INDEPTH Prague Meeting Date and Time
Title
Authors
Abstract
Prague, Czech Republic February 25th-27th 2019
INDEPTH Prague Meeting INDEPTH Prague Meeting
Welcome to Praha
On behalf of the COST-Action CA16212 “Impact of Nuclear Domains in Gene Expression and Plant Traits” (INDEPTH), we are very pleased to welcome all meeting participants to Praha.
Plant Sciences and INDEPTH in the Czech Republic By Ales Pecinka
Plant sciences have a long tradition in the Czech Republic and the biology of nucleus is one of the classical themes of plant research in the country. The key plant research institutions are concentrated in the major cities of Praha, Brno, Olomouc and Ceské Budejovice.
The Czech INDEPTH partners include several groups from the Institute of Experimental Botany of the Czech Academy of Science (IEB) in Praha and Olomouc (Doležel, Pecinka, Šimková, Honys), Central European Institute of Technology (CEITEC) in Brno (Dvorácková, Fajkus, Lysak), Palacký University in Olomouc (Šebela, Bitomský) and Biology Centre of the Czech Academy of Science in Ceské Budeeovice (Mozgová).
COST is an EU-funded programme that enables researchers to set These groups focus on the analysis of plant nuclear up their interdisciplinary research networks in Europe and beyond. organization, chromatin and chromosome structure, genome We provide funds for organising conferences, meetings, training stability and gene regulation in the model and crop plants. The schools, short scientific exchanges or other networking activities in Czech INDEPTH partners have unique expertise in flow cytomety a wide range of scientific topics. By creating open spaces where people and ideas can grow, we unlock the full potential of science. and nuclei sorting, proteomics, microscopy, (epi)genomics and genome stability, which they are happy to share with all INDEPTH Cover image: http://www.bhmpics.com/view-sunset_prague_city- members. wide.html Not for commercial use
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Meeting Information Travel information
For all meeting information please contact: [email protected] Arrival by plane
The meeting will take place at: From Václav Havel Airport please take a taxi to the hotel. This will take about 20 minutes. Orea Hotel Pyramida, Belohorská 24, 169 00 Praha 6 Czech Republic Phone: + 420 233 102 111 E-mail: [email protected] https://www.hotelpyramida.cz/en/
Arrival by train to Praha Hlavní Nádraží train station
> Take metro line C north to Vltavská (3 stops, 2minutes) >Take Tram 25 to Bílá Hora west from Vltavská to Malovanka (10 stops, 16minutes) > Walk 4minutes West to hotel
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Meeting Schedule 15:30-15.50 Gwénaëlle Détourné (Université Clermont Auvergne, France): Characterisation of a novel family
th of plant nuclear envelope associated proteins (NEAP) Monday February 25 2019 in Arabidopsis thaliana
9:00-12:00: Meeting for members of INDEPTH Management 15.50-16.10 Anis Meschichi (Swedish University of Agricultural Committee Sciences, Sweden): Characterization of chromatin mobility during DNA damage responses in 12:30-13.15: Arrival/Registration and Poster setup Arabidopsis thaliana
13:15-13:35 Welcome Speech 16.10-16.30 Valya Vassileva (Institute of Plant Physiology and Genetics, Bulgaria): Zebularine-induced DNA 13:35-14:15 Opening Keynote: Eric J. Richards (Boyce Thompson demethylation impairs auxin signalling in Institute, USA) Arabidopsis roots
14:15-16:30 WG1: Quantitative imaging and analysis of the plant 16.30-16.45 WG5 Session. Introduce dissemination activities, nucleus in 3D. published papers and future opportunities Chair: Dimiter Prodanov and Ana Paula Santos 16:45- 17.45 Pecha Kucha Talks (see page 22 for speaker list) 14:15-14.30 Dimiter Prodanov (IMEC, Belgium): Summary of WG1 activities 17:45- 19.30 Poster session
Christophe Tatout (Université Clermont 20.15- late Gala Dinner Auvergne, France): Overview of Florence WG1, Workshop, Training school 3D-FISH and OMERO repository at Florida State University
14:30-15:05 Invited speaker: Andrew French (Nottingham University, UK): Deep machine learning for plant image analysis
15:05-15:30 Coffee Break
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Tuesday 26th February 11.30- 12.00 Introduction to plant science from ITC countries: - Turkey: Ahmet Tek (Nigde Omer Halisdemir 09:00-11:05 WG2: Transcriptional regulation through association University) of chromatin domains with nuclear compartments - Russia: Elena Salina (Institute Cytology and Chair: Stefanie Rosa and Sara Farrona Genetics SB RAS) - Tunisia: Moez Hanin (University of Sfax) 09.00- 09.10 Summary of WG2 activities: Stefanie Rosa (Swedish University of Agricultural Sciences, Sweden) and 12.00- 13.00 Parallel sessions for WG meeting /round table Sara Farrona (National University of Ireland-Galway, discussion Ireland) 13:00-14:00 Lunch 09.10- 09.45 Invited Speaker Andreas Houben (Gatersleben IPK, Germany): CRISPR/Cas9-mediated labelling of 14:00-16:25 WG3: Structure of nuclear domains and the genomic sequences in living and fixed cells functional output for plant traits Chair: Ales Pecinka and Monica Pradillo 09.45- 10.05 Martina Dvorackova (CEITEC, Czech Republic): Impact of histone chaperone genes and DNA repair 14.00- 14.10 Summary of WG3 activities: Ales Pecinka (Institute pathways on rDNA stability of Experimental Botany, Czech Republic) and Monica Pradillo (Complutense University of Madrid, Spain) 10.05-10.25 Francesca Lopez (National University of Ireland- Galway, Ireland): CRISPR-Cas9 mutagenesis unravels 14.10- 14.45 Invited speaker: Julio Salinas (Centro de the role of 45s rDNA genes in A.thaliana Investigaciones Biológicas, Madrid, Spain): development To be or not to be (for plants under adverse environmental conditions). The answer is in the 10.25-10.45 Aline Probst (Université Clermont Auvergne, France): spliceosome Histone H3.3 deposition pathways in Arabidopsis thaliana 14.45- 15.05 Moez Hanin (University of Sfax, Tunisia): The RSS1-PP1 pathway and its role in plant tolerance to 10.45-11.05 Iva Mozgova (Institute of Microbiology CAS, Czech abiotic stresses Republic): Novel roles of PRC2 in photomorphogenesis and seedling establishment 15.05- 15.25 Kirsten Krause (The Arctic University of Norway, Norway): Cuscuta campestris: A plant genome under 11.05-11.30 Coffee Break the influence of a parasitic lifestyle
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15.25- 15.45 Tzion Fahima (University of Haifa, Israel): Cloning 09:10- 09:45 Invited Speaker Martin Mascher (IPK Gatersleben, of the wheat YR15 resistance gene sheds light on the Germany): Hi-C in cereals: genome assembly, plant tandem kinase-pseudokinase family structural variation and chromatin organization
15.45- 16.05 Ana Paula Santos (Universidade Nova de Lisboa, 09:45- 10:05 Dariusz Plewczynski (Centre of New Technologies, Portugal): Plasticity of Epigenetics and Chromatin Warsaw, Poland): Chromatin looping architecture of structure: concerted strategies for enhanced plant the human genome tolerance under stress 10:05- 10:25 Amanda Souza-Camara (IPK Gatersleben, Germany): 16.05- 16.25 Gianluca Teano (IBENS, France): Linker histone H1 Polymer simulations to understand the structure and dynamics control Arabidopsis nuclear reorganization dynamics of mitotic barley chromosomes in response to light signals. 10:25- 10:45 Hana Simkova (Institute of Experimental Botany, 16.25-17.00 Coffee Break Czech Republic): Wheat rRNA gene loci resolved by chromosome sorting, optical mapping and RNA-seq 17.00- 18.00 Parallel session for Core Group Meeting 10.45- 11.15 Coffee Break 17.00-18.15 Poster session 11.15- 11.45 STSM update with David Evans (Oxford 18.15-20.15 Social event: Discovering Praha by night and free Brookes University, UK) evening 11.45- 12.00 Final Update and poster prize presentation: Célia Baroux (University of Zürich, Switzerland) Wednesday 27th February 12.00- Buffet lunch or Packed lunch and Exit 09:00-10:30 WG4: Storage, Data management and integrative analysis Chair: Stefan Grob
09:00-09:10 Summary of WG4 activities: Stefan Grob (University of Zürich, Switzerland)
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13.35pm February 25th Keynote Speaker
GENETIC DISSECTION OF EPIGENETIC PHENOMENA IN PLANTS
Eric Richards, Junsik Choi, Susan Strickler, and Erika Hughes
Boyce Thompson Institute, Ithaca, New York, USA
Our group studies the mechanisms that package and contextualize eukaryotic genomes largely through forward genetics in the model plant, Arabidopsis. Our work on cytosine methylation uncovered connections between chromatin remodeling and DNA modification (Jeddelohet al., 1999), while demonstrating the importance of this common covalent nucleotide modification in stabilizing both the genome and the epigenome. In addition, study of Arabidopsis mutants has revealed that the cytosine methylation system also plays a role in maintaining heterochromatin organization in the three-dimensional space within the interphase nuclei (for example, Woo et al., 2007). This interaction between epigenetic modification and nuclear cell biology has driven us to explore more directly how nuclear organization impacts genomic activity and stability.
An ongoing mutant screen in Arabidopsis has identified a set of genes important for specifying and/or maintaining nuclear shape and size in Arabidopsis. These genes include those encoding NMCP-type proteins that are a major component of the nuclear lamina. Despite dramatic changes in chromocenter organization in these nuclear lamina mutants, heterochromatin-reinforced silencing of transposons and pericentromeric sequences remains intact. Nonetheless, a large number of genes are mis-expressed in these mutants, arguing that the three-dimensional genome organization might play a role in control of gene expression patterns in euchromatin. Our analysis, however, points to alternative models, including the possibility that the ectopic induction of plant defense responses, which predominate in these mutants, stems from nuclear lamina defects that trigger premature cell death.
Jeddeloh, J.A., Stokes, T.L., and Richards, E.J. (1999). Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nature Genetics 22, 94-97.
Woo, H.R., Pontes, O., Pikaard, C.S., and Richards, E.J. (2007). VIM1, a methylcytosine- binding protein required for centromeric heterochromatinaization. Genes Dev. 21, 267- 277.
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14.30pm February 25th Invited talk WG1
DEEP MACHINE LEARNING FOR PLANT IMAGE ANALYSIS
Andrew French
University of Nottingham, UK
Deep machine learning has seen much success in the analysis of images across many different domains. Much of the recent emergence of artificial intelligence (AI) can be credited to advances in neural-network based deep learning.
At Nottingham, we have developed such approaches for use in various types of plant image analysis, including with projects detecting features and events in confocal and light sheet data. In this talk, I will briefly introduce machine learning and deep learning, and discuss some of our recent results applied to images of plants at various scales.
Pound, M.P., Atkinson, J.A., Townsend, A.J., Wilson, M.H., Griffiths, M., Jackson, A.S., Bulat, A., Tzimiropoulos, G., Wells, D.M., Murchie, E.H., et al. (2017). Deep machine learning provides state-of-the-art performance in image-based plant phenotyping. GigaScience 6.
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15.30pm February 25th WG1 CHARACTERISATION OF A NOVEL FAMILY OF PLANT NUCLEAR ENVELOPE ASSOCIATED PROTEINS (NEAP) IN ARABIDOPSIS THALIANA
Gwénaëlle Détourné1,2, Emmanuel Vanrobays1, Katja Graumann2, Aline V. Probst1, Christophe Tatout1 and David Evans2
1-CNRS UMR6293 INSERM U1103, Génétique Reproduction et Développement (GReD), Université Clermont Auvergne, Clermont-Ferrand, France
2- Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Oxford, United-Kingdom
The nucleoskeleton underneath the nuclear envelope is needed to transmit signals to the nucleus and induce changes in chromatin organisation and ultimately gene expression. A novel family of Nuclear Envelope Associated Proteins (NEAPs) proposed to be new components of the plant nucleoskeleton has been recently evidenced in the model plant Arabidopsis thaliana. NEAP proteins are encoded by a small gene family composed of three genes (the fourth being a pseudogene) and are targeted through a nuclear localisation signal to the nucleus where they are anchored at the inner nuclear membrane through their C-terminal transmembrane domain.
NEAPs also possess several long coiled-coil domains reminiscent to the lamin structure in animals. The present work aims at performing a functional analysis of NEAPs using T-DNA insertion and CRISPr-Cas9 mutant lines. The NEAP interactome is investigated by molecular approaches (Yeast Two Hybrid, IP followed by mass spectrometry) as well as in vivo localisation and co-localisation coupled to image analyses (apFRET, acceptor photobleaching Fluorescence Resonance Energy Transfer). Results so far are suggesting that NEAP proteins can interact with each other to form homo or hetero-dimers and may interact with other components of the nucleoskeleton anchoring them at the inner nuclear membrane to maintain nuclear morphology and chromatin organisation.
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15.50pm February 25th WG1
CHARACTERIZATION OF CHROMATIN MOBILITY DURING DNA DAMAGE RESPONSES IN ARABIDOPSIS THALIANA
Anis Meschichi and Stefanie Rosa
Swedish University of Agricultural Sciences - Almas allé 8, Uppsala, Sweden
Plant cells are subject to DNA damage such as double-strand breaks (DSBs), which may arise from environmental stresses, cell metabolism or DNA replication. DSBs are a deleterious type of DNA damage, which can be repaired by two different pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Double-strand break repair by HR requires a homology search. Large-scale movement of DNA DSBs has been reported in yeast and stated to facilitate the homology search (1,2).
Yet, the precise function of these movements remains relatively obscure. Here, we investigated the mobility of a tagged locus upon genotoxic stress using the lacO/ LacI system in Arabidopsis roots. We have established a protocol to measure the mean square displacement of LacI-GFP spots in epidermal cells from the meristematic and differentiation zones. Our results have shown that differentiated cells have overall a lower global mobility compared to meristematic cells. Those results are in line with our previous studies that showed histones become less mobile and more strongly bound to chromatin with increasing differentiation states (3).
By using a DSB inducer (Zeocin) we have been able to measure the DSB site mobility by tracking RAD51-GFP spots in the nucleus. Our results have shown an increase in mobility at DSB sites as well as at the level of global chromatin upon genotoxic stress. Interestingly we also observed an increase of nucleus size, which may be suggestive of an overall chromatin decompaction upon DSBs. Altogether, our study suggests that chromatin mobility is a non- negligible factor that may contribute DSB repair by homologous recombination in order to keep genome integrity.
(1) Krawczyk, P. M., Borovski, T., Stap, J., Cijsouw, T., Cate, R. t., Medema, J. P Aten, J. A. (2012). Chromatin mobility is increased at sites of DNA double-strand breaks. Journal of Cell Science, 125(9), 2127–2133. https://doi.org/10.1242/jcs.089847 (2) Seeber, A., Dion, V., & Gasser, S. M. (2013). Checkpoint kinases and the INO80 nucleosome remodeling complex enhance global chromatin mobility in response to DNA damage. Genes and Development, 27(18), 1999–2008. https://doi.org/10.1101/gad.222992.113 (3) Rosa, S., Ntoukakis, V., Ohmido, N., Pendle, A., Abranches, R., & Shaw, P. (2014). Cell Differentiation and Development in Arabidopsis Are Associated with Changes in Histone Dynamics at the Single-Cell Level. The Plant Cell, 26(December), 1–14. https://doi.org/10.1105/tpc.114.133793
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16.10pm February 25th WG1
ZEBULARINE-INDUCED DNA DEMETHYLATION IMPAIRS AUXIN SIGNALLING IN ARABIDOPSIS ROOT
Valya Vassileva and Mariana Georgieva
Department of Molecular Biology and Genetics, Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
The eukaryotic nucleus is functionally divided into a loosely packed and actively transcribed euchromatin, and a condensed and transcriptionally silenced heterochromatin. DNA methylation is classically associated with chromatin compaction and the formation of heterochromatin. Global reduction of DNA methylation levels due to mutations in the DNA methyltransferase coding genes or mediated by DNA demethylating agents leads to transcriptional reactivation and a partial heterochromatin decondensation.
Using the model plant Arabidopsis thaliana, we investigated the effect of the DNA methyltransferase inhibitor zebularine on the interplay between chromatin condensation and auxin perception in the cells located in root apical meristem and lateral root primordia.
The methylation inhibitor zebularine has the advantages of high stability and low toxicity (Baubec et al. 2009). Employing DR5-GFP reporter with a nuclear localisation signal under a synthetic auxin-responsive promoter, and the R2D2 reporter line developed to monitor in vivo auxin levels (Liao et al. 2015), we followed zebularine effects via time-lapse confocal microscopy.
Our results demonstrate that zebularine treatment led to misregulation of auxin responses, which is likely due to chromatin accessibility changes.
