Jahresbericht V13.Indd

CEPL AS Annual Report 2015 Imprint

Published by CEPLAS – Cluster of Excellence on Plant Sciences Heinrich Heine University Düsseldorf 40204 Düsseldorf

Editors Prof. Dr. Andreas P. M. Weber (responsible) Dr. Céline Hönl Dr. Juliane Schmid

Photos p. 4/5 istock.com/© Aydın Mutlu p. 8/9 fotolia.com/© Vasiliy Koval p. 24 Thomas Wrobel p. 70/71/73/77/98 (b.) Steffen Köhler p. 82/83 Siegfried Werth p. 98 (t.) atelier caer All other pictures: © CEPLAS

Layout atelier caer, Düsseldorf CEPL AS Annual Report 2015 Annual Report 2015

1. General presentation 1

2. Organisation 5

3. Research 9

3.1 Research Area A 10

3.1.1 Construction of a phylogenetic and genomic frame-work for the study of divergence of annual and perennial life cycles A1 Genotypic and phenotypic analysis of interspecific hybrids between A. montbretiana and A. alpina 11 A2 Analysis of the roles of PERPETUAL FLOWERING 1 direct target genes in perennial flowering and their divergence in sister annual species 12 A3 Comparison of annual-perennial species pairs across the Brassicaceae 12 A4 Nutrient recycling in the perennial plant Arabis alpina 13 A5 Molecular characterisation of senescence in annual and perennial plants 13 A6 Photoperiodic control of flowering time in Arabis alpina 14 3.1.2 Elucidation of regulatory networks that determine formation and identity of meristems A7 The control of adventitious root formation in the perennial Arabis alpina 15 A8 Developmental basis for asexual reproduction in Cardamine 15 A9 Mechanisms involved in perennial life cycle of Arabis alpina: The role of auxin in perennial traits 16 A10 The regulation of inflorescence branching in Arabis alpina 17 A11 The regulation of inflorescence branching in Arabis alpina 17 A12 Analysis of axillary meristem initiation in perennial plant Arabis alpina 18 A13 Genotypic and phenotypic analysis of tiller development in cultivated (Hordeum vulgare) and wild barley species (H. v. spp. spontaneum, H. bulbosum) 18 A14 Genetic dissection of natural variation in tiller development in cultivated and wild barley 19 A15 Genetic and environmental control of inflorescence development and floret fertility in barley and wheat 19 A16 Comparative transcriptome profiling of meristem states in annual and perennial Arabis and Arabidopsis species and hybrids 20 A17 Analysis of an evolutionary conserved module regulating root system development in monocot and dicot species 21 A18 Using receptor kinase pathways to modify plant traits 21 3.1.3 Modelling regulatory modules that differ between annual and perennial plants A19 Transcriptome Data Analysis and Optimal Experimental Design 22 Summary and Outlook 23

3.2 Research Area B 24

3.2.1 Evolutionary transcriptomics of C3 and C4 species pairs

B1 Mesophyll and bundle-sheath-specific transcriptomes of 3C /C4 species pairs 25 Contents

B2 Identification of 4C genetic determinants by a comparative transcriptome analysis of C3 and C4 Flaveria spp. and by forward genetics with Arabidopsis thaliana 26

B3 Circadian control of transcriptomes and proteomes of C3 / C4 species pairs 26 B4 Unravelling the mechanisms that control bundle-sheath cell size in leaves of

C4 plants 27

B5 Establishing a functional link between leaf development and C4 photosynthesis 27 3.2.2 Genetic analyses with Arabidopsis thaliana for identifying genes involved in leaf anatomy and morphology B6 Unravelling the functional relationship between leaf anatomy and photosynthesis by mutational and synthetic approaches 28 B7 Genetic variation for leaf anatomy in Arabidopsis 29 B8 Light effects on leaf anatomy in Arabidopsis 29 B9 Differentiation of vascular veins and surrounding mesenchyme: the role of

leaf meristems within adaptations to C4 anatomy 30

B10 Generation of synthetic tools for the engineering of C4 photosynthesis 30

3.2.3 Experimental evolution towards C4 photosynthesis

B11 In silico exploration of paths towards C4 metabolism 31

B12 Experimental evolution towards C4 32

B13 Transforming Arabidopsis plants towards C4 metabolism 32

3.2.4 Regulation and metabolic interactions of C4 photosynthesis

B14 The C4 cycle and its surrounding metabolism 33

B15 Alterations to the regulation of C4 enzymes during evolution of C4 photosynthesis 34 B16 Structural evolution of phosphoenolpyruvate carboxylase kinase (PPCK) in the genus Flaveria 34

B17 Nitrogen and Sulfur metabolism in C4 plants 35 B18 Mathematical modelling of acclimation processes of the photosynthetic electron transport chain in green algae and plants 35 Summary and Outlook 36

3.3 Research Area C 38

3.3.1 Structure, function and ecology of the plant microbiome C1 Characterisation of the mycobiome of Arabis alpina 40 C2 Characterisation of oomycete and protozoan species from tissues of A. thaliana and A. alpina 40 C3 Diversity of oomycetes and protists in intimate association with Arabidopsis 41 C4 Bioinformatic analysis of endophytic flora in A. thaliana and A. alpina 41 3.3.2 Understand how biotic and abiotic factors affect the composition and activities of plant associated microbial communities C5 Shaping of the Arabis alpina microbiome by plant host interactions with environmental factors – phosphorous limitation 42 C6 Role of WRK75 in phosphate, pathogen and temperature responses 43 C7 Investigation of the effect of temperature-modulated defense homeostasis on plant-microbe symbioses in Arabidopsis thaliana 43 C8 Environmental influences on pipecolic acid biosynthesis, defense priming, and systemic acquired resistance: light, nitrogen supply, and the C/N balance 44 C9 Mutualistic interactions of pathogenic and non-pathogenic protists in foliar biofilms 44 Annual Report 2015

C10 Role of fungal lifestyle and secreted effectors in multitrophic microbe – microbe and microbe – plant interactions 45 3.3.3 Identify the genes in both host and microbe that determine interaction outcome C11 Identification of plant growth promoting genes and pathways of bacterial symbionts in the A. thaliana rhizosphere 46 C12 Evolution of biotrophy in fungal symbionts: What can the genomes reveal? 47 C13 Functional characterisation of candidate effector proteins in the Sebacinales 47 C14 Role of fungal WSC lectin-like proteins in the interaction of endophytic fungi with plant roots 48 C15 Characterisation of a leaf-specific Ustilago maydis α-L-arabinofuranosidase 48 C16 Characterisation of the molecular mechanisms underpinning fungal beneficial effects in roots during tripartite interactions 49 C17 Development of a model system for smut fungus – Brassicaceae interaction 49 C18 The cumulative impact of environmental parameters to photosynthetic eukaryotes at a molecular level 50 3.3.4 Determine reciprocal effects of host-microbe interactions on secondary metabolites C19 Exploring the coordination of indole glucosinolate metabolism and ER body formation in plant fitness 51 3.3.5 Synthetic symbioses – shaping the microbiome to increase plant performance C20 Modelling algae and bacteria consortia 52 Summary and Outlook 52

3.4 Research Area D 54

3.4.1 Plant-microbe interactions in the context of plant defense reactions and plant nutrition D1 The impact of secondary metabolites on the composition of the endophytic microflora inArabidopsis thaliana 55 D2 Mathematical models of glucosinolate metabolism in plants 56 D3 Natural variation in the interaction of plants and bacteria in the rhizosphere 57 D4 MS-based identification of secondary metabolites crucial for plant-microbe interactions 57 D5 Characterising plant-bacteria interactions that promote the uptake of nitrogen and sulfur from organic sources 58 D6 Regulatory and evolutionary aspects of stress-inducible plant metabolic pathways 58 3.4.2 Signalling molecules, metabolic modules, and synthetic microorganisms D7 Characterising plant-bacteria interactions that promote the uptake of nitrogen and sulfur from organic sources 59 D8 Exploring the phytosulfokine triterpene pathway in Arabidopsis thaliana 60 D9 Synthetic microbes for the production of plant secondary metabolites 60 D10 Programming triterpene biosynthesis pathways in cyanobacteria using synthetic RNA-based devices 61 D11 Functional expression and biochemical characterisation of plant P450s 61 D12 In vitro analysis of selected ABC transporters from plants 62 Summary and Outlook 62

3.5 Publications 64 Contents

4. Plant Metabolism and Metabolomics Labs 71

4.1 Platform Düsseldorf 72

4.2 Platform Cologne 73

5. Promotion of young researchers 75

5.1 Research internships for undergraduates 76

5.2 Bachelor Programme in Quantitative Biology 77

5.3 CEPLAS Graduate School 78

5.4 CEPLAS Postdac Programme 79

5.5 Young researchers activities 80

6. Promotion of gender equality 83

6.1 Career support 84

6.2 Family support 85

7. Key figures 87

7.1 Staff 88

7.2 Finances 91

8. Public outreach 95

8.1 Public relations work 96

8.2 Political outreach 100

8.3 Press archive 101

9. Technology transfer and cooperation management 103

9.1 Presentation to industry 104

9.2 Planned cooperation with industry 104

9.3 Non-academic careers for young scientists 105

General presentation Annual Report 2015

Who we are

The Cluster of Excellence on Plant Sciences – CEPLAS is funded in the context of the German Excellence Initiative by the German Federal and State governments with 28 Mio €. CEPLAS is a Max-Planck-Institute for Plant Breeding Research joint research project of the Universities Düssel- dorf and Cologne, the Max Planck Institute for Plant Breeding Research and the Forschungs- zentrum Jülich.

Research goals

The global demand for plant products is increas- plants to adapt to adverse environmental con- ing with unprecedented pace and it has been es- ditions and constraints. CEPLAS employs a re- timated that agricultural yields will have to dou- search strategy that is driven by comparative evo- ble by the year 2050. However, global change, lutionary analyses in combination with modern in particular, altered precipitation and tempera- synthetic biology. ture patterns, is challenging the sustained production of crops and thus the agronomic base of Specifically, CEPLAS researchers investigate the human civilisation. Simultaneously, arable land is mechanistic basis and genetic architecture of becoming scarce due to increased erosion and selected complex traits that have a crucial im- population pressure. Meeting the continuously pact on adaption to limited resources and yield increasing demand will require innovative strate- and are therefore of outstanding importance in gies for crop improvement that aim at enhancing designing and breeding the crops of the future: yield without compromising increases in the use of water, nutrients, and soil, or diminished resist- ▪ Annual and perennial life histories ance to pests. ▪ C4 photosynthesis (photosynthetic carbon conversion efficiency) CEPLAS aims at achieving a fundamental under- ▪ Plant - microbe interactions standing of the genetic mechanisms that enable ▪ Metabolic interactions

Our vision

Through transformative science and innovative training, CEPLAS contributes to meeting the grand challenge of the 21st century, maintaining a sustainable food and energy base for human societies. CEPLAS raises the awareness of the importance of plant science for societal and economic progress and it serves as a platform for collaboration and interactions between academia and private entities. CEPLAS integrates scientific disciplines to develop new paradigms in plant science.

CEPLAS members working on the concept of the renewal proposal at the internal retreat in December 2015

2 General presentation

Third year achievements

The CEPLAS research programme 2014 and approx. 300 applications is becoming progressively more were received. Forty candidates integrated across institutions and were invited for a one-week interna- research groups, as evidenced by tional selection workshop at HHU. the increasing number of joint re- The selection committee identified search projects, publications and candidates for all open positions research grants. A major achieve- and most started their tenure in CE- ment in 2015 was the approval PLAS in May. of CRC 1208 (Membrane identity and dynamics) that is head- We are excited to report that our ed by CEPLAS PI Lutz Schmitt new Bachelor Programme in Quan- and unites the research of eight titative Biology was successfully CEPLAS groups with that of non- accredited in August and a first CEPLAS scientists. More than 80 cohort of five students started the publications, most in the leading study programme in October. With journals in plant sciences and sev- the experience and feedback from eral in interdisciplinary journals the first student cohort, we aim to such as Nature, eLife, and PNAS increase enrolment to approx. 30 USA, have been published by the students over the course of the next CEPLAS groups in 2015; approx. years. Major 25 % of these by CEPLAS postdoctoral researchers and gradu- Moreover, we intensified our activ- goals ate students. Jointly, the CEPLAS ities addressing stakeholders and PIs and AIs raised more than 18 decision-makers in politics. Repre- for 2016 Mio € in supplementary funding in sentatives of CEPLAS met with a 2015, most notably approx. 8 Mio € range of members of the German through CRC 1208. parliament to emphasise the need ▪ Develop for strategic investments in plant strategy In May the midterm evaluation of sciences in Germany. This also in- and goals CEPLAS by the Scientific Advisory cluded a visit to the Swiss federal for a renewal Board was conducted, which pro- protected site for the growth of ge- proposal vided highly valuable input and netically engineered plants. suggestions for the further development of the cluster that will be We also extended our public and ▪ Assess and implemented over the course of the political outreach activities. This restructure the next years. In particular, we aim at included guided tours through a CEPLAS career achieving a closer integration of the CEPLAS-sponsored exhibition at development research programmes of Research the HHU Botanical Garden, public programme Areas A and B, as well as C and D. lecture series in the cities of Co- logne and Düsseldorf, and deep- With the appointment of Benjamin ened cooperation with primary and ▪ Increase the Stich to the W3 position in Plant secondary schools. number of Quantitative Genetics and Geno- Quantitative mics in 2015, we completed the With the help of Günter Strittmatter Biology team of new CEPLAS faculty and we tried to expand our collaboration students thereby achieved one of our major with industry by organising public goals for 2015. He will start his work information events for industry as within CEPLAS in May 2016. well as special information days re- ▪ Mentor more quested by specific companies. In CEPLAS Another major goal for 2015 was addition, we organised two excur- postdoctoral the recruitment of a new cohort of sions for our young researchers to researchers CEPLAS postdoctoral researchers. offer opportunities for insights into to obtain Fourteen positions were interna- industry research and networking independent tionally announced in December with putative future employers. fellowships

Organisation Annual Report 2015

CEPLAS is comprised of four Research Areas, As a service facility for all CEPLAS members and each headed by a research area coordinator and external collaborators, CEPLAS established two co-coordinator. The research area coordinators Plant Metabolism and Metabolomics Platforms, are responsible for the scientific development of one specialised on primary (HHU) and another on the respective research area and the distribution secondary metabolites (UoC). of the allocated funds within the research area. The Central Office, equipped with two positions, All research area coordinators are part of the is responsible for the operational business and CEPLAS Steering Committee, together with the administrative management of the cluster. It cluster speaker and deputy speaker, the equal also supports the speaker and deputy speaker, opportunity representative and one representa- the Steering Committee as well as the Scientific tive of the young researchers. Additionally, one Advisory Board. It organises the meetings of the representative of the Forschungszentrum Jülich various committees and boards within CEPLAS, is invited to the Steering Committee meetings. the General Assembly as well as conferences, workshops and symposia. The committee is responsible for the overall operation and development of the cluster, allocation The Central Office cooperates with the finance of resources and preparation of site evaluations. departments and human resources departments of the partnering institutions in administrative is- In fall 2015 the cluster’s mid-term elections took sues. In addition, it is responsible for all reporting place during of the annual General Assembly and correspondence (DFG, president’s offices), where all members were asked to vote on the for the design and organisation of the homepage official positions within the cluster. The elections and for public relation work. gave new faculty the opportunity for a stronger integration in the decision-making process of the The Equal Opportunity Office is responsible for cluster. all gender-related issues or concerns such as career support for female scientists or family sup- To support of young researchers, CEPLAS offers port. a B.Sc. in Quantitative Biology, a Graduate School and a Postdoc Programme, each headed by one (or two) faculty member(s), supported by a coordination position.

CEPLAS

Research Areas Young Plant Administration Researchers Metabolism & Research Area A Programmes Metabolomics Central Office George Coupland Laboratories HHU MPIPZ B.Sc. Quantitative Primary Graduate School Research Area B Biologie Metabolites UoC Peter Westhoff Peter Westhoff Tabea HHU HHU Mettler-Altmann Equal Stanislav Kopriva HHU UoC Opportunity Research Area C Office Secondary UoC Alga Zuccaro Graduate School UoC Metabolites Ute Höcker Sabine Metzger UoC Administration Research Area D UoC UoC Ulf-Ingo Flügge Postdoc UoC Programme (Markus Pauly HHU) Rüdiger Simon HHU

6 Organisation

Members of the SAB

CEPLAS is counse- ▪ Mike Bevan led by a Scientific John Innes Centre Norwich, UK Advisory Board (SAB) which is com- ▪ Justin Borevitz posed of scientists Australian National University, Australia from academia and industry, which ▪ Natalja Dudareva meet on an annual Purdue University, USA basis. ▪ George Freyssinet The SAB receives Bio-EZ, France and reviews the annual report ▪ Karin Herbers of the cluster BASF SE, USA and provides an assessment to the ▪ Jane Langdale Supervisory Board. University of Oxford, UK

Moreover, the SAB ▪ Steve Long is responsible for University of Illinois, USA the external evaluation of the cluster ▪ Magnus Nordborg (annual evaluation Gregor Mendel Institute of Molecular Plant Biology GmbH, Austria report, mid-term and comprehensive ▪ John Rathjen assessment) and Australian National University, Australia provides advice on hiring decisions, ▪ Kazuki Saito scientific and RIKEN Center for Sustainable Resource Science, Japan structural planning, development of ▪ Johanna Schmitt the training pro- University of California, USA grammes, implementation of equal ▪ Klaas van Wijk opportunity meas- Cornell University, USA ures, and overall development of ▪ Dabing Zhang the cluster. Shanghai Jiao Tong University, China

Research Annual Report 2015

3.1. Research Area A Elucidation and manipulation of the mechanisms that differentiate annual and perennial life histories

Plant life history varies widely, even among closely related species. In this Research Area we compare annual and perennial species and aim to ex- Coordinator plain how key traits diversify during the divergence of these life histories. Annuals evolve from perennial progenitors in response to environmental George Coupland selective pressures that reduce survival of adult perennials, and this has occurred often in the Angiosperms. Evolution of annuals affects traits such as life span, adaptation to environment, storage and recycling of metabolites, Co-coordinator propensity for clonal propagation, timing and duration of flowering as well as number of progeny. Several traits characteristic of perennials would be Rüdiger Simon beneficial in breeding crop plants, but were removed from annual crops in the early stages of domestication.

Members In this Research Area, we aim to identify regulatory modules that diverged during the evolution of annuals from perennials with the objective of engi- Maria Albani neering perennial traits in annual species. We focus on characteristic dif- Petra Bauer ferences in meristem function, flowering behaviour, nutrient recycling, root Ute Höcker growth and longevity. The groups within Research Area A concentrate their Karl Köhrer efforts on two major model systems, which are closely related Brassicaceae Markus Kollmann species, particularly in the Arabidopsis and Arabis genera, as well as Hor- Maarten Koornneef deum species related to barley. A range of approaches are used including Peter Nürnberg forward genetics, reverse genetics based on CRISPR-Cas9, transcriptom- Richard Reinhardt ics exploiting RNAseq, comparison of newly acquired genome sequenc- Laura Rose es from phylogenetically closely related species, ChIPseq for interspecies Ulrich Schurr comparison of transcription factor targets and development of algorithms Klaus Theres for inferring gene regulatory networks from transcriptome data. Miltos Tsiantis Maria von Korff Ongoing projects in the Research Area are summarised in the following Wolfgang Werr sections, so here only a few of the major developments that occurred this year are mentioned. Genomes of two further annual species (Arabis iberica and Arabis auriculata) were obtained using Pacific Biosystems sequencing methodologies, and the resulting high quality assemblies allow us to compare two independent occurrences of annual life history closely related to our perennial model species Arabis alpina. By exploiting the population generated by crossing annual A. montbretiana and perennial A. alpina, we were able to show that introgression of a single chromosomal region of A. montbretiana into A. alpina was sufficient to generate plants exhibiting a combination of flowering traits, vernalization requirement and perpetual flowering, not found in either parent. Similarly, by developing CRISPR-cas9 for use in A. alpina, we were able to generate null alleles of genes proposed to have a central role in conferring competence to flower in the context of the perennial life cycle, and thereby to test models of how this trait diverged during the evolution of annuals. We have also extended the range of species used to include Cardamine resedifolia, which shows dramatic clonal propagation of shoots from roots, a trait usually associated with perennials. This Brassicaceae species provides a practical model to study this developmental plasticity and an interesting point of comparison with the study of adventitious root formation on the aerial shoots of perennial Arabis alpina. In addition, we have initiated the examination of nutrient recycling and of senescence patterns within the perennial life cycle of Arabis alpina, which generates collaborations with Research Area D and extends our analysis beyond developmental traits.

10 Research Area A

Pleiotropic effects of mutations in different row type genes on tiller number cv. Bowman. (A) Photographs of one representative plant per genotype were taken at 92 days after sowing. (B) Tiller number was as- sessed two weeks after transplantation (50 days after sowing), at flag leaf stage, at anthesis stage and at full maturity (Liller CB, Neuhaus R, von Korff M, Koornneef M, van Esse W (2015) Mutations in Barley Row Type Genes Have Pleiotropic Effects on Shoot Branching. PloS one 10(10):e0140246.). For further details see project A13.

3.1.1 Construction of a phylogenetic and genomic frame-work for the study of divergence of annual and perennial life cycles

In this research section we apply genomics tools within a phylogenetic framework to generate platforms for studying the divergence of the annual and perennial life cycles. As well as obtaining genomic sequences from model species, this includes RNAseq of staged material and ChIPseq to compare the target genes and regulatory networks associated with key transcription factors. These tools are applied to the study of traits that characteristically differ between annuals and perennials, such as seasonal flowering patterns, senescence patterns and nutrient recycling.

A1 Genotypic and phenotypic analysis of interspecific hybrids between A. montbretiana and A. alpina

Aim of the project Researchers This project exploits plants generated by inter- Christiane Kiefer crossing annual Arabis montbretiana to perennial René Richter Arabis alpina to identify genes that confer phenotypic differences between the species. Project leaders George Coupland Results Maarten Koornneef We made inter-species hybrids between the perennial species A. alpina and its sister annu- Project type al species A. montbretiana. Using the genome Postdoc project assemblies of both species and genotyping by

11 Annual Report 2015

sequencing technology, we derived genotypes Project duration of plants generated by backcrossing and sub- 01.01.2013 - 30.04.2015 sequent selfing. Around 500 plants were geno- typed with approximately 20,000 polymorphic Cooperation (other participating CEPLAS markers. Subsequently, we constructed a series and non-CEPLAS researchers) of lines that are predominately A. alpina but Maria Albani contain a small number of short segments of the Marcel Bucher A. montbretiana genome. In this way, almost all Laura Rose of the A. montbretiana genome has been intro- Korbinian Schneeberger (MPIPZ) gressed into A. alpina. We use these to define Rüdiger Simon genes conferring annual traits, to analyse epigenetic changes that occurred during the cross and Publications to generate copy number variants using translo- Willing EM, Rawat V, Mandakova T et al. cations. (2015) Nature Plants 1(2):1-7.

A2 Analysis of the roles of PERPETUAL FLOWERING 1 direct target genes in perennial flowering and their divergence in sister annual species

Aim of the project Researcher To use ChIPseq and RNAseq to identify the tar- Vicky Tilmes get genes of the PEP1 transcription factor in perennial Arabis alpina and to compare these with Project leaders the targets of the orthologous protein FLC in an- George Coupland nual Arabidopsis thaliana. Martin Hülskamp Results Project type PEP1 and FLC are orthologous floral repressors in Ph.D. project A. alpina and A. thaliana, respectively. Their differential expression contributes to the different Project start life histories of the species. Here we compared 01.11.2013 their target genes. A relatively low proportion, about 15 %, was conserved, but this group con- Cooperation (other participating CEPLAS tained most genetically important flowering tar- and non-CEPLAS researchers) gets, including SOC1, SPL15 and SEP3. Among Julieta Mateos (Fundación Instituto Leloir, the non-conserved targets, genes involved in GA Buenos Aires, Argentinia) metabolism and cold responses were present in Eva Willing (MPIPZ) both species, but they were different genes in each case. We demonstrated that in A. alpina PEP1 has a role to repress cold-induced genes within the first few hours of cold exposure, and this seems to be important for growth when the plant is exposed to longer durations of cold.

A3 Comparison of annual-perennial species pairs across the Brassicaceae

Aim of the project Researcher Comparison of annual-perennial species pairs Andrew Heidel across the Brassicaceae

Project leaders Results Laura Rose We used comparative genomic methods to de- George Coupland termine the genes underlying perennial /annual life history transitions. Our analysis covered 12 Research Area A

four species pairs consisting of an annual and a perennial in each pair (Arabidopsis thaliana/ Project type Arabidopsis lyrata, Arabis montbretiana/Arabis Ph.D. project alpina, Draba nemorosa/Draba hispanica and Arabis verna/Aubrieta canescens) plus the annual Project duration Capsella rubella. Orthologous genes that have 01.04.2013 - 30.04.2015 been deleted or become nonfunctional in all annual species are potential candidates and we aim to find these genes. We identified families in which genes were present in perennials but miss- ing in all annuals. Gene families matching this description include a number of potential regulatory genes including transcription factors and kinases. These genes are candidates for future experimental evaluation and validation within Research Area A. A paper on this work has been submitted to BMC Genomics.

