages indicates the presence of an evolutionary selection mechanism promoting the formation of gene clusters. Biosynthetic gene clusters are also observed for other plant chemical defense compounds such as terpenoids and benzoxazinoids. We propose that gene cluster formation is driven by antagonistic ecological selection pressures.

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Posters: Plant Products

α-Onocerin is an unusual triterpene in Ononis spinosa Aldo Ricardo Almeida Robles1, Giovanni Appendino2, Lemeng Dong1 and Søren Bak1

1 Department of Plant and Environmental Sciences, University of Copenhagen 2 Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale, Novara, Italy Triterpenes constitute a structurally diverse group of metabolites derived from the cyclization of squalene derived intermediates. Triterpenes are synthesized by a plethora of plant families; however, some species can synthesize unique triterpene skeletons not yet detected in other families of the plant kingdom. Ononis spinosa is a species in the Fabaceae family. Extracts from the plant are known to have diuretic, anti-inflammatory and analgesic activities. Ononis spinosa is able to produce a unique symmetric triterpene denominated α-onocerin, although it is not known if the properties mentioned above are attributable to this compound. Rowan and Dean (1971) showed that protein extracts from leaves and roots of Ononis spinosa produce α-onocerin using squalene-2(3),22(23)-diepoxide as substrate and not 2(3)-oxidosqualene. However no specific protein(s) or genes responsible for the cyclization of squalene-2(3),22(23)-diepoxide are known. TriForC (triforc.eu) is an EU-funded collaborative project on establishing an integrative and innovative pipeline for the exploitation of plant triterpenes. In attempts to increase the available resources for triterpene industrial use, we aim to elucidate the biosynthesis of α-onocerin. Additionally, specific bioassays testing only α-onocerin are few in the literature, thus α-onocerin is being tested for several agricultural and medicinal bioassays.

28

Posters: Plant Products

Integrated analysis of metabolites, transcripts and proteins gives insight into regulation of plant defense chemistry Daniel Vik, Meike Burow, Barbara Ann Halkier

DynaMo Center of Excellence, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark The glucosinolate defense compounds of the model plant Arabidopsis constitutes a powerful model system for studying general biological phenomena. This project aims at increasing our understanding of biosynthetic pathways and their regulation at the enzyme level, i.e. translational rates, post-translational modifications and protein degradation. The glucosinolate metabolic flux is rapidly altered upon pathogen/herbivore encounter, suggesting immediate regulation of the biosynthetic enzymes at the protein level. While robust quantification methods are well-established for metabolites and transcripts, accurate quantification of proteins has previously been impossible to perform without raising protein-specific antibodies, and is even then difficult to perform in a high-throughput manner. Recent advances in mass spectrometry-based protein-identification now allow for the development of reliable protein quantification assays, otherwise known as selected reaction monitoring. Integrating the analysis of transcript, protein and metabolite under different conditions, will allow us to gain insight into the contributions of the individual regulatory levels which control metabolic flux during environmental changes.

29

Posters: Plant Products

Alternative biosynthetic gene clusters for oxime-derived compounds in the genus Phaseoulus show the evolutionary dynamics of plant chemical defence Daniela Lai, Behrooz Darbani, Fred Rook Plant Biochemistry, Dept. of Plant and Environmental Sciences, University of Copenhagen It is increasingly recognised that the biosynthetic genes for plant chemical defence compounds can be organised in gene clusters: non-homologous genes of the pathway are co-localized in the same genomic region. Gene clusters are proposed to form as a result of antagonistic selection pressures, such as the presence or absence of different herbivores and pathogens1. We previously reported the independent evolution of biosynthetic gene clusters for cyanogenic glucosides in cassava (Manihot esculenta), Sorghum bicolor, and the model legume Lotus japonicus2. In these three cases the first enzyme of the biosynthetic pathway is a member of the CYP79 family of cytochrome P450s. The second step in the biosynthesis of cyanogenic glucosides is the conversion of the oxime into a hydroxynitrile, catalysed by a second cytochrome P450 enzyme, followed by its glucosylation by a UDP-glucosyltransferase of the UGT85 family. In the biosynthesis of cyanogenic glucosides, the second cytochrome P450 producing the hydroxynitrile in L. japonicus was found to be a member of the CYP736 family, and not a member of the CYP71 family as observed in sorghum and cassava2. To investigate the evolutionary dynamics of biosynthetic gene clusters we analysed the biosynthetic pathway for cyanogenic glucosides and its genomic organisation in other legumes, such as Phaseolus lunatus (lima bean). Remarkably, the biosynthesis of cyanogenic glucosides in P. lunatus involves a member of the CYP83 family as a second enzyme, indicating that even within legumes the cyanogenic glucoside biosynthetic pathway is not conserved. The common bean, Phaseolus vulgaris, is not cyanogenic but has been reported to produce oxime volatiles following insect damage. A gene cluster containing a CYP79 and a number of closely related CYP83 genes is being investigated in both P. lunatus and P. vulgaris. Differences in the enzymatic specificity of the CYP83s are proposed to produce a variety of oxime derived defence compounds in the Phaseolus genus. More generally, members of the CYP79 family convert amino acids into oximes, which are the starting point for the synthesis of a wide range of plant defence compounds. High variability in the identity and specificity of oxime metabolising enzymes is suggested to result from the continuous adaptation of plant defence to a dynamic biotic environment. 1 Takos and Rook (2012), Trends Plant Sci 17: 383; 2 Takos et al. (2011) Plant J. 68: 273

30

Posters: Plant Products

Barley (Hordeum vulgare L.) endoprotease B2 controlled by the D-Hordein promoter is expressed and active in developing barley grains. Differential proteomics and lipidomics analysis. Giuseppe Dionisio*, Anne Lind Rosenkilde, Inger Bæksted Holme, Henrik Brinch-Pedersen Aarhus University, Faculty of Science and Technology, Dept. of Molecular Biology and Genetics, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark. *E-mail: Giuseppe.dionisio@agrsci.dk For increasing protein hydrolysis in feed, food and industrial applications, new efficient protease candidates are desirable. In barley (Hordeum vulgare L.), cysteine endoproteases are responsible for major protein degradations during germination and may have a large potential as industrial enzyme. Recombinant barley endoprotease B2 (recHvEP-B2) has been produced in Pichia pastoris (Rosenkilde et al., 2014) and successfully used for increasing protein derived nitrogen in feedstuff according to soaked barley procedures (Christensen et al., 2014). In the present work, the endosperm specific D-hordein promoter was used for driving the overexpression of a Hordeum vulgare endoprotease B2 (HvEP-B2) in the developing endosperm of barley. A total of 323 embryos were transformed with Agrobacterium and after regeneration of plants, 42 T0 plants was tested positive by PCR, giving a transformation efficiency of 13 %. By qRT-PCR, the over-expression levels in developing endosperms were found to be up to 5.6-fold higher than in untransformed control endosperms. Endoprotease activity assays on mature T1 and T2 seeds showed up to 13-fold increased activity levels, compared to non-transformed control. Functionality of the in planta produced HvEPB2 was confirmed in highly producing lines after degradation of Osborne fractionated hordeins to a degree similar to that of recombinant produced HvEP-B2. However at macroscopic level the seed analysis reveals that the endosperm has been shrunk. After Osborne fractionation and SDS-PAGE the protein composition looks altered as if most of the hordeins have been largely decreased and the albumins/globulins have been largely increased. Furthermore reduction in starch contents and increase in lipids, phenolics, soluble and insoluble fibers have been detected. Here we present data on the analysis by differential proteomics and lipidomics performed by nanoLC-MS/QTOF related to HordD::HvEP-B2 lines versus non-transformed control. Endogenous EP-B2 inhibitors, cystatins, have increased in abundance in all transformed HordD::HvEP-B2 lines vs control. References Christensen JB, Dionisio G, Poulsen HD, Brinch-Pedersen H. (2014) Effect of pH and Recombinant Barley (Hordeum vulgare L.) Endoprotease B2 on Degradation of Proteins in Soaked Barley. J Agric Food Chem. 62(34):8562-70. doi: 10.1021/jf502170v.

Rosenkilde A. L., Dionisio G., Holm P. B., Brinch-Pedersen H. (2014) Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins. Phytochemistry 97:11-9. doi: 10.1016/j.phytochem.2013.09.004.

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Posters: Plant Products

Investigation of regulatory networks controlling glucosinolate profiles Lea Møller Jensen1, Barbara Ann Halkier1, Daniel Kliebenstein1,2, and Meike Burow 1 1 DNRF Center DynaMo, Copenhagen Plant Science Centre, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark 2 Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA

95616 The major group of natural products in Brassicales is glucosinolates. The group contains an array of chemically diverse molecules, which provide chemical defense towards different kinds of insects and microbes. Consequently, the plant controls the kind and level of glucosinolates produced depending on pests in the environment. Several transcription factors influence glucosinolate production, but additionally two loci encoding biosynthetic enzymes in the pathway have a major impact on the level and structures produced. One of the enzymes in these loci is the 2-oxoglutarate-dependent dioxygenase, AOP3, which modifies glucosinolate structures. We investigate AOP3’s influence on glucosinolate production in Arabidopsis. To study whether an AOP3-induced change in glucosinolate accumulation is due to the enzymatic activity, the protein or the RNA; we generated different versions of AOP3 and introduced these into accessions without AOP3 expression in the vegetative tissue. Unexpectedly, the chosen accessions had unchanged glucosinolate levels upon AOP3 expression. We therefore hypothesized that the effect of AOP3 is depend on specific combinations of other genes. To test this we crossed plant lines with the different AOP3 versions with a WT of another accession not expressing AOP3. Analysis of the F2 population showed that AOP3 is able to increase the production of some glucosinolates in specific genetic backgrounds. Subsequently, we did genome-wide genotyping by MassArray to generate markers for QTL mapping in the F2 population. The QTL mapping identified AOP3 as a QTL for accumulation of specific glucosinolates indicating that AOP3 effects glucosinolate accumulation. The study further identified significant interactions between AOP3 and other QTLs, which enables us to elucidate components of the regulatory network surrounding AOP3.

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Posters: Plant Products

Bioprospecting of natural and aynthetic cucurbitane related compounds Lemeng Dong1, Aldo Ricardo Almeida Robles1, Mathias Brodersen1, Bekzod Khakimov1, Søren Bak1

1 Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Triterpenes constitute an extreme diverse class of natural products with a vast array of biological activities. They are derived from a limited set of characteristic skeletons and the structural diversity comes from a broad range of modification on the different carbon positions of the C30 skeletons. Many naturally occurring plant triterpenes are already being used as anticancer drugs, pesticide, and antimicrobial agents. TriForC (triforc.eu) is an EU-funded collaborative project on establishing an integrative and innovative pipeline for the exploitation of plant triterpenes. TriForC will establish a pipeline for the discovery, sustainable production and commercial utilisation of known and novel high-value triterpenes with new or superior biological activities. As one of the TriForC partners, we focus on a distinct group of triterpenes, cucurbitacins, with highly unsaturated and oxygenated properties. Cucurbitanes are naturally synthesized in the Cucurbitaceae family and derived from the cucurbitane skeleton. Cucurbitanes exhibits different bioactivity like anticancer, antidiabetic and insecticidal based on the different modification on the cucurbitane skeleton. To study the biological activity of naturally and unnaturally produced cucurbitane derived compounds, several species in the Cucurbitaceae family will be subjected to bioprospecting. For bioprodution naturally and novel cucurbitane derived compounds will be introduced to tobacco and microalgal platform by metabolic engineering.

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Posters: Plant Products

What determines product specificity of oxidosqualene cyclases in triterpenoid biosynthesis? Pernille Ø. Erthmanna, Bekzod Khakimovb, Niels Agerbirka, Søren Baka

a Department of Plant and Environmental Sciences, University of Copenhagen b Department of Food Science, University of Copenhagen In the wild crucifer Barbarea vulgaris the production of triterpenoid saponins is correlated with resistance towards crucifer crop pests and they therefore have a potential as a biopesticides1,2. The proposed biosynthetic pathway of triterpenoid saponins branches off from the sterol biosynthesis with 2,3-oxidosqualene as the shared precursor. The triterpenoid backbone structures are further modified mainly by cytochromes P450 and glycosyltransferases, thereby creating a vast structural diversity. 2,3-oxidosqualene is cyclized by oxidosqualene cyclases (OSC) to a limited number of triterpene backbone structures such as α-amyrin, β-amyrin, and lupeol. Despite high sequence identity the OSCs produce different backbone structures and also differ in the ratios of the different compounds produced. Our current research is focused on improving the activity of the saponins as biopesticides by e.g. targeting other organisms. One of the strategies is to elucidate the relationship between the structure and the activity of the OSC, as this will enable us to control the backbone structures formed by the OSC and also their ratios. 1 Kuzina et al. (2009): Identification of defense compounds in Barbarea vulgaris against the herbivore Phyllotreta nemorum by an ecometabolomic approach. Plant physiology.Vol. 151, pp. 1977-1990. 2 Agerbirk et al. (2003): A saponin correlated with variable resistance of Barbarea vulgaris to the diamondback moth Plutella xylostella. Journal of Chemical Ecology. Vol. 29, pp. 1417-1433

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Posters: Plant Products

Unlocking the code of saponin structure-activity relationships for use in pest management Qing Liu1, Pernille Ø. Erthmann1, Bekzod Khakimov2, Søren Bak1

1 Department of Plant and Environmental Sciences, 2 Department of Food Science, University of Copenhagen, Copenhagen, Denmark Saponins are steroid and triterpenoid glycosides that constitute a structurally diverse class of plant defense compounds. Due to their detergent-like properties, saponins can disrupt cell membranes of herbivores and pest, cause cell death, and ultimately kill them. Despite the promising potential of saponins for use as bio-pesticide, little is known about which saponin structures are toxic to which specific herbivores or pests. This study aims to elucidate the relationship between saponin chemical structures and their biological activities and evolution. The plant system used in this study is Barbarea vulgaris, which is the only genus in the Cabbage family known to produce saponin. Previously we have identified several biosynthetic genes of saponins as well as genome regions (QTL) that determine resistance of saponin in B. vulgaris (Augustin et al. 2012; Kuzina et al. 2011; Kuzina et al. 2009). We will use genomics, transcriptomics and metabolomics tools to elucidate the biosynthetic pathway of saponins, and metabolically engineer desired or novel saponins in model plants with combinations of biosynthetic genes. Purified saponins from engineered plants will be used in bioassays with a range of pest species, to unlock the code of which saponin structures have a given specific biological activity.

35

Posters: Plant Products

How to visualize the orchestration of glucosinolate biosynthesis? Sebastian J. Nintemann1, Tonni Grube Andersen1, Meike Burow1, Barbara Ann Halkier1

1 DNRF Center DynaMo, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Denmark Glucosinolates (GLS) are defense compounds found in brassicaceous plants such as the model plant Arabidopsis thaliana and are known to respond in abundance to environmental conditions and attack. Therefore, the biosynthetic pathways of these specialized metabolites serve as a fruitful model system for studying the dynamics of pathway organization. The GLS pathways involve several subcellular compartments. Moreover, different types of GLS are synthesized partly by pathway-specific specialized, and partly by more promiscuous enzymes. Preliminary data suggest that specific interactions between involved proteins may play a role in GLS biosynthesis. To study the location of the biosynthetic machinery at the whole plant as well as the cellular level, we have developed a toolset of fluorophore-tagged biosynthetic enzymes stably expressed in the native host Arabidopsis under the control of their native promoters. This toolset will furthermore be used to study the subcellular spatial organization of the enzymes and to investigate protein-protein interactions by in vivo FRET/FLIM measurements. In parallel, we utilize a transient expression system in Nicotiana benthamiana to express the entire GLS biosynthetic machinery, allowing us to further investigate subcellular organization, protein-protein interactions and to measure the efficiency of GLS biosynthesis. The ability to include the entire pathway and thus quantify GLS production is a major advantage compared to in vitro methods or yeast-based analyses. Together, these tools allow for investigation of protein dynamics in living plant cells. We thus aim at elucidating the GLS biosynthetic pathway organization and its plasticity in plant development and stress response by employing these techniques.