Baubec T, Pecinka A, Rozhon W, Mittelsten Scheid O. 2009. Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine. Plant Journal 57, 3, 542-554
Liao CY, Smet W, Brunoud G, Yoshida S, Vernoux T, Weijers D. 2015. Reporters for sensitive and quantitative measurement of auxin response. Nature Methods 12, 3, 207
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16.45pm: Pecha Kucha Speaker List 16.45pm: Pecha Kucha Speaker List
Bisa Andov (Oxford Brookes University, UK) Adéla Machelová (CEITEC, Masaryk University, Czech Republic) WG2.1- INVESTIGATING PROTEIN-PROTEIN INTERACTIONS BETWEEN WG2.9- GENOMIC STABILITY IN CHROMATIN REMODELLING MUTANTS ARABIDOPSIS MID-SUN PROTEINS AND THE TRANSCRIPTION FACTOR PKR1, PKR2, CHR5, CHR9 AND CHR24: Page 76 maMYB AT THE PLANT NUCLEAR ENVELOPE: Abstract page 68 Rainer Melzer (University College Dublin, Ireland) Francesco Blasio (Universidad Complutense de Madrid; Spain) WG3.7- GENOME-WIDE ANALYSIS OF MIKC-TYPE MADS-BOX GENES IN WG3.2- MEIOTIC CONSEQUENCES OF CHROMOSOME DOUBLING ON WHEAT: Page 91 MUTANTS DEFECTIVE IN NUCLEAR ENVELOPE-ASSOCIATED PROTEINS: Page 86 Miguel Montez (University of Warsaw, Poland) WG2.10- USE OF SINGLE MOLECULE FLUORESCENCE IN SITU Nadia Fernández (Universidad Complutense de Madrid, Spain) HYBRIDIZATION TO VISUALIZE SENSE AND ANTISENSE RNA TRANSCRIPTS WG1.4- A FIRST GLIMPSE OF NUCLEAR PORE COMPLEX DYNAMICS AT THE DOG1 LOCUS IN SINGLE CELLS OF ISOLATED EMBRYOS: Page 77 DURING ARABIDOPSIS MEIOSIS: Page 63 Pavla Navratilova (Centre of Plant Structural and Functional Genomics, Czech Cristian Forestan (University of Padova, Italy) Republic) WG3.4- DROUGHT STRESS AND RECOVERY PERIOD INDICATE CRITICAL WG3.9- APPROACHING DWARFING PHENOTYPE IN WHEAT BY ATAC-SEQ: EPIGENETIC CONTROL POINTS IN STRESS ADAPTATION AND FLOWERING Page 93 REGULATION, PROVIDING INSIGHTS INTO EPIGENETIC MEMORY IN ZEA MAYS: Page 88 Beáta Petrovská (Institute of Experimental Botany, Olomouc, Czech Republic) WG4.2- SETTING UP THE STAGE FOR FUNCTIONAL ANALYSIS OF NUCLEAR Chunlian Jin (Ghent University, Belgium) PROTEINS IN BARLEY: Page 100 WG2.4- MSIX, AN ESSENTIAL PROTEIN FOR MULTIPLE REPRODUCTIVE PROCESS IN ARABIDOPSIS: Page 71 Lauriane Simon (Swedish University of Agricultural Sciences, Uppsala, Sweden) WG2.14- SUVH7: A NEW ACTOR IN THE REGULATION OF ENDOSPERM Katerina Kabelácová (Institute of Plant Molecular Biology, Ceske Budejovice, DEVELOPMENT: Page 81 Czech Republic) WG2.5- POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) MODULATES Fen Yang (Institute of Experimental Botany, Olomouc, Czech Republic) PHOTOAUTOTROPHIC GROWTH OF PHYSCOMITRELLA PATENS: Page 72 WG2.16: ANALYSIS OF SMC5/6 COMPLEX MUTANT DEFECTS DURING REPRODUCTIVE DEVELOPMENT IN ARABIDOPSIS): Page 83 Kalyanikrishna (Freie Universität Berlin, Germany) WG2.6- ELUCIDATING THE ROLE OF EPIGENETIC REGULATORS AT THE NUCLEAR PERIPHERY IN ARABIDOPSIS: Page 73
Konstantin Kutashev (CEITEC, Masaryk University, Czech Republic ) WG1.5- ANALYSIS OF HISTONE H3 VARIANTS DISTRIBUTION IN THE REPETITIVE REGIONS OF A.THALIANA GENOME: Page 64
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09.10pm February 26th Invited speaker WG2
CRISPR/CAS9-MEDIATED LABELLING OF GENOMIC SEQUENCES IN LIVING AND FIXED CELLS
Solmaz Khosravi1, Takayoshi Ishii1,2 Steven Dreissig1, Holger Puchta3, and Andreas Houben3
1- Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, 06466 Stadt Seeland, Germany 2- Arid Land Research Center (ALRC) Tottori University, Hamasaka 1390, 680- 0001, Tottori-shi, Japan 3- Botanical Institute, Karlsruhe Institute of Technology, POB 6980, 76049 Karlsruhe, Germany
Visualizing the spatio-temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed tools to visualize defined genomic sequences in living and fixed cells based on a bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana (Dreissig et al., 2017).
By tracking the dynamics of telomeres visualized by CRISPR-dCas9, we reveal long range telomere movements of up to 2 µm within 30 minutes during interphase. We show that CRISPR-dCas9 can be combined with fluorescence-labelled proteins to visualize DNA-protein interactions in vivo. In addition, we developed a tool to visualize defined genomic sequences in fixed plant and animal nuclei and chromosomes based on a two-part guide RNA recombinant Cas9 endonuclease complex.
In contrast to classical FISH, CRISPR-based FISH does not require DNA denaturation and permits good structural preservation. We provide real-time visualization of the CRISPR/Cas9-mediated DNA labelling process. CRISPR/Cas9-mediated DNA labelling methods will revolutionize the field chromosome biology.
Dreissig, S., Schiml, S., Schindele, P., Weiss, O., Rutten, T., Schubert, V., Gladilin, E., Mette, M.F., Puchta, H., and Houben, A. (2017). Live-cell CRISPR imaging in plants reveals dynamic telomere movements. Plant J 91, 565-573.
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09.45pm February 26th WG2
IMPACT OF HISTONE CHAPERONE GENES AND DNA REPAIR PATHWAYS ON rDNA STABILITY
Martina Nešpor Dadejová1, Karolína Kolárová1,2, Zuzana Fusková1 and Martina Dvorácková1
1- Central European Institute of Technology (CEITEC) and Faculty of Sciences of Masaryk University, Brno, Czech Republic 2- Institute of Biophysics ASCR, v.v.i., Královopolská 135, 61265, Brno, Czech Republic
DNA loci encoding 45S rDNA ribosomal genes represent the major class of DNA repeats in Arabidopsis thaliana genome, aprox. 5% of its total size. Since rRNA form essential cellular machineries – ribosomes - responsible for protein production, the integrity of rDNA is required for plant overall plant´s fitness. An important role in the genome stability maintenance is dedicated to the balanced incorporation of individual histones into DNA and their precise epigenetic modification.
This is mediated by evolutionarily conserved proteins called histone chaperones, but large number of chromatin factors modulate this process. Their mutual interactions as well as histone binding specificity have not been fully understood yet. We focus here on the H3/H4 histone chaperones - Chromatin Assembly Factor 1 (CAF-1), H2A/H2B histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1), and NAP1-RELATED PROTEIN 1 and 2 (NRP1, NRP2). Morphological changes, decreased genome stability, progressive loss of repetitive sequences and sensitivity to DNA damaging agents were observed in plants defective for CAF1 complex (Mozgova et al., 2010).
We show here that both H3/H4 and H2A/H2B histone chaperone groups affect the stability of rDNA repeats and plant ´s viability, revealing an interesting compensation effect. We further present how individual histone chaperones interact with each other and with DNA repair genes and speculate how cells cope with the histone chaperone deficiencies. We conclude that the phenotypic features caused by fas1 mutation can be balanced by varied mechanisms, involving the modulation of the chromatin structure as well as selection of alternative DNA repair pathways.
This work was supported by The Czech Science Foundation (grant 19-11880Y), COST INDEPTH and INTERCOST (LTC18048).
Mozgova, I., Mokros, P., and Fajkus, J. (2010). Dysfunction of chromatin assembly factor 1 induces shortening of telomeres and loss of 45S rDNA in Arabidopsis thaliana. The Plant cell 22, 2768-2780.
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10.05pm February 26th WG1
CRISPR-CAS9 MUTAGENESIS UNRAVELS THE ROLE OF 45S rDNA GENES IN A. THALIANA DEVELOPMENT
Francesca Lopez1, Antoine Fort1,2, James Friel1, Peter Ryder1, Marcus McHale1, Hirofumi Ishihara3, Ronan Sulpice2 and Charles Spillane1
1- Genetics and Biotechnology Laboratory, Plant and AgriBiosciences Research Centre (PABC), School of Natural Sciences, National University of Ireland Galway, Ireland. 2- Systems Biology Laboratory, Plant and AgriBiosciences Research Centre (PABC), School of Natural Sciences, National University of Ireland Galway, Ireland. 3- Max Planck Institute of Molecular biology, Potsdam, Germany
Although 45s rDNA genes are ubiquitous to all eukaryotes, the number of repeats and their chromosomal locations are highly polymorphic across different phyla. The role of transcriptionally active and inactive 45s copies has been amply characterized in cellular biology by numerous studies linking loss of 45s copies to abnormal proliferation and cancer (Xu et al., 2017, PLOS Genet. 13, e1006771), and also to genome integrity and senescence (Obayashi and Kobayashi, 2014, Proc. Jpn. Acad. Ser. B. Phys. Biol. Sci. 90, 119–129).
In plants however, it hasn’t yet been possible to formulate a direct link between variation in 45s copy number and their function in development. Previous studies employed the Chromatin Assembly Factor-1 (CAF-1) mutants which are knock-out alleles of histone chaperones FASCIATA 1/2 , which lead to a drastic reduction in rDNA copy number (Kaya et al., 2001, Cell 104, 131–142; Mozgova et al., 2010, Plant Cell 22, 2768–2780), however it has not yet been possible to draw a causative association between the phenotypes observed and the reduction of copy number. Here, we show how CRISPR-Cas9 mutagenesis has been employed to generate A. thaliana plants with a stably inherited reduction of up to 80% of 45s rDNA genes.
We report that decrease of 45s copies is linked with aberrant seedling development, which is also associated with deregulated mechanisms of cell proliferation and meristematic activity. Since these mechanisms are established during early embryonic development, we analyzed embryogenesis in our 45s rDNA Low Copy Number lines and found severe aberrations of correct body plan establishment. Further work will be focused on investigating the mechanisms that link 45s rDNA copy number and the first cell division of embryo development.
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10.25pm February 26th WG2
HISTONE H3.3 DEPOSITION PATHWAYS IN ARABIDOPSIS THALIANA
Duc C1, Benoit M1, Le Goff S1, Desset S1, Benhamed M2, Tatout C1 and Aline Probst1
1- Université Clermont Auvergne, CNRS, Inserm, GReD, F-63000 Clermont- Ferrand, France 2 Institute of Plant Sciences Paris Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Evry, Université Paris-Saclay, Orsay, France
Histones are essential components of the nucleosome, the basic subunit of chromatin that structures linear DNA molecules and regulates access of other proteins to DNA. DNA accessibility, and in turn gene expression and higher-order chromatin organization, can be modulated through specific post-translational modifications or by incorporation of different histone variants into the nucleosome. The correct and well- timed deposition of histone variants is controlled by a network of histone chaperone complexes. While histone chaperones are highly conserved through evolution, histone variants such as H3.1 and H3.3 that are deposited in DNA synthesis-dependent or -independent manner, respectively, have emerged several times in different lineages.
We have characterized the Arabidopsis histone chaperone complexes Histone Regulator A (HIRA) and Alpha Thalassemia-mental Retardation X-linked (ATRX) and show that loss of these complexes affects nucleosomal occupancy and genome function. Arabidopsis HIRA and ATRX mutant alleles are viable, but cause severe developmental defects when combined together, suggesting that HIRA and ATRX function in complementary histone H3.3 deposition pathways. Indeed, Arabidopsis ATRX binds H3.3 and ATRX loss-of-function impacts cellular histone H3.3 pools and in consequence modulates the H3.1/H3.3 balance in the cell.
At the genome-wide scale, our data indicate that ATRX modifies gene expression through H3.3 deposition at a set of genes characterized both by elevated H3.3 occupancy and high expression levels, altogether emphasizing the role of histone chaperones in regulating chromatin dynamics and fine-tuning genome expression.
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10.45pm February 26th WG2
NOVEL ROLES OF PRC2 IN PHOTOMORPHOGENESIS AND SEEDLING ESTABLISHMENT
Iva Mozgová1-3, Helena Hönig Mondeková1-3, Tomáš Konecný1-3, Lenka Bucinská1,2, Roman Sobotka1,2, Jirí Kubásek2, Jirí Šantrucek2, Martin Tichý1, Katerina Kabelácová1-3, Eliška Kuthanová Trsková1,2, Aurelie Crepin1
1- Centre Algatech – Institute of Microbiology CAS, Trebon, Czech Republic 2- University of South Bohemia in Ceské Budejovice, CZ 3- Present address: Institute of Plant Molecular Biology, Biology Centre CAS, Ceské Budejovice, CZ Embryo-to-seedling transition is a crucial developmental and metabolic phase transition in plants. In Arabidopsis, the transition is governed by the Polycomb Repressive Complexes (PRCs) that have a well-established role in ensuring stable transcriptional repression of key embryo maturation genes (Aichinger et al., 2009; Bouyer et al., 2011; Bratzel et al., 2010; Chanvivattana et al., 2004; Chen et al., 2010).
We find that the known ectopic reversal of the embryonic program in PRC2 mutants only occurs under mixotrophic or heterotrophic growth conditions. Under standard photoautotrophic growth conditions, PRC2 is in fact not needed for stable repression of embryonic genes, completion of photomorphogenesis and establishment of vegetative growth. Overcoming the requirement for PRC2 for the embryo-to-seedling transition, we study the function of PRC2 in the establishment and maintenance of photoautotrophic growth in plants, which has so far been hampered by the developmental arrest of plants fully depleted of PRC2 activity. Combining techniques of developmental epigenetics with plant physiology and photosynthesis, we show that PRC2 moderates responses to ambient light conditions. Although the most severe (likely combined) defects associated with PRC2 dysfunction in vegetative tissue are seen in the PRC2 catalytic null mutants (clf swn), we observe particular defects with different expressivity in the single mutants. We aim to differentiate between biogenic (developmental) and operational processes governed by PRC2 in vegetative tissue. I will present our progress in identifying upstream molecular pathways that are modulated by PRC2 with the goal to establish its function in vegetative tissue.
Aichinger, E et al (2009). CHD3 proteins and polycomb group proteins antagonistically determine cell identity in Arabidopsis. PLoS Genet 5, e1000605. Bouyer, D et al (2011). Polycomb repressive complex 2 controls the embryo-to-seedling phase transition. PLoS Genet 7, e1002014. Bratzel, F et al (2010). Keeping cell identity in arabidopsis requires PRC1 RING-finger homologs that catalyze H2A monoubiquitination. Curr. Biol. 20, 1853–1859. Chanvivattana, Y et al (2004). Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131, 5263–5276. Chen, D et al (2010). The Arabidopsis PRC1-like ring-finger proteins are necessary for repression of embryonic traits during vegetative growth. Cell Res. 20, 1332–1344.
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14.10pm February 26th Invited speaker WG3
TO BE OR NOT TO BE (FOR PLANTS UNDER ADVERSE ENVIRONMENTAL CONDITIONS). THE ANSWER IS IN THE SPLICEOSOME
Julio Salinas
Department of Microbial and Plant Biotechnology; Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
The control of pre-mRNA splicing is emerging as an important layer of regulation in plant response to abiotic stress. In eukaryotes, pre-mRNA splicing is carried out by the spliceosome, a highly evolutionary conserved ribonucleoprotein (RNP) machinery composed of five small nuclear ribonucleoproteins (snRNPs) that conform its core, and hundreds of non-snRNPs. Each snRNP consists of a snRNA (U1, U2, U4, U5 or U6) and an associated heptameric protein complex. The U1, U2, U4 and U5 snRNAs associate with the Sm complex, while the U6 snRNA interacts with the related Sm-like (LSM) 2-8 complex. Several non-snRNPs have been involved in controlling abiotic stress responses in plants. Little is known, however, about the functions of the spliceosome core components in these responses.
We showed that the LSM2-8 complex differentially regulates plant responses to abiotic stresses. Recently, we have demonstrated that the Arabidopsis Sm complex also regulates differentially plant responses to abiotic stresses, although in a distinct way than LSM2-8. Interestingly, high coverage transcriptomic analyses revealed that, depending on the stress conditions, each complex performs its function by ensuring the correct splicing of a reduced number of specific pre-mRNAs corresponding to stress-related genes which, in turn, determines the appropriate patterns of gene expression required for plant adaptation.
All these findings indicate that different spliceosome core components provide different specificities to the activity of this cellular machinery, that these specificities are determined by the environmental conditions, and that they are essential for correct adaptation of plants to their surroundings.
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14.45pm February 26th WG3
THE RSS1-PP1 PATHWAY AND ITS ROLE IN PLANT TOLERANCE TO ABIOTIC STRESSES
Mariem Bradaï1,2, Habib Mahjoubi1,3, Yukata Tamari4, Vitor Amor- im-Silva5, Shin Takeda4, MA Botella5, Marie-Edith Chabouté3, Chantal Ebel1,2 and Moez Hanin1,2
1- Plant Physiology and Functional Genomics Research Unit, Institute of Bio- technology, University of Sfax, BP 1175, 3038 Sfax, Tunisia 2- Laboratoire de Biotechnologie et d’Amélioration des Plantes, Centre de Bio- technologie de Sfax, BP 1177, 3018 Sfax, Tunisia 3- Institut de biologie moléculaire des plantes, UPR 2357 du CNRS, Université de Strasbourg, 12, rue du Général Zimmer, 67084 Strasbourg Cedex, France 4. Bioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya, Japan 5. University of Malaga. Dep. Biología Molecular y Bioquímica Facultad de Ciencias Campus de Teatinos s/n 29071 Málaga, Spain
To cope with environmental constraints, plants evolved complex cellular and molecular mechanisms controling cell division and differentiation in meristematic tissues. RSS1 (Rice Salt Sensitive 1) and Type one protein phosphatase (PP1) are major regulators in the signaling pathway linking salt stress perception to meristem maintenance where RSS1 acts probably by inhibiting PP1 activity during G1/S transition. Our work shows that the wheat RSS1 counterpart TdRL1 (Triticum durum RSS-Like 1) is the functional homolog of RSS1 since it was able to complement the salt hypersensitivity of rss1 mutant. Our cytological studies show that TdRL1 is cytoplasmic in interphase and associated to the spindle during mitosis. Remarkably, TdRL1 changes its subcellular localization under salt stress with a partial nuclear accumulation, highlighting its multifunctional nature during salt stress response. On another hand, co-immunuprecipitation experiments show that TdRL1 is able to interact with a wheat PP1 (TdPP1a) and to dampen its phosphatase activity. In BiFc assays TdPP1a and TdRL1 co-localize within cortical microtubule network. Arabidopsis lines over- expressing TdPP1a::GFP showed enhanced abiotic stress tolerance and increased root growth under Brassinosteroid (BR) treatments. Interestingly, TdPP1a seems to interfere with BR signalling via its interaction with BES1 (a transcription factor that regulates the expression of BR-responsive genes) and increasing its dephosphorylated (active) level. The questions regarding the contribution of the RSS1- PP1 pathway in the maintenance of meristems under stress conditions and its connection with the BR signaling in enhancing plant stress tolerance are currently investigated.
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INDEPTH Prague Meeting INDEPTH Prague Meeting
15.05pm February 26th WG3
CUSCUTA CAMPESTRIS: A PLANT GENOME UNDER THE INFLUENCE OF A PARASITIC LIFESTYLE
Kirsten Krause
Department of Arctic and Marine Biology, UiT The Arctic University of Norway, Dramsvegen 201, N-9037 Tromsø, Norway
The genus Cuscuta is a large parasitic plant genus within the Morning Glory family (Convolvulaceae, order Solanales) that contains exclusively epiphytic parasites. The larcenous lifestyle is connected to a number of conspicuous morphological reductions such as a lack of roots and the reduction of leaves and cotyledons to microscopic scales. Genomes of Cuscuta species reportedly range from around 500 megabase pairs (Mbp) to more than 40 gigabase pairs.
A globally very abundant noxious weed, Cuscuta campestris, was recently sequenced - the first parasitic plant genome for which a sequence became available. The 581 Mbp-large genome carries signs of a whole genome duplication event that might have triggered an amplification wave of LTR retrotransposons some 1.5 to 2 million years ago. The reduced photosynthetic capacity in C. campestris is reflected by the loss of genes coding for components of the photosynthetic machinery in both, nuclear and chloroplast, genomes.
Other aspects of the parasite’s metabolism, too, are affected by gene losses, while, on the other hand, regions with extensive sequence homologies (including introns) to phylogenetically distant families suggest horizontal uptake of DNA from Cuscuta’s hosts. Losses, gains and potential neo-functionalization of genes will be presented in view of the altered regulatory and functional requirements of the parasite.
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INDEPTH Prague Meeting INDEPTH Prague Meeting
15.25pm February 26th WG3
CLONING OF THE WHEAT YR15 RESISTANCE GENE SHEDS LIGHT ON THE PLANT TANDEM KINASE-PSEUDOKINASE FAMILY
Tzion Fahima
Institute of Evolution, University of Haifa, Israel.
Yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease threatening much of global wheat production. Race- specific resistance (R)-genes are used to control rust diseases, but the apidr emergence of virulent Pst races has prompted the search for a more durable resistance. Wild emmer (WEW). Triticum turgidum ssp. dicoccoides, the tetraploid progenitor of common wheat distributed along the Fertile Crescent, has valuable residual adaptive diversity in response to various diseases, including stripe rust. Yr15 is a dominant WEW gene, located on chromosome 1BS, that confers broad-spectrum resistance to stripe rust.
Comparative genomics, chromosome walking, BAC libraries (WEW and bread wheat), whole genome assemblies, EMS mutagenesis and transgenic approaches enabled us to clone Yr15 and validate its function. Macro- and microscopic observations of fungal development and accumulation of biomass suggest that hypersensitive response plays a central role in the resistance mechanism conferred by Yr15. Analysis of Yr15 protein structure revealed a novel gene structure for R-genes in wheat with a tandem kinase-pseudokinase protein domains, that are both essential for conferring resistance to stripe rust, designated here as wheat tandem kinase 1 (WTK1). The existence of a similar gene architecture in 92 putative proteins across the plant kingdom, including the barley RPG1 and a candidate for Un8, suggests that they are members of a distinct family of plant proteins, termed here tandem kinase-pseudokinases (TKPs). The absence of the functional Yr15 allele in tested wheat varieties highlights the value of WEW germplasm as a reservoir of resistance genes for wheat.
Klymiuk V., Yaniv E., Huang L., Raats D., Fatiukha A., Chen S., Feng L., Frenkel Z., Krugman T., Lidzbarsky G., Chang W., Jääskeläinen M.J., Schudoma C., Paulin L., Laine P., Bariana H., Sela H., Sørensen C.K., Hovmøller M.S., Distelfeld A., Chalhoub B., Dubcovsky J., Korol A.B., Schulman A.H., Fahima T. (2018). Cloning of the wheat Yr15 resistance gene sheds light on plant tandem kinase family. Nature Communications 9:3735
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15.45pm February 26th WG3 PLASTICITY OF EPIGENETICS AND CHROMATIN STRUCTURE: CONCERTED STRATEGIES FOR ENHANCED PLANT TOLERANCE UNDER STRESS
Ana Paula Santos, Ferreira LF and Oliveira MM
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Genomics of Plant Stress. Av. da República, 2780-157 Oeiras, Portugal and IBET, Apartado 12, 2781-901 Oeiras, Portugal
The spatial organization of chromosome structure within the interphase nucleus, as well as the patterns of methylome and histone modifications act concertedly being flexible enough to influence gene expression and plant phenotypes. Environmental stresses are often associated with extensive chromatin rearrangements and modifications of epigenetic levels and patterns. Stress -tolerant plants can be a good tool to unveil potential connections between specific epigenetic modifications and stress tolerance capacity. We showed that abiotic stress conditions including cold, heat and mild salinity as well as 5-AC induced DNA hypomethylation can cause interphase chromatin structural changes of rice rDNA Loci. Variations occurring on global DNA methylation levels in response to salt stress were tissue - and genotype-dependent.
The salt-tolerant rice variety Pokkali was remarkable in its ability to quickly relax DNA methylation in response to salt stress. The genome wide DNA methylation analysis of this variety under salinity revealed a set of Differentially Methylated Regions (DMRs) between control and stress samples whose DNA methylation pattern showed a general tendency for demethylation events in stress samples as compared to control. We hypothesize that short regions as DMRs can shape the chromatin landscape of specific genomic regions and, therefore, may modulate the function of several genes.
More knowledge on the mechanism underlying epigenetic regulation in response to environmental factors will certainly contribute to connect chromatin and epigenetic regulation of stress responsive genes with potential application on enhancement of plant stress tolerance.
Acknowledgments: The work was supported by the FCT (Portuguese Foundation for Science and Technology)throughtheproject“Epigenetic regulation of the rice genome under environmental stresses” ‘[BIA-BCM/111645/2009]’, and through the R&D unit, UID/Multi/04551/2013 (GREEN-IT).
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16.05pm February 26th WG3
LINKER HISTONE H1 DYNAMICS CONTROL ARABIDOPSIS NUCLEAR REORGANIZATION IN RESPONSE TO LIGHT SIGNALS.
Gianluca Teano1, I. Mestiri1, C. Bourbousse1, C. Bowler1, C. Baroux2 and F. Barneche1
1- Plant and Diatom Genomics, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), UMR CNRS 8197, Paris, France 2- Plant Developmental Genetics, Department of Plant and Microbial Biology, University of Zürich, Switzerland
Being capable of rapid phenotypic adaptation in response to environmental cues, plants are characterized by a remarkable developmental plasticity, a feature presumably at the nexus of their sessile life-form. Specially, plants have the remarkable ability to sense and use light conditions as a source of information to adapt their morphology and physiology accordingly to a changing environment. For example, the first perception of light by young plantlets emerging from the soil induces radical changes in gene expression launching growth and photosynthetic activity. During this transition, genome expression reprogramming is accompanied by massive rearrangements of chromatin organization.
In darkness heterochromatin containing silent and condensed repeated elements are scattered within multiple foci in the nucleoplasm. During de- etiolation, rapid condensation of heterochromatic domains allows the neo-formation of 8-to- 10 chromocenters in which most heterochromatic elements are condensed. This phenomenology has led us to the identification of histone H1.3 as a key molecular player in triggering light- induced chromatin dynamics. Linker histones are conserved structural components of eukaryotic chromatin that contribute to higher-order chromatin organization by restricting DNA accessibility.
Here we show that heterochromatin rearrangements requires downregulation of linker histone variant H1.3. We also observed that light with low red:far-red content mimicking plant neighbor perception induces an antagonistic process of H1.3 accumulation driving chromocenters disaggregation in adult cotyledons. Expression and cytogenetic data indicate that dynamic controls of histone H1.3 variant drive these two mirroring processes, possibly under the control of common light signaling components.
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09.10pm February 27th Invited Speaker WG4
HI-C IN CEREALS: GENOME ASSEMBLY, STRUCTURAL VARIATION AND CHROMATIN ORGANIZATION
Martin Mascher1,2
1- Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstraße 3, 06466 Seeland, Germany 2- German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany
Chromosome conformation capture sequencing (Hi-C) is a method for the comprehensive interrogation of the three-dimensional (3D) organization of chromatin in the nucleus. In addition to studying genome organization, Hi-C has also become a powerful approach for genome assembly and studying structural variation at the sequence level.
In this presentation, I will report on the various uses of Hi-C in the genomics of cereal crops. We used Hi-C data to obtain chromosome-scale reference sequence assemblies of the Triticeae species wheat, barley, rye and their wild relatives. The positional information afforded by Hi-C made it possible for the first to derive a linear order of sequence scaffolds in the vast non-recombining pericentromeric regions of Triticeae chromosomes.
By aligning Hi-C data from diverse genotypes to the reference genomes of wheat and barley, we detected many large-scale variants such inversions or inter-chromosomal translocations. Finally, we used Hi-C data to compare global chromatin organization between interphase and metaphase in barley, revealing pronounced differences in contact probabilities between both stages of the cell cycle.
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09.45pm February 27th WG4
CHROMATIN LOOPING ARCHITECTURE OF THE HUMAN GENOME
Dariusz Plewczynski1,5, Michał Sadowski1, Agnieszka Kraft1,5, Przemysław Szałaj1,3,4, Michał Własnowolski1,5, Michał Kadlof1, Zhonghui Tang2 and Yijun Ruan2
1- Centre of New Technologies, Warsaw University, Warsaw, Poland 2- The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA 3- Centre for Innovative Research, Medical University of Bialystok, Białystok, Poland 4- Hasselt University, Belgium 5- Faculty of Mathematics and Information Science, Warsaw University of Technology, Warsaw, Poland
ChIA-PET is a high throughput mapping technology that reveals long-range chromatin interactions and provides insights into the basic principles of spatial genome organization and gene regulation mediated by specific protein factors. Recently, we showed that a single ChIA-PET experiment provides information at all genomic scales of interest, from the high resolution locations of binding sites and enriched chromatin interactions mediated by specific protein factors, to the low resolution of non- enriched interactions that reflect topological neighborhoods of higher-order chromosome folding.
This multilevel nature of ChIA-PET data offers an opportunity to use multiscale 3D models to study structural- functional relationships at multiple length scales, but doing so requires a structural modeling platform. Here we report the development of 3D-GNOME (3-Dimensional GeNOme Modeling Engine), a complete computational pipeline for 3D simulation using ChIA-PET data. 3D-GNOME consists of three integrated components: a graph-distance-based heatmap normalization tool, a 3D modeling platform, and an interactive 3D visualization tool.
Using ChIA-PET and Hi-C data derived from human B-lymphocytes, we demonstrate the effectiveness of 3D-GNOME in building 3D genome models at multiple levels, including the entire genome, individual chromosomes, and specific segments at megabase (Mb) and kilobase (kb) resolutions of single average and ensemble structures. Further incorporation of CTCF-motif orientation and high-resolution looping patterns in 3D simulation provided additional reliability of potential biologically plausible topological structures. Our work provides the high-resolution and multi-scale computational model of chromatin looping architecture in human genome.
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10.05pm February 27th WG4
POLYMER SIMULATIONS TO UNDERSTAND THE STRUCTURE AND DYNAMICS OF MITOTIC BARLEY CHROMOSOMES
Amanda Souza Câmara1, Petr Cápal2, Tomáš Beseda2, Jan Vrána2, Axel Himmelbach1, Nils Stein1, Andreas Houben1, Veit Schubert1, Jaroslav Doležel2, Hana Šimková2 and Martin Mascher1,3
1- Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland Germany 2- Centre of Plant Structural and Functional Genomics, Institute of Experimental Botany, Olomouc, Czech Republic 3- German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany
From interphase to metaphase the chromosomes go through intense condensation. This process, which is very conserved within eukaryotic organisms, leads to a two- to three-fold packing, enabling an accurate segregation of the chromosomes. Recent analysis from HiC data have brought light into this impressive process and are helping to unravel the more packed structure of the mitotic chromosome. Complementarily, polymer simulations can produce several models to be tested and their fit to experimental data be verified.
Together, these two techniques were able to reconcile two apparently conflicting former views: condensation arising either from loop extrusion or from helical packing. Gibcus et al. have suggested that both models may be true. Mitotic chromosome may be formed by nested loops arranged side by side in a dynamical helical scaffold. This model is in good agreement with the contact probability calculated from Hi-C experiments with human cells. Our own Hi-C data from flow- sorted chromosomes suggest a similar structure for mitotic chromosomes of barley. Despite the differences between these two organisms regarding genome size and the presence of topologically associated domains during interphase, they both seem to share the same packing strategy, relying mainly on the different roles of condensins I and II. With polymer simulations, we can also infer structural aspects specific to barley, such as the loop lengths, the height of a helix turn, or the relative concentrations of condensins.
These are all features that may help us understand how the large barley genome is organized during the cell cycle. Furthermore, de novo modelling may indicate which forces are acting on the polymer and are driving chromosome condensation. This may shed a light into the regulation of the process and how the involved proteins work and assemble during chromosome condensation.
(1) Gibcus, Johan H., et al. (2018) “A pathway for mitotic chromosome formation.” Science 359.6376
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10.25pm February 27th WG4
WHEAT rRNA GENE LOCI RESOLVED BY CHROMOSOME SORTING, OPTICAL MAPPING AND RNA-SEQ
Tulpová, Z., Toegelova, H., Kapustová, V., Navrátilová, P., Hribová, E., Vrána, J., Doležel, J. and Hana Šimková
Institute of Experimental Botany, Centre of Plant Structural and Functional Genomics - Czech Republic
Ribosomal RNA gene loci pose an indispensable part of both prokaryotic and eukaryotic genomes. In eukaryotes, they are associated with nucleolus, the largest functional domain of the nucleus and the site of ribosomal biogenesis. They are mostly organized as long head-to- tail tandem arrays spanning several hundred kilobases to multiple megabases, which precludes their complete assembling from NGS data and impedes characterization of particular loci.
In our study, we identified and analysed 26S-5.8S-18S rRNA multigene loci in bread wheat genome by Bionano genome (BNG) mapping, a technology that visualizes short sequence motives along DNA molecules of 150 kb to 1 Mb. The rDNA arrays can be recognised in the maps as a regular label pattern with ~9-kb unit. BNG maps were produced from separated rDNA-bearing chromosome arms discriminated by flow cytometry. The BNG map data enabled precise positioning of the rDNA arrays in the sequence assembly and also quantification of rDNA units in particular chromosome arms.
Locus-specific rDNA unit sequences were reconstructed from chromosome-arm specific raw Illumina data (IWGSC, 2014) and partial rDNA sequences surrounding the identified loci. This information combined with transcriptomics (RNA-seq, Iso-Seq) and epigenetics data enabled a detailed analysis of expression pattern for particular major and minor rRNA loci.
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List of Posters List of Posters
WG1.1- ON LINE PERFORMANCE EVALUATION AND OPTIMIZATION OF WG2.1- INVESTIGATING PROTEIN-PROTEIN INTERACTIONS BETWEEN IMAGE SEGMENTATION METHODS USING GENETIC ALGORITHM ARABIDOPSIS MID-SUN PROTEINS AND THE TRANSCRIPTION AND GENERIC GROUND TRUTH IN 3D MICROSCOPY IMAGING FACTOR maMYB AT THE PLANT NUCLEAR ENVELOPE Zikrija Avdagic (University of Sarajevo, Bosnia and Herzegovina) Bisa Andov (Oxford Brookes University, UK)
WG1.2- ARABIDOPSIS LINKER HISTONES REGULATE FINE-SCALE WG2.2- CHARACTERIZATION OF CATALYTIC MUTATIONS IN ARABIDOPSIS CHROMATIN ARCHITECTURE AND MODULATE DEVELOPMENTAL THALIANA BRAHMA (BRM) CHROMATIN REMODELING ATPASE DECISIONS Rafal Archacki (University of Warsaw, Poland) Celia Baroux (University of Zürich, Switzerland) WG2.3- EPIGENETIC REGULATION TO DROUGHT STRESS IN OLIVE PLANT WG1.3- SINGLE-PLANT PHENOTYPING COMBINED WITH LONG-TERM (OLEA EUROPAEA L.) DIRECT SELECTION FOR EFFICIENT RESOURCE UTILIZATION IN Birsen Cevher-Keskin (TUBITAK Marmara Research Center, Turkey) COWPEA AND THE ASSOCIATED GENOMIC AND EPIGENOMIC CHANGES WG2.4- MSIX, AN ESSENTIAL PROTEIN FOR MULTIPLE REPRODUCTIVE Dionysia A. Fasoula (Agricultural Research Institute, Nicosia, Cyprus) PROCESS IN ARABIDOPSIS Chunlian Jin (Ghent University, Belgium) WG1.4- A FIRST GLIMPSE OF NUCLEAR PORE COMPLEX DYNAMICS DURING ARABIDOPSIS MEIOSIS WG2.5- POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) MODULATES Nadia Fernández (Universidad Complutense de Madrid, Spain) PHOTOAUTOTROPHIC GROWTH OF PHYSCOMITRELLA PATENS Katerina Kabelácová (Institute of Plant Molecular Biology, Ceske WG1.5- ANALYSIS OF HISTONE H3 VARIANTS DISTRIBUTION IN THE Budejovice, Czech Republic) REPETITIVE REGIONS OF A.THALIANA GENOME Konstantin Kutashev (CEITEC, Masaryk University, Czech Republic ) WG2.6- ELUCIDATING THE ROLE OF EPIGENETIC REGULATORS AT THE NUCLEAR PERIPHERY IN ARABIDOPSIS WG1.6- IS NUCLEOLUS-ASSOCIATED TELOMERE CLUSTERING UNIVERSAL Kalyanikrishna (Freie Universität Berlin, Germany) IN CRUCIFER SPECIES? Wenbo Shan (CEITEC, Masaryk University, Czech Republic) WG2.7- ROLE OF PRC2 IN EMBRYO AND SEEDLING DEVELOPMENT Tomáš Konecný (Institute of Microbiology CAS, Czech Republic) WG1.7- CHARACTERIZATION OF CATALYTIC MUTATIONS IN ARABIDOPSIS THALIANA BRAHMA (BRM) CHROMATIN REMODELING ATPASE. WG2.8- CRISPR-CAS9 MUTAGENESIS UNRAVELS THE ROLE OF 45S RDNA Paulina Stachula (University of Warsaw, Poland) GENES IN A. THALIANA DEVELOPMENT Francesca Lopez (National University of Ireland-Galway, Ireland) WG1.8- DEVELOPMENT AND BENCHMARKING OF SEGMENTATION METHODS TO ANALYZE THE PLANT 3D NUCLEUS WG2.9- GENOMIC STABILITY IN CHROMATIN REMODELLING MUTANTS Christophe Tatout (Université Clermont Auvergne, France) PKR1, PKR2, CHR5, CHR9 AND CHR24 Adéla Machelová ((CEITEC, Masaryk University, Czech Republic)
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WG2.10- USE OF SINGLE MOLECULE FLUORESCENCE IN SITU WG3.2- MEIOTIC CONSEQUENCES OF CHROMOSOME DOUBLING ON HYBRIDIZATION TO VISUALIZE SENSE AND ANTISENSE RNA MUTANTS DEFECTIVE IN NUCLEAR ENVELOPE-ASSOCIATED TRANSCRIPTS AT THE DOG1 LOCUS IN SINGLE CELLS OF ISOLATED PROTEINS EMBRYOS Francesco Blasio (Universidad Complutense de Madrid, Spain) Miguel Montez (University of Warsaw, Poland) WG3.3- EFFECT OF SUMMER DROUGHT AND ROOT ROT ON SUGAR BEET WG2.11-IMPACT OF HISTONE CHAPERONE GENES AND DNA REPAIR YIELD IN VARIETY REGISTRATION TRIALS PATHWAYS ON rDNA STABILITY Zivko Curcic (Institute of Field and Vegetable Crops, Serbia) Martina Nešpor Dadejová (Central European Institute of Technology (CEITEC), Masaryk UniversityCzech Republic) WG3.4- DROUGHT STRESS AND RECOVERY PERIOD INDICATE CRITICAL EPIGENETIC CONTROL POINTS IN STRESS ADAPTATION AND WG2.12-RIBOSOMAL RNA GENES DISTRIBUTION AND EXPRESSION FLOWERING REGULATION, PROVIDING INSIGHTS INTO ORGANIZE NUCLEOLUS ASSPCIATED CHROMATIN DOMAINS EPIGENETIC MEMORY IN ZEA MAYS Frédéric Pontvianne (Laboratoire Génome et Dévelopement des Cristian Forestan (University of Padova, Italy) Plantes, France) WG3.5- CHARACTERIZATION OF HvDME, A BARLEY EPIGENETIC WG2.13-CHROMATIN DYNAMICS UPON DNA DAMAGE IN REGULATOR, IN RESPONSE TO DROUGHT STRESS ARABIDOPSIS THALIANA Aliki Kapazoglou (Institute of Applied Biosciences, Greece) Svenja Reeck (Swedish University of Agricultural Sciences, Sweden) WG3.6-GRAIN PROTEIN CONTENT QTLS IDENTIFIED IN A DURUM × WILD WG2.14-SUVH7: NEW ACTOR IN THE REGULATION OF ENDOSPERM EMMER WHEAT MAPPING POPULATION TESTED IN FIVE DEVELOPMENT ENVIRONMENTS Lauriane Simon (Swedish University of Agricultural Sciences, Sweden) Tamar Krugman (University of Haifa, Israel)