A4 Nutrient recycling in the perennial plant Arabis alpina

Aim of the project Researcher Nutrient recycling capabilities are an important Anna Sergeeva feature of the perennial life style that differ between annuals and perennials and affect metab- Project leaders olism at the whole plant level. We investigate Petra Bauer nutrient relocation during perennial life style Stanislav Kopriva by applying and combining biochemical experiments, physiological analysis and plant growth Project type as well as molecular analysis and next generation Ph.D. project sequencing. The project will rely on the use of model species resources established in Research Project start Area A. This project addresses goals of Research 01.02.2015 Areas A, C and D.

Cooperation (other participating CEPLAS Results and non-CEPLAS researchers) Arabis alpina wild type and flowering mutant Maria Albani lines are continuously being grown to harvest George Coupland and to analyse plant tissue samples for metab- Tabea Mettler-Altmann (Plant Metabolism olomic activities and storage compound pro- and Metabolomics Laboratory, HHU) duction. Methods for anatomical and metabolic Klaus Theres measurements have been established.

A5 Molecular characterisation of senescence in annual and perennial plants

Aim of the project Researcher This project aims to study similarities and differ- Luise Brand ences in the control of senescence between annual and perennial Brassicaceae species. Project leaders George Coupland Results Maria Albani This project started only a few months ago. How- ever, we have identified A. alpina orthologues of Project type Postdoc project 13 Annual Report 2015

A. thaliana genes involved in the control of senes- Project start cence. We are now harvesting carefully defined 01.08.2015 leaf and shoot material through extended time courses. We will then compare the expression of Cooperation (other participating CEPLAS senescence regulators between the species and and non-CEPLAS researchers) perform RNAseq for global expression analysis. Petra Bauer Our aim is to identify differences in gene regulation between the species that might contribute to the phenotypic divergence between gener- alised monocarpic senescence after flowering observed in annuals and perennials such as A. alpina that contain tissues that do not senesce and continue to grow vegetatively through subsequent years.

A6 Photoperiodic control of flowering time in Arabis alpina

Aim of the project Researcher ▪ To understand the effect of day length on Panpan Jiang flowering time and inflorescence development and their possible interactions with the Project leaders vernalization pathway/perennialism in perpe- Ute Höcker tual and seasonal A. alpina accessions. Maria Albani ▪ To analyse photomorphogenic responses in A. alpina (seedling deetiolation, shade avoid- Project type ance). Ph.D. project Results Project start We identified a perpetual A. alpina accession that 01.10.2013 flowers independently of the day length and thus behaves very differently from other perpetual A. Cooperation (other participating CEPLAS alpina accessions which flower earlier in long day and non-CEPLAS researchers) than in short day. To identify the molecular cause George Coupland underlying this phenotypic difference, we are currently analysing the expression of candidate genes in the divergent accessions. Moreover, we have initiated crosses for future QTL mapping of the responsible locus/loci.

Second, we identified A. alpina accessions with very divergent shade avoidance responses, especially when compared to Arabidopsis. These accessions are currently characterised at the molecular and phenotypic level.

14 Research Area A

3.1.2 Elucidation of regulatory networks that determine formation and identity of meristems

Annual and perennial plants show marked differences in meristem formation and identity, which contribute to divergence in life history. For example, flowering and vegetative shoots are maintained on individual perennial plants to ensure that they survive reproduction. Furthermore, perennials more often utilize clonal reproduction, which can involve formation of ectopic meristems and switches in meristem identity. In this research section, we study these processes by examining branching patterns in A. alpina and barley, adventitious root formation in A. alpina and clonal propagation in C. resedifolia.

A7 The control of adventitious root formation in the perennial Arabis alpina

Aim of the project Researcher Adventitious roots emerge from aerial plant tis- Priyanka Mishra sues and are essential for clonal propagation of horticultural and forest species. Application of Project leaders synthetic auxin on stem cuttings is used in hor- Maria Albani ticulture to increase the initiation and elongation Rüdiger Simon of adventitious roots. However, the regulation of this process has not been studied in perenni- Project type al “intact” plants. Limited studies using artificial Ph.D. project systems such as A. thaliana seedlings grown on plates highlight the role of auxin in adventitious Project start rooting. 01.10.2014 The aim of this project is to understand the regulation of adventitious rooting in A. alpina by: ▪ Identifying the molecular mechanisms regulating adventitious root formation using forward and reverse genetics ▪ Gaining insights into whether this process is developmentally regulated ▪ Studying the role of auxin

Results We developed a protocol to induce adventitious roots on soil grown plants by applying synthetic auxin using sprays. This approach revealed developmental and spatial patterns of adventitious root formation in A. alpina. Adventitious roots develop at certain positions on the plant and correlate with flowering.

A8 Developmental basis for asexual reproduction in Cardamine

Aim of the project Researcher The aim of this project is to understand the de- Chihiro Furumizu velopmental basis for vegetative propagation in the genus Cardamine. Project leaders Miltos Tsiantis Results Angela Hay Among several species examined, C. resedifolia was chosen for further investigation because it is

15 Annual Report 2015

a diploid self-compatible plant that can be read- Project type ily cultivated. Phenotypic analyses indicated that Postdoc project C. resedifolia propagates vegetatively by form- ing shoots from roots. Project start 01.05.2015 To study the genetic basis for this phenomenon we are following two complementary approaches: mutant isolation and QTL analyses. For the first approach, a population of EMS mutagenised M2 seeds is ready to be screened for relevant phenotypes. For the second, we have crossed two divergent strains of the species and verified that random markers segregate in a Mendelian fashion in the F2 generation. This material will be used for QTL mapping.

A9 Mechanisms involved in perennial life cycle of Arabis alpina: The role of auxin in perennial traits

Aim of the project Researcher Auxin is produced in expanding leaves at the Alice Vayssières shoot apex and transported basipetally through the stem. When the shoot apical meristem com- Project leaders mits to reproductive development, it becomes a Maria Albani weak auxin source and this results in the release Wolfgang Werr of apical dominance. In A. alpina, as in many perennials, axillary branches maintain vegetative Project type growth and potentially can serve as new auxin Associated Postdoc project sources on the plant.

Project start The aim of this project is: 01.08.2015 ▪ to follow the changes in auxin distribution during development ▪ to relate changes in auxin distribution with perennial traits ▪ to create a model

Results We characterised the release of apical dominance in relation to flowering. Flower buds in A. alpina initiate during vernalisation and similarly to A. thaliana this results to the activation of axillary buds. Initiated branches do not outgrow immediately but resume vegetative growth after plants return to warm temperatures. The position of these axillary branches correlate with senescence patterns, the presence of paradormant buds and adventitious roots.

We are currently developing the molecular tools to measure auxin transport and auxin levels at different developmental stages.

16 Research Area A

A10 The regulation of inflorescence branching in Arabis alpina

Aim of the project Researcher Inflorescence buds in A. alpina develop during Evelyn Obeng-Hinneh the vernalisation period and emerge when plants are transferred to warm temperatures. This pat- Project leader tern of flowering resembles that of many temper- Maria Albani ate perennials where flower initiation and emer- gence are separated in time. Project type CEPLAS funded via AG Albani* The aim of this project is: * Project also associated and co-funded by SPP1530 ▪ to characterise inflorescence development during cold (including inflorescence branch- Project start ing and outgrowth) 01.08.2015 ▪ to determine the molecular mechanisms regulating inflorescence development in A. alpina

Results We determined the length of vernalisation period required to ensure complete flowering response. To study the molecular mechanisms regulating inflorescence development, we performed a mutagenesis screen. We identified several reduced inflorescence branching mutants (rib), which also show a flowering time phenotype. Using mapping by sequencing, we identified at least 5 rib mutants carrying lesions in a novel gene.

This candidate gene encodes for an AAA-ATPase that has not been previously studied. We are currently verifying our findings in A. alpina and A. thaliana.

A11 The regulation of inflorescence branching in Arabis alpina

Aim of the project Researcher Similar to Project A10, the aim of this project is Yanhao Zhou to identify other components that regulate inflorescence branching in A. alpina by exploring ad- Project leader ditional rib mutants. Maria Albani Results Project type In progress – Project start 01.10.2015 CEPLAS funded via AG Albani* * Project also associated and co-funded by SPP1530

Project start 01.10.2015

17 Annual Report 2015

A12 Analysis of axillary meristem initiation in perennial plant Arabis alpina

Aim of the project Researcher To identify and analyse genes that regulate the Udhaya Ponraj pattern of axillary meristem formation during shoot development of Arabis alpina. Project leaders Klaus Theres Results Maria Albani Bud initiation in A. alpina was microscopically analysed at different stages of development. This Project type study revealed that buds that remain vegetative Ph.D. project are produced before vernalization, whereas buds that undergo flowering are produced after the Project start onset of vernalization. 01.10.2013 RNA in-situ hybridisation was used to compare the expression pattern of the axillary meristem regulator LATERAL SUPPRESSOR (LAS) in A. alpina with that of the Arabidopsis LAS gene. The patterns of LAS mRNA accumulation in Ara- bis and Arabidopsis are similar. To compromise AaLAS function, an RNAi construct was introduced into A. alpina plants. RNA-seq analysis of very young axillary buds is being used to study the transcriptome of emerging buds at different stages of the A. alpina life cycle.

A13 Genotypic and phenotypic analysis of tiller development in cultivated (Hordeum vulgare) and wild barley species (H. v. spp. spontaneum, H. bulbosum) Aim of the project Researcher Genetic dissection of (axillary) meristem initiation Wilma van Esse and outgrowth.

Project leaders Results Maria von Korff We demonstrated that mutations in barley row- Maarten Koornneef type genes have pleiotropic effects on shoot branching. We identified three mutant groups Project type as characterised by their relationships between Postdoc project spike branching and tillering:

Project start 1a) Mutants with a higher seed number per 01.07.2013 spike and a decrease in tillering at early developmental stages, Cooperation (other participating CEPLAS 1b) mutants with an increased seed number and and non-CEPLAS researchers) a reduced tiller number only at maturity and Laura Rossini (University of Milan, Italy) 2) mutants characterised by a reduction in Rüdiger Simon seeds per spike and in tiller number.

Publications While negative correlations between seed num- Liller CB, Neuhaus R, von Korff M et al. ber and tillering in mutant group 1b were likely (2015) PloS one 10(10):e0140246 due to resource limitations, negative and positive correlations between seed number per spike and tiller number in mutant groups 1a and 2, respectively, suggested that row-type genes also controlled tillering. 18 Research Area A

Currently, transcriptional profiling is employed to gain insight into how genes affecting spike and inflorescence branching are regulated and control downstream targets. Understanding the genetic and molecular correlation between shoot and inflorescence branching is essential to improve crop yield through the modification of these traits.

A14 Genetic dissection of natural variation in tiller development in cultivated and wild barley

Aim of the project Researcher Identification and functional characterisation of Agatha Walla the high-tillering granum-a mutant

Project leaders Results Maria von Korff Detailed phenotypic analysis of the high tillering Rüdiger Simon granum-a (gra-a) mutant showed that high tillering was caused by an altered plastochron in this Project type mutant. RNA-sequencing of two allelic variants Ph.D. project (gra-a.1 and gra-a.2) introgressed into a common genetic background combined with genetic Project start mapping experiments allowed to map the causa- 01.02.2014 tive gene to an interval of 0.4cM.

Cooperation (other participating CEPLAS Tiller development was also studied in a diverse and non-CEPLAS researchers) collection of annual and perennial Hordeum spe- Laura Rossini (University of Milan, Italy) cies. Annual species were characterised by a short period of tiller outgrowth and early whole plant senescence whereas perennial species produced tillers over a period of one year and showed selective senescence of individual tillers. The collection of annual and perennial Hordeums was subjected to exome sequencing to study natural variation of candidate genes for axillary meristem development.

A15 Genetic and environmental control of inflorescence development and floret fertility in barley and wheat

Aim of the project Researcher To elucidate the mechanisms which determine Filipa Tomé floret fertility in response to photoperiod and expression variation of Flowering Locus (FT)-like Project leaders genes in barley and wheat. Maria von Korff Andreas Weber Results We showed that variation in the expression of Project type FT-like genes in the barley leaf and meristem Postdoc project correlated with fertility. In photoperiod shift experiments from long to short days, we found that Project start long-day exposure until flowering was crucial for 01.07.2015 inflorescence development and floret fertility in

19 Annual Report 2015

wild-type plant which do not express FT under Cooperation (other participating CEPLAS short days. Under short days, the main culm in- and non-CEPLAS researchers) florescence was aborted before flowering. Intro- Jorge Dubcovsky (University of California, gression lines with increased FTexpression under USA) long days showed a higher floret fertility, while clock mutants with a photoperiod independent FT expression produced fertile florets also under short days. We subjected tissues of developing leaves and inflorescences to RNA-sequencing to identify downstream targets of FT-like genes which control floret fertility.

A16 Comparative transcriptome profiling of meristem states in annual and perennial Arabis and Arabidopsis species and hybrids

Aim of the project Researcher The aim of this project is the definition of regu- Helge Pallakies latory gene networks contributing to either an- nualism or perennialism in root development. Project leaders We therefore perform comparative phenotypic Rüdiger Simon and transcriptome analyses in two closely related George Coupland Arabis species: the perennial Arabis alpina Pa- jares and the annual Arabis montbretiana. Project type Postdoc project Results The characterisation of early root development Project duration revealed differences in root length and emer- 01.05.2013 - 30.04.2015 gence of lateral roots. From the first day up to 14 days after germination, A. alpina exhibited Cooperation (other participating CEPLAS smaller roots compared to A. montbretiana. Fur- and non-CEPLAS researchers) thermore, lateral roots emerged at later stages in Jorge Dubcovsky (University of California, A. alpina compared to A. montbretiana. USA) The observation of later developmental stages Publications up to initiation of flowering in A. montbretiana Pallakies H, Simon R (2014). Molecular revealed differences in growth speed and root plant 7(11):1619-1636. system expansion.

Furthermore, differences in lateral root angles caused by differences in gravitropic responses are indicated. Experiments with both species and wild variations (collected by S. Woetzel, MPIPZ) aimed at gravitropic responses were performed. From three early root meristem stages, transcriptomes were analysed via RNAseq. We concen- trated our analysis on genes involved in phyto- homrone signalling pathways, cell division and differentiation. A manuscript presenting this data set is in preparation.

Our approach has deepened our understanding of the factors that allow perennial plants to develop an expanded root system. We expect that the differences in expression levels we noted between annual and perennial plant roots will allow targeted modification of root growth behaviour.

20 Research Area A

A17 Analysis of an evolutionary conserved module regulating root system development in monocot and dicot species

Aim of the project Researcher Understand the genetic modules that determine Gwendolyn Kirschner root meristem growth in barley, using barley homologues of genes known to be important for Project leaders root development in Arabidopsis, maize and rice Rüdiger Simon as a starting point. Maria von Korff Results Project type We have by now achieved a characterisation Ph.D. project of the barley root meristem growth behaviour. The stem cell niche and its cell division patterns Project start were studied using EDU stainings. Homologues 01.12.2013 of regulatory genes known to be of functional importance for meristem development and the stem cell state in Arabidopsis were cloned from barley, and both transgenic knock-downs (via RNAi) and reporter lines were generated, which are currently being analysed.

The results could enable to grow plants with a root system that is more resistant to drought, water stress and could prevent soil erosion.

A18 Using receptor kinase pathways to modify plant traits

Aim of the project Researcher Seeds stem from fertilised ovules, which are initi- Nozomi Kawamoto ated from a meristematic tissue called placenta. The mechanisms that pattern the placenta, i.e. Project leader that specify individual cells as ovule anlagen, Rüdiger Simon is not understood. We aim at unravelling these mechanisms which would allow to modify seed Project type number as an important agronomic trait. Postdoc project Results Project start We performed a GWAS and QTL analysis on 01.09.2015 Arabidopsis ecotypes (Nordborg collection) and mapped chromosomal regions important for Cooperation (other participating CEPLAS ovule number and ovule density. We found that and non-CEPLAS researchers) ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 Lucia Colombo (University of Milan, Italy) are main regulators of ovule density, and iden- Keiko Torii (University of Washington, USA) tified peptide ligands that interact with these Naoyuki Uchida receptor kinases. Misexpression of ER from a placenta-specific promoter reduced the spacing between ovules, indicating that the ER pathway lays a central role in ovule spacing.

We are now testing if the key components of the stomata biogenesis pathway also act in ovule initiation.

21 Annual Report 2015

3.1.3 Modelling regulatory modules that differ between annual and perennial plants

In this work package we aim to develop methods that can be used to model regulatory modules from the genetic, RNAseq, ChIPseq data obtained in the work described above. Modelling of such networks would assist in predicting which interventions would be most effective in engineering perennial traits into annuals. At present, the modelling algorithms are tested using the extensive data sets available in yeast.

A19 Transcriptome Data Analysis and Optimal Experimental Design

Aim of the project Researcher To infer direct interactions between gene activi- Armin Sadat Khonsari ties from transcriptome data.

Project leader Results Markus Kollmann We developed a mathematical approach to infer directed links in transcriptional network on Project type genome-wide level from knockout or knockdown Ph.D. project experiments. In contrast to other methods, our method is unbiased and can work in the realistic Project start setting that not all genes are perturbed one-by- 01.04.2013 one. For a test, we applied our method to the currently largest eukaryotic knockout screen, comprising transcriptome data for ~6000 genes in ~1700 knockout mutants.

Our results show that only a minor fraction of the existing links can be inferred as consequence of the strong noise between biological replicates and the incomplete knockout set. This result is in agreement with our analytical result that network structures where only a few genes regulate many other genes are the most difficult one to infer. We confirm that the central regulators in eukaryotes are epigenetic factors and transcription factors, as expected.

However, there exists a strong discrepancy between the reported links of gene regulatory networks (CHIP-Seq experiments) and the highly significant transcriptional interactions we find from network inference, which confirms that there must exist significant additional regulation of gene expression on the level of DNA, RNA and protein activity. We are currently finalising the manuscript.

22 Research Area A

Summary and Outlook

Model systems for studying divergence of annual and perennial traits have been developed in the Brassicaceae, particularly the Arabidae, and in Hordeum around the crop plant barley. Extensive genomic platforms and genetic tools are now available for studying traits of interest. The carefully chosen biological systems being analysed allow for interesting comparisons in mechanism between different models. For example, does understanding branching patterns in A. alpina at the levels of gene expression and auxin transport help to explain differences in the control of tillering between barley and its perennial relatives? Or are there fundamental similarities in the developmental plasticity associated with clonal propagation of shoots from the roots of C. resedifolia and of adventitious roots from the shoots of A. alpina? In addition, the first examples of exploiting inter-species gene transfer or reverse genetics using CRISPR-Cas9 to test specific hypotheses of how traits diversify during evolution of annuals are now generating results and will test the effectiveness of manipulating such traits by introducing or engineering single genes. These genes were identified as candidates based on our definition of regulatory networks that differ between annuals and perennials, particularly in areas associated with competence to flower and regulation of the duration of flowering. However the fundamental molecular changes in these genes and networks that confer divergence in life history have not yet been identified.

Major next steps will be in understanding the divergence of these traits at higher resolution, at the level of amino acid changes in proteins or nucleotide changes in promoters and how these affect binding of specific transcription factors. Once such information is obtained in our model comparative system of A. alpina / A. montbretiana we can test whether the same molecular mechanisms underlie different occurrences of annuals in the Arabis, Arabidopsis and Cardamine genera using the genomic tools that we have generated. Furthermore, this depth of understanding will facilitate engineering of the trait in other species with high precision.

23 Annual Report 2015

3.2 Research Area B

Decoding function and development of a C4 leaf

Research Area B aims at understanding the molecular mechanisms under-

lying the evolutionary trajectory from C3 to C4 photosynthesis to a level of Coordinator detail that enables introducing C4 trait modules into current C3 species. The majority of our research was hence targeted at dissecting key characteris-

Peter Westhoff tics of C4 leaf anatomy and gene expression.

CEPLAS funds were complemented by research funds from the EU 3to4 Co-coordinator project, the DFG-funded International Research Training Group 1525, the DFG Research Group Promics, the DFG Priority Programme Adaptomics Martin Lercher and HHU Düsseldorf.

Work conducted in this area focussed on four different research topics: Members

(1) Transcriptome comparisons of C3 and C4 species of the genera Cleome

Oliver Ebenhöh and Flaveria were performed to identify putative regulators of C4 leaf Georg Groth differentiation. Ute Höcker Martin Hülskamp (2) Genetic analyses were pursued with Arabidopsis thaliana in order to Karl Köhrer detect genes that are involved in bundle-sheath differentiation, and the Markus Kollmann natural variation of this species was exploited to identify genes affecting Maarten Koornneef general leaf morphology and anatomy. Veronica G.Maurino Peter Nürnberg (3) Approaches of experimental evolution were initiated with Arabidopsis Richard Reinhardt to select directly for traits that should appear on the trajectory towards

Laura Rose C4. Ulrich Schurr

Kai Stühler (4) Projects were designed that targeted regulatory modules of C4 metab-

Miltos Tsiantis olism and investigated the interaction of C4 photosynthesis with other Andreas P. M. Weber metabolic pathways.

Differentiation SPL9 [miR156] BSH Cell size SAUR TINY k-means Chloroplast development CGA1 bundle-sheath defencitive protein 2 family

Vasculature DOT5

Flower time affecting Stress realted

Deconvolution of Flaveria transcriptomes based on leaf anatomy. For deconvolution, pictures of the leaf vascular structure were obtained by stitching microscope pictures of cleared leafs (left). The structure of the vasculature was used to categorise leaf area into three sections. The change in relative area of these categories during leaf development, combined with the transcriptomes of the specifi c leaf stages, was used to model area specifi c gene expression. Finally, k-means clus-

tering was performed on the modelled transcriptomes of C3 and C4 species to infer

C4 specifi c genes. For further information, please see also project B4.

24 Research Area B

3.2.1 Evolutionary transcriptomics of C3 and C4 species pairs

The presence of closely related C3 and C4 species in the genera Flaveria and Cleome offered the unique opportunity of using comparative transcriptome analyses for the identification of genes that are involved or required for the establishment and/or functioning of the C4 photosynthetic pathway. Projects combined within this research section pursue this approach for identifying putative regulators of C4 leaf differentiation and of the C4 cycle.

The transcriptome analyses included the comparisons of leaf transcriptomes at various developmental stages from the primordia up to fully matured leaves. In addition, the leaf transcriptomes of C3 (F. robusta) and C4 (F. bidentis) Flaveria species, which were grown under circadian clock entrained and free-running (constant light) conditions, were compared to infer regulators of C4 physiology.

The developmental transcriptomics studies were accompanied by detail analyses of leaf differentiation at the histological level. The two data sets were used to construct a linear model aiming of cor- relating changes in gene expression with leaf differentiation state. Two major results emanated from these studies. Firstly, in both species leaf development of the C4 species is lagging behind that of the C3 species. Secondly, a set of genes could be identified that is correlated with the differences in leaf differentiation between C3 and C4 leaves and will provide candidate genes for future engineering efforts. Furthermore, the model enables the deconvolution of cell-specific transcriptomes from whole- leaf transcriptome data sets.

B1 Mesophyll and bundle-sheath-specific transcriptomes of 3C /C4 species pairs

Aim of the project Researcher Isolation of transcriptomes from mesophyll and Stefanie Schulze bundle-sheath cells of different Flaveria species. The separation of these two cell-types in the leaf

Project leaders was a major development in the evolution of C4 Peter Westhoff photosynthesis. Therefore, a deeper understand- Markus Kollmann ing will help to unravel the unknown steps in the necessary changes. Flaveria is a dicotylodonous Project type plant and thus would provide insight into differ- Postdoc project ently expressed genes in the above-mentioned cell-types aside from monocots, which were used Project duration in most such studies. 01.04.2013 - 30.04.2015 Results

Transgenic plants of the C4 species Flaveria bidentis were generated, which can be used to isolate the translatomes of either mesophyll or bundle-sheath cells. RNAs were isolated and the enrichment of RNAs of the corresponding cell- type was confirmed with qRT PCR using known marker transcripts (e.g., PPCA for mesophyll and GLDPA for bundle sheath cells). High-throughput sequencing of triplicates with the Illumina method will be performed.

25 Annual Report 2015

B2 Identification of C4 genetic determinants by a comparative transcriptome analysis of C3 and C4 Flaveria spp. and by forward genetics with Arabidopsis thaliana Aim of the project Researcher To unravel the major genetic regulatory network

Kumari Billakurthi behind the C4 specific Kranz leaf anatomy by two approaches. The first approach uses transcrip-

Project leaders tome data sets of F. robusta (C3) and F. bidentis

Peter Westhoff (C4) leaves at different developmental stages, to Rüdiger Simon identify differences in genes expression patterns between these two species and to exploit these

Project type data for inferring candidates genes for C4 leaf de- Ph.D. project velopment.

Project start Because the C3 model plant Arabidopsis thaliana 01.10.2013 contains bundle-sheath cells whose gene regu-

latory system is conserved between C3 and C4 Cooperation (other participating CEPLAS dicots, the second approach uses Arabidopsis and non-CEPLAS researchers) thaliana as a model to isolate, by forward genet- Andrea Bräutigam ics, genes that affect bundle-sheath anatomy. Udo Gowik (HHU) Thomas Wrobel Results Comparative transcriptome data sets were pre- pared from leaf gradients of both Flaveria species using Illumina RNA sequencing. Analysis of the transcriptome data sets revealed that leaf

development of the C4 species is delayed as

compared to the C3 Flaveria. We are presently analysing the pathways and regulatory networks that may be responsible for this delay.