36

Posters: Plant Products

Resistance in insect herbivores to cyanogenic glucosides by avoidance, metabolism and excretion Stefan Pentzold 1,2, Mika Zagrobelny 1,2, Nanna Bjarnholt 1,2, Juergen Kroymann 3, Heiko Vogel 4, Carl Erik Olsen 1,2, Birger Lindberg Møller 1,2, Søren Bak 1,2* 1 Plant Biochemistry Laboratory, Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Copenhagen, Denmark 2 Villum research center “Plant Plasticity”, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark 3 Laboratoire d'Ecologie, Systématique et Evolution, Université Paris-Sud/CNRS, Paris, France 4 Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany *Corresponding author, phone: 0045 3533 3346, email: bak@plen.ku.dk Cyanogenic glucosides (CNglcs) are widespread phytoanticipins that release toxic hydrogen cyanide when hydrolyzed by specific β-glucosidases after plant tissue damage. Whereas specialist herbivores possess countermeasures to avoid the toxicity of CNglcs 1,2, non-adapted herbivores may suffer from toxic hydrolysis products of CNglcs 3. Here we reared different insect species on transgenic Arabidopsis plants either producing or lacking CNglcs 4 to analyze and compare insect development, metabolism and excretion. As insects, we used generalists and glucosinolate-specialists (in both cases lepidopteran and aphid species). The insects developed without signs of intoxication, had normal weight gain and consumed similar amounts of either plant type. We show that the lepidopteran species either kept the CNglcs intact during digestion and excreted them intact, or metabolized the CNglcs. The aphids did not take up and accumulate CNglcs in their body. We suggest that non-adapted insects can tolerate CNglcs when (i) keeping CNglcs intact during feeding and digestion for their excretion, (ii) metabolizing CNglcs, or (iii) avoiding CNglc-uptake during feeding. Key Words - Plant chemical defence, Insect herbivory, Cyanogenic glucosides, β-Glucosidases, Generalists, Specialists References 1 Pentzold, S., Zagrobelny, M., Roelsgaard, P. S., Møller, B. L. & Bak, S. The multiple strategies

of an insect herbivore to overcome plant cyanogenic glucoside defence. PLoS ONE 9, e91337, doi:10.1371/journal.pone.0091337 (2014).

2 Zagrobelny, M. et al. Sequestration, tissue distribution and developmental transmission of cyanogenic glucosides in a specialist insect herbivore. Insect Biochemistry and Molecular Biology 44, 44-53, doi:http://dx.doi.org/10.1016/j.ibmb.2013.11.003 (2014).

3 Gleadow, R. M. & Møller, B. L. Cyanogenic glycosides: synthesis, physiology and phenotypic plasticity. Annual Review of Plant Biology 65, 24.21–24.31, doi:doi:10.1146/annurev-arplant-050213-040027 (2014).

4 Tattersall, D. B. et al. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828, doi:10.1126/science.1062249 (2001).

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Posters: Plant Products

Elucidating the role of transport processes in leaf glucosinolate distribution Svend Roesen Madsen1, Carl Erik Olsen2, Hussam Hassan Nour-Eldin1 and Barbara Ann Halkier1

1 DNRF center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark 2 Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark In Arabidopsis thaliana, a strategy to defend its leaves against herbivores is to accumulate glucosinolates along the midrib and at the margin. Although it is generally assumed that glucosinolates are synthesized along the vasculature in an Arabidopsis leaf, thereby suggesting that the margin accumulation is established through transport, little is known about these transport processes. Here, we show through leaf apoplastic fluid analysis and glucosinolate feeding experiments that two glucosinolate transporters, GTR1 and GTR2, essential for long distance transport of glucosinolates in Arabidopsis, also play key roles in glucosinolate allocation within a mature leaf, by effectively importing apoplastically localized glucosinolates into appropriate cells. Detection of glucosinolates in root xylem sap unambiguously shows that this transport route is involved in root to shoot glucosinolate allocation. Detailed leaf dissections show that in the absence of GTR1 and GTR2 transport activity, glucosinolates accumulate predominantly in leaf margins and leaf tips. Furthermore, we show that glucosinolates accumulate in the leaf abaxial epidermis in a GTR-independent manner. Based on our results, we propose a model for how glucosinolates accumulate in the leaf margin and epidermis, which includes symplasmic movement through plasmodesmata, coupled with the activity of putative vacuolar glucosinolate importers in these peripheral cell layers.

38

Posters: Plant Products

Natural variation among Arabidopsis thaliana accessions in expression of miR826 and corresponding alkenyl glucosinolate synthesis Yunjia Tang1, Daniel J. Kliebenstein2, Meike Burow1

1 DNRF Center of Excellence for Dynamic Molecular Interactions (DynaMo) and Copenhagen Plant Science Centre (CPSC), Department of Plant and Environmental Sciences, University of Copenhagen, Denmark 2 Department of Plant Sciences, University of California, Davis, California, USA Naturally occurring polymorphisms of MIRNA genes have the potential to affect biogenesis efficiency of their mature microRNA (miRNA) and to contribute to phenotypic diversity among accessions within a given species. However, few studies have shown the linkage between MIRNA gene polymorphism and corresponding phenotypic variation. In this study, we surveyed miR826 expression and its associated phenotype, alkenyl glucosinolate accumulation, in twenty diverse Arabidopsis thaliana accessions. We found that MIR826 genes from these accessions can be divided into two groups according to their sequences. These groups of MIR826 form two types of primary miR826 structures, which influence the precursor processing and lead to two different levels of mature miR826 expression. Correspondingly, alkenyl glucosinolate levels among these accessions also fall into these two groups and the group with lower miR826 expression accumulates more alkenyl glucosinolates, which biosynthesis pathway known to vary on the enzyme and transcription factor. Our result exemplifies that MIRNA gene polymorphisms are able to lead to phenotypic variation through affecting mature miRNA biogenesis. Thus, this study adds an additional level to the naturally variable regulation of plant metabolism and increases our overall understanding of coordination among different regulatory levels.

39

Posters: Plants for Food and Feed

Biosynthesis of benzoxazinoids during germination in wheat and rye as well as the functional characterization of a BX6-like ortholog in rye Fariha Tanwir1, Francelini Antunes1, Inge S. Fomsgaard2 and Per L. Gregersen1 1 Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark 2 Institute for Agroecology, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark Benzoxazinoids are bioactive compounds related to defense against pathogens and pests in cereals i.e. maize, wheat and rye. Recent studies also showed their role in potential health promoting effects in humans. In the present study, biosynthesis and regulation of BX genes and their chemical contents were studied during germination both in wheat and rye. This knowledge can be applied for the better understanding of benzoxazinoids pathway and the enrichment of our food with health promoting components. Wheat and rye seeds were germinated on filter paper and the germinating embryo was collected at different time points. qPCR analysis with specific gene primers was done to analyze gene regulation during this process. Similar samples were also analyzed on LCMSMS for determining the benzoxazinoid concentrations. Furthermore, a BX6-like ortholog (previously not published in rye) was PCR amplified from rye and expressed in E.coli for functional characterization in-vitro. Our results showed that expression of BX genes (BX1-5) was up-regulated both in wheat and rye within 24-36 hours after germination with a gradual down-regulation at later stages. Similarly, DIBOA-glc-hexose, DIBOA-glc, DIBOA contents were also increased in wheat whereas DIBOA-glc hexose showed a contrasting trend in rye. Detailed overview of their biosynthesis and chemical transformations will be discussed. Rye based 2-OG dioxygenase enzyme, expressed from BX6-like ortholog, was His tag purified. The purified enzyme was capable of transforming DIBOA-glc to TRIBOA-glc when incubated in vitro. The transformation of DIBOA-glc to TRIBOA-glc was analysed with LCMSMS using LightSight® software. These findings point out that other orthologs of BX genes may also be present in rye, which are previously not studied due to lack of available sequence data for rye.

40

Posters: Plants for Food and Feed

Protein quality control regulating carotenoid biogenesis Nicola Zagari 1 2, Pablo Pulido1, Dario Leister 1 2

1 Copenhagen Plant Science Center (CPSC), Department of Plant and Environmental Sciences, University of Copenhagen, Denmark 2 Ludwig-Maximilians-Universität (LMU) Munich, Germany Chloroplast biogenesis is co-ordinated and regulated by the expression of nuclear and chloroplast genomes. Despite recent efforts in the search for key regulators few factors have been shown to critically affect chloroplast biogenesis. The Orange (Or) gene, initially isolated in cauliflower and directly involved in carotenoid accumulation demonstrates the existence of unknown mechanisms affecting the plastid formation and differentiation. The Or gene has also been shown to be of relevant value for improving food nutritional quality through increasing carotenoid content. Several unknown proteins have been found screening for Or-coregulated genes in a guilt-by-association approach. The output was obtained from proteomic databases such as ATTED combined with subcellular localization predictions. Interestingly four of the selected genes contain a J-domain for HSP70 interaction. HSP70 is an ubiquitous chaperone involved with protein folding and quality control. The co-chaperone activity of J-proteins relies on their substrate binding specificity and our aim is to identify the interaction partner and substrates. Finally the high homology between the cysteine-rich domain of the Or protein and the J-domain reinforces the hypothetical connection between plastid differentiation and protein quality control. Having a deeper understanding of the plastid differentiation will be enabling the conversion from one type of plastid to another improving the content of carotenoids of high nutritional value.

41

Posters: Plants for Food and Feed

Hydrolase inhibitors in barley and wheat grains: significance in feed Zelalem Eshetu, Claus K. Madsen, Giuseppe Dionisio and Henrik Brinch-Pedersen

Aarhus University, Department of Molecular Biology and Genetics, AU-Flakkebjerg, DK-4200 Slagelse, Denmark The digestive tract of animals is not fully efficient in utilizing feed nutrients. Livestocks excrete substantial amounts of undigested nutrients that pose a series of environmental problems. Moreover, in order to meet animal physiological requirements, feed is supplied with additional nutrients (e.g. protein, phosphate). This approach, however, increases resource use, nutrient load to the environment and total production costs even more. Improved nutritive value of foodstuffs can be achieved through the addition of exogenous feed enzymes. Feed enzymes such as microbial phytases, xylanases and proteases are used to improve feed utilization and minimize feed supplements. However, due to the presence of varying compositions and levels of inhibitors between species and cultivars, the efficiency of feed enzymes varies considerably. Our results confirmed that the addition of xylanases and proteases to different wheat and barley cultivars has resulted in significant variation of hydrolysis. Apart from xylanases and proteases, the inhibition of microbial phytases among feed crops has not been studied before. It is the aim of the current project to develop better combinations of barley and wheat cultivars and feed enzymes. In addition, the mechanisms of inhibition of different wheat and barley cultivars against various microbial phytases expressed in P. pastoris are studied.

42

Posters: Synthetic and Systems Biology

Bifunctional sedoheptulose/fructose bisphospahtase activity acts as a metabolic switch enhancing the calvin cycle and photosynthesis in cyanobacteria Alice Jara De Porcellinis1, Laura Brey1, Hanne Nøregaard1, Kati Thiel2, Eerika Vuorio2, Patrik Jones3, Joshua Heazlewood4, Pauli Kallio2, Yumiko Sakuragi1 1 Department of Plant and Environmental Sciences, University of Copenhagen, Denmark 2 Department of Biochemistry, University of Turku, Turku, Finland 3 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK 4 Joint BioEnergy Institute, Emeryville, California 94608, USA Oxygenic photosynthetic organisms including cyanobacteria, algae, and plants utilize pentose phosphate pathway (PPP) for photosynthesis and respiration. During photosynthesis the PPP is driven in the reductive direction and fix CO2 through a cycle also known as the Calvin-Benson-Bassham cycle, while during respiration the PPP is driven in the reverse oxidative direction to metabolize the fix carbons. In cyanobacteria these competing pathways co-exist in the cytosol, raising a question as to how they are controlled. Different approaches have been used during the past decades in order to enhancing CO2 fixation, with the main focus on manipulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), including insertion of extra gene copies, direct mutagenesis, and the modification of the RubisCO activase, which has led to contrasting results (Rosgaard, 2012). On the other hand the positive effect of overexpression of the bifunctional sedoheptulose/fructose bisphosphatase (hereafter S/FBPase) in plant has resulted in faster growth, higher dry weight, starch and also sucrose and hexose content in leaves (Feng, 2007; Lefebvre, 2005; Miyagawa, 2001). Here we assess two strategies, overexpression of RuBisCO via promoter swap and overexpression of S/FBPase driven by an additional gene copy under the control of a strong promoter. RuBisCO overexpression did not alter growth and photosynthetic performance, while overexpression of SBPase led to a significant increase in both the growth and photosynthesis rate as assessed by the O2 evolution analysis, with a coordinate increase in activities in the CBB cycle enzymes. Surprisingly, the RuBisCO content as well as the RuBisCO activities were elevated by 2-to-3 folds as compared to the wild-type control. In contrast, the abundance and the activity of glucose-6-phosphate dehydrogenase (G6PD), catalyzing the rate-limiting step in the oxidative PPP, was completely diminished in the S/FBPase mutant. These results indicate that the activity of S/FBPase acts as a switch between the reductive and oxidative PPP and plays a pivotal role in programing the central metabolism in cyanobacteria. All together these results corroborate and provide new insight into the importance of the role of S/FBPase, or metabolites derived from the SBPase activities, as a general regulator able to influence the activity of the PPP especially having a positive effect on RubisCO content and the overall photosynthetic capacity in cyanobacteria. S/FBPase overexpression was introduced to the cyanobacterium Synechocystis sp. PCC 6803 previously engineered to express the ethylene-forming enzyme (EFE) from Pseudomonas sp. and the impact on the ethylene formation evaluated. Preliminary analysis showed that the rate of ethylene production was increased by 10 folds in the mutant overexpressing both EFE and S/FBPase in comparison to the mutant overexpressing EFE alone. These results suggest that S/FBPase overexpression can be an effective strategy for the enhanced end-product formation in photobiological hosts in synthetic biology.

43

Posters: Synthetic and Systems Biology

Engineering cyanobacteria to produce dhurrin Artur Wlodarczyk, Thiyagarajan Gnanasekaran, Carl Erik Olsen, Agnieszka Zygadlo Nielsen, Poul Erik Jensen Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark In photosynthetic organisms like plant and cyanobacteria light provides the energy (in the form of ATP and NADPH) for the biosynthesis of basic cell building bricks and then more complex compounds. In order to produce high value compounds it is often necessary to perform highly specific and complex enzymatic reactions, which can be performed by proteins called cytochromes P450. These are monooxygenases anchored in the plant endoplasmic reticulum (ER) and are powered by single electron transfers from NADPH-dependent cytochrome P450 reductase (CPR). Expression level of these proteins is relatively low and the activity is often limited by NADPH and substrate pool. In our previous work we demonstrated that it is possible to omit these energy consumable reactions by relocating P450 dependent metabolic pathways to the thylakoid membranes of chloroplasts, where the reducing power generated by photosystem I is a cheap and never-ending source of electrons for P450s. Some plants produce defense compounds to protect them from pests and for the biosynthesis of some of them P450s appear to be essential. One example is dhurrin - a cyanogenic glucoside produced by Sorghum bicolor. Encouraged by the successful expression of the dhurrin pathway in Nicotiana benthamiana, we decided to move this pathway into cyanobacteria to prove that they are suitable for expression of foreign P450s. Current work is focused on targeting of P450s to the thylakoid membranes (by adding PetC signal sequence in front of each P450) and comparing of stable transgenic lines of Synechocystis spp. PCC6803, which express whole pathway and produce dhurrin and its intermediates.

44

Posters: Synthetic and Systems Biology

Novel unbiased derivatization methodology for broad-spectrum GC-MS metabolomic analysis of complex mixtures

Bekzod Khakimova,b, Mohammed Saddik Motawiaa, Søren Baka, Søren Balling Engelsenb

a Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, 1871 Copenhagen, Denmark b Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, Frederiksberg C, 1958 Copenhagen, Denmark Quantitative GC-MS metabolomics of complex biological mixtures requires robust and broad-spectrum derivatization methods. In this study, we describe the development of a TMSCN based derivatization method and demonstrate how it can outperform one of the most commonly used trimethylsilylation reagent MSTFA. Two complex samples, an artificial mixture of 35 metabolites, including amino acids, carbohydrates, small organic and phenolic acids, flavonoids, triterpenoids, and a phenolic extract of blueberry fruits were analyzed in GC-MS after derivatization. The TMSCN based methods depicted more rapid, sensitive and reproducible derivatization than MSTFA, both, for derivatization with and without prior methoximation. The method thus has great potential for untargeted metabolomics.