WG2.15-A PROGRESS REPORT ON MOLECULAR CLONING OF WG3.7-GENOME-WIDE ANALYSIS OF MIKC-TYPE MADS-BOX GENES IN CENTROMERE-SPECIFIC HISTONE H3 IN SAINFOIN (ONOBRYCHIS WHEAT VICIIFOLIA) Rainer Melzer (University College Dublin, Ireland) Ahmet L. Tek (Nigde Ömer Halisdemir University, Nigde, Turkey) WG3.8-SUNFLOWER REACTION TO COMBINED BROOMRAPE – DOWNY WG2.16: ANALYSIS OF SMC5/6 COMPLEX MUTANT DEFECTS DURING MILDEW ATTACK – PRELIMINARY STUDY REPRODUCTIVE DEVELOPMENT IN ARABIDOPSIS Dragana Miladinovic (Institute of Field and Vegetable Crops, Serbia) Fen Yang (Institute of Experimental Botany, Olomouc, Czech Republic) WG3.9-APPROACHING DWARFING PHENOTYPE IN WHEAT BY ATAC-SEQ Pavla Navratilova (Centre of Plant Structural and Functional Genomics, WG3.1- UNCOVERING ZEA MAYS LONG NON-CODING RNA REGULATORY Czech Republic) FUNCTION IN RESPONSE TO LONG-TERM WATER STRESS Armine Asatryan (University of Padova, Italy) WG3.10-SMC5/6 COMPLEX – LINKING DNA DAMAGE REPAIR WITH CHROMATIN ORGANIZATION Ales Pecinka (Institute of Experimental Botany, Czech Republic)
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WG3.11-THE STRUCTURAL AND FUNCTIONAL ORGANIZATION OF PROPHASE I NUCLEI IN WHEAT-RYE HYBRIDS Elena Salina (IPK, Gatersleben, Germany)
WG3.12-MORPHOLOGICAL CHANGES AND EPIGENETIC ANALYSIS OF TWO OF TWO MEDICAGO SATIVA L. GREEK VARIETIES UNDER DROUGHT STRESS CONDITIONS Eleni Tani (Agricultural University of Athens, Greece)
WG3.13-MODIFICATION OF EPIGENETIC MARKS BY THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A PROMOTES WHEAT MICROSPORE EMBRYOGENESIS Maria Pilar Vallés-Brau (Aula Dei Experimental Station, Spain)
WG3.14- IS CHROMATIN INVOLVED IN THE SHORT- AND LONG-TERM MEMORY OF WATER STRESS IN MAIZE? Serena Varotto (University of Padova, Italy)
WG3.15- APPLICATION OF FULLERENOL NANOPARTICLES MITIGATES EFFECTS OF DROUGHT IN SUGAR BEET PLANTS Ksenija Taški-Ajdukovic (Institute of Field and Vegetable Crops, Serbia)
WG4.1-TRANSGENE SILENCING IN 3D – HOW A CHROMOSOMAL KNOT CAN INACTIVATE FOREIGN DNA ELEMENTS Stefan Grob (University of Zürich, Switzerland)
WG4.2- SETTING UP THE STAGE FOR FUNCTIONAL ANALYSIS OF NUCLEAR PROTEINS IN BARLEY Beáta Petrovská (Institute of Experimental Botany, Olomouc, Czech Republic)
WG4.3- COUPLING HI-C WITH FLOW SORTING TO STUDY ARCHITECTURE OF PLANT CHROMATIN ACROSS THE CELL CYCLE Petr Cápal (Institute of Experimental Botany, Olomouc, Czech Republic)
WG5.1 GARNET: A NETWORK TO SUPPORT UK PLANT SCIENCE Geraint Parry (Cardiff University, UK)
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Poster WG1.1 Poster WG1.2 ON LINE PERFORMANCE EVALUATION AND OPTIMIZATION ARABIDOPSIS LINKER HISTONES REGULATE FINE- OF IMAGE SEGMENTATION METHODS USING GENETIC SCALE CHROMATIN ARCHITECTURE AND MODULATE ALGORITHM AND GENERIC GROUND TRUTH IN 3D DEVELOPMENTAL DECISIONS MICROSCOPY IMAGING www.biorxiv.org/content/early/2018/10/31/458364
1,# 2,# 1,3 Zikrija Avdagic, Omar Bilalovic, Vedad Letic, Merisa Golic and Kinga Rutowicz , Maciej Lirski , Benoît Mermaz , Jasmin 1 4 4 Muamer Kafadar Schubert , Gianluca Teano , Imen Mestiri , Magdalena A. Kroten2, Tohnyui Ndinyanka Fabrice1, Simon Fritz1, Stefan Grob1, University of Sarajevo, Faculty of Electrical Engineering, Department for Christoph Ringli1, Lusik Cherkezyan5, Fredy Barneche4, Andrzej Computing and Informatics, Laboratory AIB Zmaja od Bosne bb, 71000 Jerzmanowski2 and Célia Baroux1 Sarajevo, Bosnia and Herzegovina 1- Institute of Plant and Microbial Biology, University of Zürich, Switzerland Image segmentation is an essential step in image processing and determines 2- Institute of Biochemistry and Biophysics, Polish Academy of Sciences, precision in image quantification of plant nuclear domains. There are subjective and Warsaw, Poland objective methods used to evaluate segmentation methods performance. Subjective methods demand expert’s involvement, what is time consuming and expensive. We have 3- Department of Life Sciences, Imperial College London, London, UK decided to use objective methods for performance evaluation of segmentation methods 4- Département de Biologie, IBENS, Paris, France for predefined benchmark of 3D arabidopsis nucleus images. To compensate a lack of 5- Department of Biomedical Engineering, Northwestern University, USA data sets with ground truth nucleus images, we developed an algorithm to generate # equal contribution Generic Ground Truth Images (GGTI). Using GGTI images we quantified improvement in performance resulting from optimization of method parameters. Chromatin provides a tunable platform for gene expression control and epigenetic regulation. Beside the well-studied core nucleosome, H1 linker histones are We propose a system that receive 3D nucleus microscopy images and run in abundant components of chromatin with an intrinsic potential to influence its structure parallel Generic Ground Truth Images Subsystem (GGTIS) and Performance Evaluation and function. Being more than simple chromatin compaction factors, H1 variants and Optimization Segmentation Method Subsystem (PEOSMS). GGTIS produces a GGTI interplay with DNA methylation in both plants and animals. Arabidopsis possess three for each 2D raw image from 3D stack sample and PEOSMS produces 3D segmented variants: H1.1 and H1.2 are near ubiquitous variants present in somatic, but not in image. GGTIS is implemented in MATLAB comprising: Knowledge Based Optimizer reproductive tissues where its eviction coincides with dramatic and large-scale chromatin (KBO) that generates l parameters’ sets for n segmentation methods, Majority Voting reprogramming. H1.3 is a stress inducible isoform considered as a pioneer factor in Segmentation Algorithm (MVSA) that produces l 2D segmented images for any raw physiological adaptation to abiotic stress. 2D image, and Median Generator (MG) that produces one GGTI as median from l 2D segmented images. PEOSMS subsystem has two inputs: sequence of 2D raw images and We provide here an in-depth analysis of the collective function of H1 in sequence of 2D GGTIs uniquely mapped to one another. Genetic Algorithm (GA) in Arabidopsis from the nanoscopic molecular scale of chromatin organization to the adaptive mode evaluates performance of segmentation method and iteratively quantifies organismal level. Results obtained by combining high-resolution cytological analyses and improve value of metric built in GA fitness function. Output of PEOSMS subsystem is with transcriptome and nucleosome occupancy profiling showed that linker histones 3D segmented image. are necessary for heterochromatin assembly, fine-scale euchromatin packaging into regular nanodomains and for H3K27me3 maintenance. Critically, H1 enables nucleosome density features associated with, yet not epistatic to transcriptional status for most but some H1-sensitive gene and transposable elements. This suggests a dual causality between chromatin structure and gene expression. Finally, upon H1 depletion we observed a relaxed control of developmental transitions underpinning a so far unrecognized functional link between linker histones and Polycomb-Group activity in plants.
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Poster WG1.3 Poster WG1.4 SINGLE-PLANT PHENOTYPING COMBINED WITH LONG-TERM A FIRST GLIMPSE OF NUCLEAR PORE COMPLEX DYNAMICS DIRECT SELECTION FOR EFFICIENT RESOURCE UTILIZATION DURING ARABIDOPSIS MEIOSIS IN COWPEA AND THE ASSOCIATED GENOMIC AND EPIGENOMIC CHANGES Nadia Fernández and Mónica Pradillo
Dionysia A. Fasoula, Michalis Omirou and Ioannis M. Ioannides Departamento de Genética, Fisiología y Microbiología; Facultad de Ciencias Biológicas; Universidad Complutense de Madrid; Spain. Agricultural Research Institute, P.O. Box 22016, 1516 Nicosia, Cyprus The Nuclear Pore Complex (NPC) is a large structure spanning the Nuclear Cowpea is an important legume crop for Cyprus. A local cowpea landrace with Envelope (NE). In plants, it comprises at least 30 different proteins, called nucleoporins, prostrate habit is greatly appreciated for fresh pod consumption. Fresh pods are produced which are present in diverse stoichiometries. The main function of the NPC is mediating and continuously harvested for a period of about 3 months. the nucleocytoplasmic transport of macromolecules, but NPCs have also transport- independent reported roles. They provide a binding platform for the underlying chromatin The long-term experiments spanning almost a decade started with studies of and have been shown to participate in RNA processing, gene expression regulation, DNA the within-landrace variation involving field selection and single-plant field phenotyping repair, and chromosome segregation among other functions. Concerning the possible for pod yield, root and shoot biomass. The experimental approach uses innovative field involvement of NPCs in meiosis, it was primarily proposed that they could be a ‘press- selection designs and the subsequent growing of individual plants at wide distances stud’ that could contribute to the attachment of chromosomes to the NE. In this context, that exclude any interplant interference permitting also the full expression of the it is worth mentioning that during leptotene-zygotene transition, telomeres are attached corresponding phenotypes. The final comparative assessment is based on the use of to the NE and form a cluster, a configuration known as “bouquet”. Telomere clustering novel phenotyping-prognostic equations taking into account soil heterogeneity and the during the bouquet may promote pairing and synapsis of homologous chromosomes. relationships between evaluated genotypes to accurately predict performance in the next generation. Plant NPCs are densely spaced, however, their actual role in chromatin dynamics during meiosis remains elusive. To get insights into this issue, we decided The initial trials identified a number of family lines outyielding the controls. to analyze the distribution of NPCs and NE-associated proteins (such as SUN-domain A new single-plant phenotyping cycle was initiated, comparing the root, shoot and proteins) in Arabidopsis thaliana pollen mother cells (PMCs). We have observed that both pod biomass of these elite selections against the controls under different watering and SUN proteins and NPCs are spread over the NE, but they polarize during early prophase fertilization regimes in field conditions. Intriguingly, the results show that the input I. We have also analyzed the dynamics of these proteins in mutant lines defective for distribution in the initial material favours higher shoot and root development, but not either SUN proteins or different nucleoporins such as AtNUP136. Our results suggest the pod formation, while the opposite is the case in the elite materials. This is all the more involvement of NPCs in chromosome behavior during plant meiosis. interesting since there was no artificial cross-fertilization involved, so selected materials are currently analysed at the genomic and epigenomic level to provide information about the underlying differences. Results from the long-term field trials and the associated genomic and epigenomic analysis will be presented.
Fasoula ,VA (2013) Prognostic breeding: A new paradigm for crop improvement. Plant Breeding Reviews 37, 297–347.
Fasoula DA, Fasoula VA (1997) Competitive ability and plant breeding. Plant Breeding Reviews 14, 89–138.
Fasoulas AC, Fasoula VA (1995) Honeycomb selection designs. Plant Breeding Reviews.13, 87–139.
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Poster WG1.5 Poster WG1.6 ANALYSIS OF HISTONE H3 VARIANTS DISTRIBUTION IN THE IS NUCLEOLUS-ASSOCIATED TELOMERE CLUSTERING REPETITIVE REGIONS OF A.THALIANA GENOME UNIVERSAL IN CRUCIFER SPECIES?
Konstantin Kutashev1, Sofia Otero2, Crisanto Gutierrez2 and Wenbo Shan, Terezie Mandáková and Martin A. Lysak Martina Dvorackova1 CEITEC – Central European Institute of Technology, and Faculty of Science, Masaryk University, Brno, Czech Republic. 1- CEITEC MU and Faculty of Science, Masaryk University, Kamenice 753/5, 62500 Brno, Czech Republic In many plant species, chromosome territories (CTs) maintain their anaphase- 2- CBMSO, Nicolas Cabrera 1, 28049 Madrid, Spain telophase orientation with telomeres and centromeres positioned at opposite poles of a nucleus – the Rabl model. However, this model is not universal as shown for grasses Recent development of various methods for detection of protein – DNA with small genomes. In the small Arabidopsis thaliana genome (c. 160 Mb), the non-Rabl interactions and protein positioning, especially ChIP (Chromatin Immunoprecipitation), interphase organization has been described as the chromocenter (CC)-loop model or ChIP on chip or 4C methods, together with genome sequencing allowed to create rosette-like organization (Fransz et al., 2002). This pattern is characterized by centromeric genome browsers, carrying massive data on positioning of proteins and their CCs located at the nuclear periphery, emanating euchromatic loops comprising modifications. However, same operations on ribosomal genes (45SrDNA, here rDNA) chromosome arms, and telomeres usually clustered around the nucleolus. are more problematic, due to their repetitive nature and the way they are organised – a coexistence of active and inactive copies, often intermingled together. We used a cytogenomic approach to test whether the rosette-like interphase organization is universal across the mustard family (crucifers). The arrangement of Our earlier studies on CAF1 histone chaperone mutants allowed us to obtain centromeric CCs and telomeres was investigated in seven species with comparable lines with very low rDNA gene copy numbers, corresponding to active repeats only. chromosome numbers but contrasting genome size and phylogenetic position. In species These lines represent a unique material where active rDNA copies can be analysed with genome size ranging from 160 Mb to 340 Mb (Arabidopsis thaliana, Cardamine separately from inactive ones allowing to understand how cells regulate expression of amara, Chorispora tenella and Descurainia preauxiana), telomeres clustered around the particular rDNA genes. A non-canonical variant of H3 histone, H3.3 was earlier shown nucleolus in 71 to 75% interphase nuclei analyzed. In species with genomes larger than to be associated with 3’ end of actively transcribed genes. In this study we analyse the 700 Mb, telomeres were not observed clustered around the nucleolus. In Arabis cypria distribution of histone H3 variants and the chromatin organisation in active rDNA genes (780 Mb), Bunias orientalis (2 670 Mb) and Hesperis sylvestris (4 360 Mb), 89%, 98% and in A.thaliana wild type lines with low rDNA copy numbers. 95% of the analyzed interphase nuclei did not show telomere-to-nucleolus clustering. Further analyses are under way to elucidate how interphase chromosome are organized in crucifer species not showing telomere-to-the-nucleolus clustering. This work was supported by a research grant from the Czech Science Foundation to M.A.L. (grant no. 18-20134S).
Fransz, P., De Jong, J. H., Lysak, M., Castiglione, M.R., and Schubert, I. (2002). Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate. Proceedings of the National Academy of Sciences 99, 14584-14589.
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Poster WG1.7 Poster WG1.8 CHARACTERIZATION OF CATALYTIC MUTATIONS IN DEVELOPMENT AND BENCHMARKING OF SEGMENTATION ARABIDOPSIS THALIANA BRAHMA (BRM) CHROMATIN METHODS TO ANALYZE THE PLANT 3D NUCLEUS REMODELING ATPASE. Tristan Dubos1,2, Rémy Malgouyres2, Axel Poulet3, Céline Paulina Stachula1, Jaronczyk K.1, Sosnowska K.1, Wojcikowski M.2, Gonthier1, Pierre Pouchin1, Aline Probst1, Christophe Tatout1 and Siedlecki P.1,2 and Archacki R.1,2 Sophie Desset1
1- University of Warsaw, Faculty of Biology, Laboratory of Systems Biology 1- Université Clermont Auvergne, Laboratoire GReD, Clermont-Ferrand, France 2- Institute of Biochemistry and Biophysics, Polish Academy of Science 2- Université Clermont Auvergne, Laboratoire LIMOS, Aubière, France 3- Emory University, Department of Biostatistics and Bioinformatics, Atlanta, BRAHMA (BRM) is a core ATPase subunit of the Arabidopsis SWI/SNF chromatin USA remodeling complex. In plants, SWI/SNF is known to play crucial role in transcriptional control in development, growth, as well as hormonal and stress responses. BRM binds to The nucleus is a compartmentalized organelle containing distinct chromosomal different regulatory regions and can act as either activator or repressor of gene expression. domains and nuclear bodies. To link nuclear structure and function, we have developed an ImageJ plugin to quantify the 3D nuclear morphology as well as positioning and However, it is currently unknown which functions of BRM truly depend on organization of nuclear domains. This plugin named NucleusJ (Desset et al., 2018; its catalytic activity, as some of them can be attributed to protein-protein interactions Poulet et al., 2015), applies for a batch of images a modified Otsu thresholding method with other regulators. To investigate the role of ATPase activity of BRM we introduced to segment the nucleus and a 3D watershed algorithm to delimit chromatin domains by point mutations into the ATP- binding pocket of its catalytic domain. We obtained stable partitioning the nucleus. Quantitative parameters are computed including shape and size transformed lines expressing mutated versions of BRM and observed significantly different of nuclei as well as intra-nuclear objects such as chromatin domains and their position in phenotypes compared to WT Col-0 plants. respect to the nuclear periphery (Poulet et al., 2017).
We also compared the effects of catalytic mutations with those displayed in A current limitation is the time needed to delimit each bounding volume brm-1 null mutants, both at the phenotypic and gene expression level. Future studies including the considered nucleus. To overcome this manual step and automatically using BRM catalytic mutants will be discussed. capture large numbers of nuclei at various depths in the original wide field image, we have implemented an automatic detection of the nucleus (autocrop) as a starting point This work is supported by National WScioence Centre Poland grant no. of our workflow. Spatial positions of the cropped nuclei are then recorded in order to 2017/26/E/NZ2/00899 and The National centre for Research and Development grant no. estimate distance maps as a new estimator of spatial distribution of the nuclei in a whole LIDER/313/L-6/14/NCBR/2015 tissue context. The second main limitation is to segment the nucleus and their intra- nuclear objects from the background. To this aim, we are developing a new 3D gift- wrapping method in which a parameter of maximal threshold distance is applied between two vertices, which will define the final boundary. These new tools will be illustrated through our recent efforts to characterize a range of mutants affecting nuclear envelope- associated proteins in a big data context.
Desset, S., Poulet, A., and Tatout, C. (2018). Quantitative 3D Analysis of Nuclear Morphology and Heterochromatin Organization from Whole-Mount Plant Tissue Using NucleusJ. Methods Mol. Biol. Clifton NJ 1675, 615–632. Poulet, A., Arganda-Carreras, I., Legland, D., Probst, A.V., Andrey, P., and Tatout, C. (2015). NucleusJ: an ImageJ plugin for quantifying 3D images of interphase nuclei. Bioinformatics 31, 1144–1146. Poulet, A., Duc, C., Voisin, M., Desset, S., Tutois, S., Vanrobays, E., Benoit, M., Evans, D.E., Probst, A.V., and Tatout, C. (2017). The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants. J Cell Sci 130, 590–601.