Arabidopsis marker lines expressing a plastid-targeted GFP in their bundle-sheath cells were subjected to EMS mutagenesis or to activation tagging by using a leaf chlorenchyma-specific promoter. Stable mutants affected in bundle-sheath anatomy were obtained and the corresponding genes are presently being isolated by sequence-based mapping (in the case of EMS mutants) or by inverse PCR (in the case of activation tagged mutants).

B3 Circadian control of transcriptomes and proteomes of C3 / C4 species pairs

Aim of the project

Researcher Comparison of circadian gene expression in C3

Thea Pick and C4 species in the genus Flaveria.

Project leaders Results Andreas Weber We analysed the leaf transcriptomes of two Fla-

Peter Westhoff veria species employing C3 (F. robusta) and C4 (F. bidentis) photosynthesis under circadian clock Project type entrained (10 h light/14 h dark) and free-running Postdoc project (constant light) conditions. Using the JTK_Cycle algorithm (Hughes et al., 2010), we could identify 58 % of the genes as clock controlled genes 26 Research Area B

(CCGs) under entrained conditions in both spe- Project duration cies, while only 3 % were CCGs under free-running 01.04.2013 - 31.12.2014 conditions. A large proportion of CCGs overlaps between the two species. Gene expression of a Cooperation (other participating CEPLAS small group of 36 genes was found to maintain and non-CEPLAS researchers) cycling under both conditions exclusively in F. Ilka Axmann bidentis, while their circadian rhythmicity is di-

Dominik Brilhaus (HHU) minished under free-running conditions in the C3 Frank Harmon (UC Berkeley, USA) species. These genes represent good candidates

Markus Kollmann for fine tuning of circadian gene expression in 4C

plants and may support efforts to engineer C4

photosynthesis in C3 plants.

B4 Unravelling the mechanisms that control bundle-sheath cell size in leaves of C4 plants

Aim of the project To determine the developmental pattern respon-

Researcher sible for the generation of C4 specific bundles Thomas Wrobel sheath (BSH) traits and its implementation into a

C3 model organism. Project leaders Andreas Weber Results Martin Hülskamp We sequenced the transcriptomes of the closely

related species Flaveria robusta (C3) and Flaveria

Project type bidentis (C4) in leaves at different developmen- Ph.D. project tal stages. Leaf development was analysed using cleared leaves and cross-sections after araldite Project start embedding. BSH specific traits emerge during 01.10.2013 the development of the minor vasculature in the

C4 species. In order to determine the underlying Cooperation (other participating CEPLAS genetic programme, the leaf was divided into and non-CEPLAS researchers) three sections according to its anatomic features. Andrea Bräutigam A linear model was created to fit the expression Kumari Bilakhurti of genes, with the changes of relative area of the Udo Gowik (HHU) three sections in both species. Genes that corre- Sarah Richards (HHU) late with the presence of the differentiating sec-

tion in the C4 but not the C3 species are considered candidates for further analysis.

B5 Establishing a functional link between leaf development and C4 photosynthesis

Aim of the project Researcher To learn from leaf developmental transcriptomic

Otho Mantegazza series of Cleome C4 and C3 species the regula-

tory mechanisms specifying C4 leaf anatomy and Project leaders how these mechanisms can be transferred to the

Andreas Weber C3 model organism Arabidopsis thaliana, with

Matias Zurbriggen the aim of initiating a C4-like leaf development in this species. Project type Postdoc project

Project start 01.05.2015 27 Annual Report 2015

Results Cooperation (other participating CEPLAS Quantitative comparison of the transcriptomes and non-CEPLAS researchers) from different Cleome species with linear models Mara Schuler-Bermann (HHU) have been used for identifying candidate genes. Wolfgang Werr Currently, we are devising a Cas9 based strate- Thomas Wrobel gy for the direct evolution of the transcriptional and post-transcriptional regulation of candidate genes in Arabidopsis thaliana.

3.2.2 Genetic analyses with Arabidopsis thaliana for identifying genes involved in leaf anatomy and morphology

The forward genetic approaches aiming at identifying genes involved in bundle-sheath differentiation took advantage of the fact that comparative promoter analyses had demonstrated that the gene-regu-

latory systems of C3 and C4 dicots, i.e. Brassicaceae and Asteraceae, share common trans-regulatory

factors. This indicates that the bundle-sheath is not a novel invention of C4 species, but is present in

C3 species and its ontogeny and functional maintenance may therefore be studied in a genetically

tractable C3 model species i.e., Arabidopsis. EMS mutagenesis of Arabidopsis lines expressing a bundle-sheath chloroplast targeted reporter GFP resulted in the identification of two mutant lines with altered bundle-sheath anatomy. The affected genes are presently being isolated by the SHORE map approach. Similar mutant phenotypes - a total of six at present - were obtained by using activation tags containing mesophyll and/or bundle-sheath specific promoters. The affected genes were identified and are presently verified by cell-specific overexpression and their knock-down or knock-out.

In addition to forward genetics, projects assembled in this research section also used natural variation in Arabidopsis to identify genes involved in leaf anatomy and morphology.

B6 Unravelling the functional relationship between leaf anatomy and photosynthesis by mutational and synthetic approaches

Aim of the project Researcher To unravel the functional relationship between Roxanne van Rooijen leaf anatomy and photosynthesis. For this, we will first develop an Arabidopsis thaliana ref- Project leaders erence line for the concomitant visualisation of Peter Westhoff both chloroplasts and mitochondria in the bun- Andreas Weber dle sheath cells of leaf to directly quantify their numbers as well as the ratio between them. Project type Furthermore, we can quantify the ratio between Postdoc project chloroplasts /mitochondria in bundle sheath versus chloroplasts /mitochondria in mesophyll. We Project start will then perform activation tagging in this refer-

01.09.2015 ence line with a C4-derived leaf specific promoter, and check for aberrant leaf morphology phenotypes in any of the leaf morphology parameters just described. Different leaf morphology activation-tagged mutants will then be measured for photosynthetic light use efficiency to see if any leaf morphology parameter would be associated with enhanced photosynthesis efficiency resem-

bling C4. Lastly, we will characterise the function of the gene that is activated in these mutants.

Results 28 In progress - Project start 01.09.2015 Research Area B

B7 Genetic variation for leaf anatomy in Arabidopsis

Aim of the project

Researcher Identification of genes relevant to C4-photo-

Louai Rishmawi sythesis through the analysis of C3 plants. The analysis of different leaf traits (thickness, size, Project leaders chlorophyll content, and venation) in Arabidop- Martin Hülskamp sis thaliana and the identification of the relevant Maarten Koornneef genes will enable us to identify the molecular

basis for these traits in C3 plants. This will help

Project type to understand how the C4-trait can evolve from Postdoc project pre-existing genetic variation in these subtraits.

Project duration Results 01.05.2013 - 30.06.2015 In collaboration with the group of Siegfried Jahnke (Forschungszentrum Jülich), a software Cooperation (other participating CEPLAS for leaf-venation was developed (Bühler et al., and non-CEPLAS researchers) 2015). The phenotying of the following leaf traits Siegfried Jahnke (FZJ) have been finished: leaf thickness, area, chloro- Ulrich Schurr phyll content and venation. Association mapping by GWAS and by using Magic Lines was done for Publications all traits and several candidate genes were found Bühler J, Rishmawi L, Pflugfelder D et al. for each trait. These candidates need to be con- (2015) Plant physiology 169(4):2359-2370. firmed.

B8 Light effects on leaf anatomy in Arabidopsis

Aim of the project Researcher ▪ Identify loci controlling leaf anatomy in Arabi- Eva Willée dopsis using natural genetic variation ▪ Help design a leaf with superior anatomical Project leaders features for efficient photosynthesis Ute Höcker Martin Hülskamp Results We have screened Arabidopsis thaliana acces- Project type sions for variation in the leaf anatomy traits leaf Ph.D. project thickness and vein density in response to high light. We found sufficient variation among the Project start parents of the AMPRIL mapping population and 01.05.2013 have used the AMPRIL RILs provided by Maarten Koornneef for QTL mapping. Several significant Cooperation (other participating CEPLAS QTLs were identified that are currently being and non-CEPLAS researchers) fine-mapped using heterogenous inbred families Peter Westhoff (HIFs).

As a second approach, we are currently generating transcriptome data on developing and fully developed leaves that were exposed to low and high light conditions. These leaves exhibit contrasting leaf anatomy phenotypes and thus will complement the QTL analysis approach.

29 Annual Report 2015

B9 Differentiation of vascular veins and surrounding mesenchyme: the role of leaf meristems within adaptations to C4 anatomy

Aim of the project Researcher We address the molecular basis of early leaf Satish Kumar Eeda primordia differentiation. A specific goal is to understand the activity of marginal or platemer- Project leaders istem with respect to vascularisation and eluci- Wolfgang Werr date on the temporal series of events and signals Peter Westhoff between in the mesenchymal cell niche and the vascular cambium during the earliest phase of Project type leaf development. According to genetic data, this Ph.D. project interplay relates to the density of vasular veins during leaf lamina expansion and will be put into Project start the context of elaboration of the Kranz anatomy

01.10.2014 that in C4 plants typically develops at the interface of mesenchyme and vascular strands.

Results The research project in its first year has provided three major results in Arabidopis:

▪ Based on WOX3 activity in marginal and plate meristems the initiation of a new primordium starts with mesenchymal identity. ▪ There is a delay of at least 12 hours before cambial markers like AtHB8 or WOX4 are detectable, the WOX4 promoter is presently optimised. ▪ Bundle-sheath-like identity in cells surrounding vascular strands is determined significant- ly (> 36 hours) later them the cambial fate.

In addition, the WOX3::LHG4GR and pOp::GFP reporter combination shows suitability and ro- bustness of the driver/reporter system during early stages of leaf development.

B10 Generation of synthetic tools for the engineering of C4 photosynthesis

Aim of the project

Researcher The anatomical adaptation of C4 plants called Mara Schuler Bermann ‘Kranz anatomy’ is a major evolutionary prereq-

uisite to evolve the C4 carbon concentrating Project leaders cycle and represents a major engineering chal- Peter Westhoff lenge due to complexity of the trait. It is not yet Andreas Weber fully understood which developmental factors determine Kranz anatomy, but we agree that a Project type cohort of genes is needed to initiate this spe- Postdoc project cialised anatomy early in development prior to

the integration of the two-celled C4 photosyn- Project start thetic cycle. Therefore, the conversion of leaf 01.08.2015 anatomy and the integration of an entire photosynthetic pathway can only be achieved by multigene transformation (MGT) whereby every nuclear transgene requires its own promoter. The 30 Research Area B

promoter of choice should allow the expression of an array of developmental factors, transporters and photosynthetic enzymes to be targeted to the right cell-type within the leaf. Moreover, it requires a toolbox of cis-regulatory modules (CRM) to drive cell-type specific expression.

The project focuses on biological and methodo- logical aspects and the aims are 1. The functional characterisation of genetic regulators of Kranz anatomy. 2. The identification of cis-regulatory modules driving expression early in vascular development in leaf primordia. 3. The generation of an experimental system for multigene targeting to bundle sheath cell.

Results In progress - Project start 01.08.2015

3.2.3 Experimental evolution towards C4 photosynthesis

The quantitative evolutionary model of C4 photosynthesis, developed in the framework of CEPLAS

(Heckmann et al, Cell 2013; Mallmann et al, eLife 2014), indicates that the transition from C3 to C4 photosynthesis proceeded in modules and that each of the individual modules was adaptive. This implies that each step brought a small but detectable advantage in the photosynthetic capacity and suggests that it should be possible to evolve a C4-like or, at least, a C3-C4 intermediate type of photosynthesis by applying the concept of experimental evolution combined with synthetic biology. Projects combined here either attempt to refine the current model by incorporating not yet considered factors of evolutionary pressure/selection or by using experimental/mutagenic approaches to select for C3-C4 like properties by using Arabidopsis thaliana.

B11 In silico exploration of paths towards C4 metabolism

Aim of the project

Researcher C4 photosynthesis evolved over 60 times inde-

Esther Sundermann pendently from C3. Our goal is to better understand this evolutionary process with a view Project leaders towards artificial selection. We will simulate evo-

Martin Lercher lutionary trajectories towards C4 in environments Andreas Weber that differ in factors that affect photorespiration.

Project type Results Ph.D. project We created a model that considers the effects of light, temperature, and gas partial pressures on

Project start CO2 assimilation rate, which we use as a proxy for 01.06.2014 plant fitness. The light-dependent model considers an optimal energy partitioning and limitations Cooperation (other participating CEPLAS by the availability of NADPH and ATP. The effect and non-CEPLAS researchers) of temperature is approximated by an extended David Heckmann (HHU) Arrhenius equation, which models both activation and inactivation through structural instabil- ity. The model successfully predicts experimental data.

31 Annual Report 2015

B12 Experimental evolution towards C4

Aim of the project Researcher ▪ Analysis of anatomical and physiological Nadine Höcker traits of diverse Arabidopsis thaliana ecotypes grown under low (190 ppm) and high

Project leaders (700 ppm) CO2 concentrations. Andreas Weber ▪ Screen for ecotypes with striking differences Peter Westhoff over the mean for traits related to efficient carbon assimilation (biomass, photosynthetic Project type rates). Genetic analysis with focus on differ-

Postdoc project ences related to CO2 concentration mechanisms and structures. Project duration 01.05.2013 - 30.06.2015 Results 64 Arabidopsis thaliana ecotypes and polyploid- Cooperation (other participating CEPLAS iced lines have been screened and non-CEPLAS researchers) 1.) at low (180 ppm) versus ambient (390 ppm)

Fabio Fiorani (FZJ) CO2 concentrations and Niklas Körber (FZJ) 2.) at high (700 ppm) versus ambient (390 ppm)

Ulrich Schurr CO2 concentrations at short day (12/16 h, 22/18°C).

The plants grew for one month until the first

ecotypes under high CO2 conditions started bolting and committed to flowering. Data analysis of the first experiment displayed different growth and photosynthetic characteristics, measured via the automated GROWSCREEN devise. Based on this analysis a ranking of most interesting candidate ecotypes was performed and these have been selected for RNAseq, as well as for EA-IRMS.

B13 Transforming Arabidopsis plants towards C4 metabolism

Aim of the project

Researcher To generate C4 prototype from C3 plant Arabi- Yuanyuan Li dopsis. It will be a breakthrough achievement for CEPLAS if, towards the end of this project we

Project leaders succeeded in generating C3 plants performing

Veronica G. Maurino C4 photosynthesis. We aim to combine genetic Martin Lercher engineering, mutagenesis, and artificial selection

to implement C4 photosynthesis in the C3 plant Project type Arabidopsis. We will engineer Arabidopsis plants Postdoc project with RubisCo specifically expressed in bundle sheath cells. The plants will later be mutagenised Project start to screen for improved photosynthesis and are 01.04.2015 expected to respond to this artificial selection

pressure by evolving further towards C4 anatomy Cooperation (other participating CEPLAS and biochemistry. and non-CEPLAS researchers) David Heckmann (HHU) Results We have already raised the T1 transformed plants and expect to get the Arabidopsis plants with RubisCo specifically expressed in bundle sheath cells in two generations. 32 Research Area B

3.2.4 Regulation and metabolic interactions of C4 photosynthesis

The general make-up of the C4 cycle and its regulation by metabolic and environmental factors are essentially known. However, still important details in the composition and regulation of the C4 cycle are unknown, i.e. which transporters are involved shuttling metabolites between organelles and cytosol or how is regulatory specificity achieved at the molecular level. It is also not well understood how

C4 cycle is coordinated with other metabolic pathways such as sulfur or nitrogen metabolism. By using the transcriptomic data sets generated from C3/C4 species pairs of Flaveria and Cleome, the knowledge about C4 enzymes and their regulators as well as methods of structural biology the projects of this research section attempt to fill the gaps in our understanding of the 4C cycle and its regulation. This is imperative, if metabolic engineering approaches are to be successful.

B14 The C4 cycle and its surrounding metabolism

Aim of the project

Researcher To understand the integration of the C4 cycle into Andrea Bräutigam the surrounding metabolism and its evolution using high throughput data. Project leaders Andreas Weber Results

Peter Westhoff C4 photosynthesis was hypothesised to have

evolved in conditions stressful to the ancestral C3

Project type plant. To investigate transcriptional changes in C3 Associated Postdoc project plants under stress, RNA-seq datasets generated by collaborators (Martin Hülskamp – salt stress, Project duration Jürgen Zeier – biotic stress, pathogenic bacteria, 01.08.2014 - 31.12.2015 Veronica G. Maurino – salt stress, Marion Eisen-

hut / Stefan Timm – CO2 stress) were analysed in Cooperation (other participating CEPLAS detail, especially with regard to photosynthesis. and non-CEPLAS researchers)

Pascal Antoine Christin (University of Shef- The C4 photosynthesis of Alloteropsis semialata

field, UK) was studied to identify the C4 cycle components

Marion Eisenhut (HHU) and an enzyme heretofore not associated with C4 Martin Hülskamp photosynthesis was identified as part of the core Veronica G. Maurino cycle. Stefan Timm (University of Rostock) Jürgen Zeier A meta-analysis of RNA-seq experiments from

closely and distantly related C3 and C4 species Publications summarised the current status for the molecular

Steffens A, Bräutigam A, Jakoby M et al. identity of the C4 cycle components. Three trans- (2015) PLoS biology 13(7):e1002188. port proteins remain uncharacterised at the molecular level: however, the possibility that their Bernsdorff F, Döring AC, Gruner K et al. function is not necessary because of diffusion or (2016) The Plant cell 28(1):102-129. carried out by transport proteins already known exists. It is likely that only synthetic biology will resolve the question.

33 Annual Report 2015

B15 Alterations to the regulation of C4 enzymes during evolution of C4 photosynthesis

Aim of the project Researcher (1) Identify and characterise NAD-malic enzyme

Meike Hüdig (NAD-ME) of Tarenaya hassleriana (C3 plant)

and Gynandropsis gynandra (C4) as close rela- Project leaders tives to Arabidopsis thaliana. The comparison Veronica G. Maurino will help to reveal changes that occurred dur-

Peter Westhoff ing NAD-ME evolution in C4. (2) Study the role of mitochondrial malate meta- Project type bolism in A. thaliana by using loss-of-function Ph.D. project lines of NAD-ME and mitochondrial malate dehydrogenases. Project start 01.02.2014 Results (1) In vitro characterisation of heterologously Publications expressed NAD-MEs is ongoing. Compar- Hüdig M, Maier A, Scherrers I, et al. (2015) ative NAD-ME transcript analysis has been Plant & cell physiology 56(9):1820-1830. performed for Cleome species, additional

NAD-ME C3 /C4 species pairs will be analysed. Physiological characterisation in leaf material started (circadian rhythm, enzyme composition). (2) Developmental characterisation of 13 knockout lines (single, double, triple gene knock- outs) has been performed. Additional phenotyping is ongoing.

B16 Structural evolution of phosphoenolpyruvate carboxylase kinase (PPCK) in the genus Flaveria

Aim of the project Researcher To resolve the molecular basis of the preferen-

Johannes Schwabroh tial interaction of protein kinase PPCKA with C4-

PEPC and of kinase isoform PPCKB with C3-PEPC. Project leaders To this end, we will identify key residues in both Georg Groth kinases that determine their interaction and solve

Peter Westhoff high-resolution structures of C3- and C4-typical PPCKA and PPCKB isoenzymes from Flaveria by Project type X-ray crystallography. Detailed knowledge of the Ph.D. project molecular basis controlling specificity of PPCKA

for C4-PEPC will provide essential information on

Project start the regulation of the C4-module. 01.10.2014 Results PPCKs were successfully expressed in soluble form in E. coli rosetta-gami and purified to ho- mogeneity from the bacterial host. Interaction of

the purified 4C -isoform of PPCKA was analysed in

binding studies with the C4-PEPC from F. trinervia by microscale thermophoresis. Analysis of these binding studies suggest a sub-µM dissociation constant for this interaction.

34 Research Area B

B17 Nitrogen and Sulfur metabolism in C4 plants

Aim of the project Researcher To find out the biological relevance of differential

Silke Weckopp distribution of N and S metabolism in C4 plants and how this distribution affects use efficiency of Project leaders these nutrients. Stanislav Kopriva Peter Westhoff Results We revealed a gradient in accumulation of re-

Project type duced S compounds towards increased C4 char- Ph.D. project acteristics in Flaveria species. Similar gradient was seen in sulfate uptake and reduction rate. A Project start significant change in distribution of sulfate and 01.01.2015 phosphate in roots and shoots was observed be-

tween C3 and C4 species. Publications Weckopp SC, Kopriva S (2014) Frontiers in plant science 5:773.

Kopriva S, Calderwood A, Weckopp SC et al. (2015) Plant science: an international journal of experimental plant biology 241:1-10.

B18 Mathematical modelling of acclimation processes of the photosynthetic electron transport chain in green algae and plants

Aim of the project Researcher Using theoretical methods to understand how Anna Matuszynska various photosynthetic model organisms accli- mate to changing light conditions. Project leaders Oliver Ebenhöh Results Peter Westhoff In collaboration with several experimental groups dynamic mathematical models are developed for Project type various scenarios and for different species for ex- Associated Ph.D. project ample:

Project start 1. A minimal mathematical model for qE to study 01.05.2014 short-term light memory: with a minimal set of equations we can capture the dynamics of Cooperation (other participating CEPLAS a complex system and can test hypotheses on and non-CEPLAS researchers) the role of zeaxanthin in the photoprotective Federica Cariti (University of Geneva, memory (Arabidopsis); Switzerland) Gilles Curien (CEA, Grenoble, France) 2. Include light wavelength as external param- Giovanni Finazzi ((CEA, Grenoble, France) eter impact on organisms performance, be- Serena Flori (CEA, Grenoble, France) cause so far there is no dynamic model of Michel Goldschmidt-Clermont (University light photosynthetic acclimation that includes of Geneva, Switzerland) wavelengths to study how light quality affects Somayeh Heyidari (Ferdowsi University of photosynthetic activity (Chlamydomonas); Mashhad, Iran) Jun Minagawa (National Institute for Basic 3. Implement Calvin cycle demand to study sup- Biology, Okazaki, Japan) ply-demand regulation and make predictions Ryutaro Tokutsu (National Institute for regarding biomass production; Basic Biology, Okazaki, Japan) 35 Annual Report 2015

4. Dynamics of state transitions (Chlamydomo- Publications nas). Matuszynska A, Ebenhöh O (2015) Bio- chemical Society transactions 43(6):1133- These activities will support CEPLAS to optimise 1139. photosynthetic efficiency.

Summary and Outlook

Collaborative efforts within CEPLAS Research Area B provided a quantitative model of C4 evolution

that now serves as a paradigm for a stepwise introduction of C4 trait modules into current C3 species. However, the computational modelling efforts were built on the premise that anatomical precondi- tioning was already in place. Yet, these evolutionary enablers, in particular the factors controlling the establishment of an enlarged bundle-sheath rich in chloroplasts and mitochondria, are far from being

understood. A C4-like bundle-sheath appears to be essential, if experimental evolution approaches

are to be successful. Such a bundle-sheath exists in C3-like C3-C4 intermediate species such as Mori- candia arvensis, and this species may therefore represent a good model for artificial evolution.

Inferring candidate genes from evolutionary or other transcriptome studies has – to the present date

– not yet yielded validated genes for introducing typical C4 Kranz anatomy into a C3 leaf anatomy. It remains to be seen whether candidate genes identified in the framework of CEPLAS or elsewhere will solve this problem. We will therefore focus our future efforts on validation of candidate genes from these prior studies.

Forward genetics is powerful because a causal relationship between gene and phenotype is an inher- ent property of this experimental approach. Candidate genes isolated in our prior forward genetic approaches are highly promising. Hence we will concentrate on exploiting these genes for engineering of leaf anatomy. A serious limitation for such engineering work is the scarcity of available defined (synthetic) modules that allow appropriate expression of candidate genes in space and developmental time. We will therefore intensify our efforts to isolate such regulatory modules, including the design of synthetic promoters.

36 Research Area B

37 Annual Report 2015

3.3 Research Area C The molecular basis of plant-microflora interaction

Roots and leaves of healthy plants host a wide range of microbes which enhance resistance to pathogens and/or facilitate plant mineral nutrient up- Coordinator take in exchange for carbohydrates and other organic metabolites. These associations play a key role in shaping terrestrial ecosystems and are widely Alga Zuccaro believed to have promoted the evolution of land plants.

To establish compatibility with their host, plant-associated microbes have Co-coordinator evolved diverse colonisation strategies with distinct morphological, functional and genomic specialisations as well as different degrees of interde- Jane Parker pendence. These associations can be host-specific or display a broad host range and undergo long-term interactions with a large variety of plants. To establish and maintain a compatible interaction, hosts as well as microbes Members must respond and adapt to different signals. Alternative lifestyle and colonisation strategies may thus be a consequence of this adaptation to highly Michael Bonkowski variable environments. Recent findings indicate that both abiotic factors Marcel Bucher and host genotype interact to influence plant colonisation by microbes and Gunther Döhlemann that a small number of microbial taxa, named microbial “hubs,” which are Oliver Ebenhöh strongly interconnected help shape community structure. Michael Feldbrügge Martin Hülskamp The increasing number of available plant and microbe genomes, togeth- Eric Kemen er with rapid technical advances in computational biology, metagenomics, Karl Köhrer culture-independent microbe detection and manipulation of microbe-plant Maarten Koornneef interactions, offers unprecedented opportunities for new discoveries to Martin Lercher this rapidly evolving field. This is the foundation for exploring how distinct Alice McHardy microbial and host symbiosis determinants modulate interactions with dif- Peter Nürnberg ferent plants. Results from this research will help to develop sound experi- Richard Reinhardt mental strategies in more complex environments (e.g., in the rhizosphere) Laura Rose at different temporal and spatial scales. This can be seen as a contribution Paul Schulze-Lefert to the fundamental understanding of the plant-microbes metaorganism Jürgen Zeier and beneficial plant-microbe symbioses and thereby to the development Alga Zuccaro of novel strategies for plant protection and biological growth promotion.

