Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Posters - Session 1- High resolution RNA expression map of Arabidopsis root reveals alternative splicing regulation Masashi YAMADA, Song Li2, Uwe Ohler3, Philip Benfey1 1Duke University, Department of Biology and HHMI, Durham, NC, 2Virginia Polytechnic Institute and State University, Department of Crop and Soil Environmental Sciences, Blacksburg, VA, 3Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany ABSTRACT Alternative splicing is an important mechanism for increasing transcriptome plasticity and proteome diversity in eukaryotes. Alternative splicing has been well studied at functional level in Animals. It has been known that alternative splicing isoforms expressing in the specific tissues or at the specific developmental stages control animal development. However in plants, it was not clear where alternative splicing isoforms are expressing and what is a developmental role of alternative splicing isoforms. The shoot apical meristem and root apical meristem are continuously located at the growing tips of the plant and give rise to various organs. To understand the function of alternative splicing isoforms at the cell type and developmental specific level in Arabidopsis root meristem, I collected specific cells of root meristem expressing GFP driven cell type or developmental specific promoter. We did whole transcriptome analysis from nearly all cell types in Arabidopsis root. We detected almost all known and many unknown alternative splicing isoforms in the specific cell type or developmental zones of the root meristem. Alternative splicing isoforms were more differentially expressing between the developmental zones, however not so much changing between specific cell types. These data suggest that alternative splicing isoforms control root meristem development rather than cell type specific development. To confirm this idea, we did over-expression of two isoforms of two transcription factors differentially expressing between the developmental zones. Over-expression of these transcription factors changed the development of root meristem. These data showed that alternative splicing isoforms regulate root meristem development. KEYWORDS Alternative splicing, root meristem, RNA-seq Alternative splacing in plant defence Lesley FOSTER, Systems Biology DTC, University of Warwick, Senate House, Gibbet Hill Road, Coventry, CV4 7ALKatherine Denby, School of Life Sciences and Warwick Systems Biology Centre, Gibbet Hill Road, Coventry, CV4 7AL ABSTRACT Alternative splicing (AS), the mechanisms whereby introns and exons are combined in different combinations enabling the same pre-mRNA to make many different mRNA transcripts, is an important regulatory mechanism of gene expression in eukaryotes. AS is a relatively unexplored area of gene regulation in plants, however, some components of the MOS4 associated complex (MAC), an evolutionary conserved component of the spliceosome, have been shown to effect the splicing patterns of the defence related genes SNC1 and RPS4. A splicing defect of SNC1, where introns 2 and 3 are retained, results in reduced levels of the R protein being produced (Xu et al., 2012).We have utilized next generation sequencing technologies to identify Arabidopsis thaliana genes that are differentially spliced in response to Botrytis cinerea infection. To further investigate the importance of differential splicing in effective plant defence and how it may be regulated, a variety of loss of function MAC mutants were investigated. Some of these mutants demonstrated enhanced susceptibility to B. cinerea and affected the splicing patterns of identified differentially spliced genes. These results taken together show that AS plays an important role in plant defense and indicate that the MAC may be involved in the underlying mechanisms. Understanding the mechanisms behind AS may enable plant engineering to be carried out at the level of post translational control enabling production of plants with increased disease resistance and helping to maintain food security in the 21st century. REFERENCES Xu, F., Xu, S., Wiermer, M., Zhang, Y., & Li, X. (2012). The cyclin L homolog MOS12 and the MOS4-associated complex are required for the proper splicing of plant resistance genes. The Plant Journal, 70(6), 916–928. doi:10.1111/j.1365-313X.2012.04906 .x At4g25290 splicing variants differently localize in the cell Justyna. ŁABUZ 1, C. Aggarwal,2, O. Sztatelman1,3, P. Zgłobicki1, A.K. Banaś1 1Department of Plant Biotechnology, Jagiellonian University, Krakow, Poland.2Department of Gene Expression, Adam Mickiewicz University, Poznan, Poland3present address: Department of Plant Biochemistry, IBBPAN, Warsaw, Poland. ABSTRACT The At4g25290 gene encodes a putative photolyase, an enzyme responsible for the photoreactivation of cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone dimers in DNA induced by UV-B irradiation.The At4g25290 protein contains two domains: a N-terminal photolyase (functional) and a C - terminal hydrolase. The two domain structure is conserved only in organisms which contain plastids, as At4g25290 homologs are found in algae and plants. The expression of At4g25290 is high in Arabidopsis aerial organs, as compared to roots. This gene is up-regulated by light. The analysis of mRNA isolated from leaves reveals that at least three At4g25290 splicing isoforms are expressed. Western Blot analysis confirms this