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Poster WG2.1 Poster WG2.2 INVESTIGATING PROTEIN-PROTEIN INTERACTIONS BETWEEN CHARACTERIZATION OF CATALYTIC MUTATIONS IN ARABIDOPSIS MID-SUN PROTEINS AND THE TRANSCRIPTION ARABIDOPSIS THALIANA BRAHMA (BRM) CHROMATIN FACTOR maMYB AT THE PLANT NUCLEAR ENVELOPE REMODELING ATPASE
Bisa Andov, David Evans, Verena Kriechbaumer and Katja Stachula P.1, Jaronczyk K.1, Sosnowska K.1, Wojcikowski M.2, Graumann Siedlecki P.1,2 and Rafal Archacki 1,2
Department of Biological and Medical Sciences, Oxford Brookes University, 1 University of Warsaw, Faculty of Biology, Laboratory of Systems Biology Headington Campus, Oxford, OX3 0BP 2Institute of Biochemistry and Biophysics, Polish Academy of Science
Inner nuclear membrane (INM)-localised sad1/UNC-84 (SUN) proteins are BRAHMA (BRM) is a core ATPase subunit of the Arabidopsis SWI/SNF required to mediate interactions between the nucleoskeleton and cytoskeleton by chromatin-remodeling complex. In plants, SWI/SNF is known to play crucial role in interacting with outer nuclear membrane (ONM)-localised Klarsicht/ANC-1/Syne transcriptional control in development, growth, as well as hormonal and stress responses. homology (KASH) proteins. These SUN-KASH interactions are known as Linker of BRM binds to different regulatory regions and can act as either activator or repressor of Nucleoskeleton and Cytoskeleton (LINC) complexes, and are essential for carrying out gene expression. However, it is currently unknown which functions of BRM truly depend nuclear processes such as chromosome decondensation and nuclear positioning. SUN on its catalytic activity, as some of them can be attributed to protein-protein interactions proteins have previously been identified as having a highly-conserved C-terminal SUN with other regulators. domain (Cter-SUNs), but a second sub-family has been identified in plants where the SUN domain is more centrally located. These are the mid-SUN proteins and are poorly To investigate the role of ATPase activity of BRM we introduced point mutations described across all kingdoms. into the ATP- binding pocket of its catalytic domain. We obtained stable transformed lines expressing mutated versions of BRM and observed significantly different phenotypes Previous work on mid-SUN proteins in the model plant Arabidopsis thaliana compared to WT Col-0 plants. We also compared the effects of catalytic mutations with have shown that they can interact with Cter-SUNs, each other and KASH proteins, those displayed in brm-1 null mutants, both at the phenotypic and gene expression level. indicating that they are also involved in forming LINC complexes. This poster shall focus Future studies using BRM catalytic mutants will be discussed. on characterising protein-protein interactions between the transcription factor maMyb and Arabidopsis mid-SUNs AtSUN3 and AtSUN4. These interactions have been tested by This work is supported by National Science Centre Poland grant no. 2017/26/E/ using a quantitative imaging technique known as fluorescence resonance energy transfer NZ2/00899 and The National centre for Research and Development grant no. (FRET), a proven technique used to identify in vivo protein interactions in living cells. LIDER/313/L-6/14/NCBR/2015
Graumann, K., Vanrobays, E., Tutois, S., Probst, A.V., Evans, D.E., and Tatout, C. (2014) Characterization of two distinct subfamilies of SUN-domain proteins in Arabidopsis and their interactions with the novel KASH- domain protein AtTIK. Journal of Experimental Botany , 65, 6499–6512. Karapova, T.S., Baumann, C.T., He, L., Wu, X., Grammer, A., Lipsky, P., Hager, G.L., and McNally, J.G. (2002) Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. Journal of Microscopy, 209, 56-70. Murphy, S. P., Simmons, C. R. and Bass, H. W. (2010) Structure and expression of the maize (Zea mays L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants. Biomed Central Plant Biology, 10, 269. Slabaugh, E., Held, M. and Brandizzi, F. (2011) Control of root hair development in Arabidopsis thaliana by an endoplasmic reticulum anchored member of the R2R3-MYB transcription factor family. The Plant Journal, 67, 395-405.
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Poster WG2.3 Poster WG2.4 EPIGENETIC REGULATION TO DROUGHT STRESS IN OLIVE MSIX, AN ESSENTIAL PROTEIN FOR MULTIPLE REPRODUCTIVE PLANT (OLEA EUROPAEA L.) PROCESS IN ARABIDOPSIS
1 2 3 Birsen Cevher-Keskin , A. Hediye Sekmen , Nurcan Ulucay , Chunlian Jin, Burcu Nur Keçeli and Danny Geelen* Erhan Erdik2, Azime Gokce2, Ünal Kaya3, Yasemin Yildizhan1 and Aydan Sarac-Derdiyok1 Ghent University, Department Plants and Crops, Faculty of Bioscience Engineering, 9000 Gent, Belgium 1- TUBITAK Marmara Research Center, Genetic Engineering & Biotechnology Inst. PO Box:21, 41470 Gebze-KOCAELI-TURKEY MSI1–Like (MSIL) proteins are involved in many protein complexes controlling 2- Ege University, Faculty of Science, Department of Biology, Izmir-TURKEY chromatin dynamics, functioning in cell cycle, cell proliferation and developmental 3- Olive Research Institute Izmir-TURKEY processes. In contrast to fungi, insects and vertebrates, which encode only for one or two MSIL proteins, plants possess a small family that falls into three groups, suggesting subfunctionalization. Arabidopsis has six MSIL proteins (MSI1-6) (Tripathy et al., 2015). Epigenetics plays a very crucial role in modulating the expression of nuclear AtMSI4 (FVE) plays a role in regulating flowering time and is involved in cold response genes by switching on/off mechanisms in response to environmental stresses. The main (Ausin et al., 2004; Kim et al., 2004). objective of this work is a comprehensive characterization of the epigenetic mechanism to drought stress of olive leaf tissues through high throughput techniques such as MSI6 (HTD1), the most evolutionary distant member, was reported to play RNA-Seq and ChIP-seq analysis. The slow-growing character of olive trees makes field a role in heat tolerance (Kim et al., 2014). AtMSI1 functions in seeds development as trials costly and time-consuming. Therefore, it is beneficial to take advantage of those a part of the MEA (MEDEA)/FIE (FERTILIZATION-INDEPENDENT ENDOSPERM)/ FIS morphological and physiological traits relevant to drought tolerance in order to facilitate (FERTILIZATION-INDEPENDENT SEED2) polycomb group complex (Kohler et al., 2003). the breeding and selection process. Olive genotypes originating from Turkey and showing It also functions in regulating the cell cycle during male gametogenesis and acts together a different response to drought stress were selected for this project. with FAS1 and FAS2 in trichome development, both as a member of CAF1 complex (Chen et al., 2008; Exner et al., 2006). To evaluate for the agronomic traits of these genotypes under drought stress conditions, some physiological and biochemical responses to drought stress were Recent studies showed that MSI1 interacts with retinoblastoma proteins and compared. The maximum H2O2 levels were observed in Ödemis, Essek and Gemlik CUL4-DDB complex, controlling parental gene imprinting in Arabidopsis (Dumbliauskas genotypes. The sensitivity to drought stress of Ödemis, Essek and Gemlik genotpes et al., 2011; Jullien et al., 2008). To study the functions of Arabidopsis MSIL proteins in was associated decreased activities of catalase (CAT), peroxidase (POX) and ascorbate sexual reproduction, we screened different T-DNA insertion lines for each family member peroxidase (APX), as compared to their control resulting in higher H2O2 accumulation for fertility. We found several lines to show a reduction in fertility, suggesting a role in and oxidative stress-induced lipid peroxidation. Drought stress tolerance of Çekiste was meiosis or in microsporogenesis. Further experiments are under way to analyze the associated with its ability to maintain constitutive activities of SOD and induced CAT, underlying defect that causes a reduction in fertility. POX and APX. Selected genotypes from these experiments showing different responses to drought stress (by adding 2 tolerant and 1 non-tolerant genotype) will be assessed for RNA-seq and ChIP-seq experiments. This work will identify differential gene expression and altered plant phenotypes upon exposure to drought stress to decipher the role of nuclear domains and hence epigenetic regulation in strategic plant olive.
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Poster WG2.5 Poster WG2.6 POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) MODULATES ELUCIDATING THE ROLE OF EPIGENETIC REGULATORS AT THE PHOTOAUTOTROPHIC GROWTH OF PHYSCOMITRELLA NUCLEAR PERIPHERY IN ARABIDOPSIS PATENS Kalyanikrishna1, Pawel Mikulski1,2,3, Mareike Hohenstatt3 and Helena Hönig Mondeková1,2,4, Katerina Kabelácová2,4, Katarina Daniel Schubert1,3 Landberg3, Eva Sundberg3, Roman Sobotka1,2 and Iva Mozgová2,3,4 1- Institute of Biology, Freie Universität Berlin, Germany 1- Centre Algatech, Institute of Microbiology of the Czech Academy of Sciences, 2- John Innes Centre, Norwich, UK 37981 Trebon , Czech Republic 3- Institute for Genetics, Heinrich-Heine-University Düsseldorf, Germany 2- Faculty of Science, University of South Bohemia, 37005 Ceske Budejovice, Czech Republic One of the key epigenetic regulators in plants are Polycomb group (PcG) 3- Department of Plant Biology and Forest Genetics, Uppsala BioCenter, proteins which form highly conserved protein complexes (PRC1 and PRC2) and regulate Swedish University of Agricultural Sciences and Linnean Centre for Plant a series of developmental processes by facilitating chromatin mediated gene repression Biology, SE–750 07 Uppsala, Sweden 4- Present address: Institute of Plant Molecular Biology, Biology Centre of the (Förderer et al.,2016; Schubert et al., 2005). In our lab, we characterized a novel Czech Academy of Sciences, 370 05 Ceske Budejovice, Czech Republic chromatin protein family in Arabidopsis, PWWP INTERACTOR OF POLYCOMBS1 (PWO family) which consists of three members. PWO1 interacts with all three histone Polycomb Repressive Complex 2 (PRC2), a histone H3-lysine 27 tri- methyltransferase subunits of the Arabidopsis PRC2, CLF, SWN and MEA. Similar to PcG methyltransferase, is a highly conserved protein complex involved in epigenetic cellular proteins, PWO1 is expressed in diverse tissues and localizes to euchromatic regions in memory. It specifies cell fate and governs development in eukaryotes. PRC2 is crucial for the nucleus (Hohenstatt et al., 2018). multiple developmental transitions in the flowering plant Arabidopsis thaliana and for apogamy repression and leafy gametophyte development in the bryophyte Physcomitrella In the list of putative interactors of PWO1 from co-immunoprecipitation patens (Okano et al., 2009, Mosquna et al., 2009). experiment coupled with mass spectrometry, presence of nuclear lamina associated components, especially CROWDED NUCLEI 1(CRWN1) - a coiled coil analog of lamin We show that the presence or absence of exogenous sugar in the growth/ proteins in Arabidopsis - gained attention as it has prominent role in maintaining nuclear cultivation medium results in two morphologically different states of colonies of morphology (Wang et al., 2013) and chromocenter organization (Dittmer et al., 2007). null PRC2 mutants of P. patens (ΔPpCLF and ΔPpFIE) under long term cultivation. Further investigation on PWO1-CRWN1 showed a physical and genetic interaction and Exogenous sugar together with increased light intensity further promotes sporophyte- similar set of regulated genes (Mikulski et al., 2017). We have characterized several like body formation in freshly established colonies of null PRC2 mutants. In order to chromatin modifiers, which interact with PWO1 and in addition regulate nuclear size limit developmental effects of PRC2 absence, we further performed phenotypic analysis and morphology. The dynamics and interplay of these above-mentioned proteins in gene on protonema only. Both ΔPpCLF and ΔPpFIE show altered chloronema morphology regulation and nuclear periphery still needs to be unfolded. However, we speculate that compared to wild type under different light intensities, especially under low light. Using PWO1 along with its interacting partners, is a putative link between PRC2 mediated gene chlorophyll fluorescence measurement, low temperature fluorescence measurement and regulation and the nuclear periphery.
real-time RT-PCR, we found that under low light, the non-photochemical quenching Dittmer, T. A., Stacey, N. J., Sugimoto-Shirasu, K., and Richards, E. J. (2007). LITTLE NUCLEI genes affecting nuclear morphology in (NPQ) and photosystem PSI/PSII ratio is reduced, corresponding with a reduced Arabidopsis thaliana. Plant Cell 19, 2793–803. Förderer, A., Zhou, Y., Turck, F. (2016). The age of multiplexity: recruitment and interactions of Polycomb complexes in plants. Curr expression of photosynthesis-related genes in the PRC2 mutants. With increasing light Opin Plant Biol 29, 169–178. intensity, PRC2 mutants do not follow the acclimation changes that appear in the WT. Hohenstatt, L.M., Mikulski, P., Komarynets, O., Klose, C., Kycia, I., Jeltsch A., Farrona, S., and Schubert, D. (2018). PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 interacts with Polycomb-group proteins and histones and regulates Arabidopsis flowering and Mosquna, A., Katz, A., Decker, E.L., Rensing, S.A., Reski, R., and Ohad, N. (2009). Regulation of stem cell development. Plant Cell 30 (1), 117-133 Mikulski, P., Hohenstatt, L.M., Farrona, S., Smaczniak, C., Kaufmann, K., Angenent, G., and Schubert, D. (2017). PWWP INTERACTOR maintenance by the Polycomb protein FIE has been conserved during land plant evolution. Development 136: OF POLYCOMBS (PWO1) links PcG-mediated gene repression to the nuclear lamina in Arabidopsis. bioRxiv preprint, https://doi. 2433–44. org/10.1101/220541. Okano, Y., Aono, N., Hiwatashi, Y., Murata, T., Nishiyama, T., Ishikawa, T., Kubo, M., and Hasebe, M. (2009). A Schubert, D., Clarenz, O., and Goodrich, J. (2005). Epigenetic control of plant development by Polycomb- group proteins. Curr. Opin. polycomb repressive complex 2 gene regulates apogamy and gives evolutionary insights into early land plant Plant Biol. 8, 553-561. Wang, H., Dittmer, T., and Richards, E. J. (2013). Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size evolution. Proc. Natl. Acad. Sci. U. S. A. 106: 16321–16326. control and heterochromatin organization. BMC Plant Biol. 13, 200.
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Poster WG2.7 Poster WG2.8 ROLE OF PRC2 IN EMBRYO AND SEEDLING DEVELOPMENT CRISPR-CAS9 MUTAGENESIS UNRAVELS THE ROLE OF 45S rDNA GENES IN A. THALIANA DEVELOPMENT Tomáš Konecný1, Rafael Muñoz-Viana2, Juan Santos-Gonzales2, Claudia Köhler2, Lars Hennig2 and Iva Mozgová1,2 Francesca Lopez1, Antoine Fort1,2, James Friel1, Peter Ryder1, Marcus McHale1, Hirofumi Ishihara3, Ronan Sulpice2 and Charles 1- Centre Algatech – Institute of Microbiology CAS, Trebon, Czech Republic & Spillane1 University of South Bohemia in Ceské Budejovice, Czech Republic 2- Department of Plant Biology, Uppsala BioCenter, Swedish University of 1- Genetics and Biotechnology Laboratory, Plant and AgriBiosciences Research Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Centre (PABC), School of Natural Sciences, National University of Ireland Galway, Ireland. It is widely considered that PRC2 has crucial role in regulation of plant 2- Systems Biology Laboratory, Plant and AgriBiosciences Research Centre development. Without its proper function plants lack important part of epigenetic control of developmentally regulated genes. Seedlings missing PRC2 activity give raise to callus- (PABC), School of Natural Sciences, National University of Ireland Galway, like structures demonstrating failure of cell differentiation (Bouyer et al., 2011, Ikeuchi et Ireland. al., 2015). 3- Max Planck Institute of Molecular biology, Potsdam, Germany
Despite the requirement for PRC2 in the embryo-to-seedling transition and Although 45s rDNA genes are ubiquitous to all eukaryotes, the number of post- embryonic processes, its role in initial body plan establishment in Arabidopsis repeats and their chromosomal locations are highly polymorphic across different phyla. seems limited (Bouyer et al., 2011). Nevertheless, we find that a large number of genes The role of transcriptionally active and inactive 45s copies has been amply characterized are marked by H3K27me3 in the Arabidopsis early bent cotyledon-stage embryos and in cellular biology by numerous studies linking loss of 45s copies to abnormal therefore we ask what is the function of PRC2 during embryo development. Addressing proliferation and cancer (Xu et al., 2017, PLOS Genet. 13, e1006771), and also to the possibility of PRC2 involvement during embryogenesis, we analysed phenotypes that genome integrity and senescence (Obayashi and Kobayashi, 2014, Proc. Jpn. Acad. Ser. B. could indicate developmental defects in clf swn embryos compared to wild type. First, we Phys. Biol. Sci. 90, 119–129). compared numbers of aborted seeds in siliques of clf-29/+ swn-3/- and wild- type plants. In plants however, it hasn’t yet been possible to formulate a direct link between This approach was followed by morphological analysis and developmental variation in 45s copy number and their function in development. Previous studies marker gene expression analysis of embryos at the early bent cotyledon stage. To verify employed the Chromatin Assembly Factor-1 (CAF-1) mutants which are knock-out our observations, we tested for deviation from expected segregation frequencies of alleles of histone chaperones FASCIATA 1/2 , which lead to a drastic reduction in rDNA double mutant genotype in the studied lines. We next compared our results obtained by copy number (Kaya et al., 2001, Cell 104, 131–142; Mozgova et al., 2010, Plant Cell the phenotype assessment of mutant embryos with transcriptome and ChIP-seq data of 22, 2768–2780), however it has not yet been possible to draw a causative association clf-29 swn-3 and wild-type embryos and seedlings. between the phenotypes observed and the reduction of copy number. Here, we show how CRISPR-Cas9 mutagenesis has been employed to generate A. thaliana plants with a Bouyer, D., Roudier, F., Heese, M., Andersen, E.D., Gey, D., Nowack, M.K., Goodrich, J., Renou, J.-P., Grini, P.E., stably inherited reduction of up to 80% of 45s rDNA genes. Colot, V., Schnittger, A., 2011. Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition. PLoS Genetics 7, e1002014. Ikeuchi, M., Iwase, A., Rymen, B., Harashima, H., Shibata, M., Ohnuma, M., Breuer, C., Morao, A.K., de Lucas, We report that decrease of 45s copies is linked with aberrant seedling M., De Veylder, L., Goodrich, J., Brady, S.M., Roudier, F., Sugimoto, K., 2015. PRC2 represses dedifferentiation of development, which is also associated with deregulated mechanisms of cell proliferation mature somatic cells in Arabidopsis. Nature Plants 1, 15089. and meristematic activity. Since these mechanisms are established during early embryonic
development, we analyzed embryogenesis in our 45s rDNA Low Copy Number lines and found severe aberrations of correct body plan establishment. Further work will be focused on investigating the mechanisms that link 45s rDNA copy number and the first cell division of embryo development.