In Research Area C we aim to:

1. Characterise the structure, function, and ecology of the plant microbiome within and between species of the Brassicaceae.

2. Understand how biotic and abiotic factors affect the composition and activities of plant associated microbial communities. Identify and characterise microbial hubs.

3. Identify genes in both host and microbe that determine the outcome of these interactions.

4. Determine the reciprocal effects these interactions have on the production of metabolites in hosts and microbes (collaboration Research Area D).

5. Construct synthetic symbioses to shape the endophytic microbiome towards increased plant performance.

38 Research Area C

The expertise in Research Area C groups in ecology (e.g., Bonkowski, Rose), effector biology (e.g., Zuccaro, Döhlemann), plant immunity (e.g., Parker, Zeier), and reconstitution biology (e.g., Schul- ze-Lefert, Bucher, Kemen) combined with that of our well-established national and international long-term research collaborators and the availability of different central facilities located at the four partner centres within CEPLAS help us dissect plant-microbe ecology and design synthetic microbiomes. As a result, groups working in this emerging field within Research Area C are able to fill a new and highly promising research niche that is relevant from both a pure and applied scientific perspective.

Our recent research into the biology and genomics of plant-microbe associations has revealed fasci- nating insights to the phenotypic and trophic plasticity of these interactions and underlying genomic traits associated with intracellular accommodation of microbes in roots and leaves.

Comparison of 16S rRNA and shotgun metagenome abundances of Bacteria and Archaea in the barley rhizosphere (Bulgarelli D, Garrido-Oter R, Münch PC et al. (2015) Cell host & microbe 17(3):392-403). For further details see project C11.

39 Annual Report 2015

3.3.1. Structure, function and ecology of the plant microbiome

With respect to this goal, we determined which microbial species (fungi, bacteria, oomycetes and protozoans) prosper endophytically within plant tissues of Arabidopsis thaliana and Arabis alpina and how these endophytic communities differentiate from the microbes in the surrounding soil. Addition- ally, we produced data on how the microbiome differs across a larger taxonomic breadth (Schlaeppi et al., 2014; Bai et al., 2015; Bulgarelli et al., 2015; Hacquard et al., 2015).

C1 Characterisation of the mycobiome of Arabis alpina

Aim of the project Researcher Study the root-associated fungal microbiome of Juliana Almario Arabis alpina, a non-mycorrhizal plant growing in alpine environments including low phosphorus Project leaders (P) soils. Marcel Bucher George Coupland Results Using fungal ITS2 sequencing, we described Project type the fungal community colonizing the roots of Postdoc project A. alpina growing under controlled and natural conditions. From 830 fungal OTUs detected in Project start A. alpina roots, six were detected in every sam- 01.03.2013 ple. This core fungal root microbiome included plant-associated fungal genera like Mortierella, Cooperation (other participating CEPLAS llyonectria and Fusarium. Using correlation analy- and non-CEPLAS researchers) sis we identified a Rhynchosporium OTU whose Ganga Jeena relative abundance in plant roots correlated with Gregor Langen higher P accumulation in the plant shoot, sug- Jörg Wunder (MPIPZ) gesting this fungus could improve plant P up- Alga Zuccaro take. Indeed, Rhynchosporium inoculation promoted plant root growth under low P conditions, and increased plant shoot growth and P content under sterilised soil conditions. Moreover, the fungus facilitated 32P-phosphate acquisition of A. alpina plants under experimental conditions. We hypothesise that the studied A. alpina population developed a partnership with this fungus in order to adapt to local low P in soil.

C2 Characterisation of oomycete and protozoan species from tissues of A. thaliana and A. alpina

Aim of the project Researcher Eukaryotic organisms are an important, but un- Sebastian Ploch derstudied group of plant-associated organisms. These organisms may affect plant performance Project leaders and could represent novel organisms to gener- Laura Rose ate beneficial synthetic communities. The project Michael Bonkowski aims to discover and characterise the impact of oomycetes and protists in the rhizo- and phyllosphere of Arabidopsis thaliana and Arabis alpina. Cercozoa were chosen as the first protozoan group to be investigated.

40 Research Area C

Results Project type A large number of Cercozoan and Oomycete Postdoc project species closely associated with A. thaliana were discovered, including many taxonomically un- Project duration described groups. One Cercozoan taxon could 01.11.2013 - 30.04.2015 be found within plant tissue. This species will be used for further investigation.

A paper on these results is currently under review at the Journal of Eukaryotic Microbiology.

C3 Diversity of oomycetes and protists in intimate association with Arabidopsis

Aim of the project Researcher The diversity of oomycetes and protists associ- Melanie Sapp ated with Arabidopsis thaliana will be studied in great depth using Next Generation Sequencing. Project leaders Based on the results, experiments are planned Laura Rose to identify and characterise specific interactions Michael Bonkowski between beneficial protist and oomycete taxa. Ultimately we aim to manipulate microeukaryote Project type populations for the benefit of the plant. Postdoc project Results Project start The taxonomic coverage of Solexa compatible 07.09.2015 metabarcoding methods for oomycetes and cercozoa have been tested in silico and complemented by revision of a cytochrome c oxidase subunit to assay specific for oomycete communities. The libraries will be constructed and submitted for sequencing in the upcoming months.

C4 Bioinformatic analysis of endophytic flora in A. thaliana and A. alpina

Aim of project 1: Genomic analysis of Thecapho- Researcher ra seminis-convolvuli Yao Pan T. seminis-convolvuli is a little-studied fungal pathogen, which destroys seeds of the flowering Project leaders plant Calystegia. Specific aims of this part of the Alice McHardy project are: Martin Lercher 1. Set up a pipeline for T. seminis-convolvuli genome annotation; Project type 2. Study the evolutionary relationships between Postdoc project T. seminis-convolvuli and closely related species using comparative genomics. Project duration 01.04.2013 - 01.09.2014 Results 5,372 predicted protein coding genes in 22Mb genome sequence.

41 Annual Report 2015

Aim of project 2: Shotgun metagenomic analysis Cooperation (other participating CEPLAS of epiphytic and endophytic microbial commu- and non-CEPLAS researchers) nities in A. thaliana leaves infected with Albugo Michael Feldbrügge laibachii Vera Göhre A. laibachii is an oomycete pathogen of A. thalia- Eric Kemen na. Specific aims of this part of the project are: 1. Use shotgun metagenomics to study the core epiphytic and endophytic microbial community associated with Albugo laibachii; 2. Learn how this community interacts with A. laibachii during infection.

Results Epiphytic and endophytic microbial communities on Albugo laibachii infected leaves showed distinct features on both diversity and functional profiles.

3.3.2 Understand how biotic and abiotic factors affect the composition and activities of plant associated microbial communities

While genetic differences within host and endophytes are expected to play an important role in structuring microbial communities, we anticipate that the microbiome will also be strongly influenced by abiotic factors. Recent evidence indicates that environmental (abiotic and biotic) factors and host genotype interact to affect plant colonisation by microbes and that particular microbes, termed “hub microbes” due to their central position in a microbial network, are disproportionally important in shaping microbial communities on plant hosts (Agler et al., 2016). Research on these microbial hubs represents a novel direction in Research Area C (e.g., project C10 Döhlemann / Kemen).

C5 Shaping of the Arabis alpina microbiome by plant host interactions with environmental factors – phosphorous limitation

Aim of the project Researcher To study the genetic underpinnings for plant Michael Thielen factors that can shape the root associated fungal community composition. To approach this Project leaders goal, a synthetic fungal community is being es- Marcel Bucher tablished and will be utilised on A. alpina x A. Gunther Döhlemann montbretiana introgression lines to pinpoint genetic loci responsible for variation between the Project type parental mycobiomes. Ph.D. project Results Project start A time and cost efficient, semi-quantitative 01.05.2014 screening method for fungal community comparison has been adapted and established. After Cooperation (other participating CEPLAS identification of the ideal colonisation conditions and non-CEPLAS researchers) for the synthetic community, the first synthetic Cooperation with Paul Schulze-Lefert is communities have been applied to A. alpina and anticipated A. montbretiana. Testing of further isolates as Michael Feldbrügge members of the community and optimisation of Vera Göhre colonisation reproducibility is ongoing. 42 Eric Kemen Research Area C

C6 Role of WRK75 in phosphate, pathogen and temperature responses

Researcher Aim of the project Heike Wolff The A. thaliana WRKY75 (AtWRKY75) gene was published to be involved in plant responses to Project leaders phosphate (Pi) starvation and the infection by Martin Hülskamp biotrophic and necrotrophic pathogens. This Jane Parker qualified AtWRKY75 as a good study case to understand the regulatory crosslinks between biotic Project type and abiotic stress pathways. Ph.D. project The aim was to study the molecular details of the Project start role of WRKY75 in biotic and abiotic responses 01.11.2013 and to identify common routes.

Cooperation (other participating CEPLAS Results and non-CEPLAS researchers) As AtWRKY75 was reported to be transcription- Friederike Brüssow ally activated upon biotic and abiotic stress and Ute Höcker to transcriptionally regulate characteristic down- Carolin Seyfferth (MPIPZ) stream genes, we initially focused on these two Achim Tresch (UoC) aspects. We applied various published growth conditions and biotic and abiotic treatments using wild-type and two wrky75 mutants.

We could neither confirm the transcriptional response of AtWRKY75 to both biotic and abiotic stress treatments nor could we observe the AtWRKY75-dependent regulation of target genes. We therefore abandoned this project and are currently developing an alternative project to enable the Ph.D. student to finish the thesis.

C7 Investigation of the effect of temperature-modulated defense homeostasis on plant-microbe symbioses in Arabidopsis thaliana

Aim of the project Researcher We have been examining natural genetic varia- Friederike Brüssow tion in the A. thaliana defense network in response to temperature at the level of plant growth, stress Project leaders hormone pathway homeostasis and structuring Jane Parker of leaf-associated microbial communities. Paul Schulze-Lefert Results Project type Salicylic acid (SA) “chemotyping“ of leaves from Postdoc project 105 diverse A. thaliana accessions grown in soil at 20° C and 16° C uncovered natural variation Project start in plant responses to temperature at the level 01.09.2013 of SA accumulation. A GWAS analysis for loci underlying temp x SA differences identified five Cooperation (other participating CEPLAS statistically well supported peaks. Differential SA and non-CEPLAS researchers) accumulation in response to temperature corre- Eric Kemen lated poorly with growth (biomass), suggesting a high degree of plasticity regulating the stress hormone network and growth trade-offs.

43 Annual Report 2015

Selected “extreme“ SA x temp differential accessions are now undergoing detailed phenotyping for changes at the level of the stress hormone network and, by exploiting bacterial, fungal and oomycete phyllotyping data from CEPLAS groups (Schulze-Lefert and Kemen), effects on the structure of leaf microbial populations and plant fitness.

C8 Environmental influences on pipecolic acid biosynthesis, defense priming, and systemic acquired resistance: light, nitrogen supply, and the C/N balance

Aim of the project Researcher This project investigates how Arabidopsis thalia- Ziba Ajami-Rashidi na adapts the systemic acquired resistance (SAR) response to different environmental conditions Project leaders such as light quality, light quantity, and nitrogen Jürgen Zeier supply. In this context, it re-evaluates the rele- Laura Rose vance of the previously reported SAR signals DEFECTIVE IN INDUCED RESISTANCE1 (DIR1), Project type azelaic acid (AzA), glycerol-3-phosphate (G3P), Ph.D. project and methyl salicylate (MeSA) for SAR under var- ying light regimes as well as the functional inter- Project start relationship of these putative SAR signals to the 01.10.2013 SAR regulator pipecolic acid (Pip).

Cooperation (other participating CEPLAS Results and non-CEPLAS researchers) Genetic analyses with Arabidopsis mutants im- Shizue Matsubara (FZJ) paired in AzA signalling, G3P biosynthesis, and MeSA formation suggest that DIR1, AzA, and MeSA are dispensable for SAR under most experimental settings, and that GLYCEROL IN- SENSITIVE1 (GLI1)-dependent G3P production contributes to the strength of SAR without being fully required. Moreover, the pathogen-inducible biosynthesis of Pip is not affected by above-mentioned mutational defects.

C9 Mutualistic interactions of pathogenic and non-pathogenic protists in foliar biofilms

Aim of the project Researcher Microbial communities have been reported to Alfredo Mari strongly affect plant physiology and plant fitness. Protists, due to their ability in re-shaping the Project leaders bacterial communities by selectively grazing, are Eric Kemen likely to hold a top-level role within the plant-mi- Michael Bonkowski crobe interactions. In this context, significant knowledge has been acquired regarding the Project type rhizosphere, while evidences for an importance Ph.D. project of protists in the phyllosphere are lacking in several aspects.

44 Research Area C

In this study, we aim to analyse how protist com- Project start munities can impact the plant microbiome by 01.11.2014 targeting major microbial ‘hubs’ (Agler et al. 2015), namely the common A. thaliana endo- Cooperation (other participating CEPLAS and phyte / pathogen genus Albugo. non-CEPLAS researchers) Marcel Bucher The first step to pursue this goal was performing Laura Rose 18s amplicon Illumina sequencing with universal Paul Schulze-Lefert primers to be used on A. thaliana populations sampled across Europe. To dissect and verify mechanisms by use of gnotobiotic systems, we have coupled the high-throughput analysis with isolation and visualisation approaches of wild and planted samples.

Results We have developed an 18S Illumina sequencing approach to profile protists. To analyse the data, we have written and verified an analyses pipeline. Both sequencing and analyses pipeline are available and shared with other groups in CEPLAS.

For cultivation of protists, we have developed an isolation (in collaboration with M. Bonkowski) and storage protocol.

C10 Role of fungal lifestyle and secreted effectors in multitrophic microbe – microbe and microbe – plant interactions

Aim of the project Researcher Microbiome composition both on roots as well as Katharina Lentz on upper ground plant organs is crucial for plant health and immunity. Previous work within CEP- Project leaders LAS by the groups of Paul Schulze-Lefert and Eric Gunther Döhlemann Kemen demonstrated that both mutualistic and Eric Kemen antagonistic microbe-microbe interactions are defining the plant microbiome. A subspecies of Project type the yeast-like basidiomycete Pseudozyma, which Ph.D. project belongs to the family of Ustilaginaceae, was identified as an epiphyt of Arabidopsis thaliana Project start leaves. It is known that Pseudozyma can strongly 01.11.2015 influence its microbial community, for example by inhibiting the growth of powdery mildews. The project aims to study the role of Pseudozyma in the Arabidopsis thaliana phyllosphere.

Because of its close relation to pathogenic Usti- laginomycetes, we will also study the potential role of Pseudozyma as a plant pathogen and how its lifestyle influences the leaf microbiome.

Results In progress - Project start 01.11.2015

45 Annual Report 2015

3.3.3 Identify the genes in both host and microbe that determine interaction outcomes

We identified genomic and transcriptomic complex traits associated with lifestyle adaptations by comparative genomics of ecologically diverse bacterial and fungal species (Zuccaro et al., 2014; Bai et al., 2015; Kohler et al., 2015; Lahrmann et al., 2015; Fesel & Zuccaro, 2016; Hiruma et al., in press). Several candidate genes important for the endophytic associations (e.g., genes involved in metabolic functions, plant-microbe communication or in counteracting plant defence) were identified and several were functionally characterised (Wawra et al. (submitted); Redkar et al., 2015a; Redkar et al., 2015b).

C11 Identification of plant growth promoting genes and pathways of bacterial symbionts in the A. thaliana rhizosphere

Aim of the project Researcher The development and application of computa- Ruben Garrido Oter tional approaches to the analysis of genomic and phenotypic data for the study of the root and leaf Project leaders microbiotas and their interactions with their plant Alice McHardy host. Paul Schulze-Lefert Results Project type By systematic bacterial isolation procedures we Ph.D. project have established bacterial culture collections of the leaf- and root-associated microbiota of A. Project start thaliana, capturing the majority of the taxa found 01.10.2013 in their corresponding natural host organs. Com- parative phylogenomic analysis of more than 400 Cooperation (other participating CEPLAS sequenced genomes revealed a substantial tax- and non-CEPLAS researchers) onomic and functional overlap between leaf and Julia Vorholt (ETH Zurich, Switzerland) root isolates, with several differentially abundant Richard J. O’Connell (INRA Versailles, functional categories. France) Ruth Ley (Cornell University, USA) Further, a newly developed gnotobiotic reconsti- Jeff Dangl (University of North Carolina, tution system for synthetic communities, togeth- USA) er with a computational pipeline for the analysis Rob Knight (University of San Diego, USA) of high-throughput sequencing data, provides a system for the study of microbial community es- Publications tablishment and functions under laboratory con- Schlaeppi K, Dombrowski N, Oter RG et ditions. al. (2014) Proceedings of the National Academy of Sciences of the United States of America 111(2):585-592.

Bulgarelli D, Garrido-Oter R, Münch PC et al. (2015) Cell host & microbe 17(3):392- 403.

Hacquard S, Garrido-Oter R, Gonzalez A et al. (2015) Cell host & microbe 17(5):603- 616

Bai Y, Müller DB, Srinivas G et al. (2015) Nature 528(7582):364-369.

46 Research Area C

C12 Evolution of biotrophy in fungal symbionts: What can the genomes reveal?

Aim of the project Researcher Comparative genomics of ecologically diverse Ganga Jeena fungal species to understand complex traits associated with lifestyle adaptations. Genome and Project leaders transcriptome assembly and analysis of related Alga Zuccaro fungal species of root associated endophytes Marcel Bucher including orchid-mycorrhiza, Serendipita herbamans, Chaetospermum artocarpi, Chaetosper- Project type mum camelliae and the root endophyte of Arabis Postdoc project alpina: Rhynchosporium.

Project start Results 01.05.2015 De-novo transcriptome assembly of orchid mycorrhizal Sebacina sp. and Serendipita herbamans was generated. OM transcripts represented ~97 % fungal core genes and nearly 99 % eukaryotic core genes. Further analysis is in process. Rhynchosporium sp. from the lab of Marcel Bu- cher was sequenced using PacBio Platform with 85X coverage. Error correction and assembly was performed to assemble ~65Mb in <50 scaffolds. Further assembly improvement and analysis is ongoing.

C13 Functional characterisation of candidate effector proteins in the Sebacinales

Aim of the project Researcher Piriformospora indica and Sebacina vermifera Heidi Widmer belong to the ecologically widely distributed order Sebacinales. These root endophytes display Project leaders wide-host spectrum beneficial effects to the host Alga Zuccaro such as growth promotion and increased resist- Stanislav Kopriva ance to pathogens. Genetic transformation of these mycobionts has been established and bio- Project type informatics tools for comparative genomics and Associated Ph.D. project transcriptomics were used to identify the in planta-responsive genes. With respect to insights Project start into how sebacinoid fungi establishes them- 01.01.2015 selves in metabolically active root cells of different hosts and how the plants are reprogrammed for enhanced performance, we will functionally characterise candidate effector proteins in the Sebacinales to find answers to the following questions: Are there functionally conserved effectors between P. indica and S. vermifera? How do the sebacinoid effector proteins mediate host accommodation and mutualism?

Results Based on transcriptomic data and on apoplastic fluid analysis of infected roots, four putative effector proteins were selected and functional analysis are ongoing together with the establishment of a protein production system in submerged P. indica culture. 47 Annual Report 2015

C14 Role of fungal WSC lectin-like proteins in the interaction of endophytic fungi with plant roots

Aim of the project Researcher β-glucan is the most abundant cell wall polysac- Philipp Fesel charide in fungi and is perceived by the plant innate immune system as a MAMP. This project Project leaders aims to elucidate the role of β-glucan as a MAMP Alga Zuccaro during the interaction of the beneficial root en- Paul Schulze-Lefert dophyte Piriformospora indica and its experimental host plants Arabidopsis and barley. Project type Ph.D. project ▪ How are different plants species/ecotypes re- acting to elicitation with β-glucan? Project start ▪ Which plant receptors are involved in the per- 01.10.2015 ception of β-glucan signals? ▪ How is P. indica avoiding the recognition of Cooperation (other participating CEPLAS β-glucan by plants? and non-CEPLAS researchers) Markus Pauly Results Jürgen Seibel (University of Würzburg) ▪ Arabidopsis and barley recognise different β-glucans as MAMPs. Publications ▪ Different Arabidopsis ecotypes display differ- Fesel PH, Zuccaro A (2015) Fungal ent behaviour when stimulated with β-glucan genetics and biology. doi: 10.1016/j. ▪ The analysis of a receptor candidates is on- fgb.2015. going ▪ The fungal lectins FGB1 and WSC1 are able to bind β-glucan and are potentially involved in suppression of β-glucan recognition and signalling in planta.

A paper on these results is currently under review in Nature communications.

C15 Characterisation of a leaf-specific Ustilago maydis α-L-arabinofuranosidase Aim of the project Researcher Infection of Zea mays by the basidiomycete Us- Elaine Jaeger tilago maydis causes smut disease and provides an important model for biotrophic host-patho- Project leaders gen interactions. U. maydis penetrates the maize Gunther Döhlemann epidermis and leads to tumor formation in all Eric Kemen aerial organs. The infection of seedling leaves, adult leaves and tassels induces organ-specific Project type transcriptional changes in the pathogen as well Associated Ph.D. project as in the host. This project aims for a functional characterization of a leaf-specific U. maydis effec- Project start tor, which is predicted to encode an α-L-arabino- 01.01.2015 furanosidase (Ara1).

Results Ara1 is required for tumor formation in leaves. Microscopic analysis shows that mutants for Ara1 are impaired in cell-cell penetration, particularly in bundle sheath cells. Enzyme assays showed that Ara1 is a specificα -L-arabinofuranosidase and by 48 Research Area C

mutational analysis this activity was found to be required for virulence. Besides its tissue-specific activity, Ara1 is a maize line-specific virulence factor, a new finding in the Ustilago field.

C16 Characterisation of the molecular mechanisms underpinning fungal beneficial effects in roots during tripartite interactions

Aim of the project Researcher The recently sequenced Sebacina vermifera Debika Sarkar (MAFF 305830) is a root endophyte with wide- host spectrum beneficial effects while Bipolaris Project leaders sorokiniana is a serious root pathogen for bar- Alga Zuccaro ley. Recent studies in the group showed that S. Marcel Bucher vermifera is able to increase resistance of barley to B. Sorokiniana, suggesting a possible function Project type in biological control. In this project we want to Associated Ph.D. project address these two questions: Do mutualistic and pathogenic fungi utilise similar strategies to col- Project start onise barley roots? Does S. vermifera manipulate 01.01.2015 the plant immune system to increase resistance to pathogens and /or are there other mecha- Cooperation (other participating CEPLAS nisms like mycoparasitism in place? and non-CEPLAS researchers) Michael Bonkowski Results Stanislav Kopriva S. vermifera protects barley against B. sorokiniana in soil. Transcriptomic analyses for the established tripartite system have been performed. S. vermifera showed antagonistic activity against B. sorokiniana in soil through expression of fungal cell wall-degrading enzymes like chitinases and glucanases.

C17 Development of a model system for smut fungus – Brassicaceae interaction

Aim of the project Researcher We aim to establish a smut infection system con- Vera Göhre sisting of two genetically tractable partners by characterising the infection process of the endo- Project leaders phytic smut fungus Thecaphora thlaspeos infect- Michael Feldbrügge ing model Brassicaceae and establishing tools Laura Rose for its molecular engineering.

Project type With this project, we address the establishment Associated Postdoc project of a synthetic symbiosis module, the central aim of Research Area C. In collaboration with Eric Ke- Project start men, we will study its influence on host microbi- 01.10.2013 ome formation to generate a beneficial synthetic microcosm as a proof-of-principle. This fungus Cooperation (other participating CEPLAS also allows devising strategies to engineer met- and non-CEPLAS researchers) abolic pathways for the plant benefit (Research Kaitlyn Courville Area D) and understanding different infection Lamprinos Frantzeskakis strategies for annual and perennial hosts (Re- Eric Kemen search Area A).

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The host range of T. thlaspeos might extend to crop plants of the Brassicaceae family such as Brassica napus (seeds from Norddeutsche Pflanzenzucht Hans-Georg Lembke KG). Benefit for plant growth or protection might hence be directly translated to agricultural applications. Knowledge transfer is additionally funded by the BioSC Boost fund PlaMint.

Recent Results T. thlaspeos grows systemically as an endophyte. Colonisation occurs during co-germination of seeds and spores. Genome analysis showed conservation of the tetrapolar mating loci and revealed candidate effector genes.

C18 The cumulative impact of environmental parameters to photosynthetic eukaryotes at a molecular level

Aim of the project Researcher Plants are the most complex photoautotrophic Ovidiu Popa organisms able to colonise a wide range of ecological niches for larger time periods. Adaptation Project leader to particular environments is the driving force for Oliver Ebenhöh diversification and molecular evolution in all organisms. The domiciled ecological niche is de- Project type scribed by a combination of abiotic and biotic CEPLAS funded via AG Ebenhöh constraints. Successful adaptation is the result of an evolutionary process, which optimises the mo- Project start lecular character of the organisms. 01.01.2015 The aim of this project is to characterise the ef- Cooperation (other participating CEPLAS fect of environmental factors to the genome. and non-CEPLAS researchers) Ahmad Mannan (Imperial College London, Results UK) A genome wide comparison of single copy genes from 80 Arabidopsis thaliana accessions with their orthologous genes from Arabidopsis lyrata revealed a significant difference in the acquisition of mutations between functional categories and between Arabidopsis accessions.