45

Posters: Synthetic and Systems Biology

In planta assembly of multiple functional DNA fragments via homologous recombination Brian Christopher King, Konstantinos Vavitsas, Nur Kusaira Binti Khairul Ikram, Poul Erik Jensen, Henrik Toft Simonsen Copenhagen Plant Science Centre, Department of Plant Biology and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark

e-mail: brianking@plen.ku.dk

The moss Physcomitrella patens is a model non-flowering plant used extensively for plant physiology and development studies. It is considered a potential platform for biotechnological applications, as it combines a eukaryotic and photosynthetic cellular environment, few glycosylating enzymes, a fully sequenced genome and easy genetic manipulation via homologous recombination. Homologous recombination in Physcomitrella is reported to be as efficient as in yeast, and is a critical feature which distinguishes moss from higher plants. In this work we present an efficient Transformation Associated Recombination (TAR) cloning approach, directly in planta. Using multiple overlapping PCR or restriction digested DNA fragments, it is possible to insert multi-kilobase self-assembling sequences into targeted loci in the moss genome. This methodology can significantly facilitate large scale genetic manipulation of Physcomitrella for both basic and applied research, including the introduction of multistep heterologous biosynthetic pathways and redesign of endogenous metabolism.

46

Posters: Synthetic and Systems Biology

Secreted expression libraries of plant proteomes Claus Krogh Madsen1, Tomas Vlcko2, Zelalem Eshetu Bekalu1, Giuseppe Dionisio1, Henrik Brinch-Pedersen1

1 Dept. of Molecular Biology and Genetics, Section for Crop Genetics and Biotechnology, Research Centre Flakkebjerg, Denmark 2 Palacky University Olomouc, Czech Republic Expression libraries are important tools for the de novo discovery of enzymes and their proteinaceous inhibitors. The strength of the strategy lies in the fact, that no prior knowledge of DNA or protein sequence is required. Especially valuable are secreted expression libraries, since they facilitate high trough put screening strategies. Unfortunately the typical host, E. coli, fails to express many plant proteins in their active conformation. On the other hand, the superior expression host P. pastoris suffers from low transfection efficiency making it unsuitable for primary library construction. This challenge has been solved in the current project by the creation of a bacteriophage λ / P. pastoris shuttle vector. Libraries in the shuttle vector can be created with the high transfection efficiency of bacteriophage λ, subsequently amplified, converted to P. pastoris autonomously replicating plasmids and used for P. pastoris transformation. The vector relies on the α-factor signal peptide to promote secretion of the encoded plant proteins. Ideally cDNAs should be cloned preferentially in frame with the signal peptide sequence. Otherwise two thirds of the library will be inactive. A novel cDNA synthesis procedure which targets the start codon is therefore being developed. The resulting cDNAs are equipped with 5’ and 3’ adapters which enables accurate and directional cloning by Gibson assembly. Preliminary results indicate that cDNA samples of 10-20 ng can be converted to primary libraries of >5*106 pfu. P. pastoris in turn, have yielded up to 6000 cfu per transformation reaction with the empty vector. More colonies can easily be achieved by performing a series of transformations because amplification of the primary library provides a virtually inexhaustible source of plasmids.

47

Posters: Synthetic and Systems Biology

Evolutionary aspects of cyanogenic glucoside biosynthesis in butterflies and moths and their food plants Érica de Castro, Mika Zagrobelny and Søren Bak

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Larvae of butterflies that belong to the Heliconius genera (Papilionoidea) feed exclusively on leaves of Passiflora plants (Passifloraceae) and both are cyanogenic. While some Heliconius species obtain the cyanogenic glucosides linamarin and lotaustralin by sequestrations, others are known to de novo biosynthesise the compounds from amino acids [1]. However, the molecular basis for the synthesis is not known. Lianamarin and lotaustralin are also presents in Zygaena filipendulae (Zygaenoidea) moths and its host plant Lotus corniculatus (Fabaceae). Two cytochromes P450 enzymes and a glucosyl transferase (UGT) carry out the biosynthetic pathway of these aliphatic cyanogenic glucosides in Z. filipendula larvae as well as in the L. corniculatus plants [2]. A phylogenetic analysis of the underlying genes showed that though plants and Z. filipendulae synthesize cyanogenic glucosides in essentially the same way using the same enzyme systems, the pathways have evolved convergently in the two kingdoms and are thus not phylogenetically related [2]. We do not know if de novo biosynthesis of cyanogenic glucosides evolved in an ancestor to butterflies and moth or if the pathway has evolved recurrently in lepidoptera. The aim of this work is identify and characterize the enzymes involved in de novo biosynthesis of linamarin and lotaustralin in Heliconius melpomene, as well as to unravel the evolution of the cyanogenic glucoside pathway in Lepidoptera. Our preliminary studies of the Heliconius genome indicated putative homologs to both of the Zygaena cytochromes P450 from the cyanogenic glucoside pathway in the H. Melpomene genome. RT-PCR analysis showed that all these putative homologous genes are expressed in H. melpomene imagos. If the cyanogenic glucoside pathway in butterflies and moths are homologous, the pathway could have evolved in the common ancestor of Zygaenoidea and Papilionoidea, or even earlier, rendering this pathway an ancient invention. 1 Nahrstedt A, Davis R H. 1983. Occurrence, variation and biosynthesis of the cyanogenic glucosides linamarin and lotaustralin in species of the Heliconiini (Insecta, Lepidoptera). Comp. Biochem. Physiol. Volume 75B: 65-73. 2 Jensen N B, Zagrobelny M, Hjernø K, Olsen C E, Houghton-Larsen J, Borch J, Møller B L, Bak S. 2011. Convergent evolution in biosynthesis of cyanogenic defence compounds in plants and insects. Nature Communication Volume 2 : 273-280.

48

Posters: Synthetic and Systems Biology

Engineering of isopimaric acid pathway in Nicotiana benthamian; a step towards light-driven terpenoid biosynthesis Konstantinos Vavitsas, Thiyagarajan Gnanasekaran, Carl Erik Olsen, Agnieszka Janina Zygadlo Nielsen and Poul Erik Jensen Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark Terpenoids are a class of plant metabolites with applications as pharmaceuticals, food additives and cosmetics. To date, tens of thousands of reported terpenoid structures are available; structures that are complex and thus difficult or even impossible to produce via organic synthesis. Therefore, plants remain the most prominent source for these compounds. A diterpene resin acid (isopimaric acid) pathway from conifers was chosen to be studied as a model pathway. Isopimaric acid is a defense compound acting against pathogenic fungi and has potential antibacterial use. It is produced using geranylgeranyl diphosphate (GGPP) as a precursor molecule, derived from the mevalonate (MEV) and the methylerythritol phosphate (MEP) pathways. Using transient infiltration with Agrobacterium tumefaciens, we engineered the isopimaric acid pathway within Nicotiana benthamian leafs. Modifying the N-terminal part of one of the involved enzymes allows us to localize it to different cellular compartments and to obtain useful insights towards light-driven biosynthesis of valuable terpenoids.

49

Posters: Synthetic and Systems Biology

Reincorporation of heme into apo-CYP79A1

Mikael Krygera, Max Cryleb, Søren Baka

a Department of Plant and Environmental Sciences, University of Copenhagen, Denmark b Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg, Germany Cytochrome P450s are a superfamily of enzymes classified as heme containing monooxygenases, which perform their enzymatic reactions using activated oxygen. Several early studies showed that attempts of purifying membrane bound CYPs often resulted in an inactive enzyme as seen by the shift in the Soret absorbance from 450nm to 420nm1. The shift is likely caused by a protonation of the proximal thiolate ligand and does not cause any major conformational changes2. However, studies trying to replicate the inactivation using pressure induced inactivation heat denaturation etc. produced a variety of inactive forms some of which showed conformational changes 3. The results indicate that in some cases the heme may be shifted or the conformation of the active site altered which results in inactive enzyme. Furthermore, an increasing number of crystal structures have shown that the positioning of the plane of the heme and its propionate groups vary between the different P450s and hence could be important for optimizing the catalytical activity of each enzyme4. Together, these discoveries illustrate the importance of correct incorporation and positioning of the heme in the active site. In spite of this, very little focus has currently been devoted to this area of P450 research. With this is mind, our current research is focused on elucidating features of how the heme moiety is incorporated into P450s. The strategy we are using to investigate the incorporation process centers on a truncated version of CYP79A1 that is unable to incorporate heme into the active site during expression. We are then able to use this soluble apo-P450 to perform the incorporation of heme in vitro and thus examine the processes that governs correct heme incorporation into P450 enzymes. 1) Wells et al. (1992): Resonance Raman investigations of Escherichia coli-expressed Pseudomonas putida cytochrome P450 and P420. Biochemistry. Vol 31, pp. 4384-4393 2) Sun et al. (2013): Investigation of Heme Ligation and Ligand switching in cytochrome P450 and P420. Biochemistry. Vol. 52, pp. 5941-5951 3) Davydov et al. (1992): High pressure induced inactivation of ferrous cytochrome P-450 LM2 (IIB4) CO complex: evidence for the presence of two conformers in the oligomer. Biochem Biophys Res Commun. Vol 188, pp. 216-221 4) Denisov et al. (2012) Structural differences between soluble and membrane bound cytochrome P450s. Journal of Inorganic Biochemistry. Vol 108, pp. 150-158

50

Posters: Synthetic and Systems Biology

Forward screening of Lotus japonicus LORE1 insertion mutant lines

Niels Sandal, Anna Malolepszy, Junyi Su, Fatima Goiri, Marcin Nadzieja, Dorian Urbanski, Jens Stougaard, Stig Uggerhøj Andersen

Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark

E-mail: nns@mbg.au.dk

Lotus retrotransposon 1 (LORE1) can be activated during tissue culture, giving rise to plant lines with active LORE1 elements. In the founder line for the LORE1 mutant collection, a LORE1 element is active in the male germline, giving rise to independent stable insertions in the founder line progeny. On average, five new LORE1 copies are found in each line. LORE1 has an exonic preference, which results in a high incidence of knockout mutations in genes (Fukai et al., 2010, Urbanski et al., 2012). At present ~85.000 LORE1 lines have been developed. The total fraction of genes targeted with exonic insertions was 65%, and 81% of the genes with exonic lengths larger than 1 kb were targeted. The insertion data, plant line information and seed stocks are freely available at the LORE1 resource website http://carb.au.dk/lore1. We have performed forward screening of several thousand lines from this population. A number of mutant lines with phenotypes such as light green leaves, variegated leaves (leaves with white sectors), symbiotic mutants, root mutants, dwarf mutants and leaf shape mutants were found. The mutants were confirmed in the next generation. Among ~5,500 LORE1 lines, we identified ten families segregating plants with a pale leaf phenotype. Consulting the database with annotated insertions, we found candidate insertions for 6 of the lines. Co-segregation of the insertions with the mutant phenotypes was confirmed using genotyping PCR or sequence specific amplified polymorphisms (SSAP) analysis. We have also found many new alleles of known symbiotic genes through both forward and reverse genetics. Fukai et al., 2010, PLOS Genetics 6, e1000868. Urbanski et al., 2012, Plant J. 69, 731-741.

51

Posters: Synthetic and Systems Biology

Microbial biosynthesis of secondary metabolites involved in biocontrol Rosanna C Hennessy1, Charlotte F Michelsen2, Stefan Olsson1, Peter Stougaard1

1 Section for Genetics and Microbiology, Department of Plant and Environmental Sciences, University of Copenhagen 2 Infection Microbiology, Department of Systems Biology, Technical University Denmark The impact of microbial diseases on crop production coupled with increasing antibiotic resistance emphasizes the need for alternative agricultural methods that can reduce environmental impact and do not rely on chemical pesticides. Microbial biocontrol agents (mBCAs) could potentially provide effective and safe strategies to overcome current constraints. The Greenlandic soil-bacterium Pseudomonas fluorescens In5 is a promising biocontrol agent that has previously been shown to produce bioactive compounds against fungal pathogens1,2. Genome sequencing and analysis of In5 identified large secondary metabolite biosynthesis gene clusters. A combination of random and targeted mutagenesis, together with MALDI-TOF imaging mass spectrometry, linked two non-ribosomal peptides (NRPs) designated nunapeptin and nunamycin respectively, to antifungal activity against Rhizoctonia solani, Pythium aphanidermatum and Fusarium graminearum1, 2. In order to unravel the complex genetic regulation of these large NRP synthetase gene clusters, antisense RNAs (asRNAs) and CRISPR/Cas9 based systems are being tested and developed as tools to target transcripts of interest and elucidate gene function3, 4. To investigate the effect of purified nunamycin and nunapeptin at the omics level against pathogenic fungi, an NRP production platform is being developed which, could additionally provide a source of antifungal compounds for industrial applications (e.g. food production, pharmaceutical, personal care). Methods for direct cloning based on either linear-linear homologous recombination (LLHR) or direct assembly methods (e.g. Gibson assembly) will be used to capture, clone and refactor In5 secondary metabolite gene clusters5. The overall aim of this project is to understand how mBCAs communicate in complex communities, the conditions under which these antifungal peptides are synthesised and the compound effects on fungal pathogens. The ultimate goal is to develop a novel and sustainable biocontrol technology. References 1 Michelsen CF, Stougaard P (2011) A novel antifungal Pseudomonas fluorescens isolated from potato soils in Greenland. Current Microbiology 62:1185-92. 2 Michelsen CF (2013) Beneficial Rhizobacteria: Bioactive compounds from a Greenlandic Pseudomonas fluorescens PhD Thesis, University of Copenhagen, ISBN 978-87-996290-0-8. 3 N. Nakashima and T. Tamura (2012) Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors. Letters in Applied Microbiology 56, 436-442. 4 Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31, 233-239. 5 Ryan E Cobb and Huimin Zhao (2012) Direct cloning of large genomic sequences. Nature Biotechnology 30, 405-406.

52

Posters: Synthetic and Systems Biology

Improving the efficiency of electron transfer for light-driven cytochrome P450 reactions Silas Busck Mellor, Agnieszka Zygadlo Nielsen, Birger Lindberg Møller, Poul Erik Jensen

Copenhagen Plant Science Centre, Dept. Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark Background: Photosynthetic organisms are interesting candidates as production hosts for specialized metabolites with interest to the pharmaceutical industry due to their phototrophic nature, which allows high metabolic activity with very little input other than light. Our lab has demonstrated that biosynthetic pathways can be coupled directly to photosynthetically generated reducing power by relocating the enzymes involved in the synthesis of the sorghum-derived cyanogenic glucoside dhurrin to the chloroplast of N. benthamiana. In our model system the first enzyme in the dhurrin pathway, the cytochrome P450 (CYP) CYP79A1, obtains reducing power directly from photosynthesis by interacting with ferredoxin (Fd), to drive the conversion of tyrosine into p-hydroxyphenylacetaldoxime (Nielsen et al, 2013).

Aim: The aim of this project is to improve the partitioning of reducing equivalents towards natural product synthesis for pathways relocated to the chloroplast. To achieve this, CYP-Fd fusion proteins are designed. CYP-reductase fusions are found in nature and generally achieve high catalytic efficiency, presumably by placing the small, promiscuous proteins that serve as electron shuttles close to its site of interaction on the CYP, thereby introducing a kinetic bias towards this interaction. We wish to introduce such a bias into our synthetic system by coupling cytochrome to the major photosynthetic Fd2.

Methodology: Transient expression of fusion constructs in tobacco is followed by purification of thylakoids or intact chloroplasts containing overexpressed proteins for enzyme assays and protein quantitation. We use western blotting to determine CYP amounts relative to non-fused control CYP79A1. Tyrosine turnover is measured by autoradiography of enzyme products separated by thin layer chromatography.

Results & Discussion: Fusion of Fd2 to either C- or N-termini of CYP79A1 yields active enzyme capable of sustaining light-driven activity even in the absence of exogenously added Fd. Activity is lower than for non-fused CYP79A1 in the presence of Fd, suggesting that interaction between Fd and CYP is not optimal in the fusion enzymes. Future work aims at investigating the effect of varying linker length or ferredoxin isoforms in allowing optimal productive electron transfer to occur.

References Nielsen AZ, Ziersen B, Jensen K, Lassen LM, Olsen CE, Møller BL, Jensen PE (2013) Redirecting Photosynthetic Reducing Power toward Natural Product Synthesis. ACS Synth. Biol. 2: 308-315.