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Poster WG2.9 Poster WG2.10 GENOMIC STABILITY IN CHROMATIN REMODELLING USE OF SINGLE MOLECULE FLUORESCENCE IN SITU MUTANTS PKR1, PKR2, CHR5, CHR9 AND CHR24 HYBRIDIZATION TO VISUALIZE SENSE AND ANTISENSE RNA TRANSCRIPTS AT THE DOG1 LOCUS IN SINGLE CELLS OF Adéla Machelová1, Eva Sýkorová2 and Martina Dvorácková1,2 ISOLATED EMBRYOS
1. Central European Institute of Technology (CEITEC) and Faculty of Sciences of Miguel Montez1, Szymon Swiezewski1 and Stefanie Rosa2 Masaryk University, Brno, Czech Republic 2. Institute of Biophysics ASCR, v.v.i., Královopolská 135, 61265, Brno, Czech 1- University of Warsaw, Poland Republic 2- Swedish University of Agricultural Sciences - Almas allé 8, Uppsala, Sweden Chromatin remodelling largely affects genome stability as it is necessary for DELAY OF GERMINATION 1 (DOG1) was identified 15 years ago as a DNA replication, transcription and double-strand breaks repair. Repetitive sequences quantitative trait locus involved in the control of seed dormancy in Arabidopsis thaliana. are the least stable genomic sites (so called fragile sites) because they represent difficult Since then, a number of studies have highlighted important DOG1 functions in seed templates for DNA polymerases during replication. We employ here on telomeres and dormancy which rely on a fine regulation of DOG1 expression at the molecular level. ribosomal DNA (45S rDNA). Telomeres are nucleoprotein complexes protecting the Our lab has significantly contributed for the current understanding of how DOG1 gene ends of chromosomes, while ribosomal DNA encodes genes for ribosomal RNA, a key is regulated including the discovery of a long non-coding DOG1 antisense transcript component of ribosomes. Thus these repeats are ideal sites to study the mechanisms (asDOG1) that negatively regulates DOG1 sense expression in cis. responsible for genome integrity maintenance. Our findings suggest that as DOG1 is a key element sensing internal stimuli and In this work we focus on Chromatin Remodelling genes (so called CHR genes) environmental cues that feeds back directly on DOG1 by changes in its expression levels that effect accessibility of chromatin by sliding, nucleosome eviction or spacing. CHR and chromatin landscape. Yet, the molecular mechanism by which asDOG1 represses genes are ATPases from the complex SNF2 and they are classified into several groups DOG1 is still puzzling. In this work we used smFISH (single molecule Fluorescence In according to their structure and properties. Situ Hybridization) to visualize sense and antisense RNA transcripts at the DOG1 locus in single cells of isolated embryos. This imaging technique helped us to dissect the basic Arabidopsis PICKLE (PKL) related genes - CHR4 (PICKLE RELATED 1, PKR1), features of sense and antisense transcription at DOG1 locus behind seed dormancy. Our CHR5 and CHR7 (PKR2) - from CHD group (Chromodomain Helicase DNA-binding) are results support with experimental evidences what were previously assumptions about poorly described. PKL is involved in multiple processes including development, flowering some of the transcriptional features of sense and antisense DOG1. or signalling pathways, while PKR1 is expected to play role in homologous recombination and DNA repair. Whereas PKR2 might play a role in plant development. It was discovered Importantly, our work also provide novel insights on the transcription and post- that PKR1 and PKR2 are redundant with PKL and CHR5 showed to act antagonistically. transcription regulation of these sense and antisense transcripts including clues about Another possible DNA repair genes we investigated belong to the ERCC6-like group transcription bursts as a putative important source of cell-to-cell asDOG1 variability, (Excision Repair Cross-Complementation group 6) – CHR9 and CHR24. and a fine regulation of DOG1 mRNA nuclear retention/export buffering stochastic fluctuations in transcripts abundancy from the nucleus to the cytoplasm. Overall, our We observed the telomere shortening and rDNA loss in first generation in chr4 work generated valuable preliminary results that consist in a good starting point to a and chr24 mutants. Chr5 mutant showed rDNA loss while telomeres remain intact. future understanding of the intricate molecular regulation of gene expression at the seed Telomere shortening was also seen in the first generation in chr7 mutant together with the dormancy master regulator locus. increased rDNA copy number. Mutant chr9 showed heterogeneity in rDNA copy number and in telomere lengths. In the second generation in all mutants elevation in number of rDNA copies was observed.
This work was supported by The Czech Science Foundation (grant 16-04166Y) and COST INDEPTH, and INTERCOST ( LTC18048).
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Poster WG2.11 Poster WG2.12 IMPACT OF HISTONE CHAPERONE GENES AND DNA REPAIR RIBOSOMAL RNA GENES DISTRIBUTION AND EXPRESSION PATHWAYS ON rDNA STABILITY ORGANIZE NUCLEOLUS ASSPCIATED CHROMATIN DOMAINS
Martina Nešpor Dadejová1, Karolína Kolárová1,2, Zuzana Fusková1 Picart-Picolo A.1,2, Picault N.1,2, Picart C.1,2 and Pontvianne F1,2 and Martina Dvorácková1 1- CNRS, Laboratoire Génome et Développement des Plantes, UMR 5096, 58 1- Central European Institute of Technology (CEITEC) and Faculty of Sciences of Avenue P. Alduy, 66860 - Perpignan, France Masaryk University, Brno, Czech Republic 2- Univ. de Perpignan Via Domitia, Laboratoire Génome et Développement des 2- Institute of Biophysics ASCR, v.v.i., Královopolská 135, 61265, Brno, Czech Plantes, UMR 5096, 58 Avenue P. Alduy, 66860 - Perpignan, France Republic Genome interactions can occur beyond chromosome territories and organize around nuclear bodies. Recent studies revealed how the nucleolus organizes high order DNA loci encoding 45S rDNA ribosomal genes represent the major class of chromatin structure of certain chromosome regions displaying heterochromatic features. DNA repeats in Arabidopsis thaliana genome, aprox. 5% of its total size. Since rRNA We are currently studying the mechanisms involved in the heterochromatin organization form essential cellular machineries – ribosomes - responsible for protein production, the and their impact on gene expression using Arabidopsis thaliana as model organism. integrity of rDNA is required for plant overall plant´s fitness. An important role in the We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified genome stability maintenance is dedicated to the balanced incorporation of individual Nucleolus-Associated chromatin Domains (NADs) by deep sequencing. In wild-type, histones into DNA and their precise epigenetic modification. NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding This is mediated by evolutionarily conserved proteins called histone chaperones, genes. but large number of chromatin factors modulate this process. Their mutual interactions as well as histone binding specificity have not been fully understood yet. We focus here However, NADs also include active ribosomal RNA (rRNA) genes and the entire on the H3/H4 histone chaperones - Chromatin Assembly Factor 1 (CAF-1), H2A/H2B short arm of chromosome 4 adjacent to them. Analyses of NADs in mutant affected histone chaperone NUCLEOSOME ASSEMBLY PROTEIN 1 (NAP1), and NAP1-RELATED in rRNA genes expression or in lines with different amount of rRNA genes reveal how PROTEIN 1 and 2 (NRP1, NRP2). Morphological changes, decreased genome stability, affecting rRNA genes nuclear positioning and expression affect NADs identity. In the light progressive loss of repetitive sequences and sensitivity to DNA damaging agents were of our data and additional studies made in animal cells, we will discuss the potential role observed in plants defective for CAF1 complex (Mozgova et al., 2010). of the ribosomal RNA chromosome location in the establishment of inactive chromatin association with the nucleolus. We show here that both H3/H4 and H2A/H2B histone chaperone groups affect the stability of rDNA repeats and plant ´s viability, revealing an interesting compensation effect. We further present how individual histone chaperones interact with each other and with DNA repair genes and speculate how cells cope with the histone chaperone deficiencies. We conclude that the phenotypic features caused by fas1 mutation can be balanced by varied mechanisms, involving the modulation of the chromatin structure as well as selection of alternative DNA repair pathways.
This work was supported by The Czech Science Foundation (grant 19-11880Y), COST INDEPTH and INTERCOST (LTC18048).
Mozgova, I., Mokros, P., and Fajkus, J. (2010). Dysfunction of chromatin assembly factor 1 induces shortening of telomeres and loss of 45S rDNA in Arabidopsis thaliana. The Plant cell 22, 2768-2780.
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Poster WG2.13 Poster WG2.14 CHROMATIN DYNAMICS UPON DNA DAMAGE IN SUVH7: NEW ACTOR IN THE REGULATION OF ENDOSPERM ARABIDOPSIS THALIANA DEVELOPMENT
Svenja Reeck, Anis Meschichi and Stefanie Rosa Lauriane Simon1, Guifeng Wang1,2 and Claudia Köhler1
Swedish University of Agricultural Sciences - Almas allé 8, Uppsala, Sweden 1- Swedish University of Agricultural Sciences & Linnean Center for Plant Biology, Uppsala BioCenter, Almas Allé 5, Uppsala, Sweden Plant cells are subject to high levels of DNA damage resulting from dependence 2- present address: Henan agricultural university; 63 Nongye Rd, Jinshui Qu, on sunlight for energy and the associated exposure to environmental stresses. Double- Zhengzhou Shi, Henan Sheng, China strand breaks (DSBs) are a particularly deleterious type of DNA damage, but how DSBs are repaired at the higher chromatin level in Arabidopsis thaliana remains unclear. Our The endosperm of most angiosperms is a triploid tissue containing two maternal team adapted a live-cell imaging technique to visualize the motion of a chromatin locus and one paternal genome. This tissue transfers nutrients to the embryo and is essential its using a lacO/LacI-GFP reporter system in Arabidopsis. development, similar to the placenta in mammals. Genomic imprinting is an epigenetic phenomenon taking place predominantly in the endosperm, causing parental alleles to be This approach revealed alterations in chromatin mobility upon DNA damage. differentially expressed. Changing the balance of parental genomes causes deregulation Currently we are testing if the observed differences in DNA mobility are associated with of imprinted genes and endosperm collapse, a phenomenon referred to as “triploid changes in histone dynamics as judged by fluorescence recovery after photobleaching block”. Mutations in several imprinted genes can bypass the triploid block, revealing the (FRAP). It is now well established that post-translational modifications of histones are able central importance of balanced imprinted gene expression. to modulate histone-DNA interactions. We also aim to characterise how different histone marks are involved in DNA damage repair and more specifically how they are linked to One strong suppressor of the triploid block is the paternally expressed gene DNA mobility. SU(VAR)3-9 HOMOLOG 7 (SUVH7) (Wolff et al., 2015). Here, we show that increased expression of SUVH7 in the endosperm of triploid seeds correlates with increased Lastly, we are also interested in determining cell-to-cell variability in DNA deposition of the heterochromatic modification H3K9me2 on transposable elements damage response and we are now using single molecule RNA FISH for known DNA (TEs). Conversely, triploid seeds lacking SUVH7 function are depleted of H3K9me2. repair genes to detect possible differences between cell types and developmental stages. We furthermore demonstrate that SUVH7 interacts with ADMETOS (ADM), a DNAJ Overall, our study aims to elucidate how chromatin mobility and dynamics are involved protein that previously was shown to cause increased H3K9me2 deposition in triploid in DNA damage repair. seeds (Jiang et al., 2017). Importantly, loss of ADM and SUVH7 affects the same set of TEs, strongly suggesting that they act together to regulate H3K9me2 deposition in the
endosperm.
Jiang, H., Moreno-Romero, J., Santos-González, J., De Jaeger, G., Gevaert, K., Van De Slijke, E., and Köhler, C. (2017). Ectopic application of the repressive histone modification H3K9me2 establishes post-zygotic reproductive isolation in Arabidopsis thaliana. Genes Dev.
Wolff, P., Jiang, H., Wang, G., Santos-Gon Alez, J., and Ohler, C. Paternally expressed imprinted genes establish postzygotic hybridization barriers in Arabidopsis thaliana.
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Poster WG2.15 Poster WG2.16 A PROGRESS REPORT ON MOLECULAR CLONING OF ANALYSIS OF SMC5/6 COMPLEX MUTANT DEFECTS DURING CENTROMERE-SPECIFIC HISTONE H3 IN SAINFOIN REPRODUCTIVE DEVELOPMENT IN ARABIDOPSIS (ONOBRYCHIS VICIIFOLIA) Fen Yang1,2,3, Nadia Fernandez-Lopez4, Martina Tucková1, Jan Ahmet L. Tek and Sevim D. Kara Vrána1, Mariana Diaz3,5, Monica Pradillo4 and Ales Pecinka1,3
Department of Agricultural Genetic Engineering, Ayhan Sahenk Faculty of 1- Institute of Experimental Botany of the Czech Academy of Science, Centre Agricultural Sciences and Technologies, Nigde Ömer Halisdemir University, of the Region Haná for Biotechnological and Agricultural Research (CHR), Nigde, Turkey Šlechtitelu 31, Olomouc, Czech Republic. 2- Department of Cell Biology and Genetics, Palacký University Olomouc, Centromeres are attachment points of spindle apparatus and serve as primary Šlechtitelu 27, Olomouc, Czech Republic. constriction for proper segregation of genetic material during cell division (Houben and Schubert, 2003). Centromeres are functionally defined by the presence of a centromere 3- Max Planck Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, specific histone H3 variant known as CenH3. Unlike histone H3, CenH3 is highly Cologne, Germany. variable across species (Malik and Hennikoff, 2003). We aim to clone the CenH3 in 4- Complutense University of Madrid, Calle José Antonio Novais, 12, 28040 legume sainfoin (Onobrychis viciifolia). Madrid, Spain
Our initial search for identification of sainfoin CenH3 sequences did not provide Structural maintenance of chromosomes 5/6 (SMC5/6) complex has a crucial any significant match in NCBI GenBank. We designed several primer pairs conserved function in the organization of chromatin, control of genome stability and DNA across legumes, specific to soybean CenH3 (Teket al. 2010), lentil and Lathyrus CenH3 damage repair. However, precise molecular mechanisms of SMC5/6 complex functions (Neumann et al. 2015). Here we report our initial attempt to clone sainfoin CenH3 are unknown in plants. We found that mutations in HPY2, E3 SUMO ligase subunit genomic DNA fragments using primers from orthologous legume genes. of the SMC5/6 complex, cause uniparentally-inherited abnormal seed development characterized by poor embryo development and liquid endosperm. Houben, A., and Schubert, I. (2003). DNA and proteins of plant centromeres. Current Opinion in Plant Biology 6, 554–560. By searching for the cause of this defect, we noticed that hpy2 plants have Malik, H.S., and Henikoff, S. (2003). Phylogenomics of the nucleosome. Nat Struct Biol 10, 882–891. reduced pollen viability and produce pollen of variable sizes. Because large size of Neumann, P., Pavlíková, Z., Koblízková, A., Fuková, I., Jedličková, V., Novák, P., and Macas, J. (2015). Centromeres off the hook: massive changes in centromere size and structure following duplication of plant organs is often associated with higher polyploidy, we measured ploidy of the CenH3 gene in Fabeae species. Mol Biol Evol 32, 1862–1879. hpy2 offspring and found that some plants are triploid. This suggested that SMC5/6 Tek, A.L., Kashihara, K., Murata, M., and Nagaki, K. (2010). Functional centromeres in soybean include two complex has unknown function in gametogenesis and seed development leading to distinct tandem repeats and a retrotransposon. Chromosome Res 18, 337–347. polyploidization of the progeny plants. We will show molecular and developmental phenotypes and will discuss possible causes of these defects.
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Poster WG3.1 UNCOVERING ZEA MAYS LONG NON-CODING RNA REGULATORY FUNCTION IN RESPONSE TO LONG-TERM WATER STRESS
Armine Asatryan and Serena Varotto
Department of Agronomy, Food, Natural Resources, Animals and the Environment; University of Padova, Italy
Environmental stresses, often associated with climate changes, impose widespread deleterious effects to agricultural systems, resulting in the reduction of yield and food quality for most crops. Zea mays (maize) is a model plant, one of those lucrative crops that is highly affected by water stress, including in the territory of all EU Countries (Bänziger and Araus, 2007). Only the knowledge of the genetic and epigenetic mechanisms governing plant adaptation to the environment can provide smart molecular tools for crop improvement and sustainable production of maize populations.
lncRNAs as novel riboregulators of gene expression are versatile molecules able to perform numerous tasks during both in normal and in stressed cells through binding of proteins, DNA or other RNA molecules. Despite this, lncRNAs can act as epigenetic mediators and lead to changes in chromatin structure without altering the underlying DNA sequence. Instead, alterations in chromatin architecture govern gene expression programs and regulate cellular function. Although the importance of both chromatin regulation and lncRNAs in plant stress response is generally recognized (Xu et al., 2017), understanding of the functions and mechanisms of chromatin modifications and precise role of non-coding RNAs in chromatin processes remains largely unknown and therefore, becomes the major goal of current project. Following presentation in INDEPTH COST Consortium will delineate CORN-UniPd project scheme, as well as, it will underline proposed collaboration need within Mobility Program in the frame of INDEPTH COST Action.
Bänziger M., Araus JL. (2007) Recent Advances in Breeding Maize for Drought and Salinity Stress Tolerance. In: Jenks M.A., Hasegawa P.M., Jain S.M. (eds) Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops. Springer, Dordrecht, pp 587-601. Xu, J., Wang, Q., Freeling, M., Zhang, X., Xu, Y., Mao, Y., Tang, X., Wu, F., Lan, H., Cao, M., Rong, T., Lisch, D., … Lu, Y. (2017). Natural antisense transcripts are significantly involved in regulation of drought stress in maize. Nucleic acids research, 45(9), 5126- 5141.