Further, we can show that these differences are correlated with environmental properties, suggesting the result of adaptation to particular conditions.

50 Research Area C

3.3.4 Determine reciprocal effects of host-microbe interactions on secondary metabolites

This is a new direction in Research Area C which developed from close cooperation with Research Area D. Tryptophan-derived, indolic metabolites possess diverse functions in Arabidopsis innate immunity to microbial pathogen infection. We demonstrated the functional role and regulatory characteristics of indolic metabolism in Arabidopsis systemic acquired resistance (SAR) and its importance in maintaining a beneficial interaction with endophytes (Lahrmann et al., 2015; Stahl et al., 2016; Hiruma et al., in press). Based on these results a new project was initiated to explore the coordination of indole glucosinolate metabolism and ER body formation. We expect to expand this field of study on metabolic interactions addressing the question: how does the (primary and secondary) metabolic status of the host affect and control complex traits during microbial interactions?

C19 Exploring the coordination of indole glucosinolate metabolism and ER body formation in plant fitness

Aim of the project Researcher To address the role of indole glucosinolates and Ryohei Thomas Nakano ER bodies in interactions with soil-borne microbes, including root endophytic fungi such as Project leader Colletotrichum tofieldiae and Piriformospora in- Paul Schulze-Lefert dica.

Project type Results Postdoc project We have shown that Colletotrichum tofieldiae (Ct) is an endemic endophyte in natural A. thalia- Project start na populations in Central Spain, in contrast to 01.07.2015 many other Colletotrichum species that are de- structive pathogens. Cooperation (other participating CEPLAS and non-CEPLAS researchers) By utilising GFP-expressing transgenic Ct and Marcel Bucher RFP-expressing Arabidopsis, I found that Ct forms Kei Hiruma (NAIST, Japan) both inter-cellular and intra-cellular hyphae in Richard O’Connell (INRA, France) roots to establish intimate associations with host Soledad Sacristan (Universidad Politécnica cells. Ct transfers the macronutrient phosphorus de Madrid, Spain) to shoots, promotes plant growth and increases Alga Zuccaro fertility only under phosphorus-deficient conditions. The host’s phosphate starvation response Publications (PSR) system controls Ct root colonization and is Hiruma K, Gerlach N, Sacristán S, et al. needed for plant growth promotion (PGP). PGP (2016) Cell. doi 10.1016/j.cell.2016.02.028. also requires PEN2-dependent indole glucosinolate metabolism, but not the ER body system.

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3.3.5 Synthetic symbioses – shaping the microbiome to increase plant performance

The composition and functional properties of microbial communities have a significant influence on plant performance. We aim to elucidate the details of these interactions and their underlying mechanisms with the long-term goal of engineering artificial associations in major crops. As a proof of concept an additional project was recently added in this section.

C20 Modelling algae and bacteria consortia

Aim of the project Researcher To build computational models able to repro- Antonella Succurro duce the dynamics of symbiosis between algae and bacteria. Project leader Oliver Ebenhöh Exploiting the available information from sequenced genomes of a variety of organisms, Project type optimality of consortia can be explored in silico, Associated Postdoc project reducing time and resource waste from failed attempts of establishing synthetic communities. Project start Such a model (extendable to higher organisms 01.05.2014 like plants) allows for identification of metabolic pathways playing key roles in mutual interactions Cooperation (other participating CEPLAS between organisms, therefore representing a and non-CEPLAS researchers) powerful tool for Research Area C. Ilka Axman Alison Smith (University of Cambridge, UK) Results The current status of the model is an open source python code where two key features have been developed to supplement the common con- straint based analyses performed on genome scale models. This preliminary version is able to include cofactor dynamics in flux balance analysis and a paper is in preparation. Next steps include experimental validations and extensions to multiple organisms.

Summary and Outlook

From our recent discoveries based on working with beneficial and pathogenic microbes of plants, we start to address one of the grand challenges in our field, which is to develop an integrated molecular concept that explains how plants simultaneously manage pathogenic and beneficial interactions to ensure plant survival and maximise plant fitness. The current framework of the innate immune system of plants is conceptually quite mature and can explain the molecular recognition of pathogenic microorganisms and activation of plant immune responses to limit or terminate pathogen growth. However, it leads to an apparent paradox as it falls short explaining how plants can discriminate pathogenic from beneficial microbes to both eliminate foes and accommodate friends. There is accumulating evidence that at least part of the innate immune system is necessary for the accommodation of beneficial microbes. This calls for a conceptual realignment or even re-definition of evolutionary paths and functions of the innate immune system.

In Research Area C, we feel we are on the right track for CEPLAS to make a novel and distinctive contribution to resolve this paradox over the next several years.

52 Research Area C

53 Annual Report 2015

3.4 Research Area D Plant metabolism: from biotic challenges to synthetic biology

Plants produce a great variety of biochemically unexplored secondary me- Coordinator tabolites with plethora of functions. The chemical diversity of plant secondary metabolites can be shaped by environmental stressors as well as in Ulf-Ingo Flügge response to microbial interactions. Our aim is the identification and func- (Markus Pauly) tional analysis of plant secondary metabolites which are decisive for the interaction of plants with the root microbial community (cooperation with Research Area C). Research Area D focuses on two main interconnected Co-coordinator themes:

Karl-Erich Jaeger Firstly, we study plant-microbe interactions in the context of plant defense reactions and plant nutrition.

Members Here special emphasis is given to the study of the regulatory network leading to the synthesis of glucosinolates and tryptophan-derived indolic com- Ilka Axmann pounds. These secondary metabolites play an important role in plant de- Michael Bonkowski fense against pests and diseases and also in the establishment of systemic Oliver Ebenhöh acquired resistance. In addition, we are using Arabidopsis ecotypes and a Thomas Drepper genome-wide association study to identify genes responsible for the ob- Tamara Gigolashvili served genotype-specific differences in the rhizosphere microbial activity. Stanislav Kopriva Sabine Metzger Candidate genes as well as the differences in root exudates in the selected Markus Pauly Arabidopsis ecotypes and mutants will be further characterised, and their Lutz Schmitt impact on plant-microbe interactions will be studied. For these types of ex- Vlada Urlacher periments, a microbe collection will be used which covers the entire range Jürgen Zeier of root-derived and sequenced bacteria strains (provided by RA C).

Secondly, our aim is the identification and characterization of signaling molecules involved in plant-microbe interactions and the production of such key compounds in synthetic microbes.

This approach intends to understand reciprocal metabolic signaling in plant-microbe interactions, and to characterise further components of regulatory modules controlling the biosynthesis and transport of secondary metabolites in roots and in the rhizosphere. Special emphasis is given to the biosynthesis of terpenoids.

We aim at a step-wise assembly of biosynthetic modules leading to the biosynthesis of triterpenes in synthetic microbes, e.g. Rhodobacter capsulatus and eventually “synthetic plants”. This will have a significant impact on developing next generation agricultural products and may also lead to biotechnological and pharmaceutical innovations.

54 Research Area D

Col-0 myb34/51/122 cyp79B2/B3

Pc2127

PcBMM

Disease symptoms of Arabidopsis thaliana mutants defective in the biosynthesis of indolic glucosinolates (myb34/51/122) and, additionally, tryptophan-derived indolic compounds (cyp79B2/B3). The plants were infected with two different isolates of a fungal pathogen. (Frerigmann H, Pislewska-Bednarek M, Sanchez-Vallet A, Molina A, Glawischnig E, Gigolashvili T, Bednarek P (2016) Regulation of pathogen triggered tryptophan metabolism in Arabidopsis thaliana by MYB transcription factors and indole glucosinolate conversion products. Molecular plant. doi 10.1016/j.molp.2016.01.006). For further details see project D1.

3.4.1 Plant-microbe interactions in the context of plant defense reactions and plant nutrition

This field of research deals, on the one hand, with the impact that glucosinolates and tryptophan-derived indolic compounds have on plant-microbe interaction, by using mutants deficient in the production of specific classes of glucosinolates and indolic substances. It also aims to develop predictive mathematical models to simulate secondary metabolite biosynthetic pathways, e.g. the biosynthesis of methionine-derived glucosinolates. On the other hand, plant genes will be identified and characterised which are involved in the ability to shape the microbiome in response to nutrient deficiency. It aims to identify corresponding compounds by the analysis of root exudates of Arabidopsis accessions and mutants grown under different nutritional conditions and co-cultivated with a selection of defined root-derived bacteria.

D1 The impact of secondary metabolites on the composition of the endophytic microflora in Arabidopsis thaliana

Aim of the project Researcher The production of secondary metabolites is an Henning Frerigmann important feature of plants to interact with their biotic environment. The biosynthesis of glucosi- Project leaders nolates and the indolic phytoalexin camalexin is Ulf-Ingo Flügge well studied in the model system A. thaliana and Marcel Bucher can therefore serve as a tool to understand the regulation of a complex biosynthetic network of Project type secondary metabolites. Postdoc project

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This project contributes to a better understand- Project start ing of the regulatory network of secondary me- 01.01.2013 tabolites crucial for plant-microbe interactions. The deeply characterised regulon might serve in Cooperation (other participating CEPLAS the future as a good target to generate a synthet- and non-CEPLAS researchers) ic plant with altered ability for specific plant-mi- Tamara Gigolashvili crobe interactions. AG Schulze-Lefert Alga Zuccaro Results Pawel Bednarek (Polish Academy of A group of MYB transcription factors (MYB34/ Sciences, Poland; MPIPZ Cologne) 51/122) represents the main regulators of indolic Erich Glawischnig (TU München) glucosinolates. Although multiple myb-mutants contain less camalexin upon abiotic treatments, Publications we could show that these three MYB transcrip- Frerigmann H, Pislewska-Bednarek M, tion factors do not directly regulate genes for Sanchez-Vallet A et al. (2016) Molecular these indolic phytoalexins but provide the pre- plant. doi 10.1016/j.molp.2016.01.006) cursor indole-3-acetaldoxime, which is shared with the biosynthesis of indolic glucosinolates. Frerigmann H, Glawischnig E, Gigolashvili T (2015) Frontiers in plant science 6:654.

Lahrmann U, Strehmel N, Langen G, et al. (2015) The New phytologist 207(3):841- 857.

D2 Mathematical models of glucosinolate metabolism in plants

Aim of the project Researcher Glucosinolates are an important class of sul- Suraj Sharma fur-containing plant secondary metabolites that play an important role in defence against path- Project leaders ogens. The understanding of the biosynthesis Oliver Ebenhöh is thus a key for deciphering plant-microbe in- Stanislav Kopriva teraction. We develop mathematical models to explain the factors governing and regulating the Project type diversity and biosynthesis of these metabolites. Ph.D. project Project start Results 01.10.2014 We have developed a prototype model of the chain-elongation process of aliphatic glucosinolates. The model can address fundamental questions like which factors govern the chain- length distribution of methionine-derived glucosinolates, one of the biosynthetic key processes. Also, one common phenomenon in secondary metabolisms is the promiscuity of enzymes and transporters, which can be explained by our model. Additionally, we have developed a method based on concepts of machine learning and natural selection to find the optimal values of model parameters.

Our models will support CEPLAS in the targeted design of pathways producing specialised compounds. 56 Research Area D

D3 Natural variation in the interaction of plants and bacteria in the rhizosphere

Aim of the project Researcher We use natural variation in Arabidopsis acces- Anna Koprivova sions to study the basis of interaction of plants and bacteria in the rhizosphere. Enzymatic re- Project leader action of sulfatase in the rhizosphere is used as Stanislav Kopriva a measure of how the different ecotypes shape the bacterial community. The data is used for ge- Project type nome-wide association study the identify genes CEPLAS funded via AG Kopriva underlying this variation.

Project start Thematically, the project is in the centre of RA 01.11.2013 D. It will identify genes underlying the ability of plants to shape their microflora. Genes involved Cooperation (other participating CEPLAS and in synthesis of secondary plant metabolites help non-CEPLAS researchers) selection of suitable compounds, enzymes, and Irene Klinkhammer transporters analysed in other projects of RA D. Achim Schmalenberger (University of Limerick, Ireland) Results We analysed 360 Arabidopsis accessions and observed at least fivefold difference in the sulfatase activity in the rhizosphere of individual accession. The genome-wide association study resulted in 55 candidate genes, which are currently being tested in corresponding T-DNA lines.

D4 MS-based identification of secondary metabolites crucial for plant- microbe interactions

Aim of the project Researcher The interaction between plant roots and soil Manuela Peukert microbes is regulated by multiple genetic and metabolic factors. Interactions strongly depend Project leaders on the plant genotype and microbial strains and, Stanislav Kopriva presumably, on the exuded metabolic cocktail Alga Zuccaro and the recognition by interaction partners. Ulf-Ingo Flügge The aim of the project is to identify (secondary) Project type metabolites that are crucial for the interaction of Postdoc project plants with plant growth promoting microbes. This will be achieved by analysis of root exudates Project start of Arabidopsis accessions and mutants under 01.08.2015 different nutritional conditions and co-cultivation with bacteria (see D5). This project is central for Cooperation (other participating CEPLAS RA D and utilises also resources generated within and non-CEPLAS researchers) RA C. Sabine Metzger (CEPLAS MS platform) Udo Seiffert (IFF Magdeburg) Results Michael Kertesz (University of Sydney, Experimental set-ups for exudate collection were Australia) established and tested.

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D5 Characterising plant-bacteria interactions that promote the uptake of nitrogen and sulfur from organic sources

Aim of the project Researcher This project seeks to deepen our understand- Richard Jacoby ing of how bacterial metabolism supports plant nitrogen and sulfur nutrition from organic mol- Project leaders ecules. Approximately 20 diverse bacterial iso- Ulf-Ingo Flügge lates will be chosen from the MPIPZ’s culture col- Stanislav Kopriva lection covering the entire range of root-derived bacteria. Also, ten Arabidopsis accessions will be Project type chosen according to their capacity to stimulate Postdoc project microbial activity (see D3). Co-cultivation experiments will be undertaken with the ~200 different Project start combinations of 10 plants × 20 microbes, in hy- 01.06.2015 droponic media where the only source of nitrogen and sulfur is organic (delivered via protein Cooperation (other participating CEPLAS extract). Metabolic flux pathways in top and bot- and non-CEPLAS researchers) tom performing plant × microbe combinations Oliver Ebenhöh will then be analysed, with mathematical expert Paul Schulze-Lefert Oliver Ebenhöh who is collaborating with data analysis and interpretation. Also, genetic approaches will be undertaken to define key gene variants between contrasting genotypes.

Results Genomic sequences from isolated bacterial strains have been analysed, to define a set of ten bacterial strains with contrasting metabolic com- petencies. Also, a plant-microbe co-cultivation system has been set up and tested.

D6 Regulatory and evolutionary aspects of stress-inducible plant metabolic pathways

Aim of the project Researcher The research project intends to identify new me- Elia Stahl tabolites which are synthesised in plants after pathogen-infection, investigate their function in Project leaders inducible plant defense, and define a regulatory Jürgen Zeier network of pathogen inducible metabolite pro- Vlada Urlacher duction in plants.

Project type Results Ph.D. project We demonstrated that basal levels and induced Project start production of tocopherols, a class of lipid-soluble 01.04.2013 antioxidants with vitamin E activity synthesised exclusively by photosynthetic organisms, contrib- Cooperation (other participating CEPLAS ute to Arabidopsis basal resistance against Pseu- and non-CEPLAS researchers) domonas syringae (Psm) by preventing oxidative Ulf-Ingo Flügge damage of lipids. Furthermore, we found that Henning Frerigmann tryptophan-derived indole metabolism is activated in Arabidopsis after Psm inoculation. Our data Publications suggest that indole accumulation is positively Stahl E, Bellwon P, Huber S, et al. regulated by the immune regulatory metabolites (2016) Molecular plant. doi 10.1016/j. salicylic acid and pipecolic acid, contributes to molp.2016.01.005 non-host resistance against non-adapted pathogens but is not required for the establishment of 58 systemic acquired resistance. Research Area D

3.4.2 Signalling molecules, metabolic modules, and synthetic microorganisms

Many bacteria use quorum sensing to co-ordinate their behaviour and to regulate a diverse variety of physiological processes including cell-population density. While Gram-negative bacteria produce and secrete mainly acetylated homoserine lactones (AHLs), Gram-positive bacteria use oligopeptides to communicate. In plants, it is well established that sulfated-oligopeptides including phytosulfokines (PSKs) have growth-promoting effects. It is hypothesised that plant-associated bacteria (provided by RA C) use similar signal molecules to modulate plant growth and/or induce the formation of sulfated peptides. We could demonstrate that plant triterpenes (e.g., marneral and thalianol) act downstream of these signaling peptides. The aim is to explore the PSK-triterpene pathway in plants and to identify bacteria acting on this pathway.

Terpenoids have important roles for various plant-microbe interactions. This project further aims to develop synthetic modules for the production of plant terpenoids (sequiterpenes, triterpenes and products derived thereof) in microbes, e.g. Rhodobacter capsulatus. Such modules comprise, in addition to biosynthetic components, modifying enzymes such as cytochrome P450 monooxygenases and finally transporters, e.g. ABC transporters for the export of the produced secondary compounds. This collaborative project combines the expertise of several groups to achieve the goals.

D7 Characterising plant-bacteria interactions that promote the uptake of nitrogen and sulfur from organic sources

Aim of the project Researcher The project aims to identify changes in plant root Katharina Sklorz architecture and exudation in response to microbial signals. Project leaders Michael Bonkowski Results Ulf-Ingo Flügge We could show that specific signal molecules (Acyl-Homoserine Lactones, AHLs) of Gram-neg- Project type ative bacteria are perceived by A. thaliana and Ph.D. project induce specific phenotypic changes in root architecture. AHLs also influence root exudation, Project start thus indicating a direct feed-back of plants on 01.03.2013 the microbial community in the rhizosphere. Us- ing hydroponic systems with sterile A. thaliana Cooperation (other participating CEPLAS plants, we could identified several specific com- and non-CEPLAS researchers) pounds in root exudates upon addition of certain Henning Frerigmann AHLs. The unknown nature and the high num- Tamara Gigolashvili ber of metabolites in root exudates pose analytic Stanislav Kopriva, challenges. Further investigations using Pyroly- Sabine Metzger sis-Field-Ionization MS will enable the identifica- Paul Schulze-Lefert tion of the differing metabolic classes. Peter Leinweber (Rostock University) In a second approach, effects of AHLs on exudation of mutants impaired in glucosinolate biosynthesis are being investigated. Additionally, we study the impact of A. thaliana mutants of on natural bacterial communities. The mutants lack either specific glucosinolates or camalexin or both and the feedback of their exudates on the microbial community composition is being investigated by phospho-lipid fatty acid (PLFA) profiling. The PLFA method targets the membrane composition of microorganisms and is a sensitive and quantitative indicator of soil microbial communities. 59 Annual Report 2015

D8 Exploring the phytosulfokine triterpene pathway in Arabidopsis thaliana

Aim of the project Researcher We hypothesise that plants and beneficial soil Dorian Baumann bacteria communicate using signaling peptides. Especially Gram-positive bacteria such as Actino- Project leader bacteria and Bacilli use secreted peptides as au- Ulf-Ingo Flügge toinducers during quorum sensing. This project Tamara Gigolashvili addresses the role of the plant-derived peptides phytosulfokines (PSK) and root growth factors Project type (RGF) in plant-bacteria communication and plant Ph.D. project growth promotion. Furthermore, we will elucidate how the growth-promoting effect of PSK is Project start connected to the biosynthesis of the triterpenes 02.01.2015 marneral and thalianol, and how plant secondary metabolites might be involved in plant-bacteria Cooperation (other participating CEPLAS communication. For this, a fully-sequenced mi- and non-CEPLAS researchers) crobiota collection (RA C) will be employed (see Henning Frerigmann D4, D5). Richard Jacoby Tabea Mettler-Altmann Results Sabine Metzger We previously showed that genes involved in Margret Sauter (University Kiel) the biosynthesis of marneral and thalianol are induced in response to exogenous PSK. Exoge- nous PSK application induces plant root growth and root growth is inhibited in mutants lacking PSK or the secondary metabolite marneral.

D9 Synthetic microbes for the production of plant secondary metabolites

Aim of the project Researcher This project seeks to develop synthetic modules Anita Loeschcke for the production of plant terpenoids in microbes. We focus on the biosynthesis and func- Project leaders tional analysis of terpenoids together with part- Karl-Erich Jaeger ners from RAs C and D. Thomas Drepper Results Project type We constructed a triterpenoid precursor biosyn- Postdoc project thesis module which enabled the biosynthetic production of squalene and 2,3-oxidosqualene Project start in the carotenogenic bacterium Rhodobacter 01.01.2013 capsulatus. Production of squalene, which is also a valuable compound for biomedical appli- Cooperation (other participating CEPLAS cations, was optimised to yield 13 mg/L. Model and non-CEPLAS researchers) triterpene biosynthesis modules with A. thaliana Ilka Axmann oxidosqualene cyclases were constructed and Marcel Bucher evaluated, identifying this step as the critical bot- Ulf-Ingo Flügge tleneck for plant triterpenoid biosynthesis in bac- Tamara Gigolashvili teria. Different strategies are currently explored, Stanislav Kopriva including improvement of precursor supply and Sabine Metzger triterpene synthesis via various oxidosqualene Lutz Schmitt cyclase enzymes of bacterial origin. Further- Vlada Urlacher more, the types of target terpenoids have been extended by including novel putative and as yet Publications non-characterised plant sesquiterpenoid syn- Loeschcke A, Thies S (2015) Applied micro- thases selected for functional expression in bac- biology and biotechnology 99(15):6197- terial hosts. The respective compounds might be 60 6214. involved in shaping the root microbiome. Research Area D

D10 Programming triterpene biosynthesis pathways in cyanobacteria using synthetic RNA-based devices

Aim of the project Researcher Engineering biosynthesis of plant triterpenes Dennis Dienst in cyanobacteria and development of universal synthetic regulatory (RNA-based) modules for Project leader optimising the microbial pathway flux (also for Ilka Axmann plant-associated bacteria).

Project type Results CEPLAS funded through support of The squalene accumulating strain ∆shc of Syn- AG Axmann echocystis served as host for Co2+-inducible co-expression of squalene epoxidase and two Project start separate oxidosqualene cyclases (OSC). The 01.08.2014 corresponding mRNAs were detected and the OSC transcripts accumulated constitutively. The Cooperation (other participating CEPLAS conversion of squalene to oxidosqualene was and non-CEPLAS researchers) demonstrated by GC-MS. An LC-MS-MS-based Thomas Drepper detection workflow is currently established at Sabine Metzger UoC, already indicating synthesis of thalianol Vlada Urlacher and marneral. Engineering of promoter-based Pia Lindberg (Uppsala University, Sweden) logic gates for tunable production is in progress. Sven Findeiß (University of Vienna, Austria) A platform for measuring regulatory RNA activi- André Estévez-Torres (CNRS Paris, France) ty in Synechocystis is being established and will be exploited for the logic gate installation. Met- abolic network modelling combined with a pho- tobioreactor setup monitoring highly controlled growth of producer strains will help to characterise and optimise the process.

D11 Functional expression and biochemical characterisation of plant P450s

Aim of the project Researcher Development of synthetic modules for the pro- Sarah Kranz duction of plant triterpenes and their oxygen- ated derivatives in microbes. Plant cytochrome Project leaders P450 monooxygenases (CYPs) will be produced Vlada Urlacher and characterised. Karl-Erich Jaeger In the context of CEPLAS, the project aims to Project type (i) Understand the biosynthesis of triterpenoids Ph.D. project (ii) Transfer synthetic modules into microbes (RAs C and D). Project start 01.04.2013 Results CYP71A16 and CYP705A12 from the marneral Cooperation (other participating CEPLAS pathway, the thalianol hydroxylating CYP708A2, and non-CEPLAS researchers) cytochrome P450 reductase and triterpenoid Ulf-Ingo Flügge synthases from A. thaliana were expressed in E. Tamara Gigolashvili coli. Various constructs allowing the functional Lutz Schmitt coexpression of CYPs with cyclases and/or reductase were generated. Marnerol was synthesised and applied for biochemical characterisation of CYP71A16. In addition, bacterial CYP mutants with activity towards α-amyrin, β-amyrin and lupeol were identified.

61 Annual Report 2015

D12 In vitro analysis of selected ABC transporters from plants

Aim of the project Researchers Identification of the substrate spectrum of plant Katharina Gräfe ABC transporters from A. thaliana roots. Kalpana Shanmugarajah Results Project leaders We have established heterologous expression Lutz Schmitt and purification protocols for the ABC transport- Andreas Weber ers PDR2, PDR8 (PEN3) and ABCG1 (WBC1) from Karl-Erich Jaeger A. thaliana. Reconstitution in artificial liposomes has been initiated. Project type Ph.D. project In parallel, transport studies aiming to identify the natural substrate(s) of these transporters Project start by mass spectrometry have been started. Here, 01.06.2013 we will employ plasma membrane vesicles (PMVs) containing the wild-type transporter or an Cooperation (other participating CEPLAS ATPase deficient mutant. PMVs will be incubated and non-CEPLAS researchers) with the cytosol of plant roots in the presence of Ulf-Ingo Flügge ATP. Stanislav Kopriva After isolation of PMVs, the luminal content of wild type and an ATPase-deficient mutant will be determined by mass spectrometry (MS). Dif- ferences in the MS spectra should provide first insights into the nature of the substrate(s).