53

Posters: Synthetic and Systems Biology

Towards glyco engineered plants – using precise genetic engineering Svenning Rune Moeller1, Xueying Yi1, Pernille Louise Munke Hansen1, Bodil Jørgensen1, Yang Zhang2, Eric P. Bennett2, Nicholas Baltes3, Henrik Clausen2, Hans H. Wandall2, Bent Larsen Petersen1

1 Department of Plant and Environmental Sciences, University of Copenhagen, Denmark 2 Department of Cellular and Molecular Medicine, Copenhagen Centre for Glycomics, University of Copenhagen, Denmark 3 University of Minnesota, USA The expanding cross field between glycobiology and immunology has open up for novel approaches of targeting a range of diseases using immune modulatory strategies. Designed carbohydrate mediated targeting of key immune cells is a novel promising approach in treatment of allergy, cancers and autoimmune diseases and has the potential of becoming a new generation of targeted glycoprotein therapeutics with immune stimulatory/modulatory properties. Our recent knowledge of the genetic regulation of glycosylation machineries in plants and mammalian cells has enabled the use of precise genetic engineering technologies to glycoengineer cells for production of proteins with compatible or immune stimulatory glycans targeting specific receptors of the human innate immune system with the long term aim of conferring tolerance (allergy) or immunity (cancer).

Our first attempt to glyco engineer Arabidopsis by means of TALE nucleases (TALENs) appeared to be futile. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, however, was shown to functionally restore a reporter gene disrupted with sgRNA glycogene targets by HR when transiently expressed in N. benthamiana thus providing a means of testing CRISPR sgRNA targets and ultimately generating precisely glyco engineered plants.

54

Posters: Signalling and Cellular Trafficking

Identifying novel regulators of photosynthetic acclimation to light changes Anna Holzwarth1, Dario Leister2

1,2 Copenhagen Plant Science Center (CPSC), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

2 Plant Molecular Biology, Department of Biology I, Ludwig-Maximilians-University Munich, Großhaderner Str. 2, D-82152 Planegg-Martinsried, Germany In a natural environment plants are constantly exposed to variations in light conditions. These challenge the plant with excess light energy and uneven distribution of light energy between the photosystems. It has been shown that the photosynthetic apparatus has multiple strategies of adaption to keep the light reaction efficient. This includes short term strategies like non-photochemical quenching and state transitions as well as long term changes in thylakoid architecture and the photosystem ratio. Whereas the main proteins that constitute the light reaction have been identified many unknowns remain in our knowledge of the regulational pathways of photosynthesis. The guilt-by-association approach is based on the observation that most photosynthetic proteins share a unique transcriptional signature and are evolutionarily conserved through the green photoautotroph organisms. This approach was used to identify potentially novel regulators which will be screened for a mutant phenotype in Arabidopsis thaliana. The mutant lines with a phenotype indicating photosynthesis impairment will be further characterized using proteomic tools.

55

Posters: Signalling and Cellular Trafficking

Cross-talk between Methionine Biosynthesis and Aliphatic Glucosinolates in Arabidopsis thaliana Christoph Crocoll1, Michael Reichelt2 Barbara Halkier1

1 DNRF Center of Excellence for Dynamic Molecular Interactions (DynaMo), Section of Molecular Plant Biology, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, DK-1871 Frederiksberg, Denmark 2 Max Planck Institute for Chemical Ecology, Biochemistry Department, D-07745 Jena, Germany Methionine is not only a simple building block for protein biosynthesis but also a precursor or intermediate in several other pathways. In fact, 80% of free methionine is found as the metabolite S-adenosyl-methionine (SAM) which is a major methyl-group donor in trans-methylation reactions[1]. The central role of methionine and its metabolites is reflected in a tight regulation and very limited availability of free methionine in plants (~5-15 nmol/g fresh weight in Arabidopsis). Plant species of the Brassicaceae family, including the model plant Arabidopsis, have succeeded in adding another level of complexity by producing methionine-derived specialized bioactive compounds known as aliphatic glucosinolates (GLs). GLs can also be derived from other amino acids and are important key players in the plant’s natural defense system against herbivores and microorganisms. A key player in methionine regulation has been identified as the first committed step in de novo biosynthesis of methionine which is catalyzed by cystathionine gamma-synthase (CGS). Two regulatory motifs have been identified in the N-terminal part of the CGS[2]. Mutations in one of the regulatory motifs result in over-accumulation of methionine to up to 60-fold higher levels than found in wild-type Arabidopsis plants[3]. Analysis of the levels of aliphatic glucosinolates in these mutants showed an increase of up to 4-fold compared to levels in wild-type plants while GLs derived from tryptophan showed no such over-accumulation. Expectantly, other methionine metabolites such as SAM also over-accumulated in these Arabidopsis mutants. Currently, we are investigating Arabidopsis lines expressing CGS-YFP fusion proteins under control of the native promoter. Comparison of CGS localization with the localization of proteins involved in glucosinolates biosynthesis will enable us to elucidate potential spatial and/or temporal patterns that are responsible for the re-allocation of methionine from the tightly regulated methionine metabolism into the biosynthesis of aliphatic glucosinolates. First results indicate a distinct pattern for CGS localization in different tissue. 1. Ravanel, S., et al., Methionine metabolism in plants: chloroplasts are autonomous for de novo

methionine synthesis and can import S-adenosylmethionine from the cytosol. Journal of Biological Chemistry, 2004. 279(21): p. 22548-57.

2. Hacham, Y., T. Avraham, and R. Amir, The N-terminal region of Arabidopsis cystathionine gamma-synthase plays an important regulatory role in methionine metabolism. Plant Physiology, 2002. 128(2): p. 454-62.

3. Inaba, K., et al., Isolation of an Arabidopsis thaliana Mutant, mto1, That Overaccumulates Soluble Methionine (Temporal and Spatial Patterns of Soluble Methionine Accumulation). Plant Physiology, 1994. 104(3): p. 881-887.

56

Posters: Signalling and Cellular Trafficking

VPS9a mediates pre- and post-invasive disease resistance against powdery mildew infection Mads Eggert Nielsen1, Gerd Jürgens2, Hans Thordal-Christensen1

1 Plant and Soil Science Section, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark

2 Developmental Genetics, Center for Plant Molecular Biology, University of Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen, Germany Plants defend themselves against pathogenic microbes by establishing several chemical and physical barriers at the time of attack which effectively prevents certain pathogens from spreading. Resistance to powdery mildew fungi are some of the best described disease resistance processes where several of the components involved have been identified. Here we describe VPS9a, the guanine-nucleotide exchange factor for Rab5 like GTPases, as a new player in this type of innate immunity, required for both pre- and post-invasive resistance. We have found that VPS9a mediates a new pre-invasive resistance pathway which acts in concert with the previously described pathways defined by PEN1 and PEN2. Moreover, VPS9a is required for the formation of a post-invasive barrier, named the encasement, which forms around the invading fungus. For the first time, we show here that the encasement serves a direct role in post-invasive resistance to both adapted- and non-adapted powdery mildews. Formation of the encasement is a direct consequence of the activation of the VPS9a substrate, ARA7, which is a key process regulating a number of developmental cues. In contrast, VPS9a dependent pre-invasive resistance is not dependent on ARA7 activation showing that the pre- and post-invasive resistance require different requirement for VPS9a most likely by involving activation of an unidentified Rab GTPase.

57

Posters: Signalling and Cellular Trafficking

Specific activation of MYB transcription factors is regulated by subcellular protein-protein interactions in Arabidopsis Marie Pireyre, Barbara Halkier, Meike Burow

DynaMo Center of Excellence, Copenhagen Plant Science Center, Department of Plant and Environmental Sciences, University of Copenhagen Plants are sessile organisms and have developed complex regulatory networks to cope with their ever-changing environment. In those networks, transcription factors are keys to integrate information and trigger specific transcriptional programs. In response to external cues, six R2-R3 MYB transcription factors induce transcription of the biosynthetic machinery of chemical compounds called glucosinolates. Glucosinolates are Brassicales-specific specialized metabolites involved in defense and immunity. Approximately 35 different glucosinolate structures have been described in Arabidopsis. Individual MYBs induce production of different classes of glucosinolates in a trigger specific manner, and their interplay controls ratios and quantities. This is thought to be due to their-specific binding to DNA elements through the R2R3 domain. While the R2R3 domain is conserved among the six MYBs, the C-terminal domain is highly variable. Thus, we propose that MYB specificity could be modulated by trigger-specific protein-protein interactions (PPIs) and subcellular relocalization rather than by DNA binding specificity only. To learn more about the influence of the DNA binding and the PPI domains on the control of distinct glucosinolate profiles, we have generated stable plants expressing chimeric MYB proteins in myb knock-out backgrounds and determined the relative abundance of glucosinolate classes induced by each chimeric MYB. Under the hypothesis that binding partners provide additional functional specificity, we have conducted an untargeted Yeast-Two-Hybrid screen. This approach has identified membrane-bound partners that might retain the MYBs in the cytoplasm until rapid activation of the glucosinolate pathway is required. Finally, mimicking plant’s defense compounds accumulation allowed the identification of factors interacting with the MYBs and potentially involved in the regulation of their transcriptional activity. Combining different methods led to identification of numerous interactors that may provide response specificity in transcription factor activity. In contrast to recent findings, we did not detect any other transcription factors but rather proteins involved in post-transcription regulation and subcellular organization, suggesting a more complex layer of regulation than DNA-binding alone. Understanding those mechanisms is important to have a better view on how the plants modulate their chemotype and thus increase performance towards pathogens and herbivores.

58

Posters: Signalling and Cellular Trafficking

Discovering the mechanisms behind cross-kingdom RNA transfer: Host-Induced Gene Silencing (HIGS) in the Arabidopsis powdery mildew fungus Golovinomyces orontii

Marijn Knip, Hans Thordal-Christensen Defence Genetics Group, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg, Denmark In recent years it has come to light that many closely associated species, often from very different branches of the tree of life, can exchange RNA molecules that can influence gene expression. This phenomenon has been especially well studied in plant-pathogen interactions, where it has been used to induce gene-silencing in the pathogens, by expression of double-strand RNA-generating constructs in the infected plants. This system is called Host-Induced Gene Silencing (HIGS) and has been shown to be effective in down-regulating important genes in various pathogens, such as the barley powdery mildew fungus (Blumeria graminis), Fusarium graminearum, different oomycetes, insects and nematodes. Thereby, it can reduce the growth of pathogens and pests, and be used as resistance strategy. It appears that HIGS involves that RNA is selected for transfer, packaged and transported to the adjacent organism. However, how this occurs is not known. We are creating an experimental setup aiming to lift the veil on the processes involved in HIGS. For this we have shown that HIGS can occur between Arabidopsis thaliana and the powdery mildew fungus (Golovinomyces orontii), and we have identified different plants that display resistance to G. orontii by expression of an RNA-hairpin generating construct, targeting the P450 gene CYP51 in the fungus. We will use this platform to do a forward-genetic screen to identify HIGS-related genes.

59

Posters: Signalling and Cellular Trafficking

AtGTR3 is an indole-specific glucosinolate transporter responsible for controlling root/shoot indole glucosinolate distribution Morten Egevang Jørgensen, Xu D, Olsen CE, Nour-Eldin HH and Halkier BA

DynaMo Centre of Excellence, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, 1871 Frederiksberg C, Denmark Glucosinolates (GLS) are essential defense compounds and especially indole GLS have recently received substantial attention in a plant-pathogen context [1, 2]. Two GLS transporters, NPF2.10 (AtGTR1) and NPF2.11 (AtGTR2), are responsible for translocation of GLS to the seeds and for shaping the root/shoot profile of aliphatic GLS by phloem and xylem mediated bi-directional transport [3, 4]. Intriguingly, the root-shoot profile of indole GLS was not determined by AtGTR1 and AtGTR2 as evidenced by grafting experiments with stocks and scions from biosynthetic null and transport mutants [3]. This indicates the existence of additional GLS transporters in A. thaliana specific for indole GLS. We have identified a transporter responsible for controlling the indole GLS root/shoot profile in planta by employing a functional genomics approach in Xenopus laevis oocytes. Characterization of the identified transporter by Two Electrode Voltage Clamp (TEVC) electrophysiology and LCMS based uptake assays in X. laevies shows high-affinity active uptake of indole GLS in contrast to aliphatic GLS which were not actively transported. Micrografting of GLS transporter and biosynthesis knockout mutants demonstrate a role for this newly identified transporter in shaping the root/shoot indole GLS profile. The physiological role of an indole GLS specific transporter is discussed in the context of AtGTR1 and AtGTR2.

1. Bednarek, P., et al., A Glucosinolate Metabolism Pathway in Living Plant Cells Mediates Broad-Spectrum Antifungal Defense. Science, 2009. 323(5910): p. 101-106.

2. Halkier, B.A. and J. Gershenzon, Biology and biochemistry of glucosinolates. Annual Review of Plant Biology, 2006. 57: p. 303-333.

3. Andersen, T.G., et al., Integration of Biosynthesis and Long-Distance Transport Establish Organ-Specific Glucosinolate Profiles in Vegetative Arabidopsis. Plant Cell, 2013.

4. Nour-Eldin, H.H., et al., NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature, 2012.

60

Posters: Signalling and Cellular Trafficking

Chloroplastic isoprenoid biosynthesis is specifically regulated by the protein quality control formed by chaperones and proteases Pablo Pulido1,3, Dario Leister1,2, Manuel Rodriguez-Concepcion3

1Copenhagen Plant Science Centre, PLEN, UCPH, (Denmark),

2Ludwig-Maximilians-

Universität München (Germany), 3Centre for Research in Agricultural Genomics (Spain)

Plastids provide plants with biochemical pathways that are not found in other eukaryotic kingdoms. For example, isoprenoid precursors are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids, whereas animals and fungi synthesize these essential metabolites using a completely unrelated pathway which is also used by plants to produce cytosolic and mitochondrial isoprenoids. MEP-derived isoprenoids include compounds essential for photosynthesis (such as carotenoids and the side chain of chlorophylls, tocopherols, plastoquinone and phylloquinones) and growth regulation (including the hormones gibberellins, cytokinins, strigolactones and abscisic acid). Many plastidial isoprenoids also have nutritional and economic relevance. A fine control of MEP pathway enzyme levels takes place at the post-transcriptional level, but little is known about the specific mechanisms behind this regulation.

We recently showed that the Arabidopsis thaliana J-protein J20 interacts with inactive forms of the first enzyme of the MEP pathway, deoxyxylulose 5-phosphate synthase (DXS), to deliver them to the Hsp70 chaperone for eventual activation (which involves folding or refolding) or degradation (which involves unfolding prior to proteolytic cleavage) (Pulido et al. 2013). The mechanism behind this dual role of the J20/Hsp70 system, however, remained unknown.

Because Hsp100 chaperones have been shown in other systems to participate in both refolding and degradation of protein clients, here we investigated whether the four Hsp100 isoforms found in Arabidopsis chloroplasts (ClpB3, ClpC1, ClpC2 and ClpD) participated in the J20/Hsp70-mediated control of DXS folding and accumulation. We found that Hsp70 and ClpB3 chaperones are indeed able to interact and collaborate in the refolding of DXS. After demonstrating that DXS is a target of the Clp protease complex (but not of other plastidial proteases such as Lon, Deg, or FtsH), we next showed that interaction of Hsp70 with ClpC1 (another Hsp100 chaperone that, unlike ClpB3, harbors a domain to bind to Clp protease subunits) contributes to the Clp-mediated degradation of DXS independent of ClpS, the only Clp protease adaptor reported to date. Besides demonstrating the existence of a J-protein/Hsp70 pathway delivering substrates to the Clp protease in Arabidopsis chloroplasts, our data reveal the molecular components of the protein quality control pathways involved in maintaining appropriate levels of active DXS enzyme inside plastids, hence allowing individual plastids to finely adjust the MEP pathway flux to their changing metabolic requirements. References: Pulido P, Toledo-Ortiz G, Phillips MA, Wright LP, Rodriguez-Concepcion M (2013). Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control. Plant Cell 25, 4183-4194.