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Poster WG3.2 Poster WG3.3 MEIOTIC CONSEQUENCES OF CHROMOSOME DOUBLING ON EFFECT OF SUMMER DROUGHT AND ROOT ROT ON SUGAR MUTANTS DEFECTIVE IN NUCLEAR ENVELOPE-ASSOCIATED BEET YIELD IN VARIETY REGISTRATION TRIALS PROTEINS Zivko Curcic1, Ksenija Taski Ajdukovic1, Nevena Nagl1 and Francesco Blasio, Nadia Fernández, Bianca Martín, Juan L. Santos Katarina Zarubica2 and Mónica Pradillo 1- Institute of Field and Vegetable Crops, Maksima Gorkog 30, Serbia, Novi Sad Departamento de Genética, Fisiología y Microbiología; Facultad de Ciencias 21000 Biológicas; Universidad Complutense de Madrid; Spain. 2- Ministry of Agriculture and Environment, Republic of Serbia, Nemanjina 22- 26 – Serbia, Belgrade 11000 In autopolyploids, the presence of more than two homologous chromosomes produces complex synaptic interactions involving synaptic partner switches (SPSs) during Climate changes negatively affect the sugar beet production, especially in the prophase I. SPSs are preferentially located near the chromosome ends (subterminal southern and central parts of the Europe. In agroecological conditions of Serbia during or distal regions). These associations are frequently consolidated by chiasmata and the last few years the largest losses in yield and quality of sugar beet comes from high contribute to multivalent formation. These phenomena compromise meiotic stability, percent of root rot caused by extreme drought in second period of sugar beet vegetation. since they may result in unbalanced segregation of chromosomes during the first meiotic Before a new variety can be placed on the market in Serbia, it must be on the List of division, leading to aneuploidy and reduced fertility. registered varieties of agricultural plants. The registration of a variety is decided by the Ministry of Agriculture and Environment. SUN proteins are inner nuclear envelope (NE) elements that control chromosome movements in meiosis. During early prophase I, these proteins connect In order to be approved, new varieties are evaluated regarding their agronomic, the telomeres with the force generating mechanisms in the cytoplasm. In Arabidopsis technological and environmental value in the national testing network. Field trials thaliana, the absence of the C-terminal SUN domain proteins (AtSUN1 and AtSUN2) for sugar beet hybrid registration so far include an assessment of root yield, inverted affects meiotic progression. Indeed, the double mutant Atsun1 Atsun2 fails to complete sugar, resistance to most significance diseases in Serbia Rhizomania,( Cercospora synapsis and presents a reduction in the mean cell chiasma frequency. In addition, this beticola, Rhizoctonia solani) and percent of root rot. In 2018, thirteen sugar beet hybrids mutant has an increase in the number of synapsis initiation points (SIPs) at early zygotene underwent evaluation in registration field trials. Data analysis showed no difference and unresolved interlock-like structures at late prophase I in comparison to wild-type between hybrids for potential yield, but ANOVA for root rot percent, estimated yield and plants. yield loss revealed significant differences (p>0.001) between hybrids. Intensive drought in August and September had negative effect on sugar beet production and probably In this study, we analyze the consequences of doubling chromosomes when contributed to occurrence of high root rot. AtSUN1 and AtSUN2 are not functional. Studying meiosis in these circumstances may help to gain a better understanding of the importance of the NE-associated meiotic The results indicate that, in order to reduce yield losses, future sugar beet chromosomal movements when more than two homologous chromosomes are available. hybrids registered for growing in this environmental conditions must have increased tolerance to extreme drought and root rot.
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Poster WG3.4 Poster WG3.5 DROUGHT STRESS AND RECOVERY PERIOD INDICATE CHARACTERIZATION OF HvDME, A BARLEY EPIGENETIC CRITICAL EPIGENETIC CONTROL POINTS IN STRESS REGULATOR, IN RESPONSE TO DROUGHT STRESS ADAPTATION AND FLOWERING REGULATION, PROVIDING 1# 1 1 INSIGHTS INTO EPIGENETIC MEMORY IN ZEA MAYS Aliki Kapazoglou , Vicky Drossou , Anagnostis Argiriou , and Athanasios Tsaftaris1,2,3 Cristian Forestan1, Farinati S.1, Pavesi G.2, Rossi V.3 and Varotto S.1 1- Institute of Applied Biosciences (INAB), CERTH, Thermi-Thessaloniki, GR- 1- Department of Agronomy Animals Food Natural Resources and Environment 57001, Greece (DAFNAE), University of Padova, Viale dell’Università 16, 35020 Legnaro (Italy) #current address: Institute of Olive tree, Subtropical Crops and Viticulture, 2- Department of Biosciences, University of Milan, Via Celoria 26, 20133 Hellenic Agricultural Organization (Demeter), Lykovrysi, Athens, GR-14123, Milano (Italy) Greece 3- CREA - Unità di Ricerca per la Maiscoltura, Via Stezzano 24, 24126 Bergamo 2- Department of Genetics and Plant Breeding, Aristotle University of (Italy) Thessaloniki, Thessaloniki, GR-54124, Greece 3- Perrotis College, American Farm School, Thessaloniki, Greece During their lifespan, plants are exposed to a multitude of stressful factors that affects their development and reproductive fitness. When subjected to stressful The response of plants to abiotic stress like drought is regulated in part by conditions, plants rapidly respond and adapt through a sophisticated variety of epigenetic mechanisms such as DNA methylation, histone modifications and the action physiological, biochemical, transcriptional, and epigenetic mechanisms. Dynamic of small RNAs which modify chromatin structure and profoundly affect gene expression changes in chromatin structure and concomitant transcriptional variations play an and cellular function. important role not only in stress response, but are also involved in epigenetic memory mechanisms. Histone marks and gene expression patterns could be indeed stably Barley is a crop of high agronomic importance in Europe and worldwide. In maintained during cell division, once the triggering stimulus has been removed. order to investigate the molecular mechanisms that underlie epigenetic modifications There is good evidence that chromatin may play a pivotal role in somatic memory upon exposure to drought stress a series of chromatin modifiers have been characterized phenomena and although many progresses have been made in understanding chromatin in barley. Among a number of genes investigated in the past a gene encoding a DNA modifications implicated in plant response to environmental triggering conditions, we are glycosylase, HvDME, was found to be markedly induced under conditions of drought still far from connecting molecular genetics and developmental data around environment stress in a cultivar-dependent manner. In addition, a barley miRNA, HvmiR1126, was and chromatin. shown to exhibit near 100% complementarity to a region within an intron in the HvDME gene body, pointing to a putative miRNA target site and suggesting gene expression Through the integration of RNA-Seq and ChIP-Seq data from maize plants control by miRNA mechanisms. subjected to a mild and prolonged drought stress and after the complete recovery from the stress, we identified several stress- responsive genes in which stress-induced Moreover, the DNA methylation status in the promoter and gene body of transcriptional and histone marks variations persist after the stress removal and the HvDME was examined in order to further explore epigenetic regulation of a barley DNA recovery stage. The stress-induced stable chromatin environments and expression of these demethylase homologue in response to drought stress. genes could represent a coordinated strategy for plants to cope with drought stress and rapidly adapt to recurring stresses. Drought stress caused transcriptional and chromatin Understanding epigenetic cellular processes that take place under drought will changes also at many genes involved in flowering regulation and inflorescences enhance our knowledge of the molecular mechanisms associated with the response to patterning. Particularly interesting is the stress induced delay of chromatin marks level abiotic factors in barley and other cereals and may be utilized for the development of variation that are normally associated to the increase in expression levels of two flowering improved cultivars able to better cope with stresses and the ongoing climatic change. associated MADS-box coding genes: both chromatin marks and the associated mRNA levels persist after the stress removal, underlining stress memory and resulting in the stress induced alteration of flowering time and inflorescence development.
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Poster WG3.6 Poster WG3.7 GRAIN PROTEIN CONTENT QTLS IDENTIFIED IN A DURUM × GENOME-WIDE ANALYSIS OF MIKC-TYPE MADS-BOX GENES WILD EMMER WHEAT MAPPING POPULATION TESTED IN FIVE IN WHEAT ENVIRONMENTS Susanne Schilling1, Alice Kennedy1,2, Sirui Pan1, Lars Jermiin1,3 and Andrii Fatiukha1, Naveh Filler1, Gabriel Lidzbarsky1, Abraham B. Rainer Melzer1 Korol1, Curtis J Pozniak2, Tzion Fahima1 and Tamar Krugman1 1- School of Biology and Environmental Science, University College Dublin, 1- Institute of Evolution and the Department of Evolutionary and Environmental Ireland Biology, Faculty of Natural Sciences, University of Haifa, Haifa, Israel 2- Institute for Microbiology and Plant Biology, Katholieke Universiteit Leuven, 2- University of Saskatchewan, Saskatoon, SK, Canada. Belgium 3- Research School of Biology, Australian National University, Australia Wild emmer wheat (WEW) (Triticum turgidum ssp. dicoccoides, genome BBAA) was shown to confer high values of grain protein content (GPC) and therefore harbors Wheat (Triticum aestivum) is one of the most important crops worldwide. great potential for improvement of cultivated wheat nutrition value. A recombinant inbred Facilitating wheat breeding and the fine-tuning of important traits such as stress line (RIL) population derived from a cross between T. durum var. Svevo and WEW (acc. resistance, yield and plant architecture are of great importance, given a growing global Y12-3) was used for QTL mapping of GPC. Genotyping of 208 F6 RILs yielded 4,166 population paired with more challenging climate and therefore cultivation conditions. polymorphic SNP markers that were used to construct an ultra-dense genetic map. MADS-box genes encode for transcription factors that regulate virtually all aspects of plant development. Especially MIKC-type (type II) MADS-box genes are involved in The map contained 1,510 skeleton markers, with a total length of 2,169 cM and flowering time control, plant and root architecture, flower and seed development as an average distance of 1.5 cM between SNPs. GPC was evaluated in the RIL population well as stress responses. Further on, the family of plant MADS-box genes with their large grown in range of five environments. These included rain-shelter greenhouse and open number of genes is ideal to study evolution of genes after gene duplication. field with low annual precipitation, in which plants were grown in water-limited and well-watered conditions. Total of 13 GPC QTLs were mapped, with LOD ranging from We present a detailed overview of number, phylogeny, and expression of 3.6 to 27.8, and PEV ranging from 0.8 to 24.4%. For 12 of the QTLs the favorable allele MIKC-type MADS-box genes in Triticum aestivum (bread wheat). We find that wheat was contributed by WEW. has 201 type II MADS-box genes and has members in all major monocot MIKC-type clades. Using in silico analyses we deduced a relatively strong conservation of expression Significant effects were obtained for 10 QTLs under all environments, and pattern within each subfamily, indicating functional similarity among closely related three under greenhouse conditions only. Major QTLs with favorable alleles from WEW homologs. Furthermore, chromosomal location appears to be correlated to putative were identified on chromosomes 4BS, 5AS, 6BS and 7BL. The QTLs showed high LODs pseudogenization and tandem duplication. and short intervals, therefore reducing number of candidate genes. The number of high confidence genes within QTL intervals ranged from 5 to 653 genes. These results We will use the identified MADS-box genes to screen for sequence variation demonstrate the importance of WEW gene pool as a source for novel alleles to improve among different wheat lines. This will provide a starting point to reveal how allelic GPC in cultivated wheat. variation in MADS-box genes may affect agronomically important traits in wheat.
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Poster WG3.8 Poster WG3.9 SUNFLOWER REACTION TO COMBINED BROOMRAPE – APPROACHING DWARFING PHENOTYPE IN WHEAT BY DOWNY MILDEW ATTACK – PRELIMINARY STUDY ATAC-SEQ
Dragana Miladinovic1, Ivan Citakovic2, Boško Dedic1, Sandra Pavla Navratilova Cvejic1, Aleksandra Radanovic1, Siniša Jocic1, Milan Jockovic1, Ana Marjanovic Jeromela1 and Jelena Samardžic3 Centre of Plant Structural and Functional Genomics Slechtitelu 31 CZ-78371 Olomouc - Holice, Czech Republic 1- Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad Serbia Genetic manipulation of Gibberelin (GA) metabolic pathway is a succesfully 2- Faculty of Biology, University of Belgrade, Studentski trg 16, Belgrade, Serbia used method to improve agricultural features of crops. Previously generated semidwarf 3- Institute for Molecular Genetics and Genetic Engineering, University of durum wheat cultivar Icaro has been characterized as a GA-insensitive mutant with Belgrade, Vojvode Stepe 444a, Belgrade, Serbia increased degradation of GA precursor caused by significant overexpression of GA 2-oxidase. As the nucleotide sequences of the predicted ORF of GA 2-oxidase were During the evolution plants have developed a large set of defense mechanisms identical in the mutant Icaro and the original tall durum wheat variety Anhinga, we against pathogens. Defense responses have been studied in details in model plant species hypothesize that increased gene expression of this enzyme could result from disrupted such as Arabidopsis. However, to complete our understanding of resistance mechanisms, transcriptional regulation involving non-coding DNA. it is important to study defense responses in non-model plants, such as sunflower. In our experiment we aimed to study expression of defense-related genes in the sunflower host Common methods to assess the regulatory landscape of a locus involve plant during combined infection with downy mildew and broomrape and elucidate their methylome and histone modification profiling, characterizing open chromatin regions in role in sunflower reaction to attack of different pathogens. order to map DNA regulatory elements and revealing long-range interactions between promoters and cis-regulatory DNA. We plan to employ previously developed assay for Downey-mildew-resistant genotype was inoculated with the pathogen and transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), a method planted into the broomrape inoculated soil. For gene expression analysis, leaf samples in which hyperactive Tn5 transposase loaded with DNA adaptors selectively inserts the were taken at different stages from plants with combined broomrape-downy mildew adaptors into accessible regions within living cells. Comparison of the ATAC- seq peak infection, plants only infected with broomrape, and control plants. Expression of defense profiles of the GA-2 oxidase loci in the mutant semidwarf Icaro and tall Anhinga wheat related genes is analyzed using RT-PCR, with special focus on defensin. varieties could help to decipher the causative variation of chromatin organization. v Acknowledgements This study was supported by Ministry of Education, Science and Technological Development of Republic of Serbia, project TR31025, Provincial Secretariat for Higher Education and Science of Vojvodina, project 114-451-2126/2016-03, and COST Action CA 16212.
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Poster WG3.10 Poster WG3.11 SMC5/6 COMPLEX – LINKING DNA DAMAGE REPAIR WITH THE STRUCTURAL AND FUNCTIONAL ORGANIZATION OF CHROMATIN ORGANIZATION PROPHASE I NUCLEI IN WHEAT-RYE HYBRIDS
Ales Pecinka Loginova D.B, Schubert V., Houben A., Elena Salina and Silkova O.G. Institute of Experimental Botany AS CR, Šlechtitelu 31, Olomouc, Czech Republic ICG SB RAS, Lavrentjev ave.10., Novosibirsk, Russia, 630090 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Eukaryotic Structural maintenance of chromosomes (SMC) complexes include Gatersleben, Germany cohesin, condensin and SMC5/6. Cohesin connects sister chromatids during cell divisions and helps forming regulatory chromatin loops. Condensin complexes mediate chromatin In intergeneric wheat hybrids, there is no chromosome pairing. In this regard, and chromosome compaction throughout cell cycle. SMC5/6 complex is involved in the goal of the present study was to understand the structural and functional organization maintenance of genome stability and DNA damage repair. All three complexes are of prophase I nuclei in wheat-rye hybrids with different genetic background [1]. essential for functions and survival of eukaryotic cells. Combination of immunostaining with antibodies against ASY1, CENH3, and ZYP1 with high-resolution microscopy (3D-SIM) enable to study the centromere dynamics and We will present our efforts in characterization of SMC5/6 complex functions in organization as well as organization of the synaptonemal complex (SC). plants. In Arabidopsis, this complex consists of SMC5 and SMC6 (SMC6A and SMC6B paralogs) heterodimeric backbone to which six NON-SMC ELEMENTS are connecting At early leptotene CENH3 signals differed in number, size and shape. During and perform different functions. While it is known that this complex is required for leptotene a different degree of centromere condensation has been observed between the normal levels of homologous recombination and sister chromatid pairing upon DNA individual chromosomes. The formation of 8 to 16 (mostly 14) centromere clusters were damage in plants, recent studies suggest that SMC5/6 complex influences many other, observed in 1Rv(1A)xR, 6R(6A)xR and 2R(2D)xR hybrids. In 2R(2D)xR, some of CENH3 including developmental and stress response, processes. signals (mostly 4) differed in size (bigger) and had irregular shapes (star- shaped), possibly due to the combination of several centromeres. We analysed two uncharacterized NSE4A subunits of this complex in Arabidopsis and demonstrate that one of the paralogs is essential, while the other is The SC component loading at leptotene and at early zygotene, as well as the SC expressed only during specific stages of reproductive development. Furthermore, NSE4A dynamics at diplotene until diakinesis in wheat is similar to the SC dynamics described and NSE4B are partially functionally diversified at the protein level. Interestingly, we for rye, but differs from that of rye during pachytene [2]. Disappearance of anti-ASY1 after found that SMC5/6 complex contributes to heterochromatin organization in Arabidopsis full loading of ZYP1 at zygotene and pachytene was observed also in the meiocytes of the and is essential for seed development in Arabidopsis and barley. This makes SMC5/6 wheat-rye hybrids 2R(2D)xR, 1Rv(1A)xR and 6R(6A)xR. Despite the lack of homologues complex universal player connecting maintenance of genome stability with chromatin in the wheat-rye hybrids, the loading of ZYP1 occurred. Multiple long extended (linear) organization. signals of ZYP1 appeared. However, at diakinesis almost all chromosomes are univalent, which indicates desynapse and the normal functioning of Ph-genes.
The work was supported by COST-STSM (CA16212 – 41305).
[1] Silkova, O.G., and Loginova, D.B. (2016). Sister chromatid separation and monopolar spindle organization in the first meiosis as two mechanisms of unreduced gametes formation in wheat-rye hybrids. Plant Reprod 29(1- 2),199- 213. DOI:10.1007/s00497-016-0279-5 [2] Hesse, S., Zelkowski, M., Mikhailova, E., Keijzer, K., Houben, A., Schubert, V. (2018). Ultra structure and dynamics of synaptonemal complex components during meiotic pairing of rye A and B chromosomes. Frontiers In Plant Science, in press
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Abstract WG3.12 Poster WG3.13 MORPHOLOGICAL CHANGES AND EPIGENETIC ANALYSIS OF MODIFICATION OF EPIGENETIC MARKS BY THE HISTONE TWO OF TWO MEDICAGO SATIVA L. GREEK VARIETIES UNDER DEACETYLASE INHIBITOR TRICHOSTATIN A PROMOTES DROUGHT STRESS CONDITIONS WHEAT MICROSPORE EMBRYOGENESIS
Y.Ventouris1, Eleni Tani1, E. Avramidou2, D. Vlachostergios3, S. Maria Pilar Vallés, I. Valero, S. Allúe, A. Costar and A. M. Castillo Chorianopoulou4 and E. Abraham5 Aula Dei Experimental Station, The Council for Scientific Research (EEAD-CSIC), 1- Agricultural University of Athens, Department of Crop Science, Laboratory of Avda Montañana 1005, 50059 Zaragoza, Spain Plant Breeding and Biometry, Iera Odos 77, 11855, Athens, Greece 2- Hao Demeter, Institute of Mediterranean & Forest EcosystemsTerma Histone acetylation is a reversible epigenetic mechanism that plays a key role in the regulation of plant development, defense and adaptation. This histone modification Alkmanos, 11528, Athens modulates the structure of the chromatin, acting on its degree of condensation and 3- HAO-Demeter, Institute of Industrial and Forage Crops. Theofrastou Street 1, determining the activation or silencing of a genomic region. In microspore embryogenesis 41335,Greece (ME), microspores triggered by stress deviate from the gamethopytic towards the 4- Agricultural University of Athens, Department of Crop Science, Laboratory of embryogenic developmental pathway leading to doubled haploid plant production. Plant Physiology and Morphology, Iera Odos 77, 11855, Athens, Greece Histone acetylation levels have been associated with the ability of microspore 5- Faculty of Agriculture, Forestry and Natural Environment, School of reprogramming (Rodríguez-Sanz et al., 2014; Pandey et al., 2017). The efficacy of new Forestry and Natural Environment, Aristotle University of Thessaloniki, 54124 strategies for microspore embryogenesis induction based on the application of histone Thessaloniki, Greece deacetylase inhibitors has been described (Li et al., 2014; Jiang et al., 2017).