result. The shortest splice variant, named micro, has only the photolyase domain, which is still functional. The longer isoform, named mini, may use an alternative start codon, as compared to full length At4g25290. Depending on the translation start it encodes only a hydrolase or a functional photolyase domain. Full length At4g25290 is expressed in chloroplasts and at the plasma membrane. The splice variants are differentially localized. Micro, the shortest isoform forms clumps at the plasma membrane and in the cytoplasm. Thus, it shows miss-localization. Mini, the longer one is found in the nucleus, at the plasma membrane and in chloroplasts in case of the photolyase domain. The splice variant containing only the hydrolase domain localizes in cytoplasm and nucleus. Role of AtNTR1 in transcription and splicing Grzegorz BRZYZEK1, Yanwu Guo2, PolandJakub Dolata3, Adam Mickiewicz4, Szymon Świeżewski5 1Institute of Biochemistry and BiophysicsPolish Academy of Sciences, Warsaw, Poland, 2Institute of Biochemistry and BiophysicsPolish Academy of Sciences, Warsaw, 3 Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, 4University in Poznan, Poland, 5Institute of Biochemistry and BiophysicsPolish Academy of Sciences, Warsaw, Poland ABSTRACT RNA splicing is multistage and complex process involving vast number of proteins and RNA molecules that form a dynamic complex - spliceosome. One of proteins involved the last step of splicing – disassembly and recycling of spliceosomal subunits is NTR1. NTR1 protein is involved in activation of PRP43 helicase, and is highly conserver across all eukaryotes.In higher eukaryotes like Arabidopsis thaliana, tightly controlled splicing is one of crucial steps in pre-mRNA maturation since most of genes harbor multiple introns. In addition alternative splicing can generate different mRNAs from single gene largely contributing proteome diversity.Majority of splicing occur co-transcriptionally allowing for kinetic coupling of transcription elongation rate and alternative splicing. So that polymerase transcription rate affects selection of alternative splice sites. atntr1 mutant plants show multiple phenotypes like low seed dormancy level and circadian clock defects. Alternative splicing profiling and PolII Chip data show that atntr1 mutant have splicing defects and PolII occupation changes consistent with kinetic coupling model. PolII occupation over the tested genes in atntr1 mutant were significantly reduced on the alternative splice sites suggesting that AtNTR1 protein lower the polymerase elongation rate on alternative splice sites and in turn affects more frequent selection of 3’ alternative splice sites. Our data suggest that AtNTR1 may play central role in kinetic coupling. Nevertheless true nature of AtNTR1 mediated PolII processivity changes is still unclear. Characterising RBP expression patterns and alternative splicing in response to cold temperature Nikoleta A. TZIOUTZIOU 1, Cristiane P. G. Calixto1, Runxuan Zhang 2, Allan B. James3, Craig G. Simpson2, Wenbin Guo1,2, Eduardo Eyras4, Hugh G. Nimmo3 and John W. S. Brown1,2 1University of Dundee, UK; 2James Hutton Institute, Dundee, UK; 3University of Glasgow, UK; 4Universitat Pompeu Fabra and ICREA, Barcelona, Spain. Abstract Gene expression patterns in plants change dramatically in response to environmental stimuli. Alternative splicing is highly implicated in the regulation of such changes at the posttranscriptional level, mediated by RNA binding proteins (RBPs). Previous studies demonstrated the importance of AS in regulating the expression of core clock genes under low temperature. However, the underlying mechanisms and factors that control coldsensitive AS are unknown. In Arabidopsis, more than 300 RBPs have been identified with serine/arginine rich proteins (SRs) and heterogeneous nuclear ribonucleoprotein particle proteins (hnRNPs) being the two main families. In order, ultimately, to build splicing networks to identify key splicing factors which are involved in the regulation of temperature-dependent AS, we have performed deep RNA-seq on a high resolution timecourse of Arabidopsis plants transferred from 20°C to 4°C. To quantify alternatively spliced transcript variants, a comprehensive non-redundant reference transcript dataset was constructed to analyse the RNA-seq data. Dynamic data for the expression of individual transcripts have been generated, giving us the opportunity to study the effect of low temperature on the AS and expression changes of Arabidopsis RBPs, SFs and spliceosomal protein genes. So far, RBPs with either no change in expression and/or AS or major changes in both expression and AS in response to cold have been identified, including isoform switches that occur rapidly after transfer to cold. Furthermore, the dynamic RNA-seq dataset has allowed the identification of genes which have not previously been associated with the cold response. The RNA-seq data is currently being prepared for construction of transcription and co-splicing networks aiming to identify genes that may regulate alternative splicing in response to low temperature, with emphasis given to the core clock genes. Identification of Regulators of Polypyrimidine Tract Binding Protein Splicing Factors in Arabidopsis Tanja DÖTSCH (1) , Stauffer Eva (1) , Wachter Andreas (1) (1) Center for Plant Molecular Biology (ZMBP), University of Tübingen, Auf der Morgenstelle 32, 72076 Tübingen, GERMANY Abstract Alternative