61

Posters: Signalling and Cellular Trafficking

Secondary metabolites in almond flowers Jorge Del Cueto Chocano1,2, Carl Erik Olsen2, Birger Lindberg Møller2, Federico Dicenta1 and Raquel Sánchez-Pérez2 1 Plant Breeding Department, CEBAS-CSIC, P.O. Box 164, 30100 Campus Universitario de Espinardo, Murcia, Spain 2 Plant Biochemistry Lab, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark *e-mail: rasa@plen.ku.dk Secondary metabolites i.e. cyanogenic glycosides are present in more than 3,000 plant species, acting as defense compounds against herbivores. Upon disruption of tissue containing cyanogenic glucosides, the toxic compound hydrogen cyanide is released. The two cyanogenic glucosides present in almond are prunasin and amygdalin. Amygdalin is accumulated in the bitter almonds and, prunasin, its precursor, in immature seeds and in the vegetative parts of the tree as leaves, stems, and roots. For the first time, prunasin has been found during flowering, starting in the dormant stage in almond, suggesting a new function for these secondary metabolites. From November 2013 to March 2014 in the experimental orchard of CEBAS-CSIC, in Santomera (Murcia, South-East Spain), the content of cyanogenic glucosides was analyzed by LC-MS/MS using five almond cultivars differing in their flowering time: Achaak and Desmayo (early flowering time), S3067 and Lauranne (late flowering time), and Penta (extra late flowering time). The two main cyanogenic glucosides, prunasin and amygdalin, were found in all the varieties analyzed, and the highest was found in the bitter variety (S3067). Moreover, the highest concentration of prunasin was observed right after flowering took place for all the five cultivars, suggesting that this compound could play an important role in flower development. Acknowledgements This study was financed by the project “Almond breeding” by the Spanish Ministry of Economy and Competiveness and the Villum Foundation. Keywords: Prunasin, amygdalin, dormancy, flowering time, LC-MS/MS

62

Posters: Signalling and Cellular Trafficking

COP1 E3 ligase protects HYL1 to retain microRNA biogenesis Seok Keun Cho, Samir Ben Chaabane, Pratik Shah, Christian Peter Poulsen & Seong Wook Yang Laboratory of Plant Biochemistry, Department of Plant and Environmental Sciences, Center for UNIK Synthetic Biology, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1817 Frederiksberg C, Copenhagen, Denmark E-mail: swyang@plen.ku.dk Constitutive photomorphogenic 1 (COP1) is a RING-finger E3 ligase that plays a central role in photomorphogenesis by destabilizing many light-regulated transcription factors and photoreceptors. Here, we reveal a novel function for COP1 E3 ligase in controlling global miRNA biogenesis in Arabidopsis thaliana. In cop1 mutants, the level of miRNAs is dramatically reduced because of the diminution of HYPONASTIC LEAVES 1 (HYL1), an RNA-binding protein required for precise miRNA processing. HYL1 is destabilized by an unidentified protease, which we tentatively call protease X, that specifically cleaves the N-terminal region from HYL1, thus neutralizing its function. Our results further show that the cytoplasmic partitioning of COP1 under light is essential to protect HYL1 against protease X. Taken together, we suggest a novel regulatory network involving HYL1, protease X, COP1 and light signalling that is indispensable for miRNA biogenesis in Arabidopsis thaliana.

63

Posters: Signalling and Cellular Trafficking

In vivo calcium signaling induced by secreted RALF peptide hormones. Sisse K. Gjetting1,2, Khalid Mahmood3 and Anja T. Fuglsang1,2

1 Section for Transport Biology, Dept. of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 2 PUMPKIN Research Centre for membrane pumps in cells and disease 3 Dept. of Agroecology, Flakkebjerg, Aarhus University RALF (rapid amplification factor) is a family of small, secreted peptide hormones with great impact on plant growth and development. They are plant-specific and characterized by their ability to cause an instant and dramatic increase of apoplastic/extracellular pH. Recently, the receptor FERONIA was shown to inactivate the plasma membrane H+-ATPase upon interaction with RALF1 by phosphorylation of the Ser899 residue (Haruta 2013). How the signal is transduced from FERONIA to AHA2 is still unresolved, but may include a brief, rapid increase in cytosolic calcium, as observed in previous experiments (Haruta 2008). Research in our lab showed that the secreted peptide PSY1, which activates AHA2 via the receptor kinase PSY1R, caused a transcriptional upregulation of two RALF-like isoforms, RALFL33 and RALFL35 in Arabidopsis. PSY1 is involved in control of root cell elongation, and we suggest that the upregulated RALF isoforms may act in a feedback mechanism with PSY1 to control root cell elongation. To investigate this hypothesis, we have taken a live imaging approach to dissect the timing of cellular ion-dynamics in response to external RALF application in Arabidopsis roots. Using genetically encoded biosensors for calcium (YC3.6) and pH (apo-pHusion), and a root-perfusion confocal microscope setup (Gjetting et al. 2012), we demonstrate a detailed, cell- and isoform-specific cytosolic Ca2+ response to RALF perfusion. In response to RALF33 perfusion, an instant Ca2+-spike, traveling basipetally was seen, followed by a second Ca2+-wave in the epidermis and cortex, which correlated with apoplastic alkalinization, whereas a third, much slower wave was seen in the endodermis/stele. RALF35 perfusion resulted in one Ca2+-wave corresponding to the second epidermis/cortex wave of RALF33. The spatiotemporal variation indicates that RALF33 and RALF35 isoforms could be involved in different cellular responses, but may both contribute to calcium-mediated inactivation of AHA2, and subsequent apoplastic alkalinization. The localization of the responses to the proximal elongation zone could also be in agreement with a role as a brake for PSY1-mediated cell elongation. In addition, highly variable Ca2+ signals in response to the two RALF isoforms were also observed in leaves and guard cells, where oscillations, waves or spiking is seen in some areas of cells whereas others remain unresponsive.

64

Posters: Signalling and Cellular Trafficking

Monitoring of Hormone Dynamics in the Plant Body of Arabidopsis thaliana Sophie Lambertz1,2, Morten Egevang Jørgensen1,2, Meike Burow1,2, Dietmar Geiger3, Barbara Ann Halkier1,2, Hussam Hassan Nour-Eldin1,2

1 DNRF Center DynaMo, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvadsensvej 40, 1871 Frederiksberg C, Denmark 2 CPSC, Department of Plant and Environmental Sciences, Faculty of Science, University of

Copenhagen, Thorvadsensvej 40, 1871 Frederiksberg C, Denmark 3 Julius-von-Sachs-Institute, Molecular Plant Physiology and Biophysics, University of

Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany Plants’ growth, development, and interaction with their changing environment rely on finely-tuned signaling mediated by phytohormones. Crucial regulatory elements are spatio-temporal hormone composition in different tissues and organs, ultimately leading to appropriate responses upon stimuli. Transport elements, including long-distance and intercellular movement of the signaling molecules in the plant, are essential components of the composition. In an in vitro approach, we identified a transport protein from Arabidopsis thaliana that appears capable of facilitating multiple phytohormones simultaneously. Phenotypical analyses of a knock-out line indicate that this transporter is involved in hormone homeostasis upon external stimuli. We are therefore monitoring the composition of different phytohormones with LC-MS/MS approaches and hormone signaling activity with high spatio-temporal resolution.

65

Posters: Signalling and Cellular Trafficking

Idenfication of novel plant defence components using pen1 syp122 as a tool

Wenjun Xie, Andrea Lenk, Carsten Pedersen and Hans Thordal-Christensen

Dept. of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C

Email xie@plen.ku.dk

Plants protect themselves from pathogens by activating a defence signalling network. The Arabidopsis double mutant pen1 syp122 is tiny and mimics a response as if it was attacked by pathogens. Using EMS as mutagen on pen1 syp122, a triple mutant scan was performed in the M2 generation. More than 200 partially rescued pen1 syp122 ssd (suppressor of syntaxin-related death) homozygous lines were collected. SSD genes are typically required for pathogen defence. In one triple mutant, 9s9, a novel defence component SSD6 was identified by Mutmap. Knocking out SSD6 in pen1 syp122 by T-DNA insertion and overexpression of SSD6 in 9s9 confirmed that mutating of this gene rescues pen1 syp122. Four other ssd6 allelic mutants were identified by complementation test and sequencing. All plant species have homologues of SSD6, but none of them has been studied. We currently make subcellular localization of wild-type and mutant versions of SSD6 in order to help unraveling its function.

66

Posters: Biomass production and processing

Cultivation of photoautotrophic plant suspension cultures in photobioreactors Anna Segečová1, Jan Červený1, Thomas Roitsch1,2

1 Department of Adaptation Biotechnologies, Global Change Research Centre AS CR, v.v.i., Drásov 470, 664 24 Drásov, Czech Republic 2 Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre,

University of Copenhagen, Højbakkegård Allé 13, 2630 Taastrup, Denmark Plant suspension cultures represent reduced model of a complex and heterogenous system of higher plants. They are often used in biotechnology for production of highly valuable secondary metabolites. They consist of single cells or small clusters forming uniform population directly accessible to exogenous stimuli which makes them ideal for experimental use. Plant suspension cultures thus pose a significant and promising experimental and biotechnological system.

Most of the plant suspension cultures used in biotechnology are heterotrophic, because of their higher growth rate and a variety of secondary metabolites produced. However, they require sugar for their growth, which makes them more susceptible to contamination and resources demanding. On the other hand, photoautotrophic suspension cultures are able to grow without sugars, with CO2 as the sole carbon source, but in addition require light source. The presence of chloroplasts makes it possible to use this cultures for production of secondary metabolites with biosythesis pathways located in plastids. The presence of chlorophyll enables to use the highly sensitive method of pulse amplitude modulated chlorophyll fluorescence to assess perturbations of primary carbohydrate metabolism. Photoautotrophic plant suspension cultures require elevated concentration of CO2 for their growth. In classical cultivation setup, this is usually ensured by carbonate buffer located in a flask joint to another flask with the culture. This makes the cultivation and maintenance laborious and less attractive to researchers. In our poster we present a novel and efficient approach to cultivation of photoautotrophic plant suspension cultures in photobioreactors originally designed for cultivation of microalgae. Photobioreactors allow us to cultivate photoautotrophic suspension cultures in sterile and controlled conditions under CO2 enriched atmosphere, and to monitor several growth and cultivation parameters online. This makes the instrument ideal for both optimization and standardization of the cultures growth. Cultivation of photoautotrophic cultures in photobioreactors also provides larger amounts of homogenous biomass than the standard cultivation techniques. This biomass can be further used for subsequent analytical or upscaling experiments.

67

Posters: Biomass production and processing

Modified brassinosteroids accumulation affects saccharification in Brachypodium distachyon Daniele Silvestro, Jozef Mravec, Hong-Zhang Chen, Louise Nancke, Jesper Harolt, William Willats, Poul Erik Jensen Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Large effort is currently made to tailor the cell wall-derived biomass by manipulating intrinsic regulatory mechanisms. Brassinosteroids (BR) are crucial plant hormones especially determining the size, architecture, fertility and overall productivity of plants and are also coupled to the cell wall-sensing signaling. Here we show that endogenous modification of BR in the grass model Brachypodium leads to the synthesis of cell walls with significantly lower recalcitrance while not compromising the plant growth and sensitivity to pathogens. The biological base of this observation is the role of BR in secondary cell wall formation demonstrated on the BR receptor mutants and by in silico approach. We generated a new type of genetically designed cell walls with superb performance both in cellulose yield and saccharification efficiency via the overexpression of a BR biosynthesis gene in Poales-belonging crops. This could represent a potential tool for improved second generation biofuel production.

68

Posters: Biomass production and processing

Plant Factory Hybrid Biopolymers for Future Materials-Related Applications Domenico Sagnelli1,2, Kim H Hebelstrup2, Massimiliano Carciofi2, Shahnoor S Shaik1, Andreas Blennow1 1 Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen 2 Aarhus University, Faculty of Agricultural Sciences, Department of Genetics and Biotechnology Bio-derived polymers, like starch, form the basis for the next generations of advanced and environmental friendly materials and prebiotic ingredients. Plasticized starch (TPS, thermoplastic starch) is processable using conventional plastic processing techniques, such as extrusion and injection molding. Mechanical performance and water resistance are improved by blending with other polymers (Averous, 2004), e.g. Mater-Bi™ TPS contains synthetic polycaprolactone. Alternatively an in planta designed bio-grafted hybrid starch alone or combined with lipid complexes, for water resistance, can be sufficiently functionalised. This method can provide a technological break-through and transform raw starch into more plasticized, yet stable, states. Starch films with superior form stability can be produced using transgenic approach (Gillgren et al., 2011) and we generated over-expressor and suppressor barley lines with radically improved starch, including a water resistant amylose-only, AmOn, (Carciofi et al., 2011; 2012) and a pre-amorphisised hyper-phosphate (HiPho) starch (Carciofi et al., 2011). Employing a combined USER cloning and novel callus phenotype screening approach these starches will be exploited to generate hybrid fructan-starch polysaccharides by expressing designer fructosyl transferases (EC 2.4.1.21) to generate biomaterials with thermoplastic (TPS) and shape memory assets and compared to native starch and traditional TPS. For hybrid enzymes catalytic efficiency must be enhanced and local substrate concentration will be increased by introducing starch binding domains (SBDs) to enhance activity at the starch granule surface.

L. Avérous (2004). “Biodegradable multiphase systems based on plasticized starch: a review.” J. Macromol. Sci. Polym. Rev. C4/3, 231-274

M. Carciofi, S.S. Shaik, S.L. Jensen, A. Blennow, J.T. Svensson, É. Vincze, K.H. Hebelstrup

(2011), Hyperphosphorylation of cereal starch, J. Cereal Sci., 54/3, 339-346,

M. Carciofi, A. Blennow, S.L. Jensen, S.S. Shaik, A. Henriksen, A. Buléon, P.B. Holm, K.H. Hebelstrup (2012). Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules. BMC Plant. Biol. 12, 223.

69

Posters: Biomass production and processing

A systems biology analysis of early-stage sugar beet storage root development Eric van der Graaff1, Alexandra Jammer2, Alfonso A. Albacete3, Wolfgang Koch4, Britta Schulz4 & Thomas Roitsch1,5

1 Department of Plant and Environmental Sciences, Crop Science Section, Copenhagen University, Taastrup, Denmark 2 Institute of Plant Sciences, Karl-Franzens-University Graz, Austria 3 CEBAS-CSIC, Campus de Espinardo, Espinardo, Murcia, Spain 4 KWS SAAT AG, Einbeck, Germany 5 Global Change Research Centre, CzechGlobe AS CR, Drásov, Czech Republic In sugar beet (Beta vulgaris L.), sucrose is not only the major transport form of assimilates, but it also accumulates at high concentrations in storage roots. Since sugar beet studies so far concentrated on source-sink relations in mature plants with a fully developed storage root, the metabolic changes occurring during the initial phase of sugar beet storage root development have not been systematically addressed. In the presented study, a systems biology analysis, centered around physiological phenotyping, was conducted for early-stage sugar beet storage root development. The activities for different key enzymes of carbohydrate metabolism were analyzed in developing storage roots over the first 80 days after sowing, complemented with an in situ localisation of selected enzyme activities, expression analyses for the respective transcripts, anatomical investigations, and soluble sugar, hexose-phosphate and phytohormone profiles. Based on the accumulation dynamics of biomass and sucrose, as well as on anatomical parameters, the early phase of storage root development can be subdivided into two stages (prestorage stage; secondary growth and sucrose accumulation stage), each of which is characterised by distinct metabolic, transcriptional and phytohormonal signatures. The onset of secondary growth and sucrose storage is preceded by a phase of metabolic transition. Acknowledgements: The financial support by KWS SAAT AG (Einbeck, Germany) to A.J. is gratefully acknowledged. .

70

Posters: Biomass production and processing

Structural cell wall proteins of winter wheat (Triticum aestivum) affected by nitrogen status Jan J. J. Van Hecke, Christian Bukh, William G. T. Willats and Jan K. Schjørring Department of Plant and Environmental Science, SCIENCE, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg DK E-mail: jvh@plen.ku.dk Cell wall polysaccharides (sugars) and lignin are essential parts of the mature plant cell wall. Cell wall proteins on the other hand are mostly overlooked due to the fact that they only contribute 1% to the cell wall dry matter of monocots[1]. However, cell wall proteins facilitate diverse integral functions e. g. during plant development, defense and in response to environmental cues[2]. Cell wall proteins are the only substances within the cell wall which directly contain nitrogen. This, therefore, suggests a link between the abundance of cell wall proteins and the nitrogen status of the plant. Structural proteins, a subgroup of cell wall proteins, help to maintain the physical properties of cell walls by interlinking cell wall components with each other[3]. In this newly started Ph.D. project we investigate the role of structural proteins with respect to cell wall properties and their response to different nitrogen regimes. The main aim of the project is to create fundamental plant knowledge about the formation, distribution and interaction of structural cell wall proteins in response to changes in plant nitrogen status. The experimental work will focus on Brachypodium distachyon and wheat. Brachypodium is chosen in order to take advantage of the knowledge in this species about genes involved in cell wall biosynthesis as well as the availability of mutants with modified cell wall properties. The results will be validated in winter wheat which will be grown with different nitrogen applications under field conditions and harvested at several growth stages from flowering to maturity. Changes in the abundance and localization of cell wall proteins in response to nitrogen fertilization will be investigated by immunochemistry. In addition high throughput screening of biomass (modified CoMMP) will be performed in order to create a tool to help breeders to select for favored cell wall protein traits. Acknowledgement: The study is funded under The Bio-Value Strategic Platform for Innovation and Research (www.biovalue.dk), co-funded by the The Innovation Foundation, case no: 0603-00522B [1] Vogel, J. Unique aspects of the grass cell wall. Current opinion in plant biology 11, 301–7 (2008).