Summary and Outlook

The interlinked projects are grouped around the identification and characterisation of plant secondary metabolites involved in plant-microbe interactions. They encompass the characterization of plant defense compounds as well as plant signaling molecules which are decisive for shaping the root microbiome with respect to plant nutrition and plant growth. Furthermore, it is envisaged to produce promising candidate compounds, in particular terpenoids, in “synthetic microbes” and to further exploit their bioactive potential.

62 Research Area D

63 Annual Report 2015

1. Wang Q, Hasson A, Rossmann S, Theres K (2016) Divide et impera: boundaries shape the plant body and initiate new meristems. New Phytologist 209(2):485-498.

2. Stahl E, Bellwon P, Huber S, Schlaeppi K, Bernsdorff F, Vallat-Michel A, Mauch F, Zeier J (2016) Regulatory and functional aspects of indolic metabolism in plant systemic acquired resistance. Molecular plant. doi 10.1016/j.molp.2016.01.005.

3. Sheehan H, Moser M, Klahre U, Esfeld K, Dell‘Olivo A, Mandel T, Metzger S, Vandenbussche M, Freitas L, Kuhlemeier C (2016) MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation. Nature genetics 48(2):159-166.

4. Schlüter U, Weber APM (2016) The Road to C4 Photosynthesis: Evolution of a Complex Trait via Intermediary States. Plant & cell physiology. doi 10.1093/pcp/pcw009.

5. Le CTT, Brumbarova T, Ivanov R, Stoof C, Weber E, Mohrbacher J, Fink-Straube C, Bauer P (2016) ZINC FINGER OF ARABIDOPSIS THALIANA12 (ZAT12) Interacts with FER-LIKE IRON DEFICIEN- CY-INDUCED TRANSCRIPTION FACTOR (FIT) Linking Iron Deficiency and Oxidative Stress Re- sponses. Plant physiology 170(1):540-557.

6. Koprivova A, Kopriva S (2016) Hormonal control of sulfate uptake and assimilation. Plant molecular biology. doi 10.1007/s11103-016-0438-y.

7. Hiruma K, Gerlach N, Sacristan S, Nakano RT, Hacquard S, Kracher B, Neumann U, Ramirez D, Bucher M, O’Connell RJ, Schulze-Lefert P (2016) Root Endophyte Colletotrichum tofieldiae Confers Plant Fitness Benefits that Are Phosphate Status Dependent. Cell. doi 10.1016/j. cell.2016.02.028

8. Frerigmann H, Pislewska-Bednarek M, Sanchez-Vallet A, Molina A, Glawischnig E, Gigolashvili T, Bednarek P (2016) Regulation of pathogen triggered tryptophan metabolism in Arabidopsis thaliana by MYB transcription factors and indole glucosinolate conversion products. Molecular plant. doi 10.1016/j.molp.2016.01.006.

9. Brumbarova T, Le CT, Ivanov R, Bauer P (2016) Regulation of ZAT12 protein stability: the role of hydrogen peroxide. Plant signaling & behavior. doi 10.1080/15592324.2015.1137408.

10. Brilhaus D, Bräutigam A, Mettler-Altmann T, Winter K, Weber AP (2016) Reversible Burst of Tran- scriptional Changes during Induction of Crassulacean Acid Metabolism in Talinum triangulare. Plant physiology 170(1):102-122.

11. Bräutigam A, Gowik U (2016) Photorespiration connects C3 and C4 photosynthesis. Journal of experimental botany. doi 10.1093/jxb/erw056.

12. Bernsdorff F, Döring AC, Gruner K, Schuck S, Bräutigam A, Zeier J (2016) Pipecolic Acid Orches- trates Plant Systemic Acquired Resistance and Defense Priming via Salicylic Acid-Dependent and -Independent Pathways. The Plant cell 28(1):102-129.

13. Ball SG, Bhattacharya D, Weber AP (2016) EVOLUTION. Pathogen to powerhouse. Science 351(6274):659-660.

14. Agler MT, Ruhe J, Kroll S, Morhenn C, Kim ST, Weigel D, Kemen EM (2016) Microbial Hub Taxa Link Host and Abiotic Factors to Plant Microbiome Variation. PLoS biology 14(1):e1002352.

15. Abdallah HB, Bauer P (2016) Quantitative Reverse Transcription-qPCR-Based Gene Expression Analysis in Plants. Methods in molecular biology 1363:9-24.

16. Zhu L, Bu Q, Xu X, Paik I, Huang X, Höcker U, Deng XW, Huq E (2015) CUL4 forms an E3 li- gase with COP1 and SPA to promote light-induced degradation of PIF1. Nature communications 6:7245.

64 Publications

17. Zhao Y, Wang J, Liu Y, Miao H, Cai C, Shao Z, Guo R, Sun B, Jia C, Zhang L, Gigolashvili T, Wang Q (2015) Classic myrosinase-dependent degradation of indole glucosinolate attenuates fumoni- sin B1-induced programmed cell death in Arabidopsis. The Plant journal : for cell and molecular biology 81(6):920-933.

18. Weber AP (2015) Discovering New Biology through Sequencing of RNA. Plant physiology 169(3):1524-1531.

19. Vom Dorp K, Hölzl G, Plohmann C, Eisenhut M, Abraham M, Weber AP, Hanson AD, Dörmann P (2015) Remobilization of Phytol from Chlorophyll Degradation Is Essential for Tocopherol Synthe- sis and Growth of Arabidopsis. The Plant cell 27(10):2846-2859.

20. Vijayakumar V, Liebisch G, Buer B, Xue L, Gerlach N, Blau S, Schmitz J, Bucher M (2015) Integrat- ed multi-omics analysis supports role of lysophosphatidylcholine and related glycerophospholip- ids in the Lotus japonicus-Glomus intraradices mycorrhizal symbiosis. Plant, cell & environment 39(2):393-415.

21. Strand DD, Livingston AK, Satoh-Cruz M, Froehlich JE, Maurino VG, Kramer DM (2015) Activa- tion of cyclic electron flow by hydrogen peroxide in vivo. Proceedings of the National Academy of Sciences of the United States of America 112(17):5539-5544.

22. Steffens A, Bräutigam A, Jakoby M, Hülskamp M (2015) The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabili- zation and Localization. PLoS biology 13(7):e1002188.

23. Soto D, Cordoba JP, Villarreal F, Bartoli C, Schmitz J, Maurino VG, Braun HP, Pagnussat GC, Za- baleta E (2015) Functional characterization of mutants affected in the carbonic anhydrase domain of the respiratory complex I in Arabidopsis thaliana. The Plant journal : for cell and molecular biology 83(5):831-844.

24. Somssich M, Ma Q, Weidtkamp-Peters S, Stahl Y, Felekyan S, Bleckmann A, Seidel CA, Simon R (2015) Real-time dynamics of peptide ligand-dependent receptor complex formation in planta. Science signaling 8(388):ra76.

25. Simon S, Rühl M, de Montaigu A, Wötzel S, Coupland G (2015) Evolution of CONSTANS Reg- ulation and Function after Gene Duplication Produced a Photoperiodic Flowering Switch in the Brassicaceae. Molecular biology and evolution 32(9):2284-2301.

26. Redkar A, Hoser R, Schilling L, Zechmann B, Krzymowska M, Walbot V, Doehlemann G (2015a) A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors. The Plant cell 27(4):1332-1351.

27. Redkar A, Villajuana-Bonequi M, Doehlemann G (2015b) Conservation of the Ustilago maydis Effector See1 in related smuts. Plant signaling & behavior 10(12):e1086855.

28. Rast-Somssich MI, Broholm S, Jenkins H, Canales C, Vlad D, Kwantes M, Bilsborough G, Dello Ioio R, Ewing RM, Laufs P, Huijser P, Ohno C, Heisler MG, Hay A, Tsiantis M (2015) Alternate wir- ing of a KNOXI genetic network underlies differences in leaf development of A. thaliana and C. hirsuta. Genes & development 29(22):2391-2404.

29. Pokhilko A, Ebenhöh O (2015) Mathematical modelling of diurnal regulation of carbohydrate allocation by osmo-related processes in plants. Journal of the Royal Society, Interface / the Royal Society 12(104).

30. Pokhilko A, Bou-Torrent J, Pulido P, Rodriguez-Concepcion M, Ebenhöh O (2015) Mathematical modelling of the diurnal regulation of the MEP pathway in Arabidopsis. The New phytologist 206(3):1075-1085.

65 Annual Report 2015

31. Pohlmann T, Baumann S, Haag C, Albrecht M, Feldbrügge M (2015) A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking. eLife 4.

32. Pires MV, Junior AA, Medeiros DB, Daloso DM, Pham PA, Barros KA, Engqvist MK, Florian A, Krahnert I, Maurino VG, Araujo WL, Fernie AR (2015) The influence of alternative pathways of res- piration that utilize branched-chain amino acids following water shortage in Arabidopsis. Plant, cell & environment. doi 10.1111/pce.12682.

33. Ort DR, Merchant SS, Alric J, Barkan A, Blankenship RE, Bock R, Croce R, Hanson MR, Hibberd JM, Long SP, Moore TA, Moroney J, Niyogi KK, Parry MA, Peralta-Yahya PP, Prince RC, Redding KE, Spalding MH, van Wijk KJ, Vermaas WF, von Caemmerer S, Weber AP, Yeates TO, Yuan JS, Zhu XG (2015) Redesigning photosynthesis to sustainably meet global food and bioenergy demand. Proceedings of the National Academy of Sciences of the United States of America 112(28):8529-8536.

34. Ordonez-Herrera N, Fackendahl P, Yu X, Schaefer S, Koncz C, Höcker U (2015) A cop1 spa mutant deficient in COP1 and SPA proteins reveals partial co-action of COP1 and SPA during Arabi- dopsis post-embryonic development and photomorphogenesis. Molecular plant 8(3):479-481.

35. Mulki MA, von Korff M (2015) CONSTANS controls floral repression by upregulating VERNALIZA- TION 2 (Vrn-H2) in barley. Plant physiology 170(1):325-337.

36. Mentink RA, Tsiantis M (2015) From limbs to leaves: common themes in evolutionary diversification of organ form. Frontiers in genetics 6:284.

37. Maurino VG, Engqvist MK (2015) 2-Hydroxy Acids in Plant Metabolism. The Arabidopsis book / American Society of Plant Biologists 13:e0182.

38. Matuszynska A, Ebenhöh O (2015) A reductionist approach to model photosynthetic self-regulation in eukaryotes in response to light. Biochemical Society transactions 43(6):1133-1139.

39. Mateos JL, Madrigal P, Tsuda K, Rawat V, Richter R, Romera-Branchat M, Fornara F, Schneeberger K, Krajewski P, Coupland G (2015) Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis. Genome biology 16:31.

40. Mai HJ, Lindermayr C, von Toerne C, Fink-Straube C, Durner J, Bauer P (2015) Iron and FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR-dependent regulation of proteins and genes in Arabidopsis thaliana roots. Proteomics 15(17):3030-3047.

41. Lyu MJ, Gowik U, Kelly S, Covshoff S, Mallmann J, Westhoff P, Hibberd JM, Stata M, Sage RF, Lu H, Wei X, Wong GK, Zhu XG (2015) RNA-Seq based phylogeny recapitulates previous phylogeny of the genus Flaveria (Asteraceae) with some modifications. BMC evolutionary biology 15:116.

42. Loeschcke A, Thies S (2015) Pseudomonas putida-a versatile host for the production of natural products. Applied microbiology and biotechnology 99(15):6197-6214.

43. Lo Presti L, Lanver D, Schweizer G, Tanaka S, Liang L, Tollot M, Zuccaro A, Reissmann S, Kahmann R (2015) Fungal effectors and plant susceptibility. Annual review of plant biology 66:513-545.

44. Liller CB, Neuhaus R, von Korff M, Koornneef M, van Esse W (2015) Mutations in Barley Row Type Genes Have Pleiotropic Effects on Shoot Branching. PloS one 10(10):e0140246.

45. Lenders MH, Weidtkamp-Peters S, Kleinschrodt D, Jaeger KE, Smits SH, Schmitt L (2015) Direc- tionality of substrate translocation of the hemolysin A Type I secretion system. Scientific reports 5:12470.

66 Publications

46. Le Roux C, Huet G, Jauneau A, Camborde L, Tremousaygue D, Kraut A, Zhou B, Levaillant M, Adachi H, Yoshioka H, Raffaele S, Berthome R, Coute Y, Parker JE, Deslandes L (2015) A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity. Cell 161(5):1074-1088.

47. Langner T, Özturk M, Hartmann S, Cord-Landwehr S, Moerschbacher B, Walton JD, Göhre V (2015) Chitinases are essential for cell separation in Ustilago maydis. Eukaryotic cell 14(9):846- 857.

48. Langner T, Göhre V (2015) Fungal chitinases: function, regulation, and potential roles in plant/ pathogen interactions. Current genetics. doi 10.1007/s00294-015-0530-x.

49. Lahrmann U, Strehmel N, Langen G, Frerigmann H, Leson L, Ding Y, Scheel D, Herklotz S, Hilbert M, Zuccaro A (2015) Mutualistic root endophytism is not associated with the reduction of sap- rotrophic traits and requires a noncompromised plant innate immunity. The New phytologist 207(3):841-857.

50. Kottb M, Gigolashvili T, Grosskinsky DK, Piechulla B (2015) Trichoderma volatiles effecting Arabi- dopsis: from inhibition to protection against phytopathogenic fungi. Frontiers in microbiology 6:995.

51. Kopriva S, Talukdar D, Takahashi H, Hell R, Sirko A, D‘Souza SF, Talukdar T (2015) Editorial: Fron- tiers of Sulfur Metabolism in Plant Growth, Development, and Stress Response. Frontiers in plant science 6:1220.

52. Kopriva S, Calderwood A, Weckopp SC, Koprivova A (2015) Plant sulfur and Big Data. Plant science : an international journal of experimental plant biology 241:1-10.

53. Kopriva S (2015) Partitioning of sulfur between primary and secondary metabolism. Sulfur Metabo- lism in Plants – Molecular Physiology and Ecophysiology of Sulfur, ed De Kok LJ, Hawkesford, M., Rennenberg, H., Saito, K., Schnug, E (Springer), pp 11-20.

54. Kopriva S (2015) Plant sulfur nutrition: from Sachs to Big Data. Plant signaling & behavior 10(9):e1055436.

55. Kolar K, Wischhusen HM, Müller K, Karlsson M, Weber W, Zurbriggen MD (2015) A synthetic mammalian network to compute population borders based on engineered reciprocal cell-cell communication. BMC systems biology 9(1):97.

56. Kluth M, Stindt J, Dröge C, Linnemann D, Kubitz R, Schmitt L (2015) A Mutation within the Ex- tended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3. Journal of Biological Chemistry 290(8):4896-4907.

57. Khonsari AS, Kollmann M (2015) Perception and regulatory principles of microbial growth control. PloS one 10(5):e0126244.

58. Karkar S, Facchinelli F, Price DC, Weber AP, Bhattacharya D (2015) Metabolic connectivity as a driver of host and endosymbiont integration. Proceedings of the National Academy of Sciences of the United States of America 112(33):10208-10215.

59. Hüdig M, Maier A, Scherrers I, Seidel L, Jansen EE, Mettler-Altmann T, Engqvist MK, Maurino VG (2015) Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehy- drogenase. Plant & cell physiology 56(9):1820-1830.

67 Annual Report 2015

60. Hemetsberger C, Mueller AN, Matei A, Herrberger C, Hensel G, Kumlehn J, Mishra B, Sharma R, Thines M, Hückelhoven R, Doehlemann G (2015) The fungal core effector Pep1 is conserved across smuts of dicots and monocots. The New phytologist 206(3):1116-1126.

61. Hacquard S, Garrido-Oter R, Gonzalez A, Spaepen S, Ackermann G, Lebeis S, McHardy AC, Dangl JL, Knight R, Ley R, Schulze-Lefert P (2015) Microbiota and Host Nutrition across Plant and Animal Kingdoms. Cell host & microbe 17(5):603-616.

62. Haag C, Steuten B, Feldbrügge M (2015) Membrane-Coupled mRNA Trafficking in Fungi.Annual review of microbiology 69:265-281.

63. Göhre V (2015) Immune responses: Photosynthetic defence. Nature Plants 1(6):15079.

64. Frerigmann H, Glawischnig E, Gigolashvili T (2015) The role of MYB34, MYB51 and MYB122 in the regulation of camalexin biosynthesis in Arabidopsis thaliana. Frontiers in plant science 6:654.

65. Fesel PH, Zuccaro A (2015) beta-glucan: Crucial component of the fungal cell wall and elusive MAMP in plants. Fungal genetics and biology. doi 10.1016/j.fgb.2015.12.004

66. Facchinelli F, Weber APM (2015) Analysis of Cyanophora paradoxa tells important lessons on plastid evolution. Perspectives in Phycology 2(1):3-10.

67. Engqvist MK, Schmitz J, Gertzmann A, Florian A, Jaspert N, Arif MA, Balazadeh S, Mueller-Roe- ber B, Fernie AR, Maurino VG (2015) GOX3, a glycolate oxidase homologue of yeast L-lactate cytochrome c oxidorreductase, supports L-lactate oxidation in roots of Arabidopsis thaliana. Plant physiology 169(2):1042-1061.

68. Eisenhut M, Hocken N, Weber AP (2015) Plastidial metabolite transporters integrate photorespiration with carbon, nitrogen, and sulfur metabolism. Cell calcium 58(1):98-104.

69. Domröse A, Klein AS, Hage-Hülsmann J, Thies S, Svensson V, Classen T, Pietruszka J, Jaeger KE, Drepper T, Loeschcke A (2015) Efficient recombinant production of prodigiosin in Pseudomonas putida. Frontiers in microbiology 6:972.

70. Digel B, Pankin A, von Korff M (2015) Global Transcriptome Profiling of Developing Leaf and Shoot Apices Reveals Distinct Genetic and Environmental Control of Floral Transition and Inflo- rescence Development in Barley. The Plant cell 27(9):2318-2334.

71. Desouki AA, Jarre F, Gelius-Dietrich G, Lercher MJ (2015) CycleFreeFlux: efficient removal of thermodynamically infeasible loops from flux distributions. Bioinformatics 31(13):2159-2165.

72. de Vries S, Kloesges T, Rose LE (2015) Evolutionarily dynamic, but robust, targeting of resistance genes by the miR482/2118 gene family in the Solanaceae. Genome biology and evolution 7(12):3307-3321.

73. Czyzewicz N, Wildhagen M, Cattaneo P, Stahl Y, Pinto KG, Aalen RB, Butenko MA, Simon R, Hardtke CS, De Smet I (2015) Antagonistic peptide technology for functional dissection of CLE peptides revisited. Journal of experimental botany 66(17):5367-5374.

74. Cui H, Tsuda K, Parker JE (2015) Effector-triggered immunity: from pathogen perception to robust defense. Annual review of plant biology 66:487-511.

75. Chen S, Lory N, Stauber J, Hoecker U (2015) Photoreceptor Specificity in the Light-Induced and COP1-Mediated Rapid Degradation of the Repressor of Photomorphogenesis SPA2 in Arabidop- sis. PLoS genetics 11(9):e1005516.

76. Cartolano M, Pieper B, Lempe J, Tattersall A, Huijser P, Tresch A, Darrah PR, Hay A, Tsiantis M (2015) Heterochrony underpins natural variation in Cardamine hirsuta leaf form. Proceedings of the National Academy of Sciences of the United States of America 112(33):10539-10544.

68 Publications

77. Butenko MA, Simon R (2015) Beyond the meristems: similarities in the CLAVATA3 and INFLORES- CENCE DEFICIENT IN ABSCISSION peptide mediated signalling pathways. Journal of experimental botany 66(17):5195-5203.

78. Bühler J, Rishmawi L, Pflugfelder D, Huber G, Scharr H, Hülskamp M, Koornneef M, Schurr U, Jahnke S (2015) phenoVein-A Tool for Leaf Vein Segmentation and Analysis. Plant physiology 169(4):2359-2370.

79. Brych A, Mascarenhas J, Jaeger E, Charkiewicz E, Pokorny R, Bölker M, Doehlemann G, Batschau- er A (2015) White collar 1-induced photolyase expression contributes to UV-tolerance of Ustilago maydis. MicrobiologyOpen. doi 10.1002/mbo3.322

80. Baumann S, Takeshita N, Grün N, Fischer R, Feldbrügge M (2015) Live cell imaging of endosomal trafficking in fungi. Methods in molecular biology 1270:347-363.

81. Banhara A, Ding Y, Kühner R, Zuccaro A, Parniske M (2015) Colonization of root cells and plant growth promotion by Piriformospora indica occurs independently of plant common symbiosis genes. Frontiers in plant science 6:667.

82. Bai Y, Müller DB, Srinivas G, Garrido-Oter R, Potthoff E, Rott M, Dombrowski N, Münch PC, Spaepen S, Remus-Emsermann M, Hüttel B, McHardy AC, Vorholt JA, Schulze-Lefert P (2015) Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528(7582):364-369.

83. Badia MB, Arias CL, Tronconi MA, Maurino VG, Andreo CS, Drincovich MF, Wheeler MC (2015) Enhanced cytosolic NADP-ME2 activity in A. thaliana affects plant development, stress tolerance and specific diurnal and nocturnal cellular processes. Plant science : an international journal of experimental plant biology 240:193-203.

84. Aghajanzadeh T, Kopriva S, Hawkesford MJ, Koprivova A, De Kok LJ (2015) Atmospheric H2S and SO2 as sulfur source for Brassica juncea and Brassica rapa: impact on the glucosinolate composition. Frontiers in plant science 6:924.

85. Adlakha N, Pfau T, Ebenhöh O, Yazdani SS (2015) Insight into metabolic pathways of the potential biofuel producer, Paenibacillus polymyxa ICGEB2008. Biotechnology for biofuels 8:159.

86. Abedpour N, Kollmann M (2015) Resource constrained flux balance analysis predicts selective pressure on the global structure of metabolic networks. BMC systems biology 9(1):88.

Plant Metabolism and Metabolomics Platform Annual Report 2015

The CEPLAS Plant Metabolism and Metabolo- Coordination mics Laboratories provide expertise and instru- mentation for the qualitative and quantitative Dr. Tabea Mettler-Altmann (HHU) analysis of plant metabolism. Our services are Dr. Sabine Metzger (UoC) open to all CEPLAS members as well as external collaborators. Technical support The aim of the laboratories is to apply routine Maria Graf, Elisabeth Klemp, Katrin Weber methods and establish new methods for the ex- (HHU), Christina Lucas (till May 2015), traction and subsequent analysis of primary and Dr. Vera Wewer (since November) (UoC) secondary metabolites, mainly via liquid and gas chromatographic separation methods that are Devices hyphenated with mass spectrometry detection.

GC-MS, Agilent Technologies The CEPLAS Plant Metabolism and Metabolo- UHPLC-MS/MS, Agilent Technologies mics Laboratory at the Heinrich Heine Universi- EA-IRMS, Isoprime ty in Düsseldorf, led by Tabea Mettler-Altmann, UHPLC-DAD, Agilent Technologies focuses on measuring compounds of the primary LC-QTRAP, AB Sciex metabolism, whereas the one at the University QTOF, Bruker Daltonics of Cologne, led by Sabine Metzger, focuses on ICP-MS, Agilent Technologies measuring metabolites of the secondary metabolism.