Alfalfa (Medicago sativa L. subsp. sativa) is considered as the most important In this study, blocking histone deacetylase activity by the application of forage crop worldwide, providing fodder of high nutritional value. Although alfalfa is trichostatin A (TSA) enhanced wheat microspore embryogenesis. The effect of TSA generally characterised as drought resistant species, the study of its response to drought depended on the genotype, the dose and the way it is applied: phase of the process stress, along with the development of tolerant to water deficit varieties are of a great and its combination with different stress treatment. The application of TSA during a importance. The induction of certain epigenetic modifications such as cytosine (C) starvation and high osmotic pressure treatment with mannitol had the greatest effect. methylation across the genome plays a crucial role in plant drought stress acclimation In these conditions, TSA produced a higher increase in the microspore induction rate through the formation of “epigenetic memory”. The aims of this study were : First, the in a recalcitrant genotype than in a model one. Up to 5.5-fold increase in the number evaluation of two Greek alfalfa varieties (namely “Lamia” and “Cheronia”) under water of divisions and 3.6-fold in the number of embryos and green plants was obtained as deficient conditions, and second the investigation of whether any kind of post-exposure compared with mannitol treatment. “epigenetic memory” persists in plants. Further research is under way to understand the histone deacetylases For this purpose, the following parameters were measured: root and shoot dry mechanism underlying microspore embryogenic induction and the different biological weight, root and shoot water content, the shoot/ root dry weight ratio. Also, a genome- processes associated. wide assessment of the methylation status of the CCGG sequence was conducted using Rodríguez-Sanz, H. et al (2014). Changes in Histone Methylation and Acetylation during Microspore the MSAP (Methylation Sensitive Amplification Polymorphism) method. Our results Reprogramming to Embryogenesis Occur Concomitantly with BnHKMT and BnHAT Expression and Are Associated manifest that although “Lamia” was capable of sustaining relatively high biomass with Cell Totipotency, Proliferation, and Differentiation in Brassica napus. Cytogenetics and Genomic Research, Vol.143, No. 1-3a. production under stress, “Cheronia” demonstrated superior acclimation to water deficient Pandey, P et al (2017). Dynamics of post-translationally modified histones during barley pollen embryogenesis in conditions. Concerning epigenetics, and DNA methylation in particular, there is clear the presence or absence of the epi-drug trichostatin A. Plant Reproduction 30:95-105. evidence that pre-exposure to drought results in the generation of “epigenetic memory” in Li, H. et al (2014). The Histone Deacetylase Inhibitor Trichostatin A Promotes Totipotency in the Male stressed plants. This was the case particularly for “Cheronia”. Gametophyte. The Plant Cell, Vol. 26: 195–209. Jiang, F. et al. (2017). Trichostatin A increases embryo and green plant regeneration in wheat. Plant Cell Reports 1-6.
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Poster WG3.14 Poster WG3.15 IS CHROMATIN INVOLVED IN THE SHORT- AND LONG-TERM APPLICATION OF FULLERENOL NANOPARTICLES MITIGATES MEMORY OF WATER STRESS IN MAIZE? EFFECTS OF DROUGHT IN SUGAR BEET PLANTS
Cristian Forestan, Silvia Farinati, Vincenzo Rossi,Giulio Pavesi and Ksenjia Taski-Ajdukovic1, I. Icevic Borisev2, D. Arsenov2, M. Serena Varotto Zupunski2, M. Borisev2, Z. Curcic1 and A. Djordjevic2
Department of Agronomy Animals Food Natural Resources and Environment 1- Institute of Field and Vegetable Crops, Maksima Gorkog 30, Novi Sad, Serbia (DAFNAE), University of Padova, Viale dell’Università 16, 35020 Legnaro (Italy) 2-Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 2, Novi Sad, Serbia. To study the effect of the water stress at epigenetic level we used a ChIP-Seq approach, focusing our attention on drought stress (that based on transcriptome analysis Drought is the major environmental factor limiting yield loss for the sugar beet was the most effective one) and on these histone modifications: H3K4me3, H3K9Ac and (Beta vulgaris L.) crop. However, mechanisms for reducing drought stress are lacking, H3K27me3, the first two modifications are associated to gene transcription activation since sugar beets are mostly produced without irrigation and drought tolerant varieties the third one to gene silencing of developmental regulated genes. Stress was applied for are not available. There are reasons to believe that fullerenol nanoparticles (FNPs) ability 10 days and after a period of 7 days recovery, the leaf transcriptome and the dynamics to form hydrogen bonds with water molecules makes this nanoparticle a potential of H3K9ac, H3K4me3 and H3K27me3 were analyzed in the leaf of treated and control intracellular water depot, which can be used if osmotic stress occurs. plants. The research was conducted to determine whether FNPs could mitigate drought In stressed plants compared to control, we observed variations in gene effects on sugar beet. Foliar application of 0 (control), 0.01 (F1 treatment) and 0.001 (F2 expression correlated to histone modification dynamics, which effects were proven to treatment) nmol fullerenol solution was performed after four months of sugar beet plant determine phenotypic alteration in flowering time. What this study is telling us is that growth in greenhouse. Moderate (20-30%) and severe (10-20%) water deficit regimens in maize leaf under stress, epigenetic memories can involve some genes, which among were used with a control of 60-70% soil water capacity. The results show that drought others comprised some important developmental regulated transcription factors able stress significantly increased proline content in control plants, but not in FNP treated to possibly delay flowering time after the recovery from the stress. Stress recovery was plants. particularly important for observing the stress memory at transcriptional and epigenetic level within the same plant generation. Because we were interested in evaluating if a The activity of antioxidant enzymes and the expression of their genes, as well memory of the stress could be inherited in following generations by using a Single Seed as malondialdehyde and reduced glutathione content indicate that foliar application of Descent (SSD) procedure we produced some maize accumulation mutation lines. fullerenol can alleviate oxidative stress in plants under drought conditions. Although further studies are necessary to elucidate the impact of FNPs on plants; results indicate These lines were subjected for five successive generations to the same stress that FNPs can mitigate drought stress on sugar beet plants and by doing so, may provide a protocol: we finally grown the sixth generation and re-sequenced the genomes of some tool for reducing the economic loss caused by dry conditions during crop production. lines/plants in collaboration with IGA Institute in Udine Italy. We are currently validating some indels mutations that we identified, although no significant structural variations were observed in the genome of the plants.
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Poster WG4.1 Poster WG4.2 TRANSGENE SILENCING IN 3D – HOW A CHROMOSOMAL SETTING UP THE STAGE FOR FUNCTIONAL ANALYSIS OF KNOT CAN INACTIVATE FOREIGN DNA ELEMENTS NUCLEAR PROTEINS IN BARLEY
Stefan Grob and Ueli Grossniklaus Beáta Petrovská, Zdenek Perutka, Tomáš Vleko, Nicolas Blavet, Jan Vrána, Ludmila Ohnoutková, Jaroslav Doležel, Marek Šebela and Institute of Plant and Microbial Biology, University of Zurich Aleš Pecinka
Cells require elaborate mechanisms to efficiently pack chromosomes in the nucleus, while still allowing access to the genetic information. In addition, three- Centre of the Region Haná for Biotechnological and Agricultural Research, dimensional (3D) chromosome architecture is linked to epigenetic processes and Institute of Experimental Botany AS CR, Šlechtitelu 31, Olomouc, Czech transcriptional activity. Despite progress in the field, well-established cases of functional Republic relationships between transcription and 3D chromatin architecture remain rare. We previously identified a 3D chromatin structure in Arabidopsis termed the KNOT, in which Chromatin functions are controlled by complexes of nuclear proteins and ten genomic regions (KEEs) physically contact each other. nucleic acids. However, populations of nuclear proteins and their dynamics in different cells remain poorly understood in plants. We determined proteins in G1, S and G2 Here we show that KEEs are involved in the silencing of transgenes. Transgenes stages of root meristematic cells in barley (Hordeum vulgare) using combination of flow- integrated in the genome can fold towards the KNOT, coinciding with their transcriptional associated cell sorting and mass spectrometry. The initial data released in the UNcleProt silencing. Thus, transgene integration can lead to significant perturbation of 3D database (http://barley.gambrinus.ueb.cas.cz/), using the barley draft genome, were chromosome architecture. Regions adjacent to the insertion sites are not subjected to reanalysed and refined by adding more samples and comparison with the high quality silencing, despite their dislocation within the nucleus. This novel silencing mechanism, barley reference sequence. termed KNOT-linked Silencing (KLS) may act independently of previously described silencing mechanisms, as we cannot observe any significant contribution of small RNAs These data provide extensive view on the composition of barley nuclear and DNA methylation. KLS is heritable across generation and shows trans-silencing proteome and its dynamics during cell cycle in root tissues. With this, the UNcleProt effects, as the introduction of KNOT-silenced transgenes can lead to the silencing of database aims to be a resource for understanding the functions of nuclear proteins, previously active transgenes. nuclear architecture and their relationships to genome functions.
To apply the data generated within the UNcleProt database, we selected candidate proteins for functional analysis using Arabidopsis and barley mutants. The barley mutants are generated using CRISPR/Cas9 system and we will share our experience in using genome editing in this important crop. Our most advanced mutant candidate is NSE2/MMS21/HPY2, an evolutionary conserved E3 SUMO ligase of the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex, which is involved in maintenance of genome stability. We will present our initial data concerning NSE2 mutant characterization in barley.
This work was supported by the grant No. CZ.02.1.01/0.0/0.0/16_019/000082 7 (Plants as a Tool for Sustainable Global Development) from the Operational Programme Research, Development and Education, MEYS“.
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Poster WG4.3 Poster WG5.1 COUPLING HI-C WITH FLOW SORTING TO STUDY GARNET: A NETWORK TO SUPPORT UK PLANT SCIENCE. ARCHITECTURE OF PLANT CHROMATIN ACROSS THE CELL CYCLE Geraint Parry and James Murray
Petr Cápal1, Jan Vrána1, Veronika Kapustová1, Martin Mascher2, Sir Martin Evans Building, School of Biosciences, Cardiff University, Cardiff, CF10 3AX Tomáš Beseda1, Axel Himmelbach2, Nils Stein2, Jaroslav Doležel1 and Hana Šimková1 GARNet is a BBSRC sponsored network that aims to ensure the UK Arabidopsis and wider plant research community remains competitive and productive at the national 1- Centre of Plant Structural and Functional Genomics, Institute of Experimental and international level by helping researchers make the best use of available funding, Botany, Olomouc, Czech Republic tools and resources. GARNet represents UK Arabidopsis researchers via a committee of 2- Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, elected members.
Germany GARNet acts as information hub for the community via its newsletter, mailing list and provides a point of contact for researchers and funding agencies. Working with Hi-C is a method to study chromatin packaging in cell nuclei or mitotic other groups, GARNet helps to promote interactions between fundamental and applied chromosomes based on proximity ligation. Interacting DNA fragments obtained after plant sciences. GARNet also works to increase opportunities for UK Arabidopsis scientists chromatin crosslinking are sequenced and analyzed, resulting in interaction-frequency at the international level. We actively respond to UK Government calls for evidence on maps across the genome, which can be used to model chromatin organization. policy areas relevant for the GARNet community, including on topics related to use of big data, gene- editing technology or on the impact of Brexit on UK Science and Innovation. Aiming to study plant chromatin dynamics during the cell cycle, we coupled the in situ Hi-C protocol with flow sorting, which enables purifying metaphase chromosomes The GARNet coordinator is also the current secretary for the Multinational or nuclei at particular phases of the cell cycle. Root tip meristems of barley (Hordeum Arabidopsis Steering Committee (MASC), which is an organisation that promotes global vulgare L.) cv. Morex was selected as source material, as it allows purification of both Arabidopsis research through support for a range of technical subcommittees, publication mitotic chromosomes and nuclei from the same tissue, treated exactly the same way. of a annual report on the state of Arabidopsis work and by organisation of the annual International Conference on Arabidopsis Research (ICAR). http://arabidopsisresearch.org/ index.php/en/
Geraint Parry is the Science Communication manager of the COST Action INDEPTH: ‘Impact of Nuclear Domains On Gene Expression and Plant Traits’ where he co-leads the dissemination and training opportunities for Action participants. https://www. brookes.ac.uk/indepth/
Website: https://www.garnetcommunity.org.uk/ Blog: http://blog.garnetcommunity.org.uk/ Twitter: @GARNetweets
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INDEPTH Prague Meeting INDEPTH Prague Meeting
Workshop Attendees Workshop Attendees Eleni ABRAHAM Rita ABRANCHES Valerie GAUDIN Danny GEELEN GREECE PORTUGAL FRANCE BELGIUM [email protected] [email protected] [email protected] [email protected] Bisa ANDOV Rafal ARCHACKI Tania GONZALEZ-OVIN Stefan GROB UK POLAND COST ASSOCIATION SWITZERLAND [email protected] [email protected] [email protected] [email protected] Armine ASATRYAN Zikrija AVDAGIC Moez HANIN David HONYS ARMENIA BOSNIA AND HERZEGOVINA TUNISIA CZECH REPUBLIC [email protected] [email protected] [email protected] [email protected] Célia BAROUX Francesco BLASIO Andreas HOUBEN Chunlian JIN SWITZERLAND SPAIN GERMANY BELGIUM [email protected] [email protected] [email protected] [email protected] Petr CAPAL Birsen CEVHER KESKIN Katerina KABELACOVA Kalyanikrishna KALYANIKRISHNA CZECH REPUBLIC TURKEY CZECH REPUBLIC GERMANY [email protected] [email protected] [email protected] [email protected] Zivko CURCIC Gwénaëlle DETOURNE ALIKI KAPAZOGLOU Solmaz KHOSRAVI SERBIA FRANCE GREECE GERMANY [email protected] [email protected] [email protected] [email protected] Martina DVORACKOVA David EVANS Tomas KONECNY Laszlo KOZMA-BOGNAR CZECH REPUBLIC UK CZECH REPUBLIC HUNGARY [email protected] [email protected] [email protected] [email protected] Tzion FAHIMA Sara FARRONA Kirsten KRAUSE Tamar KRUGMAN ISRAEL IRELAND NORWAY ISRAEL [email protected] [email protected] [email protected] [email protected] Dionysia FASOULA Nadia FERNANDEZ Ivona KUBALOVA Konstantin KUSTASHEV CYPRUS SPAIN CZECH REPUBLIC CZECH REPUBLIC [email protected] [email protected] [email protected] [email protected] Franziska FISCHER Cristian FORESTAN Vedad LETIC Francesca LOPEZ FRANCE ITALY BOSNIA AND HERZEGOVINA IRELAND [email protected] [email protected] [email protected] [email protected] Paul FRANSZ Andrew FRENCH Martin LYSAK NETHERLANDS UK CZECH REPUBLIC [email protected] [email protected] [email protected] 104 105
INDEPTH Prague Meeting INDEPTH Prague Meeting
Workshop Attendees Workshop Attendees Adela MACHELOVA Terezie MANDAKOVA Stefanie ROSA Elena SALINA CZECH REPUBLIC CZECH REPUBLIC SWEDEN RUSSIA [email protected] [email protected] [email protected] [email protected] Martin MASCHER Rainer MELZER Julio SALINAS Ana Paula SANTOS GERMANY IRELAND SPAIN PORTUGAL [email protected] [email protected] [email protected] [email protected] Sarah MERMET Anis MESCHICHI Daniel SCHUBERT Marek SEBELA FRANCE SWEDEN GERMANY CZECH REPUBLIC [email protected] [email protected] [email protected] [email protected] Christos MICHAILIDIS Dragana MILADINOVIC Wenbo SHAN Hana SIMKOVA CZECH REPUBLIC SERBIA CZECH REPUBLIC CZECH REPUBLIC [email protected] [email protected] [email protected] [email protected] Miguel MONTEZ Iva MOZGOVA Lauriane SIMON Amanda SOUZA CAMARA POLAND CZECH REPUBLIC SWEDEN GERMANY [email protected] [email protected] [email protected] [email protected] Pavla NAVRATILOVA Martina NESPOR DADEJOVA Paulina STACHULA Ioanna STAVRIDOU CZECH REPUBLIC CZECH REPUBLIC POLAND COST ASSOCIATION [email protected] [email protected] [email protected] [email protected] Geraint PARRY Ales PECINKA Szymon SWIEZEWSKI Eleni TANI UK CZECH REPUBLIC POLAND GREECE [email protected] [email protected] [email protected] [email protected] Beata PETROVSKA Dariusz PLEWCZYNSKI Ksenija TASKI-AJDUKOVIC Christophe TATOUT CZECH REPUBLIC POLAND SERBIA FRANCE [email protected] [email protected] [email protected] [email protected] Frédéric PONTVIANNE Mónica PRADILLO Gianluca TEANO Ahmet TEK FRANCE SPAIN FRANCE TURKEY [email protected] [email protected] [email protected] [email protected] Aline PROBST Dimiter PRODANOV Maria Pilar VALLES BRAU Serena VAROTTO FRANCE BELGIUM SPAIN ITALY [email protected] [email protected] [email protected] [email protected] Svenja REECK Eric RICHARDS Valya VASSILEVA Fen YANG SWEDEN USA BULGARIA CZECH REPUBLIC [email protected] [email protected] [email protected] [email protected] 106 107
SOCIETY FOR EXPERIMENTAL BIOLOGY PRESENTS: IMPACTINDEPTH OF CHROMATIN Prague DOMAINS Meeting INDEPTH Prague Meeting ON PLANT PHENOTYPES C E L L A N D P L A N T S E C T I O N S Y M P O S I U M 9 - 1 1 D E C E M B E R 2 0 1 9 REAL CENTRO UNIVERSITARIO ESCORIAL-MARIA CRISTINA, EL ESCORIAL, MADRID, SPAIN S E BIO LO G Y.O R G #ICDPP19
SEB POSTER
NUCLEUS IMPACT
ORGANISED BY INVITED SPEAKERS CONT’D • MONICA PRADILLO (COMPLUTENSE UNIVERSITY OF MADRID, SPAIN) • JIM MURRAY (CARDIFF UNIVERSITY, UK) • GERAINT PARRY (GARNET, CARDIFF UNIVERSITY, UK) • MOUSSA BENHAMED (INSTITUTE OF PLANT SCIENCES • A L I N E P R O B S T ( U N I V E R S I T É C L E R M O N T A U V E R G N E , F R A N C E ) PARIS-SACLAY, FRANCE) • CHRISTOPHE TATOUT (UNIVERSITÉ CLERMONT AUVERGNE, FRANCE) • N I L S S T E I N ( I P K G A T E R S L E B E N , G E R M A N Y ) • SILIN ZHONG(CHINESE UNIVERSITY OF HONG KONG, CHINA) INVITED SPEAKERS • STEVEN SPOEL (UNIVERSITY OF EDINBURGH, UK) • A L E S P E C I N K A ( I N S T I T U T E O F E X P E R I M E N T A L B O T A N Y O F T H E C Z E C• H WENDY BICKMORE (UNIVERSITY OF EDINBURGH, UK) ACADEMY OF SCIENCES, CZECH REPUBLIC) • XUEHUA ZHONG (UNIVERSITY OF WISCONSIN-MADISON, USA) • ARP SCHNITTGER (UNIVERSITY OF HAMBURG, GERMANY) • CÉLIA BAROUX (UNIVERSITY OF ZÜRICH, SWITZERLAND) IN COLLABORATION WITH • CRISANTO GUTIERREZ (CBMSO, SPAIN) • DARIUSZ PLEWCZYNSKI (UNIVERSITY OF WARSAW, POLAND) • EIRINI KAESERLI (UNIVERSITY OF GLASGOW, UK) • IRIS MEIER (OHIO STATE UNIVERSITY, USA) 108• I S A B E L B Ä U R L E ( U N I V E R S I T Ä T P O T S D A M , G E R M A N Y )