[2] Albenne, C., Canut, H. & Jamet, E. Plant cell wall proteomics: the leadership of Arabidopsis thaliana. Frontiers in Plant Science 4 (2013).

[3] Allan M. Showalter. Structure and function of plant cell wall proteins. The Plant Cell Vol. 5, 9-23 (1993).

71

Posters: Biomass production and processing

Impact of Manganese stress on Photosystem I and II in Arabidopsis thaliana Ken Suszkiewicz Krogholm, Søren Husted and Poul Erik Jensen

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Future sustainable improvement in the productivity of cropping systems depends on novel resource-efficient plant genotypes designed to match site-specific soil and climatic conditions. Deficiency in essential mineral micronutrients such as manganese (Mn), iron, zinc, copper and boron is a significant problem for crop productivity in major parts of the world. Mn is an important microelement in plant nutrition since it is involved in the function of more than 30 enzymes but especially in the oxygen evolving complex (OEC) in photosystem II. Mn is primarily located in the thylakoids where it forms the Mn cluster (Mn4CaO5) which is part of the OEC of Photosystem II. Therefore, Mn deficiency is a serious plant nutritional disorder, occurring mostly on alkaline soils that reduce the bioavailability of this metal. To investigate how the photosystems are affected by Mn stress (both Mn deficiency and Mn excess) a number of selected Arabidopsis thaliana mutants have been grown in a hydroponic system. We have tested the effect of Mn stress on the relative abundance of the proteins of the photosystems and on photosynthetic performance. Induction of Mn deficiency in the Arabidopsis thaliana mutants stn7, stn8, stn7stn8, psbp2 and psbq1psbq2 did not lead to visual nutrient deficiency symptoms, but all Arabidopsis thaliana mutants tested displayed clear phenotypes when grown under Mn stress conditions. A significant decrease in relative protein abundances of nearly all proteins of the photosystems was observed when subjected to Mn deficiency. The aim of our work is to analyze the general effects of Mn stress in Arabidopsis thaliana. In that context we employ chlorophyll fluorescence measurements in combination with an array of biochemical analyses as well as in depth study of the photosynthetic apparatus.

72

Posters: Biomass production and processing

Cell wall acetylation and plant fitness: suppressor screening of the REDUCED WALL ACETYLATION 2 reveals mutants with wild type surface permeability Lorenzo Fimognari1, Majse Nafisi1, Henrik Vibe Scheller2, Yumiko Sakuragi1

1 Copenhagen University, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, Frederiksberg 1871, Denmark 2 Joint BioEnergy Institute, 5885 Hollis St., Emeryville, California 94608, USA The goal of the project is to elucidate the role of plant cell wall acetylation in plant fitness. Acetylation of plant cell wall polysaccharides influences the production of bioethanol, and efforts are being made to alter the content of cell wall acetylation in plants. Recently the gene REDUCED WALL ACETYLATION 2 (RWA2) was isolated from the model plant Arabidopsis thaliana and was shown to be responsible for acetylation of pectins and hemicellulose (Manabe et al. 2011). The rwa2 mutant (hereafter rwa2) contained a reduced level of acetylation and, in addition, showed increased resistance to the necrotrophic fungal pathogen Botrytis cinerea (Manabe et al. 2011) as well as higher surface permeability (Nafisi M., unpublished). These findings suggested that the RWA2 gene could be a target for future genetic engineering for improved biomass quality for lignocellulosic biofuel production. However, the increased surface permeability needed to be overcome for the mutant to grow robustly in the field. To study this, we randomly mutagenized 30,000 rwa2 seeds and searched for suppressor mutants that overcome the increased permeability phenotype and successfully isolated 7 suppressor lines. This suggests that the increased surface permeability may be suppressed by secondary mutations, indicating that may exist a previously unknown genetic interaction between cell wall acetylation and surface permeability. These mutants hold keys to revealing genetic markers for better performances with respect to biofuel production without compromising plant fitness. We are currently carrying out genetic as well as physiological characterizations of these mutants.

73

Posters: Biomass production and processing

GWAS of winter wheat cultivars – identification of genes relevant for biomass and grain yield Pernille L. M. Hansen1, Jan K. Schjørring1, Birger Eriksen2, Ole Andersen2, Andrea Bellucci1, Christian Bukh1, and Søren K. Rasmussen1 1 Department of Plant and Environmental Science, SCIENCE, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg DK 2 Sejet Plant Breeding, Nørremarksvej 67, 8700 Horsens DK E-mail: plmh@plen.ku.dk Background: Wheat is grown worldwide and provides food for about 30% of the world’s population [1]. IPCC (2014) expect food demands to raise toward 2050 and together with climate changes it increases the demands for wheat breeding. Wheat straw has a huge potential as a sustainable feedstock for the production of high-value products such as functional components used in the chemical industry [2]. Considerable variation exist among genotypes with respect to straw yield and quality (levels of cellulose, hemicellulose and lignin), which have been shown to affect the sugar accessibility of mature wheat straw biomass (yellow biomass) [2-4]. The variation is genetically inherited and the degradability of straw is not linked with grain yield [3, 4] indicating that breeding for yellow biomass traits could be established without compromising yield. We aim at making breeding programs for wheat more efficient by developing single-nucleotide polymorphism (SNP) markers that can select for high biomass quality (parameters important for biorefining purposes) and high straw yield while the targets for grain yield and lodging resistance are maintained. Method: 96 winter wheat lines are grown at Sejet for a 3 year period (around 50% of the lines will be grown repeatedly). The cultivars will be SNP genotyped and phenotyped for grain and straw yield, plant height, lodging degree, sugar release ability, and a compositional analysis of the cell wall (lignin, cellulose and hemicellulose). The traits and the SNPs will then be investigated for any significant association using a genome wide association study (GWAS). GWAS results will also be used to select cultivars showing a high or low straw yield to create mapping populations, which will be analyzed by QTL mapping or linkage studies. Perspective: We expected to identify SNP markers linked with traits for increasing yields and quality of the yellow biomass and hopefully to identify the respectively candidate genes. Acknowledgement: The study is funded under The Bio-Value Strategic Platform for Innovation and Research (www.biovalue.dk), co-funded by the The InnovationFoundation, case no: 0603-00522B [1] Eversole K, Feuillet C, Mayer KF, Rogers J. Slicing the wheat genome. Introduction. Science 2014;345:285-7. [2] Collins SRA, Wellner N, Bordonado IM, Harper AL, Miller CN, Bancroft I, et al. Variation in the chemical composition of wheat straw: the role of tissue ratio and composition. Biotechnol Biofuels 2014;7. [3] Jensen JW, Magid J, Hansen-Moller J, Andersen SB, Bruun S. Genetic variation in degradability of wheat straw and potential for improvement through plant breeding. Biomass Bioenerg 2011;35:1114-20. [4] Lindedam J, Andersen SB, DeMartini J, Bruun S, Jorgensen H, Felby C, et al. Cultivar variation and selection potential relevant to the production of cellulosic ethanol from wheat straw. Biomass Bioenerg 2012;37:221-8.

74

Posters: Biomass production and processing

Low silicon Brachypodium mutant as a tool for investigation of silicon effects on cell wall composition Sylwia E. Głazowska, Emiko Murozuka, Christian Bukh, William G.T. Willats, Jan K. Schjoerring Department of Plant and Environmental Sciences, Thorvaldsensvej 40, Copenhagen, 1871 Frederiksberg C, Denmark (glaz@plen.ku.dk) Members of Poaceae family are important sources of food and feed. Recently they’ve been also proposed as promising lignocellulose biomass for energy production. However grasses occur to actively transport and accumulate silicon at a high level constituting even up to 10% of the plant dry matter. Silicon plays role as beneficial element in plants by improving their growth and resistance towards biotic and abiotic stresses, but also has a negative effect on the enzymatic digestibility and thus reduces the conversion efficiency of lignocellulosic biomass to monomeric sugars. Cell wall is known deposition site of silicon, but knowledge about the specific binding forms of Si, functional properties in relation to cell wall structure and composition is still limited. On this background, the objective of our research is to understand Si deposition mechanism and interactions with cell wall components in grass species. In order to investigate these aspects we have isolated and characterized Brachypodium mutants defective in silicon influx (bdLsi1-1). Wild type (wt) plants and mutants were grown in soil until maturity and the elemental content of dry matter, including Si, measured by inductively coupled plasma optical emission spectrometry (ICP-OES). Finally cell wall composition was determined by comprehensive microarray polymer profiling (CoMPP). This method enables fast mapping of cell wall components by using monoclonal antibodies designed to recognize specific glycan-epitopes. The Si concentration in bdlsi1-1 mutants was on average 0.17% of the dry matter, which was 82% lower compared to the wt. Main deposition site of silicon occur to be leave/sheath fraction comprising 47% and 59% of total silicon in mutant and wt respectively. The CoMPP intensity profile reviled several alterations both in extracted pectin and hemicellulose fractions. The highest signals and the most pronoun modifications were observed for pectin antibodies in leave/sheath. At the same time signal for hemicellulose antibodies was more equally distributed between the samples both mutant and wild type, as well within tissue types. Our preliminary data shows that Brachypodium distachyon bdlsi1-1 mutant is a suitable tool for further studies of Si deposition and interactions with cell wall components, as well as the consequences for enzymatic degradability of cell walls. We are currently investigating these relationships in further details, focusing on soil grown plants with a larger span in cell wall Si concentrations between wt and mutants.

75

Posters: Plant Breeding – Quality, Productivity and Diseases

Peptide aptamer-mediated resistance to barley powdery mildew Agnieszka Siwoszek1, Carsten Pedersen1, Paul KoFerrigno2, Hans Thordal-Christensen1

1 Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Denmark 2 Visiting Professor, Leeds Institute of Molecular Medicine, Leeds University/Chief Scientific Officer, Avacta Life Sciences, UK Barley powdery mildew disease is caused by a biotrophic fungus Blumeria graminis f.sp. hordei (Bgh). Resistant varieties of spring barley are widely used; however their exploitation has been hampered by pleiotropic effects of resistance genes (necrosis, reduced grain yield). In this study, we propose alternative resistance to barley powdery mildew, based on peptide aptamer technology. Peptide aptamers are recombinant proteins that bind to and inactivate a protein of interest. In case of barley powdery mildew the common feature of barley powdery mildew secreted proteins is YxC motif. Therefore, we chose the YxC motif of effector candidates (CSEPs) as a target for peptide aptamers to achieve a durable resistance against barley powdery mildew. We did a yeast two-hybrid screen to identify peptide aptamers targeting the YxC motif of CSEPs. This interaction was confirmed by bimolecular fluorescence complementation assay in Nicotiana benthamiana. We made point mutations within the YxC motif, which confirmed the interaction to be motif specific. This suggests that the identified peptide aptamers should be able to target the majority of CSEPs. Localization studies showed that both peptide aptamers (YFP-tagged) and CSEPs (mCherry-tagged) are localized to the plant cytosol and nucleus and coexpression of both does not affect the localization of either of proteins. Interestingly, when YFP-tagged peptide aptamer was fused to a nuclear localization signal and coexpressed with mCherry-tagged CSEP, we could see both proteins localizing to the nucleus. This suggests that the peptide aptamer is forming a complex with the fungal effector and the binding is strong enough to change the localization of the effector protein. Transient overexpression of selected peptide aptamers in barley epidermal cells resulted in lower susceptibility to barley powdery mildew. Arabidopsis thaliana Bgh-susceptible mutants were used to create overexpression lines of selected peptide aptamers. Those plants will be used to verify disease resistance mediated by peptide aptamers.

76

Posters: Plant Breeding – Quality, Productivity and Diseases

Functional analyses of barley powdery mildew effector candidates Carsten Pedersen, Ali Ahmed, Geziel Aguilar, Wen-Jing Zhang and Hans Thordal-Christensen Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen Previous studies have identified around 500 Bgh effector candidates (CSEPs) from the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) and they are believed to manipulate host defence response and facilitate susceptibility (Spanu et al. 2010, Pedersen et al. 2012), but functional studies are hampered by the fact that Bgh is an obligate biotroph and recalcitrant to genetic transformation. To study the function of effector candidates we therefor employ particle bombardment as a transient technique for silencing effector candidate genes in the pathogen by host-induced gene silencing (HIGS). We use the Yeast Two-hybrid system for screening for putative effector targets in the host and confirm interactions in planta by BiFC in N. benthamiana (Zhang et al. 2012). To further examine the interaction we use transient gene silencing (TIGS) against putative effector targets in the host to see if silencing these targets increases susceptibility. Likewise, we employ over-expression of CSEPs or host-targets to test whether this increases or reduces susceptibility. We are now trying to implement virus-based techniques for both silencing and over-expression to overcome limitations with the single-cell based particle bombardment technique. Here we will give an overview and update on the functional studies of CSEPs in our lab. Spanu, P. D., Abbott, J. C., Amselem, J., et al. including Pedersen C (2010) Genome Expansion and Gene Loss in Powdery Mildew Fungi Reveal Tradeoffs in Extreme Parasitism. Science 330: 1543-1546. Zhang W-J, Pedersen C, Kwaaitaal M, Gregersen P L, Mørch S M, Hanisch S, Kristensen A, Fuglsang A T, Collinge D B, Thordal-Christensen H (2012) Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c. Mol Pl Pathol 13: 1110-1119. Pedersen, C., E. V. van Themaat, L. J. McGuffin, J. C. Abbott, T. A. Burgis, G. Barton, L. V. Bindschedler, X. Lu, T. Maekawa, R. Wessling, R. Cramer, H. Thordal-Christensen, R. Panstruga, and P. D. Spanu. 2012. Structure and evolution of barley powdery mildew effector candidates. BMC genomics 13: 694.

77

Posters: Plant Breeding – Quality, Productivity and Diseases

Puccinia effector candidate PEC6 suppresses PAMP triggered immunity and targets adenosine kinase Changhai Liu, Carsten Pedersen, Hans Thordal-Christensen Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Stripe rust, caused by the biotrophic fungus, Puccinia striiformis f. sp. tritici (Pst), is globally the most prevalent and damaging disease on wheat. It is well known that pathogens employ effectors to facilitate infection or interfere with host defence responses. However, no effector from stripe rust has been identified until now but genome and transcriptome sequencing has revealed many effector candidates (Cantu et al. 2013 and Garnica et al. 2013).

We selected some stripe rust effector candidates, which are highly expressed in haustoria and tested their function in tobacco (Nicotiana benthamiana) and wheat (Triticum aestivum) by delivering effectors into plant cells using the type three secretion system (T3SS) in Pseudomonas fluorescens. In N. benthamiana, PEC6 (Puccinia Effector Candidate 6) can significantly suppress the ROS accumulation and callose deposition induced by P. fluorescens. Similarly, PEC6 also compromised defence induced by Pfo in wheat and enhanced susceptibility to stripe rust. Localization analysis showed that PEC6 localized at nuclear and cytoplasm in wheat and N. benthamiana leaves after transient expression by particle bombardment and agro-infiltration, respectively. By Y2H (yeast two-hybrid) and BiFC (Bimolecular fluorescence complementation), PEC6 interacts with ADK (adenosine kinase) which is involved in cytokinin interconversion.

We speculate that PEC6 might work in the PTI signalling pathway by interfering with ADK which leads to changes of cytokinin pool and compromises plant defence response. So, the next challenge is to study if PEC6 is influencing the cytokinin content and what ADK does during wheat stripe rust interaction.