4.1 Platform Düsseldorf

In 2014, two new systems purchased from Agi- total carbon and nitrogen content of their bio- lent were installed: a gas chromatography device logical material and we analysed >1.000 samples coupled to a time-of-flight mass spectrometer from 2014 till now. For this purpose, we use an (GC-QTOF) and a liquid chromatography device elemental analyzer (EA) from Elementar. Due to coupled to a triple quadrupol mass spectrome- the coupling of the EA to an isotope-ratio mass ter (LC-MS/MS). The GC-QTOF is primarily used spectrometry (IRMS) we are additionally able to for routine quantification of >50 metabolites in- quantify the 12C relative to the 13C content in our cluding sugars, organic acids and amino acids in samples. The carbon isotope ratio (δ13C) allows various tissues and species. A second protocol distinguish different pathways of carbon fixation

established in our lab allows the absolute quan- such as C3 and C4 photosynthesis due to very tification of fatty acid methyl esters (FAMEs). In small differences in the isotope discrimination of 2014 and 2015 applying these two protocols, we the different carbon fixing enzymes. measured over 5.700 samples in extracts of various tissues such as leaves, seeds, cell cultures In total more than 9.000 samples were analysed and blood plasma in species ranging from yeast, in our lab since 2014 in collaboration with more cyanobacteria, green algae, higher plants to ver- than ten research groups within CEPLAS, several tebrates. international and other national groups. This far, our collaborative work resulted in two accepted A new protocol is currently under development publications, one book chapter and four submit- on the LC-MS/MS machine to absolutely quanti- ted manuscript. We have also received positive fy mainly phosphorylated compounds and there- feedback for a CEPLAS Scientific Course we fore complementing the selection of metabolites organised and delivered last September for six measured by GC-MS. Also, there are some ami- CEPLAS Ph.D. and post-doctoral researchers. no acids that cannot be separated or detected by GC-MS. For this reason a protocol was es- This year, we were also able to obtain a BOOST tablished in 2014 to quantify all twenty canoni- grant from Bioeconomy Science Center (BioSC) cal amino acids by ultra-high performance liquid of North Rhine-Westphalia state to work on own chromatography (UHPLC) coupled to a diode ar- research topics. Within the AlgalFertilizer project, ray detector (DAD). So far, we measured >1.100 we aim to understand phosphate metabolism in samples with this method. green algae to develop algal material as phosphate, a fossil resource, fertilizer for crop plants. In addition to single metabolite pools, for sever- A new postdoctoral researcher will start by the al research topics it is important to quantify the end of this year to work on this project. 72 Plant Metabolism and Metabolomics Platform

4.2 Platform Cologne

In the field of targeted analysis we extend our ent homoserine-lactones on the composition of routine quantification method to analyse fla- root exudates from Arabidopsis thaliana and the vonoid aglycons to specific flavonoid aglycons amounts of the substances. A project of AG Flüg- in lotus japonicus. Our LC-MS method now in- ge and AG Kopriva is focused on plant-microbe cludes 28 different flavonoids aglycons that we interactions. Root exudation has been shown can identify and quantify parallel. Applying this to affect plant-microbe interactions, and is sup- method we analysed extracts from Arabidopsis posed to depend on the plant genotype as well thaliana, Lotus japonicus, Petunia and Calibra- as the bacterial strain. We support the project by choa. finding the right growing conditions (Vermiculit, mineral wool) and sample preparation for root Triterpenoids were the focus of the cooperation exudates with the focus on compatibility with project with the AG Axmann and AG Jaeger. mass spectrometry. Special attention is also paid Here we have started to establish an isolation and to the sample tubes required to depletion of the LC-MS method to analyse triterpenoids like amy- bacteria. rins, lupeol, lanosterol, cycloartenol, squalene and oxidosqualene isolated from the two orga- For these untargeted approaches we used our nisms Synechocystis and Rhodobacter capsu- high resolution mass spectrometer with its ac- latus. The main challenge is the identity of the curate mass to identify and characterise com- molecular composition of these compounds. All pounds of interest. the substances have the same sum formula and are very similar in their structure. This similarity is In addition to the metabolite analysis we give sup- also reflected in a similarity of the fragment spec- port in analytical questions. Together with the AG tra. To distinguish between these triterpenoids or Urlacher we successful established an LC-method to identify new ones we had to study the frag- to isolate marneral and marnerol from yeast ex- mentation behaviour and adapt the MS-method tracts in mg quantities. Starting from a LC-method to these findings. First experiments with the two to detect marneral and marnerol we scaled up ionisation methods electrospray ionisation and this method to isolate and clean these com- atmospheric chemical ionisation show different pounds in a large scale. These two substances behaviour in between the substances. We have are not commercially available and were needed successfully established an LC-MS method, to as standards for method development. separate ten isobaric triterpenoids standards. This method is currently being applied to meas- From January till December 2015, all over 2350 ure extracts from Synechocystis and Rhodobacter samples were measured in the Cologne platform capsulatus in order to screen for accumulating in collaboration with more than ten CEPLAS re- triterpenoids. search and other international groups. As a result of our long term collaboration with the Institute The analysis of root exudates is the focus of two of Plant Science at the University of Bern a paper other projects within CEPLAS. AG Bonkowski is was published in Nature Genetics. interested in the effect of treatment with differ- 73

Promotion of young researchers Annual Report 2015

Introduction

To optimally prepare young researchers for their future career in academia and in industry, CEPLAS established a comprehensive programme for the promotion of young scientists from the undergraduate to the postdoctoral level. The programme focuses on two major aspects that play a crucial role in the preparation of young researchers for a future career in a scientific environment.

▪ Sensitising young researchers already at a very early stage for putative career options (by coaching, career days, networking with industry, career-relevant transferable skills) and actively assist in career planning and development.

▪ Preparing young researchers optimally for the current and future requirements in the area of plant sciences and life sciences in general (Bachelor Programme in Quantitative Biology, scientific training programme for doctoral researchers and postdocs).

The programme consists of four lines:

▪ Research internships for undergraduates

▪ Bachelor Programme in Quantitative Biology

▪ CEPLAS Graduate School

▪ CEPLAS Postdoc Programme

CEPLAS Postdoc Programme

CEPLAS Graduate Programme

B.Sc. Programme Research internships in Quantitative Biology for undergraduates

5.1 Research internships for undergraduates

CEPLAS awards fellowships to undergraduates During their 3-week research projects, students in Cologne and Düsseldorf to foster interest of receive full-time contracts. After successful com- excellent and talented students in plant scienc- pletion of the project, which includes a written es at an early career stage. Each year 12 fellow- report, students are awarded a research certifi- ships are offered to 3rd semester B.Sc. Biology cate. The programme is very well accepted and and Biochemistry students. In addition, students we can see that we reached our aim to increase with a background in Math, Physics or Computer interest in plant sciences as six students decided Science and interest in Plant Biology are highly to continue with their Bachelor thesis in the lab welcome to join one of the interdisciplinary where they did their internship or in neighbour- CEPLAS groups. ing CEPLAS labs.

76 Promotion of young researchers

5.2 Bachelor Programme in Quantitative Biology

In recent years, technological progress and the To advertise the new programme, we organised resulting introduction of many new, high-through- information lectures and distributed flyers and put methods producing big data have strongly posters at both universities already in March. changed the requirements that biologists have to Moreover the programme was advertised via the meet. Therefore, CEPLAS has established a nov- universities’ and CEPLAS website and social me- el undergraduate programme in Quantitative Bi- dia. ology that responds to this challenge. In addition to classical biology, the study programme focus- Because not all applicants fulfilled the terms of es on Mathematics, Biostatistics, Biophysics, and admission, our first cohort of students consists of Computational Biology. five students, four from HHU, and one from UoC.

In August this year, the study programme was To attract more students from both Universities officially accredited by the Agency for Quality for the next round, we will improve the promotion Assurance AQAS, and was thus ready to start in of the programme by organising whole workshop October. days on Quantitative Biology for prospective students. In addition, we will provide a promotional booklet about the programme, together with up- dated websites, posters and flyers.

First cohort of B.Sc. Quantitative Biology students

Nathalie Mütze Ronja Johnen Tim Rose

I decided to choose the I have chosen Quantitative With this study pro- study programme Biology to improve my gramme I have found the Quantitative Biology current mathematical skills possibility to combine to complement my and keep up with technical biology with my interests biological knowledge progress. Better profession- in Mathematics. Besides, with experience in Math al perspectives were also the small study groups of and informatics. I assume a crucial argument for my the programme will allow that such competences decision. a tight contact between will be very helpful during the students and the my career. teaching scientists.

77 Annual Report 2015

5.3 CEPLAS Graduate School

The CEPLAS Graduate School offers a structured, 3-year programme with a focus on plant molecular biology, genetics, biochemistry and computa- Speaker tional biology. A broad training programme in scientific and career-rele- Graduate vant transferable skills completes the programme. The training will on one School hand allow doctoral researchers to broaden their scientific knowledge; on the other hand, transferable skills training will prepare them for their future Ute Höcker career.

Furthermore, CEPLAS encourages doctoral researchers to attend confer- Coordinator ences and therefore is providing yearly funding for young scientists. Graduate School Development of the Graduate School

Esther Jawurek By the end of 2015, the number of doctoral researchers within the CEPLAS (Sira Groscurth and Graduate School has increased to 33, the highest number since the found- Petra Fackendahl ing of the Graduate School in 2013. during maternity leave) Apart from 29 regular members, 4 Ph.D. students, financed from other sources, applied and were accepted to join the CEPLAS Graduate School as associated members. Ph.D. representatives The first graduates of the CEPLAS Graduate School are expected in spring 2016. Thomas Wrobel Meike Hüdig (deputy) Training measures

Scientific training programme Current number CEPLAS is aiming to not only pro- had an additional emphasis on per- of doctoral vide the best research opportunities spectives for plant geneticists in a researchers for their doctoral researchers, but plant breeding company. to also offer a broad variety of scientific courses (e.g. Bioinformatics, Furthermore, CEPLAS provides fun- 33 Biostatistics, Non-Photochemical ding for doctoral researches in case Quenching) to train the young sci- they would like to attend specific Proportion entists in several additional meth- courses at other institutions. ods and techniques. of female Upon application, young research- scientists Most of the scientific courses were ers have the possibility to obtain offered by principal investigators additional funding from the CEPLAS 70 % and postdocs within CEPLAS, e.g. mobility fund to attend summer Analysis of Primary Metabolites by schools, external courses or ex- GC-MS or Advanced Methods for change programmes. Thus, several Internationality Fluorescence Imaging. Thus, those doctoral researchers attended inter- courses not only provide valuable national summer schools on specif- 36 % international knowledge but as well support netic topics that were highly related to students from seven working within the cluster. their research project, e.g. Meta- countries (China, bolic Pathway Analysis in Prague or Iran, India, Italy, As a response to the suggestions Synthetics and Systems Biology in Poland, Switzerland made by the Ph.D. researchers, we Sicily. and Spain) organised a course on Applied Plant Genetics, which was conducted by Dr. Günter Strittmatter in cooperation with KWS SAAT AG. The course

78 Promotion of young researchers

Career training programme

Besides being an excellent investigator, it is also variety of courses, of which this year several important for the doctoral researchers to improve young scientists chose Discussing Science or Get their career-relevant transferable skills. Therefore into Teaching for Doctoral Researchers. it is mandatory for them to attend the courses Good Scientific Practice, Scientific Presentation To train our young researchers to communicate and Scientific Writing, which are offered in col- their science in clear words to the public, the laboration with iGRAD at HHU. CEPLAS Graduate School organised a course on Successfully communicating your research – The Furthermore, the doctoral researchers have to basics of science communication and journalistic attend at least two additional courses, which fit writing for scientists, given by an external trainer their needs. They can choose between a broad from EMBL, Heidelberg.

5.4 CEPLAS Postdoc

Programme Speaker Postdoc Programme

The CEPLAS Postdoc Programme aims at pro- Rüdiger Simon viding our postdoctoral researchers with the best possible skills, expertise and network to allow them to achieve their personal career Coordinator Postdoc Programme goals. Programme components are supervision and mentoring, scientific and career train- Juliane Schmid ing, as well as contact to industry. Postdoc representatives Development of the programme Filipa Tomé By the end of 2014, CEPLAS internationally an- Luise Brand (deputy) nounced 14 open postdoc positions. In total we received more than 300 applications, most of them from abroad. After careful selection, Current number of postdoctoral we invited 35 candidates (13 from overseas, 10 researchers from Europe and 12 from Germany) to present their previous work within a public mini- 23 (including two postdocs who are currently symposium, followed by individual interviews on parental leave) with the selection committee. Project leaders showed their labs to the respective candidates and a joint dinner gave applicants and Proportion of female scientists project leaders the possibility to get to know each other. Most new postdocs started their 74 % position between April and September. In addition, one postdoc financed by other funds was associated to the postdoc programme Internationality and the cluster in 2015. Thus, at the end of this year, 23 postdocs are members of the CEPLAS 70 % international postdocs from eleven postdoc programme - two of them are current- countries (Australia, China, Columbia, France, ly on parental leave. Out of our first group, a India, Italy, Japan, Netherlands, Portugal, total of ten postdoctoral researchers have left Switzerland, USA) CEPLAS, two of them at the end of 2014, eight in 2015. Two stayed in academia, three started a position in other research institutions and one joined a company. Alumni are listed on the CEPLAS website.

79 Annual Report 2015

Training measures

To further develop their skills and competences, the four CEPLAS institutions offer a wide range of training courses for postdocs. Participation is on a voluntary basis and postdocs can choose courses that fit their needs. This year, several postdocs individually participated in courses such as Leadership Skills or Safety in Gene Technology. In addition, based on the demands of the postdocs, we organised courses on social networking, grant application, research communication and a course on how to lead and coach Ph.D. candidates.

Coaching

To sensitise young researchers already at an early stage for putative career options and to assist in career planning and development we offer individual coaching with a professional trainer. The postdocs who took this opportunity had up to three hours of individual consultation. Due to the positive feedback, we plan to offer individual coaching again in 2016.

5.5 Young researchers activities

Excursions to industry

To bring our young researchers into contact with industry and to raise awareness of the differences between academic and industry-driven research, CEPLAS organises two industry excursions per year. The first excursion in March this year took place at Qiagen, a company located in Hilden, Germany. A group of 24 young researchers visited this spin-off company of the Heinrich Heine University Düsseldorf that was founded in 1984. Qiagen has developed into a worldwide leader for the development and production of innovative technologies and products for the pre- analytical sample preparation and molecular CEPLAS young researchers visited the company tests for research within the life sciences and mo- “Nunhems“ in the Netherlands lecular diagnostics.

In September, 25 doctoral researchers and postdocs joined this year’s second excursion to Key- gene and Nunhems in the Netherlands. On the first day, the young researchers visited the company Keygene in Wageningen, which offers trait platforms as well as innovative technologies for plant breeding. At this company, they were able to get insights into the impressive sequencing facility and the phenotyping lab. On the second day, the company Nunhems (part of Bayer Crop Science) was visited. At the large company, the participants got to see different labs and afterwards the giant cucumbers in the greenhouse, used for seed production. During the excursion, the young researchers had the opportunity to present their own research projects and to dis- Young Researchers Retreat 2015, Bad Honnef cuss issues of mutual interest.

80 Promotion of young researchers

Young Researchers Retreat 2015

With more than 80 participants, the third young researchers retreat took place in Bad Honnef in October. Doctoral and postdoctoral researchers presented their work to all principal and associated investigators, to get feedback on their projects and progress. The programme was split into various sessions, each chaired by one of our young researchers. Between the sessions there was enough time for scientific discussions and networking.

Besides scientific presentations and poster sessions, group work on different topics such as improvement of public outreach activities as well as some socialising and networking events were included in the programme. Young Researchers Retreat 2015, Bad Honnef

Network meeting with Ph.D. students from Planter’s Punch Wageningen University The monthly Planter’s Punch is a short contribu- Together with the International Max Planck Re- tion on a specific aspect of the CEPLAS research search School in Cologne, the CEPLAS Graduate programme written by one of the young research- School hosted a 2-day Ph.D. meeting with a ers. The aim of this feature is to communicate the group of 20 Ph.D. students from Wageningen science that is done in CEPLAS labs to the public. University in April this year. The meeting took All contributions are available in English and in place at the Max Planck Institute for Plant Breed- German and are posted on the CEPLAS website, ing Research and the Cologne Biocenter and our facebook page and in a newsletter. included presentations and posters from both, German and Dutch students.

Young Researchers Seminar Outlook

At the young researchers seminar, CEPLAS doc- In 2016, again two excursions to industry will toral and postdoctoral researchers meet on a be offered for the CEPLAS young research- monthly basis to discuss their research and ers. A visit at Metanomics, Berlin, Germany programme issues. The young researchers them- is scheduled for February 2016 and a visit to selves are responsible for organisation and Bayer AG Leverkusen, Monheim, Germany is content of the seminar. The seminar includes planned for June 2016. scientific talks and flip-chart sessions and offers a forum for troubleshooting, scientific exchange For April 2016, the young researcher them- and networking among the young researchers. selves organise a “speed dating with industry” event. With the help of Dr. Günter Strittmatter, they have invited more than 10 Mobility fund employees from companies to spend a day with a group of CEPLAS young research- CEPLAS young researchers can apply for special ers who would like to learn more about the funding to cover their expenses for research stays working conditions in companies, to get at facilities outside of CEPLAS. This year, eight more routine in presenting themselves and requests were approved. The young researchers to expand their network. received financial support for lab visits, meetings with cooperation partners and participation in international summer schools.

Promotion of gender equality Annual Report 2015

Aims

As in academia and industry women are still underrepresented – especially in leading positions – the major aim of the CEPLAS equal opportunity programme is to increase the proportion of female scientists.

To achieve this aim, CEPLAS focuses on two major aspects:

▪ Career Support ▪ Family Support

6.1 Career support Career Day

On July 1st, the third Women‘s Career Day took place at the University of Cologne. Four exciting speakers from different fi elds in industry and academia were invited and presented their work and 3. WOMEN‘S CAREER DAY career path. JULI 1ST 2015 First speaker was Dr. Pim Lindhout, who worked UNIVERSITY for several years for De Ruiter Seeds and Mon- OF santo and fi nally founded his own company COLOGNE “Solynta” where he is now director of R&D. His

talk was followed by a presentation by Dr. Gesa orning eSSion M S Open for 09.15 am Dr. Pim Lindhout everybody! Behnken, Head of New Technologies at Bayer Director of Research & Development, Solynta, Netherlands MaterialSciences (now Covestro), who spoke 09.45 am Dr. Gesa Behnken Head of New Technologies, BAYER MaterialSciences, Germany about the challenges to balance family and work. Coffee break 10.40 am Dr. Aurelie Huser Mrs Behnken gave honest insights on how to fi nd Manager Technology Aquisition & Collaborations, BASF CropDesign, Belgium

11.10 am Prof. Dr. Paula Elomaa the balance between her position at the scientifi c Department of Agricultural Sciences, University of Helsinki, Finland department and being a mother, caring for her Location: University of Cologne, Biocenter, Zülpicher Str. 47b, Lecture Hall

family. The third speaker, Dr. Aurelie Huser from WoMen‘S Coffee Break France did her Ph.D. at MPIPZ and afterwards 02.00 pm In the afternoon all interested women from CEPLAS and non-CEPLAS labs are welcome to join an informal meeting with the speakers to discuss scientific career issues.

started directly as a licensing manager at VIB Registration required due to limited seats ([email protected])!

Ghent. She gave insights in how to enter a fi eld Location: University of Cologne, Cologne Biocenter, Seminar room 4.004 of work which is different from your educational

CEPLAS – Cluster of Excellence on Plant Sciences (EXC 1028) is funded by the DFG background. After working several years at VIB Max-Planck-Institut für in the context of the Excellence Initiative. Pflanzenzüchtungsforschung she is now a Manager for Technology Acquisi- tion & Collaborations at BASF Plant Science. The last speaker Prof. Paula Elomaa from University of Helsinki fi nished the morning session with an I’m really grateful for the opportunity to insightful talk on how to raise four children in par- talk for so long with the speakers. It was allel with a successful academic career. really helpful for me.

In the afternoon session, female CEPLAS post- I liked most that the speakers were talking docs and doctoral researchers had the opportu- about their personal experiences and how nity for an informal meeting with all speakers to to handle career and family. discuss career and family matters, the problems young scientists face when trying to balance It helped me a lot to get an idea about what work and family, and possible solutions to these possibility I may see after my Ph.D. challenges. The invited speakers named some problems that might arise in your career path if going a similar way like they did and gave advice on how to solve them.

84 Promotion of gender equality

Career development workshops

In collaboration with the Equal Opportunity offic- leadership skills. Others focused on communi- es at HHU and UoC, CEPLAS provided a diverse cation skills with special emphasis on getting a variety on career development workshops which better understanding of male corporate culture were specially designed for female scientists. The since men and women communicate in very dif- courses are aiming to make participants more ferent ways. In all workshops, external trainers aware of their own abilities, while also showing conveyed valuable specialised information and them how to apply these in different professional support which is relevant for female scientists at fields. The workshops covered numerous topics different career levels. In total, 13 CEPLAS mem- starting from career planning and optimising for bers participated in female career development researchers over academic management and workshops.

Coaching for junior professors/group leaders

The academic career path is full of challenges scientists to take part in an individual coaching and hurdles: Getting your own funding, starting programme and, hence, is covering the costs. to build-up your own research group, getting ac- The Equal Opportunity offices at HHU and UoC cepted by other researchers in the same field and support the coachee by finding a personal coach above all, combining these challenges with your who has the required experience for their individ- family issues. To support female group leaders ual situation. In 2015, two group leaders / junior and junior professors at this difficult stage of their professors took advantage of this offer. academic career, CEPLAS encourages female

6.2 Family support Helping hands programme

For pregnant women, parents on parental leave professors. Especially for female group leaders and CEPLAS fellows with young children, we it is difficult to maintain their lab running dur- provide funding for student helpers in order to ing maternity leave. Therefore, it is an important relief them from routine tasks at work and/or to issue within the CEPLAS gender programme to ensure continued operation of their research dur- strongly support female group leaders during ing leave. In 2015, the demand for support by this difficult period. Hence, two female group student helpers increased substantially when leaders received funding for a full-time postdoc compared to 2014 and was abundant among position during their absence from the lab. Ph.D. researchers, postdocs as well as junior

Child Care service during CEPLAS events

Child care is always provided during CEPLAS CEPLAS events in 2014. Additionally, we offer events. With the help of the Family Support CEPLAS members with very small infants to bring Services of the Universities, child care by quali- a family member, e.g. during retreats or excur- fied child-care workers was provided during all sions.

After hour Child Care service / Parents-child room

Parents within CEPLAS have access to funding difficult due to a number of regulations. At the that covers the expenses for after-hours child Universities Cologne and Düsseldorf parents care, and costs for day-care of toddlers under the have access to a parents-child room. Both rooms age of three according to the DFG rules, though are equipped with child care needs and computer implementation of these measures has been desks.

Key figures Annual Report 2015

7.1 Staff

Overview

Since the funding start in 2012/13, the cluster had a continuous increase in personnel. As shown in the figure, the doctoral researchers and postdocs represent the largest group with 31, respectively 27 members. In total, almost 90 people are directly financed by the cluster. However, currently around 200 researchers are working in CEPLAS labs on CEPLAS relevant topics.

90 W3 80 70 W2

60 W1 50 Postdocs 40 30 Ph.D. students 20 Technical/Administra- 10 tive Assistance 0 2012 2013 2014 2015 Administration

Development of personnel between 2012 and 2015

Proportion of female scientists

10 Male 5 Female 0 Doctoral researchers Postdocs New faculty Number of female researchers in different academic groups

88 CEPLAS – Key figures

New faculty 2015

By the end of 2015, we finally succeeded in filling all faculty positions that were announced in the context of CEPLAS. In October 2015, Benjamin Stich accepted the call for the last remaining position in Plant Quantitative Genetics and Genomics (W3).

Markus Pauly

Position W3 Plant Cell Biology and Biotechnology

Start 01.01.2016

Research focus

The long term goal of the Pauly lab is to fully understand the carbon deposition/ biosynthesis of plant cell walls and its regulation using a combination of carbohydrate analytical, genetic, molecular biological, biochemical and synthetic approaches.

Using high-throughput analytical screening technologies we have identified plant feedstocks (such as maize, sorghum, and agave) that have an enhanced lignocellulosic composition for an optimized biomass conversion to biofuels or commodity chemicals.

On a molecular level we have identified the biosynthetic machinery of the plant cell wall polysaccha- ride xyloglucan using forward, reverse and deep sequencing approaches. Corresponding genetic experiments gave us insights into the function of this major hemicellulose in plant growth and development in particular the evolutionary diversity of side-chain substitutions. Within CEPLAS I will contribute with our capabilities of detailed wall polymer analysis allowing us to investigate the difference in wall architecture between C3 and C4 specific tissues.

Short CV

Since 01/2016 Professor (W3) in Plant Cell Biology and Biotechnology, CEPLAS, Heinrich Heine University Düsseldorf, Germany

Since 07/2015 Full Professor in Plant and Microbial Biology/ Fred Dickinson Chair in Wood Science and Technology, Energy Biosciences Institute, University of California, Berkeley, USA

2010 Associate Professor in Plant and Microbial Biology/ Fred Dickinson Chair in Wood Science and Technology, Energy Biosciences Institute, University of California, Berkeley, USA

2006 Associate Professor in Biochemistry & Molecular Biology/ Genetics, DOE-Plant Research Laboratory, Michigan State University, East Lansing, USA

2001 Independent Research Group Leader, Max Planck Institute for Molecular Plant Physiology (MPI-MP), Golm, Germany

1998 Assistant Research Professor at the Royal Veterinary and Agricultural University, Copenhagen, Denmark

1998 Dr. rer. nat. (Ph.D. in Science), RWTH Aachen, Germany

1993 Diplom (Masters in Biology), RWTH Aachen, Germany 89 Annual Report 2015

Matias Zurbriggen

Position W2 Synthetic Biology

Start 01.10.2015

Research focus

Our research perspective is to apply synthetic biology approaches to control and understand eukaryotic signalling processes and regulatory networks in a quantitative and spatiotemporally resolved manner. To this aim we follow an interdisciplinary approach at the interface of engineering and life sciences, focusing on synthetic signalling networks and metabolic pathways, biological sensors, and chemical and optical switches (optogenetics) in animals and plants.