78

Posters: Plant Breeding – Quality, Productivity and Diseases

The effect of the hemoglobin level in barley (Hordeum vulgare) on the interaction with both compatible and incompatible races of Blumeria graminis Chris K. Sørensen1, Massimiliano Carciofi2, Mogens S. Hovmøller1, Kim H. Hebelstrup2 1 Department of Agroecology, Aarhus University, Flakkebjerg, Denmark

2 Department of Molecular Biology and Genetics, Aarhus Universitet, Flakkebjerg, Denmark

The pathogen Blumeria graminis which causes the mildew disease on important cereals has historically represented a major threat to the barley production in Europe and still causes severe problems in other parts of the world. The problems has to a large extend been alleviated by the integration of the resistance gene Mlo in modern varieties. Approximately seventy-five percent of the Danish barley areas were in 2010 protected by this one gene. However, the experience from most host-pathogen systems is that the effect of individual resistance genes has a limited time frame in particular when the gene(s) are used in large scale. One of the most significant resent examples is the breakdown of the stem rust resistance gene Sr31 which has the potential to become a serious threat to wheat production world-wide. In order to minimize the risk of losses due to breakdown of resistance there is a great need to increase our understanding of the interaction between important pathogens and their host(s), with a particular focus on host resistance and how this is regulated and manipulated by the pathogen. Recent research have shown that the production of Nitric oxide (NO) play an important role during infection. NO seems to act as a signaling molecule in host pathways leading to a resistant reaction against the possible intruder. The study presented here is a part of a project that seeks to investigate the role of plant hemoglobin in host-pathogen interaction through the regulation of the NO-level in the plants. Previously developed transgenic Barley lines with either over- or under-expression of hemoglobin were together with wild type plants of the barley variety Golden promise inoculated with three different races of Blumeria graminis. Two of the races were barley-adapted (i.e. forma specialis hordei) and one race was wheat-adapted (forma specialis tritici). Of the two barley-adapted races one was compatible and one incompatible with the variety Golden promise due to the presence of the resistance gene Mla8 in this variety. The barley-adapted races thus interact with the barley plants in a gene-for-gene manner whereas the wheat-adapted race is expected to interact with the barley plants at a more basic PAMP-triggered level. The hypothesis is that fungal development in both the over- and under-expressing lines will differ from the wild type due to a decrease or increase of the NO-level in the leaves of these lines, respectively. Further we expect to see a difference between the two formae speciales due to the difference in mode of interaction.

79

Posters: Plant Breeding – Quality, Productivity and Diseases

Public Private Partnership for pre-breeding in perennial ryegrass: developing cultivars with suitable adaptation to future climates in the Nordic countries Cristiana Paina1, Stephen Byrne1, Petter Marum2, Christer Persson3, Anders S. Larsen4, Mika Isolahti5, Merja Veteläinen5, Rene Aavola6, Áslaug Helgadóttir7, Gintaras Brazauskas8, Muath Alsheikh2, Bo Gertsson3, Klaus K. Nielsen4, Odd Arne Rognli9, Torben Asp1 1Department of Molecular Biology and Genetics, Aarhus University, Denmark, 2Graminor, Norway, 3Lantmännen, Sweden, 4DLF-TRIFOLIUM A/S Denmark, 5Boreal, Finland, 6Jõgeva Plant Breeding Institute, Estonia, 7Faculty of Animal and Land Resources, Agricultural University of Iceland, 8Lithuanian Research Centre for Agriculture and Forestry, 9Department of Plant and Environmental Sciences, Norwegian University of Life Sciences Perennial ryegrass (Lolium perenne L.) is the main forage grass species in Denmark and south-wards in Europe due to its superior feed quality and productivity. However, at present this species is at the border of its adaptation when grown north of a line Oslo to Helsinki. The expected climate changes in Northern Europe will result in new growth conditions for forage production under an extended growth season combined with milder and rainier autumns and winters. Perennial ryegrass is expected to expand further north due to milder winters with shorter periods of snow cover. The main weaknesses are susceptibility to low-temperature pathogens, inadequate growth cessation in the autumn to allow for sufficient cold hardening and winter survival, with low persistency as the result. In order to prepare for the predicted climate changes, a Public Private Partnership for pre-breeding program has been initiated, aimed at selecting perennial ryegrass plant material for the development of cultivars with suitable adaptation to future climates in the Nordic countries. We are exploring genetic diversity and genetic structure using a collection of 383 perennial ryegrass accessions originating from different countries. The populations have been genotyped by sequencing and genetic variation in genic regions is being assessed. The accessions are phenotyped for important agronomical traits in Denmark, Sweden, Norway, Finland, Iceland, Estonia, and Lithuania. Data being generated in this project will enable us to explore genotype by environment interactions in perennial ryegrass populations.

80

Posters: Plant Breeding – Quality, Productivity and Diseases

Barley plants over-expressing the NAC transcription factor gene HvNAC005 shows an early senescence and drought-mimicking phenotyp Dagmara Podzimska-Sroka1, Collette Matthewman1, Michael W. Christiansen1, Charlotte O’Shea2, Søren Lindemose2, Karen Skriver2, Per L. Gregersen1

1 Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark, 2 Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhage N, Denmark The NAC transcription factor family constitutes one of the largest transcription factor families in plants, with more than one hundred members in Arabidopsis. Several NAC genes have been reported to be involved in regulation of developmental and stress processes, in particular hormone-related processes taking place e.g. during drought responses and during senescence. We have studied senescence-associated NAC genes in barley (1,2) and report here on results from one of these, HvNAC005. This gene is highly up-regulated during natural senescence at an early stage, but not during dark-induced senescence. In addition, it is induced by ABA, also supported by putative ABA-responsive elements in its promoter region. The transcriptional activity of the C-terminal part of the HvNAC005 TF was verified in a yeast one-hybrid system. A protein binding microarray study showed binding of the NAC domain of HvNAC005 to oligos with the consensus core sequence TTnCGT, also known from other NAC TFs (3). We generated a number of transgenic barley lines over-expressing HvNAC005, which all showed a typical phenotype with short tillers, early senescence and poor seed set. This phenotype mimicked an ABA induced drought response. We performed a microarray study on seedlings from an over-expressing line, showing up-regulation, particularly in roots, of a range of senescence-associated genes and genes involved in ABA signaling and response. qRT-PCR confirmed this up-regulation for a number of selected genes, e.g. a nuclease, a protein phosphatase 2C, and a saccharopine dehydrogenase gene. Our data demonstrated that HvNAC005 takes part in the regulation of senescence, probably by controlling genes in the ABA signaling pathway and cellular degradation pathways. It remains to elucidate its putative role during drought responses and its association with ABA signaling. 1. Christiansen MW, Holm PB, Gregersen PL (2011) Characterization of barley (Hordeum vulgare L.)

NAC transcription factors suggests conserved functions compared to both monocots and dicots. BMC Research Notes 4: 302.

2. Christiansen MW, Gregersen PL (2014) Members of the barley NAC transcription factor gene

family show differential co-regulation with senescence-associated genes during senescence of flag leaves. J Exp Bot. 65(14): 4009-22.

3. Lindemose S, Jensen MK, Van de Velde J, O'Shea C, Heyndrickx KS, Workman CT, Vandepoele

K, Skriver K, De Masi F (2014) A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana. Nucleic Acids Res. 42(12): 7681-7693.

81

Posters: Plant Breeding – Quality, Productivity and Diseases

Hunting QTLs for heat tolerance from exotic wheat cultivars Dew K. Sharma1, Eva Rosenqvist2, Carl-Otto Ottosen3, and Sven B. Andersen1

1 Department of Plant and Environmental Sciences, Section for Plant and Soil Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark 2 Department of Plant and Environmental Sciences, Section for Crop Sciences, University of Copenhagen, Hojbakkegaard Allé 9, 2630 Taastrup, Denmark 3 Department of Food Science, Aarhus University, Kirstinebjergvej 10, 5792 Aarslev, Denmark

Wheat (Triticum aestivum L.) feeds about one third of the world population. The future climate is predicted to be more variable with higher frequency of heat waves and dry spells. Wheat is a heat sensitive crop but naturally existing genetic variation would potentially provide a source of genes towards improving stress tolerance in wheat.

In the present study, a combined approach of physiological phenotyping based on chlorophyll fluorescence parameter, Fv/Fm, that measures the maximum quantum efficiency of photosystem II (a heat stress sensitive process in the light reaction of photosynthesis) and its quantitative genetics was used to dissect the complex nature of heat tolerance into photosynthesis related traits with a top-to-bottom (forward) approach.

The naturally existing variation amongst 1274 wheat cultivars belonging to different regions of the world were phenotyped repeatedly at increasing severity of heat stress, to be able to select cultivars that are extreme for Fv/Fm in the first step. Subsequently, these extreme cultivars were also found to be different in terms of overall photosynthesis and dry matter accumulation under moderate heat stress condition mimicking natural heat waves.

In order to hunt for quantitative trait loci (QTL) associated with such physiological differences, three bi-parenal populations (F2) segregating for Fv/Fm were generated from the four contrasting parents (3 heat tolerant parents originated from Pakistan & Afghanistan and a common heat sensitive parent originated from Germany). A total of 140 F2 plants in each population were phentoyped under heat stress by Fv/Fm and genotyped by around 5000 polymorphic single nucleotide polymorphism (SNP) markers (DArTseq, Australia) for linkage analysis and QTL mapping.

As a result, three QTLs, one each in three populations were identified. Two of these QTLs were located on 3B and one QTL on 1D chromosome in the hexaploid wheat genome. Each QTL explains about 12-16% of the phenotypic variation for Fv/Fm and the positive allele was donated by the heat tolerant parents. Future works will reveal how useful these identified QTLs are in terms of improving heat tolerance in wheat via higher photosynthesis.

82

Posters: Plant Breeding – Quality, Productivity and Diseases

Physiological phenotyping by determination of phytohormone and enzyme activity signatures Dominik K. Großkinsky1, Eric van der Graaff1, Thomas Roitsch1,2

1 Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen, Højbakkegård Allé 13, 2630 Taastrup, Denmark 2 Global Change Research Centre, Czech Globe AS CR, v.v.i.., Drásov 470, Cz-664 24 Drásov, Czech Republic The availability of novel germplasm and cost efficient molecular techniques for whole genome sequencing, mapping and genomic selection as well as complementary techniques to image the whole plant phenome non-destructively, give access to information unimaginable just a few decades ago. However, to be able to generate cost efficient, reliable and robust non-invasive predictors for yield and quality it will be essential to link the current high-throughput genotyping and phenotyping of whole plants to the underlying physiological processes. Only the physiology of a plant integrates various levels of regulation of the expression of the genetic information as well as the impact of the environment and agricultural management. The activities for enzymes involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance and crop yield, as well as abiotic and biotic stress responses. Since enzyme activities integrate several modes of regulation at the mRNA and protein level, the analysis of enzyme activities is very suited to analyse the physiological state of plants. This work assessed the establishment of a simple, fast and cost-effective method to determine activities for 13 key enzymes involved in carbohydrate metabolism, mainly using coupled spectrophotometric kinetic assays. These assays proved to be robust and highly suitable for the characterisation and the diagnosis of the physiological state for various plant species, including several crop species. Plant growth and development depend on a strong regulatory network of phytohormones. Only in the recent years it became evident that the central stress signalling backbone in response to pathogen infection, comprising ethylene, jasmonic and salicylic acid, is also modulated by other phytohormones. Due to extensive interactions of abscisic acid, auxin and newly also cytokinins with the stress signalling compounds, it is required to quantify spatial and temporal dynamics of the whole phytohormone spectrum, but typically established method only determine individual, single phytohormones. Based on published procedures, we developed an easy, rapid extraction and analysis method enabling determination of abscisic, indole-3-acetic, jasmonic and salicylic acid as well as eight cytokinin derivatives within the same extract and analytical run on a UHPLC-MS/MS device. Furthermore, certain phytoalexins, especially important as additional defence compounds within generalized pathogen responses and also cytokinin induced resistance, can be determined in the very same extract. Parallel determination of these phytohormones is also essential for physiological phenotyping to assess the function of various regulatory processes in plants. We successfully applied the established method to determine complex phytohormone profile in five model and crop plant species and also different cell suspension cultures.

83

Posters: Plant Breeding – Quality, Productivity and Diseases

Genome-wide analysis of pollen allergen gene families in perennial ryegrass (Lolium perenne L.) Istvan Nagy, Stephen Byrne, Torben Asp Department of Molecular Biology and Genetics, Aarhus University, Research Centre Flakkebjerg, Forsøgsvej 1, DK-4200 Slagelse Wind pollinated plant species release pollen grains in a great abundance during their flowering season, leading to allergic symptoms in susceptible individuals. The majority of pollen allergenicity is attributed to a limited number of proteins and glycoproteins that are rapidly released after hydration. Pollens from Poaceae species - especially from ryegrass - are one of the most important airborne allergen sources in the temperate zone. In perennial ryegrass two major pollen allergens, Lol p I and Lol p5 have been identified. Similar pollen allergens have been found in other grass species as well, and Lol p I and Lol p5 proteins (also known as Group 1 and Group 5 allergens) are believed to contribute to 80 to 90% of grass pollen allergenicity. Lol p I is an acidic glycoprotein belonging to the expansin B protein family involved in cell wall loosening, while Lol p 5 is a non-glycolysated protein with ribonuclease activity. Major and minor pollen allergens belonging to various other protein families have been identified in a wide range of plant species. Most of these proteins have been shown to exist as multiple isoforms, encoded by diverse members of multigene families. Some of these pollen allergen coding gene families exhibit a relative high sequence diversity, while others are highly conserved across distantly related species. Beside their health-related importance, their diversity, complexity and organ specificity render pollen allergens to be excellent and interesting subjects for genome-wide representation and transcriptomic analyses. After collecting a large number of reference protein sequences from the SwissProt/UniProt databases, representing nearly all known types of pollen allergens from a wide range of plant taxa, including grasses, trees, conifers and weeds, we conducted Hidden Markov Model (HMM) profile based scans on a set of protein sequences consisting of 40 K high-confidence proteins from ab initio and evidence based gene predictions using genomic contigs from a high-quality perennial ryegrass genomic draft, recently accomplished by our group. The availability of transcriptome data from different vegetative and generative organs (including leaves and pollens) made us possible to perform quantitative expression profiling for each identified gene product. We conducted detailed structural analysis on each hit using a stand-alone InterProscan pipeline. Altogether, nearly 400 pollen allergen homologs belonging to 16 different protein families could be identified in the perennial ryegrass genome. The size and complexity of this protein families vary greatly: While more than 50 Lol p I (Group 1) homologs were found, Group 5 pollen allergens are represented by less than ten sequences in perennial ryegrass. We found that the largest pollen allergen-related protein family in perennial ryegrass is the family of EF-hand calcium-binding proteins (Group 7 allergens), with more than 140 members. The fact, that relatively highly conserved homologs to exotic pollen allergens typical for weeds or trees (like the Amb a I allergen from ragweed, or the Bet V allergen from birch) can be found in the ryegrass genome as well - and some of them are showing high pollen specific expression - indicates that grass pollen allergenicity might be a more complex issue than assumed previously.

84

Posters: Plant Breeding – Quality, Productivity and Diseases

QTL Analysis by Sequencing of Total Glycoalkaloid (TGA) Content in Potato Kacper Piotr Kaminski1, Kirsten Kørup2, Mads Sønderkær1, Mathias Neumann Andersen2, Mette Sondrup Andersen1, Hanne Grethe Kirk3, and Kåre Lehmann Nielsen1

1 Department of Chemistry and Bioscience, Aalborg University 2 Department of Agroecology, Aarhus University 3 LKF Vandel Potatoes are good source of carbohydrates, proteins, fiber and vitamins, but also produce biologically active secondary metabolites called glycoalkaloids (GAs). Steroidal GAs (SGAs) evolved in potato most probably as part of defense mechanism and due to anticholinesterase activity and cell membrane disruption have antimicrobial, fungicidal, antiviral and insecticidal properties. GAs are however, for the same reason, toxic to animals and humans and common symptoms of excessive ingestion include nausea, vomiting, fever or disorientation and in extreme cases can be fatal. Compared to wild species of potato the elite cultivars mainly used for consumption have low contents of GAs. Breeding for pathogen resistance, however, often requires use of wild species with high GA content and it offspring having high contents of GA quite frequently arises. Several molecular markers connected to increased GA content have been identified so far. Both dominant and recessive traits have been found to influence GA content. So far identification of underlying genes and has only been done in a few cases. The major purpose of this study was to identify genomic regions and candidate genes responsible for differential GA accumulation within diploid potato mapping population (n=90). QTL analysis was employed by use of novel method including next generation genome sequencing where prior knowledge of molecular markers is not a prerequisite for analysis. Allelic segregation between potatoes of low and high glycoalkaloid content was determined. Genomic regions with candidate genes directly connected to GAs synthesis were identified.

85

Posters: Plant Breeding – Quality, Productivity and Diseases

An essential model plant for food and bioenergy crops – Brachypodium distachyon Louise de Bang, Niels A. F. Olsen, Sylwia E. Głazowska, Andrea Bellucci, Anna Maria Torp and Søren K. Rasmussen

Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark flagstad@plen.ku.dk, niels_alvin@yahoo.dk Brachypodium distachyon is an essential model plant for the monocotyledons and especially for the cereals wheat, barley, rye and oat, because it evolutionary is very closely related to these. The dicotyledon plant Arabidopsis thaliana is the most widespread and well established model plant in research, but for the monocotyledons it is not a good model because mono- and dicots evolutionary are very distinct (Draper et al., 2001). The evolutionary distance between the two is evident in the cell wall composition which is incomparable. The cell wall is specifically targeted for 2. Generation biofuels and a close related model plant is essential for research in this area (Marriott et al., 2014). Many other agronomical important traits such as yield, stress tolerance, root growth, development, and plant-pathogen interactions are also distinct between mono- and dicots (Brkljacic et al., 2011). Brachypodium distachyon was selected as a model plant in the beginning of this century and subsequently resourceful tools such as genomic sequence, transformation methods, T-DNA lines and mutant populations have been developed in the model plant system (Thole et al., 2012). In our group we have a Brachypodium distachyon mutant population Brachylife which has been valuable in several projects for both forward and reverse genetic screens. The PCR based reverse genetic approach TILLING (Targeting Induced Local Lesions IN Genomes) has been developed for the population, which makes a gene specific screen easy and rapid (Till et al., 2006). Brkljacic, J., Grotewold, E., Scholl, R., Mockler, T., Garvin, D. F., Vain, P., Brutnell, T., Sibout, R.,

Bevan, M., Budak, H., Caicedo, A. L., Gao, C., Gu, Y., Hazen, S. P., Holt, B. F., 3rd, Hong, S. Y., Jordan, M., Manzaneda, A. J., Mitchell-Olds, T., Mochida, K., Mur, L. A., Park, C. M., Sedbrook, J., Watt, M., Zheng, S. J., and Vogel, J. P. (2011). Brachypodium as a model for the grasses: today and the future. Plant Physiol 157, 3-13.

Draper, J., Mur, L. A. J., Jenkins, G., Ghosh-Biswas, G. C., Bablak, P., Hasterok, R., and Routledge, A. P. M. (2001). Brachypodium distachyon. A New Model System for Functional Genomics in Grasses. Plant Physiology 127, 1539-1555.

Marriott, P. E., Sibout, R., Lapierre, C., Fangel, J. U., Willats, W. G., Hofte, H., Gomez, L. D., and McQueen-Mason, S. J. (2014). Range of cell-wall alterations enhance saccharification in Brachypodium distachyon mutants. Proc Natl Acad Sci U S A 111, 14601-6.

Thole, V., Peraldi, A., Worland, B., Nicholson, P., Doonan, J. H., and Vain, P. (2012). T-DNA mutagenesis in Brachypodium distachyon. J Exp Bot 63, 567-76.

Till, B. J., Zerr, T., Comai, L., and Henikoff, S. (2006). A protocol for TILLING and Ecotilling in plants and animals. Nat Protoc 1, 2465-77.

86

Posters: Plant Breeding – Quality, Productivity and Diseases

Two Argonaute genes are necessary for the control of cellular functions during the vegetative stage in Blumeria graminis f. sp. hordei Maria E. Constantin, Marijn Knip, Hans Thordal-Christensen

Department of Plant and Environmental Sciences, Faculty of Sciences, University of Copenhagen Obligate biotrophic fungi, such as powdery mildew fungi, are important pathogens that can cause disease on a large variety of crops of agronomical importance such as barley and wheat. Despite of the agronomical importance of obligate biotrophic fungi little is known about the genes and factors that are important for their pathogenesis and virulence. In the case of Blumeria graminis f. sp. hordei (Bgh), causing the powdery mildew disease affecting barley, only a few genes have been characterized. In recent years, a new tool, which allows the study of gene function of biotrophic fungi by silencing genes of interest, was developed (Nowara et al., 2010). The method is known as Host-Induced Gene Silencing (HIGS) and consists of targeting pathogen genes by expressing RNA interference (RNAi) constructs in its host. Bioinformatically, we identified three Bgh Argonaute genes: Bgh00436, Bgh00834 and BghQDE-2. By using the HIGS method, we studied the importance of these Argonaute proteins. Our results show, that two of them, Bgh00834 and BghQDE-2, are closely related, and both are important fungal components during the vegetative stage of the fungus. However, we could not show that Bgh00436 is important at this stage. We believe this is because it is expressed primarily during the sexual stage of the fungus.

87

Posters: Plant Breeding – Quality, Productivity and Diseases

Regulation of the hemoglobin/NO cycle in barley infected with powdery mildew or yellow (stripe) rust Massimiliano Carciofi1, Chris Khadgi Sørensen2, Ian Max Møller1, Mogens Støvring Hovmøller2, Kim H. Hebelstrup1 1 Aarhus University, Department of Molecular Biology and Genetics, Section of Crop Genetics and Biotechnology, Research Center Flakkebjerg, Slagelse, Denmark 2 Aarhus University, Department of Agroecology, Section of Entomology and Plant Pathology, Research Center Flakkebjerg, Slagelse, Denmark Nitric oxide (NO) is an important cellular signaling molecule in plants. It is involved in a range of physiological functions in plants. NO plays a particular central role in plant responses to biotic stresses, where it seems to function at different molecular levels including formation of nitrosylated proteins and cross-interference with various reactive oxygen species. Plant hemoglobins are important modulators of the NO signal, presumably by an oxidative mechanism including the direct reaction with O2. When biotrophic pathogens infect plants, the pathogen and the plant are struggling to take control over gene expression towards either compatibility or incompatibility. Our previous studies have shown that artificial up-regulation or silencing of endogenous hemoglobins in plants can modulate NO levels during pathogen infection to an extent where susceptibility levels are severely changed. We here show how barley plants infected with either powdery mildew (Blumeria graminis f. sp hordei) or yellow (stripe) rust (Puccinia striiformis f. sp. hordei) are struggling with the respective pathogen to control hemoglobin gene expression.

88

Posters: Plant Breeding – Quality, Productivity and Diseases

Light-dependent lesion formation might rather correlate with resistance than with susceptibility in the pathosystem barley <-> B. sorokiniana Nele Gjendal, Michael F. Lyngkjær

Department of Plant and Environmental Sciences, University of Copenhagen, Denmark Necrotic spots or lesions due to spotting diseases reduce photosynthetic area and cause premature leaf senescence. Therefore, the level of necrotic spotting is seen as an indicator of disease severity. In the case of spot blotch disease, caused by the fungus Bipolaris sorokiniana, the level of necrotic spots is dependent on light intensity. Hence, light should induce increased susceptibility as well, but microscopic investigations could report symptomless growth of this fungus (Schäfer et al., 2004). We investigated necrotic spots and fungal biomass after B. sorokiniana-infection and the possible effects of light conditions in 5 different barley genotypes and their mlo daughter lines. By manipulating light conditions we observed that no symptoms occurred on the infected leaves after incubation in darkness. Surprisingly, these symptomless plants had substantial higher amount of fungal biomass. Likewise, inhibition of PSII with DCMU under light conditions led to a significant increase of fungal growth. PSII plays a crucial role in plant response due to H2O2 production at different steps of the ps reaction center. Toxin infiltration alone of B. sorokiniana- culture filtrate into barley leaves leads to necrosis formation (Kumar et al., 2001). In our investigations, necrosis formation after toxin infiltration turned out to be light-dependent as well. B. sorokiniana toxins have been suggested to inhibit PSII (Adeishvili, 1981), but this has never been followed up. Our results show that light is crucial for lesion formation in this pathosystem, but that the fungus prefers to grow in plants where no necrotic lesions occur. Enhanced light-induced resistance to this pathogen due to a step in psII is therefore very likely. This is in contrast with the definition of the biology of this fungus and the role of its toxin. Until now, the assumption was that cell death and H2O2 facilitate colonization by all toxin producing fungi. The mlo mutation in barley leads to more stress response, and Kumar et al. (2001) could determine more and faster accumulation of H2O2 in the mlo mutant after B. sorokiniana infection in one genetic background. In our studies, the mlo mutation contributed only in one case to increased susceptibility. After 48 hours in darkness, three mlo daughter lines were significantly more infected compared to the mother plant. We therefore suggest that mlo mutants in general are not more susceptible to B. sorokiniana, and that variation in symptoms and fungal biomass is mainly due to the genetic disposition and environmental effects.

References Adeishvili, T.S., G.G. Simonyan, G.A. Tarabrin & Y.N. Fadeev (1987): Vol. 36:1, pp. 572-150-153. Jarosch, B., Kogel, K., & Schaffrath, U. (1999). 12(6), 508–514. Kumar, J., Hückelhoven, R., Beckhove, U., Nagarajan, S., & Kogel, K. H. (2001). Phytopathology,

91(2), 127–33. doi:10.1094/PHYTO.2001.91.2.127 Schäfer, P., Hückelhoven, R., & Kogel, K.-H. (2004). Molecular Plant-Microbe Interactions : MPMI,

17(4), 366–73. doi:10.1094/MPMI.2004.17.4.366.

89

Posters: Plant Breeding – Quality, Productivity and Diseases

Characterization of erectoides barley mutants ert-c and ert-d Qiongxian Lu1, Christoph Dockter2, Per Gregersen1, Mats Hansson 1 Dept. of Molecular Biology and Genetics, Aarhus University 2 Carlsberg Laboratory

Induced mutation research in plant was started in early 20th century and soon became widely used in increasing the genetic variation and developing valuable resources for crop improvement. During the past years, 10,000 different morphological and physiological barley mutants have been identified and tested. The erectoides (ert) barley mutants, which belong to this large collection, have more compact and erect spikes. The straw is shorter and stiffer. They are potential resources for development of lodging resistant barley. Currently, we are working with two recessive loci ert-c and ert-d. ert-c was mapped to 3HL centromere region based on 233 F2 population lines from the crossing of Bowman and BW305 (ert-c.1). It co-segregates with SNP marker 2_1472, 1_1283 and 1_1530. A Bowman and BW305 (ert-d.7) F2 population was originally developed for genetic mapping of ert-d. Allelic tests show that ert-c.1 and ert-d.7 are at the same locus. The ert-d.7 population lines will be pooled into the ert-c.1 population. More F2 lines from Bowman and BW305 (ert-c.1) were developed and will be added to refine the genetic map position. Genome zipper and Gramene were also used for candidate gene hunting. Flowering or development related barley genes (MLOCs) in the candidate region are under testing.

90

Posters: Plant Breeding – Quality, Productivity and Diseases

Resistance gene specificity in cereals Remy Kronbak1, Christina Rønn Ingvardsen1, Inger Bæksted Holme1, Mogens Støvring Hovmøller2, Per Langkjær Gregersen1

1 Department of Molecular Biology and Genetics, Science and Technology, Aarhus University, Denmark 2 Department of Agroecology, Science and Technology, Aarhus University, Denmark Nucleotide binding site and leucine rich repeat (NB-LRR) proteins are innate immune receptors that provide cultivar-specific disease resistance in crops and other plants. When they recognize effectors, i.e. race-specific basal plant defense inhibitors of pathogens, the pathogen in question is combated. The effector is then termed an avirulence factors (Avr). Known DNA sequences of R genes, which can be matched to specific pathogens, constitute a valuable resource in crop breeding, as the growing of disease resistant crops often is a preferred strategy to reduce pesticide usage. In the dicot species Arabidopsis and tobacco, P-loop disrupted NB-LRR proteins of the Toll/interleukin 1-like receptor (TIR) subtype act dominant negatively to the corresponding wild type protein in transgenic lines. Adopting this phenomenon in monocot crop species like cereals, where NB-LRR genes are of the coiled coil (CC) subtype rather than TIR, we are developing a method to match newly discovered R gene candidates to pathogens. The barley cv. ‘Golden Promise’ harboring the CC-NB-LRR subtype powdery mildew R gene Mla8 is used for proof-of-concept. In transgenic lines, the P-loop mutated Mla8GK206,207AA controlled by the barley heat shock protein HvHsp17 promoter was highly expressed at least 24 h after 2 h treatment in water at 38 °C evaluated at mRNA level from real-time qPCR. Lower temperatures or shorter treatment were not sufficient. Ongoing experiments should reveal, whether heat treated leaf fragments become susceptible to powdery mildew caused by the otherwise non-compatible fungus Blumeria graminis f. sp. hordei isolat Race I, which holds the Mla8 avirulence factor AvrMla8. It will thus be revealed, whether CC-NB-LRR proteins act dominant negatively on the corresponding wild type like TIR-NB-LRR proteins do. Later, transgenic lines with Mla8GK206,207AA constitutively expressed from the maize ubiquitin promoter Ubi-1 will be available. By means of the method based on dominant negative P-loop mutants we aim to reveal pathogen specificity of a cultivar-specific collection of novel NB-LRR gene candidates. From transcriptome sequencing in the wheat cv. ‘Bobwhite SH 98 26’ we found full-length R gene candidates of the NB-LRR type including a possible homoeolog of the yellow rust R gene Yr10 of cv. ‘Moro’.

91

Posters: Plant Breeding – Quality, Productivity and Diseases

Partitioning SNPs identified by GBS into genome annotation classes and calculating variance components for heading date and disease resistance from the resulting genomic relationship matrices Stephen Byrne1, Fabio Cericola2, Luc L. Janss2, Just Jensen2, Dario Fé2, Bilal Ashraf2, Adrian Czaban1, Morten G. Pedersen3, Ingo Lenk3, Christian S. Jensen3, Torben Asp1 1 Department of Molecular Biology and Genetics, Aarhus University, Research Centre Flakkebjerg, Forsøgsvej 1, 4200 Slagelse, Denmark 2 Department of Molecular Biology and Genetics, Aarhus University, Research Centre Foulum, Blichers Allé 20, 8830 Tjele 3 DLF-TRIFOLIUM A/S, Højerupvej 31, 4600 Store Heddinge, Denmark A draft sequence assembly of the perennial ryegrass genome was annotated with the aid of RNA-seq data from various genotypes, plant components, and treatments. We predicted 39,795 high quality proteins originating from 28,182 genetic loci. There was an average of 5.9 exons per protein, and an average protein Annotation Edit Distance (AED) of 0.14. Genotyping-By-Sequencing (GBS) data was generated after genome complexity reduction with ApeKI for 995 breeding families. Data was aligned against the annotated sequence assembly, and we identified variants at over 1.8 million positions, which were partitioned according to genomic feature. Variants were found in 20,594 (73%) of the gene loci, and these had on average 22.3 SNPs per gene. SNPs were partitioned according to the following annotation features; exon (277,404 SNPs), CDS (221,364 SNPs), 5’UTR (32,551 SNPs), 3’UTR (44,781 SNPs), gene (458,273 SNPs), genes with NB-ARC domains (9,056 SNPs), intron (168,023 SNPs), and inter-genic (1,420,866 SNPs). Genomic relationship matrices were created for each annotation class and SNP-explained variances for heading date and disease resistance were calculated.

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Plant Biotech Denmark

Plant Biotech Denmark (PBD) is a non-profit network of Danish public plant biotechnology research groups. The objective is to strengthen plant biotechnology research. Steering Committee

Henrik Brinch-Pedersen (chairman) Senior Scientist Department of Molecular Biology and Genetics Aarhus University hbp@mbg.au.dk Svend Christensen Head of Department Department of Plant and Environmental Sciences University of Copenhagen svc@plen.ku.dk Søren K. Rasmussen Professor Department of Plant and Environmental Sciences University of Copenhagen skr@plen.ku.dk Birte Svensson Professor Department of Systems Biology Technical University of Denmark bis@bio.dtu.dk Kåre Lehmann Nielsen Associate Professor Department of Chemistry and Bioscience Aalborg University kln@bio.aau.dk PBD secretariat

Solveig Krogh Christiansen Senior Advisor c/o University of Copenhagen soc@plen.ku.dk

Andrea Lenk Research Coordinator c/o University of Copenhagen anle@plen.ku.dk

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