Short CV

Since 10/2015 Professor (W2) in Synthetic Biology, CEPLAS associated investigator, Heinrich Heine University Düsseldorf, Germany

02/2012 Group Leader (Akademischer Rat) Synthetic Biology in Mammalian and Plant Cell Systems, Faculty of Biology, University of Freiburg, Germany

04/11- 01/12 Alexander von Humboldt Foundation Georg Forster Research Fellow at Prof. Dr. Wilfried Weber´s Lab, Synthetic Biology, BIOSS – Faculty of Biology, University of Freiburg, Germany

04/09- 03/11 Postdoc Plant Biotechnology at Institute of Molecular and Cell Biology of Rosario (IBR), Argentina (Group Prof. N. Carrillo) in collaboration with Leibniz-Institute für Pflanzengenetik and Kulturpflanzenforschung (IPK), Gatersleben, Germany (Group Dr. M. Hajirezaei and Prof. Dr. N. von Wirén). Research stay at the John Innes Centre (JIC), Norwich, UK

04/04 - 03/09 Ph.D. Thesis work in Plant Molecular Biology and Biotechnology with Prof. Dr. N. Carrillo at Institute of Molecular and Cell Biology of Rosario (IBR), Argentina in collaboration with Leibniz-Institute für Pflanzengenetik und Kulturpflanzenforschung (IPK), Gatersleben, Germany. “Development of stress tolerant transgenic plants”

08/02-03/04 Diploma Thesis work at IBR, National Research Council of Argentina

03/97- 03/04 Diploma in Molecular Biology and Biotechnology, National University of Rosario, Argentina

Guest scientists

▪ Prof. Gerald Schönknecht, Oklahoma State University, USA, DAAD/CEPLAS guest professor, May 2014 - Sept 2015 ▪ Prof. Dr. Sanjib Kumar Panda, Assam University, India, Oct-Dec 2015 ▪ Dr. Pilar Cubas, Spanish National Biotechnology Centre, Sept 2015 ▪ Dr. Aashish Ranjan, UC Davis, USA, Feb-March 2015 ▪ Kai Xun Chan, Australian National University Canberra, Sept 2015 ▪ Garret Early, North Carolina State University, DAAD Rise Scholar May-July 2015 Mirjam Garske, Michigan State University, DAAD Rise Scholar May-July 2015 90 ▪ CEPLAS – Key fi gures

7.2 Finances

Granted funds and total spending

Total spending in 2015 (k€)

Total allowance 2015 Actual spending (calculations 03/2016)

6,040 5,989

Overview total spending 2015 (% of total budget)

Central funds 10,0

Equal opportunity measures 1,0

Core facilities 4,4

New faculty 53,7

Research Area A 8,9

Research Area B 9,4

Research Area C 5,7

Research Area D 7,0

Overview central funds 2015 in % (total budget central funds k€ 599)

Administrative staff 50,9

Guest scientists 3,5

Symposia /retreats 9,7

Publications 2,7

Travel costs 1,5

Mobility fund 6,4

Recruiting 5,0

Public outreach 17,3

Young researchers programmes 2,9

91 Annual Report 2015

Overview research areas (k€)

Amount spent Additional funds % of total budget (03/2016) by universities

Research Area A 531 9 2 Ph.D. positions

Research Area B 561 9 3 Ph.D. positions

Research Area C 339* 6 4 Ph.D. positions

Research Area D 417 7 1 Ph.D. position

Total 1,848 31 10 Ph.D. positions

* Two postdocs on maternity leave and one staffing delayed.

92 CEPLAS – Key figures

Third-party funding

Additional third-party funding raised in 2015 (k€)

Funding body Number Funding amount

German Research Foundation (DFG)

Research grants 7 1,690

Collaborative Research Centres 1 8,957

Priority Programmes (subprojects) 3 568

Research Units 1 428

Large Equipment 1 763

Total 12,406

Federal Ministry of Education and Research (BMBF) 4 1,766

Ministry of Innovation, Science, Research and Technology of North Rhine Westphalia

NRW strategic project Bioeconomy Science Centre (BioSC) 19 2,355

EU 3 797

DAAD 2 72

Alexander von Humboldt Foundation 2 160

Bill and Melinda Gates Foundation 2 438

Industry 2 140

Others 5 658

Total 52 18,792

Public outreach Annual Report 2015

8.1 Public relations work

Public lecture series: “Vom Urweizen der Steinzeit zu den Genpflanzen der Zukunft“

One more time we organised our public lecture series: “Vom Urweizen der Steinzeit zu den Prof. Dr.Genpflanzen Peter Westhoff… der Zukunft”. This year the lecture ...bekleidet seit 1988 den Lehrstuhl für Entwicklungs- und Molekularbiologie der Pfl anzen an der Heinrich-Heine-Universität Düsseldorf und fungiert seit November 2014series als Prorektor took für Forschung place und Innovation. in Cologne. Peter Westhoff and NutzPFLANZEN Neben einerAndreas Vielzahl weiterer Funktionen Weber im Rahmen gaveinternationaler a Pfl an-comprehensive overview zenforschungsprojekte war Prof. Westhoff von 2000 bis 2004 Mitglied im PFLANZENnutzen Fachausschuss Botanik der Deutschen Forschungsgemeinschaft und saß von 2006 bis 2012from in deren Senat.the Im vergangenenbeginning Jahr wurde Prof. of Westhoff agriculture and the meth- in die Nationaleods Akademie of der Wissenschaftenplant breeding(Leopoldina) aufgenommen. to the challenges of the Sommersemester 2015 future. Similar to last year, the mixed audience VORTRAGSREIHE Prof. Dr.was Andreas very Weber…interested and appreciated the oppor- ... ist seit 2007 Inhaber des Lehrstuhls für Biochemie der Pfl anzen an der Vom Urweizen Heinrich-Heine-Universitättunity Düsseldorf.for subsequent discussions and questions. der Steinzeit Prof. Weber ist Sprecher des Exzellenzclusters für Pfl anzenwissenschaften Beteiligte Institutionen CEPLAS undThe Direktor desfirst Zentrums fürlecture Synthetische Lebenswissenschaften was broadcasted on the radio zu den Genpfl anzen Düsseldorf.

Max-Planck-Institute for and podcasts of all lectures are available on ourPlant Breeding Research der Zukunft homepage.

CEPLAS “Forschertage“

In July, 13 students from three different schools in and around Cologne participated in the first CEPLAS “Forschertage“. In this 3-day event the students participated in short lectures, lab experiments and workshops. The first two days took place at the Cologne Biocenter, where the students worked on photosythesis and plant-microbe interactions. On the third day, they visited the Wissenschaftsscheune of the Max Planck Institute for Plant Breeding Research where they learned about the origin of our crop plants and about the evolution of wheat and maize.

The aim of this event was to show how fascinat- ing plants are and how important they are for our daily life. Due to the positive feedback, we plan on repeating the programme in the summer holi- days 2016.

Planter’s Punch

To communicate more of the science that is done What can a mathematician tell you about plants? in the CEPLAS labs to the public, Planter’s Punch was established in 2014. Each month one special aspect of the CEPLAS research programme. All contributions are published in English and Ger- man via our homepage and facebook page.

In addition to informing a wider audience about our research, this feature trains our young researchers in communicating their science in a con- cise, easy to understand manner.

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CEPLAS “Research and Education“ Project

The project’s goal is to activate and maintain stu- the programme. In cooperation with the ZfL and dents-directed discovery of plant-environment the Institut für Biologiedidaktik “teaching pack- interactions through inquiry based research ex- ages“ for selected CEPLAS research topics will perience at B.Sc. and secondary school level. be developed by scientists and students to sup- To this end, we developed a workflow enabling port teachers with an educational design. These the sustainable knowledge transfer from signif- teaching packages will subsequently be tested icant CEPLAS research fields to practical teach- with the secondary-school students. ing modules. The initiative started in November 2014 in a kick-off meeting with delegates from The concept was developed and accepted in the Zentrum für LehrerInnenbildung – ZfL (Center August 2015 by the participating institutions. It for Teacher Training) at University of Cologne, will be carried out every semester in the format the Institut für Biologiedidaktik at UoC, rectors of a “vocational field internship” (Berufsfeldprak- and teachers of secondary schools. tikum) and is officially announced in the course catalogue of the University of Cologne (starting This initiative brings CEPLAS scientists together in 2015/2016). The scientific relevance of this with (1) University students who are in training programme will be evaluated in a concomitant to become biology teachers (BSc Education, Ph.D. thesis that is carried out in cooperation with 5th semester), (2) students of advanced second- the graduate school for teacher training (Gradu- ary-schools as well as (3) their teachers, aiming iertenschule für LehrerInnenbildung) under the to increase the awareness of complex traits in- supervision of Kirsten Schlüter (Head of Insti- volved in plant-environment interactions. tut für Biologiedidaktik) and Marcel Bucher. This initiative is supported by CEPLAS and in the The selected research topics meet the require- frame of the “UoC Excellent Research Support ments of the university-entrance diploma (Abi- Program”. To increase the number of participating tur). In the pilot phase teachers and students of institutions and ensure its long-term continuation two selected secondary schools, Ursulinenschule we are in contact with the Technical University of Hersel and Ville-Gymnasium Erftstadt, partici- Cologne (Technische Hochschule Köln), the pro- pate in the development and implementation ject Modularer Modellgarten Köln and the zdi of the programme. Outstanding and highly mo- (Zukunft durch Innovation, Ministry for Innova- tivated secondary-school students will have the tion, Science and Research, NRW) to apply for possibility to do their mandatory “school-practi- support by external funding partners in order to cal” (Facharbeit) in March 2016 in the frame of establish Biology Science Labs for school classes.

Newsletter

We started a Newsletter in January 2015 based personnel, general information and important on suggestions from our internal evaluation. The dates. To reach out to the public, the newsletter newsletter is published four times a year and is bilingual and published on the CEPLAS home- contains information on cluster activities, new page.

Participation in the lecture course “The Future of Agriculture” of the German National Academy of Sciences (Leopoldina)

In April Peter Westhoff participated as an ex- During this lecture course, 15 selected journal- pert in the lecture course for science journalists ists of the science departments of various high- “Tauchgänge in die Wissenschaft” of the Ger- rank print media and public broadcasters were man National Academy of Sciences (Leopoldina). informed about the basics of animal farming and This lecture course is organised by the Leo- agriculture, and discussed with the experts the poldina and the Robert Bosch Stiftung. The future global challenges with respect to popula- two-week course aims at informing and train- tion pressure, climate change and the potential ing journalists on scientific topics of high social of GMOs or organic farming. relevance.

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Fascination of Plants Day 2015

Under the heading “Why plants glow” the Insti- tute for Quantitative and Theoretical Biology, headed by Oliver Ebenhöh organised a workshop for pupils between age 8 and 11. The action took place in the course of the Fascination of Plants Day, an European-wide action day to In addition, there were also other activities inspire people for the fascination and importance organised by CEPLAS members around the of plant sciences. The workshop was well attend- Fascination of Plants Day at the Forschungs- ed with participation from more than 240 kids. zentrum Jülich and in Cologne.

Botanical Garden

In June, the new crops section at the Botanical and cultural history. CEPLAS contributes to the Garden Düsseldorf was re-opened with the ex- exhibition with a section on (energy) plants of the hibition Nutzpflanzen: gestern | heute | morgen. future. From July to September, several guided The large exhibition shows the development of tours were offered. wild to domesticated crop species, their usage

18th GPZ Genome Research Conference

In autumn, CEPLAS hosted this year’s conference of the Genome Research Working Group of the German Society for Plant Breeding provided: „New targets for crops of the future“. Various national and international speakers presented new hot topics for tomorrow’s plant breeding. The conference provided a comprehensive overview of new approaches currently discussed and relevant for plant breeding. CEPLAS young researchers had the opportunity to showcase their work in presentations and a poster session in the Botanical Garden Düsseldorf.

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Nacht der Wissenschaft

End of September, the “Nacht der Wissenschaft“ CEPLAS contributed with an exhibition on the took place in the centre of Düsseldorf. The event development, importance and future of crop was organised by the City of Düsseldorf, HHU plants as well as with a lecture by Andreas Weber. and HS Düsseldorf. Researchers of these institu- CEPLAS members fielded questions from over tions presented more than 30 actions, lectures one hundred interested people about various and workshops to give the public audience a topics from molecular biology to plant breeding comprehensive overview of research at their in- and consumption behaviours. stitutions. More than 4.500 people visited the event from 5 pm till midnight.

Key note lecture by Pamela Ronald

In October, Pamela Ronald (UC Davis) accepted Her 2015 Ted talk on plant genetics, food secu- the invitation from our young researchers and rity and sustainable agriculture has been viewed visited CEPLAS for a talk in Düsseldorf. Together more than 1 million times. After the presentation with her working group at UC Davis and the In- on her research work and engagement in educa- ternational Rice Research Institute at the Philip- tional work on GMO issues, young researchers pines she discovered the flood tolerant Sub1 rice had the opportunity for an informal meeting with variety which in 2013 was given to more than 4 her for further discussions. million farmers for free use.

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8.2 Political outreach

Visit at the protected site for GMO field trials in Reckenholz, Switzerland

In May, a delegation of CEPLAS members to- The site is operated by Agroscrope, the Swiss gether with scientists from the Forschungs- centre of excellence for agricultural research and zentrum Jülich and representatives of the Projekt- is affiliated with the Federal Office for Agriculture träger Jülich visited the protected site in Recken- (FOAG). The aim of the meeting was to obtain holz Switzerland. The protected site was set-up more information on the structure, organisation to give researchers working with GMO the op- and utilisation of the site, and to discuss possibi- portunity to perform experimental field trails lities for cooperation. without the threat of vandalism.

CEPLAS meets politics

In April, members of CEPLAS met with Patrizia To emphasise the importance of plant sciences Lips, (member of the German Parliament, chair- in Germany CEPLAS and the German Society women of the Committee on Education, Re- for Plant Sciences (DBG) invited members of the search and Technology Assessment) to discuss German Parliament, the Federal Ministry of Edu- the importance of plant science for bioeconomy cation and Research (BMBF) and the Federal and for Germany as a research site. In follow-up Ministry of Food and Agriculture (BMEL) to par- meetings, CEPLAS representatives met with ticipate in a parliamentary breakfast in Berlin other members of the Committee on Education, in December. Eight members of different po- Research and Technology Assessment and of the litical parties and the BMBF accepted the invi- Committee on Food and Agriculture for further tation and took the time to speak with CEPLAS discussions. and DBG representatives. Andreas Weber, Paul Schulze-Lefert and Karl-Josef Dietz, the president of the DBG, gave introductory speeches to open up the discussion.

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8.3 Press archive

▪ Press release of the Heinrich Heine University Düsseldorf 17.12.2015: Prof. Dr. Markus Pauly zum W3 Professor ernannt ▪ Press release of the Heinrich Heine University Düsseldorf 09.12.2015: Prof. Dr. Andreas Weber zum Leopoldina-Mitglied gewählt ▪ INTERN Informationen für die Beschäftigten der Heinrich-Heine-Universität Düsseldorf 12/2015: ZSL: Grundstein für neues Forschungsgebäude ▪ Düsseldorf Express 21.11.2015: So lösen wir das Welternährungs-Problem ▪ Rheinische Post 09.11.2015: Ein Zentrum für Spitzenforscher entsteht an der Universität ▪ Press release of the Heinrich Heine University Düsseldorf 06.11.2015: HHU und UKD legen Grund- stein für neues Forschungsgebäude ▪ Magazin der Heinrich-Heine-Universität Düsseldorf 11/2015: HHU und UKD legen Grundstein für neues Forschungsgebäude ▪ Press release of the Heinrich Heine University Düsseldorf 28.10.2015: Reisforschung für eine sichere Ernährung in der Dritten Welt ▪ Press release of the Heinrich Heine University Düsseldorf 12.10.2015: Selbstschutz: Acker-Schmal- wand erzeugt Vitamin E aus Stoffwechselprodukten ▪ INTERN Informationen für die Beschäftigten der Heinrich-Heine-Universität 07/2015: Ausstellung „Nutzpflanzen – Gestern – Heute – Morgen“ im Botanischen Garten ▪ Rheinische Post 15.07.2015: Wo die Supermarkt-Ware herkommt. ▪ Press release of the Heinrich Heine University Düsseldorf 13.07.2015: Botanischer Garten zeigt Nutzpflanzen – Ausstellung bis Ende 2016 ▪ Westdeutsche Zeitung 10.07.2015: Wenn Blumenkohl und Porree blühen. ▪ Press release of the University of Cologne 08.07.2015: Neuartiges Studienangebot für künftige Biowissenschaftler/innen. ▪ Press release of the Heinrich Heine University Düsseldorf 23.06.2015: Neue B.Sc. Studiengangs- Variante „Quantitative Biologie“. ▪ Press release of the Heinrich Heine University Düsseldorf 28.05.2015: Aha-Effekte für wachsende Begeisterung bei kleinen Pflanzenforschern ▪ Die Brücke zur Welt, Leben Gesellschaft und Kultur am Wochenende, Wochenendausgabe der Stuttgarter Zeitung Nr. 117, 23.05.2015: Schöne neue Pflanzenwelt ▪ Press release of the Heinrich Heine University Düsseldorf 12.05.2015: HHU bringt Pflanzen zum Leuchten – Aktionstag „Faszination der Pflanzenwissenschaften“. ▪ Radiobeitrag DRadio Wissen 09.05.2015: Biotechnik in der Steinzeit ▪ Pflanzenforschung.de 02.04.2015: Photosynthese Tuning – Kann dies ein Schlüssel zu ausreichend Nahrung für alle werden? ▪ Press release of the University of Cologne 01.04.2015: NutzPflanzen – Pflanzen nutzen: Vortrags- reihe des Exzellenzclusters CEPLAS über die Geschichte der Pflanzenzucht ▪ Sonderausgabe der Rheinischen Post, 50 Jahre HHU 21.01.2015: Mit Genetik gegen den Hunger, 50 Jahre HHU ▪ Agri-pulse Communications Inc 22.12.2014: 2015: The Year of Soils, Opinion by Marshall Matz

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Technology transfer and cooperation management Annual Report 2015

The translation of research results into economic exploitation and the cooperation with the private sector are major goals of the CEPLAS programme. For this purpose, the presentation of CEPLAS as an attractive cooperation partner in the field of plant sciences and its CEPLAS Meets interaction with the private sector have been intensified in 2015. A Industry specific focus of CEPLAS is also set on making young scientists familiar with career opportunities in non-academic areas. In addition, various Innovative approaches for activities have been started to bring the socio-economic value of plant crop improvement sciences and the need to strengthe this scientific field in Germany to the attention of the political dialogue.

9.1 Presentation to industry

To present of CEPLAS to potential cooperation panies. Interactions with the regional business partners from industry two events were organ- platforms “BioNRW” and “BioRiver”, both loca- ised. First the one-day event “CEPLAS Meets ted in Düsseldorf, have been established with Industry” with representatives from six different the goal of mediating contacts to the regional internationally active companies and the Ger- biotech and start-up funding business. man Plant Breeders Association, and second a “CEPLAS Day” for the leading German breeding A compendium addressing application-relevant company, KWS SAAT SE, held at the University of aspects within the CEPLAS programme, and op- Düsseldorf in January and March, 2015, respec- portunities for cooperation as well as exploitation tively. of research results has been finalised, comprising “project profiles” for the CEPLAS activity of each Furthermore, the CEPLAS programme was pre- Principal Investigator and Associate Investigator. sented to KeyGene (Wageningen, The Neth- This compendium will be used to intensify the erlands) and to Bayer CropScience Vegetable discussions with potential partners from the pri- Seeds (Nunhem, The Netherlands), in the course vate sector. It will also facilitate the extension of of an excursion of young scientists to these com- such discussions at the international level.

9.2 Planned cooperation with industry

As a result of the presentations of CEPLAS to potential partners from the private sector, three CEPLAS Meets Industry | January 28, 2015 comprehensive research projects which included Programme part-ners from companies have been submitted 13:00 – 14:00 Arrival of participants | Reception with business lunch within the framework of the new BMBF funding 14:00 – 14:30 Overview on organization and research activities of CEPLAS

initiative “Plant Breeding Research for the Bioec- Prof. Dr. Andreas Weber

onomy”. The decision on the funding of projects … is Chair of the Institute for Plant Biochemistry at the Heinrich Heine University Düsseldorf and will be released in spring 2016; project funding Speaker of CEPLAS.

will start in fall 2016. 14:30 – 15:15 Photosynthesis – a target for yield improvement

Prof. Dr. Peter Westhoff In addition, we succeeded in gaining support by … is Chair of the Institute for Developmental and NPZ for a project on the improvement of plant Molecular Biology of Plants at Heinrich Heine University Düsseldorf. health and productivity via plant-microbe inter- 15:15 – 15:45 Coffee Break actions in the frame of the Bioeconomy Science 15:45 – 16:30 Structure, functions and (potential) application of the plant Center (BioSc). microbiota

Prof. Dr. Paul Schulze-Lefert

… is Director of the Max Planck Institute for Plant Breeding Research Cologne and Head of the De- partment of Plant Microbe Interactions.

16:30 – 17:00 Discussion and concluding remarks

17:00 – 18:00 Get together and individual discussions of participants 104 Cooperation Partners Technology transfer and cooperation management

9.3 Non-academic careers for young scientists

Young CEPLAS scientists were introduced to the field of applied plant sciences and the working environment of plant scientists in industry by a two-day seminar, which included presentations from representatives of KWS SAAT SE.

This introduction was complemented by one-day excursions to Qiagen N.V., KeyGene and Bayer CropScience Vegetable Seeds.

Postdoctoral Fellows interested in a professional career in industry were personally advised, addressing opportunities and requirements for entering such a career as well as the application process. Outlook

We will extend contacts to companies outside Germany and beyond the plant breeding business has been defined.

CEPLAS will also intensify its interaction with regional business platforms such as “Industrie- und Handelskammer Rhein- land” (joint meeting for the presentation of CEPLAS in February 2016), and increase its participation in biotechnology fairs.

The repeated presentation of CEPLAS activities and results at companies already previously addressed will continue.

Cooperation contracts for joint research projects within the new BMBF funding initiative and within bilateral relationships beyond this public funding initiative still need to be established.

The coaching of young scientists interested in professional careers outside academia, and the interaction with politics, in order to gain stronger support for plant sciences, will remain as areas of engagement.

Finally, the strategic positioning of technology transfer and cooperation with the private sector will be integrated into the planning of the application of CEPLAS for the next “Excellence Initiative”.

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Principal Investigators

1 Jun.-Prof. Dr. Maria Albani Cologne Biocenter, UoC at MPIPZ 2 Jun.-Prof. Dr. Ilka Axmann Institute of Synthetic Microbiology, HHU 3 Prof. Dr. Marcel Bucher Cologne Biocenter, UoC 4 Prof. Dr. George Coupland MPIPZ 5 Prof. Dr. Gunther Döhlemann Cologne Biocenter, UoC 6 Jun.-Prof. Dr. Oliver Ebenhöh Institute of Quantitative and Theoretical Biology, HHU 7 Prof. Dr. Michael Feldbrügge Institute of Microbiology, HHU 8 Prof. Dr. Ulf-Ingo Flügge Cologne Biocenter, UoC 9 Prof. Dr. Ute Höcker Cologne Biocenter, UoC 10 Prof. Dr. Martin Hülskamp Cologne Biocenter, UoC 11 Prof. Dr. Karl-Erich Jaeger Institute of Molecular Enzyme Technology, HHU 12 Prof. Dr. Markus Kollmann Institute of Mathematical Modelling of Biological Systems, HHU 13 Prof. Dr. Maarten Koornneef MPIPZ 14 Prof. Dr. Stanislav Kopriva Cologne Biocenter, UoC 15 Prof. Dr. Martin Lercher Institute of Informatics, HHU 16 Prof. Dr. Alice McHardy Institute of Informatics, HHU 17 Prof. Dr. Jane Parker MPIPZ 18 Prof. Dr. Markus Pauly Institute of Plant Cell Biology and Biotechnology, HHU 19 Prof. Dr. Laura Rose Institute of Population Genetics, HHU 20 Prof. Dr. Lutz Schmitt Institute of Biochemistry, HHU 21 Prof. Dr. Paul Schulze-Lefert MPIPZ 21 Prof. Dr. Ulrich Schurr Institute of Bio and Geosciences-2 (IBG-2), FZJ 23 Prof. Dr. Rüdiger Simon Institute of Developmental Genetics, HHU 24 Prof. Dr. Miltos Tsiantis MPIPZ 25 Jun.-Prof. Dr. Maria von Korff Schmising Institute for Plant Genetics, HHU at MPIPZ 26 Prof. Dr. Andreas P. M. Weber Institute of Plant Biochemistry, HHU 27 Prof. Dr. Peter Westhoff Institute of Plant Molecular and Developmental Biology, HHU 28 Prof. Dr. Jürgen Zeier Institute of Plant Molecular Ecophysiology, HHU 29 Prof. Dr. Alga Zuccaro Cologne Biocenter, UoC

Associated Investigators

1 Prof. Dr. Petra Bauer Institute of Botany, HHU 2 Prof. Dr. Michael Bonkowski Cologne Biocenter, UoC 3 Prof. Dr. Juliette de Meaux Cologne Biocenter, UoC 4 Dr. Thomas Drepper Institute of Molecular Enzyme Technology, HHU 5 Dr. Tamara Gigolashvili Cologne Biocenter, UoC 6 Prof. Dr. Georg Groth Institute of Biochemical Plant Physiology, HHU 7 Dr. Eric Kemen MPIPZ 8 Prof. Dr. Karl Köhrer Centre for Biological and Medical Research (BMFZ), HHU 9 PD Dr. Veronica G. Maurino Institute of Plant Molecular and Developmental Biology, HHU 10 Prof. Dr. Peter Nürnberg Cologne Center for Genomics (CCG), UoC 11 Prof. Dr. Uwe Rascher IBG-2, Plant Sciences, FZJ 12 Dr. Richard Reinhardt Max Planck Genome Centre, MPIPZ 13 Prof. Dr. Kai Stühler Centre for Biological and Medical Research (BMFZ), HHU 14 Prof. Dr. Klaus Theres MPIPZ 15 Prof. Dr. Vlada B. Urlacher Institute of Biochemistry, HHU 16 Prof. Dr. Wolfgang Werr Cologne Biocenter, UoC 17 Prof. Dr. Matias Zurbriggen Institut for Synthetic Biology, HHU

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