splicing (AS) is widespread in plants, affecting more than 60 % of all introncontaining genes in Arabidopsis thaliana, and has been linked to fundamental aspects of plant development. The AS outcome depends on the action of splicing factors, only few of which have been examined in plants so far. Among those, the Polypyrimidine Tract Binding Proteins (PTBs) AtPTB1 and AtPTB2, members of the heterogeneous nuclear ribonucleoproteins (hnRNPs), have been demonstrated to regulate several hundred AS events in A. thaliana. Given that AS decisions are typically the result of an interplay of diverse factors, we are interested in the identification of modulators of PTB activity. In an unbiased approach, we identified Transportin1 as an interaction partner of AtPTB2. The interaction was independently validated and its impact on PTB function was addressed. Furthermore, as the Serine/Arginine-rich proteins (SRs) have been described as antagonists of hnRNPs, we are determining their effect on the PTB-mediated AS of a reporter construct. Current research aims at the identification of common splicing targets of PTB2 and antagonistically acting SRs. The results of these studies are expected to contribute to a better understanding of the complex mechanisms controlling AS, and thereby to provide novel insight into the regulation of plant development and adaptation to stress. sta1-1 modifier screening suggests the presence of common and specific factors in mRNA and microRNA processesings Si-in Yu, Yerim Kwon, Hemasundar Alavilli, Ji-ha Lee, and Byeong-ha LEE* Department of Life Science, Sogang University, Seoul 121-742, Korea([email protected]) Abstract The Arabidopsis STABILIZED1 (STA1) gene encodes a protein homologous to human U5 snRNP-associated 102-kDa protein (PRPF6), and the yeast pre-mRNA splicing factors, PRP1p (fission yeast) and Prp6p (budding yeast). The sta1-1 mutant defective in the STA1 gene was originally isolated using bioluminescent Arabidopsis harboring the stressinducible RD29A promoter-driven luciferase (RD29A-LUC) transgene, because sta1-1 showed higher luminescence under cold stress than the wild type. In addition to its hypersensitivity to temperature stresses, sta1-1 was indeed defective in pre-mRNA splicing. Moreover, sta1-1 showed lower accumulation of microRNA mostly because of defective primary microRNA splicing, which suggested that STA1 is involved in both mRNA and microRNA processesings. In order to identify the genetic factors in mRNA and microRNA processings, we carried out sta1-1 genetic modifier screening. Through the genetic modifier screening, we found the genetic enhancers and suppressors that affected not only each RNA processing specifically, but also both mRNA and microRNA processesings. These results suggested that mRNA and microRNA processesings share the STA1-realted common processing factors with some factors still being specific for each RNA processing. Knockdown efficiency too low? Don_'t panic, check alternative splicing Armin FUCHS, Max F. Perutz Laboratories, Medical University of Vienna, AustriaEzequiel Petrillo, Max F. Perutz Laboratories, Medical University of Vienna, AustriaStefan Riegler, Max F. Perutz Laboratories, Medical University of Vienna, AustriaMaria Kalyna, Department of Applied Genetics and Cell Biology, BOKU, Vienna, AustriaAndrea Barta, Max F. Perutz Laboratories, Medical University of Vienna, Austria Abstract RNA interference (RNAi) revolves around short RNA sequences, such as short interfering RNAs (siRNAs) and microRNAs (miRNAs), which are able to bind and repress mRNAs. Due to alternative splicing, several mRNA variants may be produced from a single gene, though not every splice variant is protein-coding. siRNAs and artificial miRNAs (amiRNAs) are usually designed to target all alternative splice variants of a gene. Knockdown efficiency is determined either on the level of total mRNA or, in case an antibody is available, on the protein level.To study functions of Arabidopsis thaliana splicing factors RS31a, RS41 and SR30, we used amiRNAs to knockdown expression of these genes. As the genes coding for these splicing factors are themselves alternatively spliced, we designed amiRNAs that would target all splice variants. Determination of knockdown efficiencies by RT-qPCR showed that total mRNA levels were down by only about 50%. However, RT-qPCR analyses of the protein-coding splice variants revealed knockdown efficiencies of up to 95%. Further investigations showed that amiRNA-targeted alternative splice variants of RS31a, RS41 and SR30 exhibit differential sensitivities to amiRNA-mediated silencing. We will discuss potential mechanisms that allow alternative splice variants to escape the silencing machinery. In addition, we found that amiRNAs can be employed to change splicing of target genes in plants, presumably by interfering with the splicing machinery inside the nucleus. Light signals regulating alternative splicing in Arabidopsis Ezequiel PETRILLO 1, Maria Kalyna2, Andrea Barta1 1. Max F. Perutz Laboratories, Medical University of Vienna, A-1030 Vienna, Austria.2. Department of Applied Genetics and Cell Biology, BOKU, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Abstract Light is a source of energy and also a regulator of plant physiological adaptations. We recently show that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. We are now trying to identify the nature of the light signals that communicate the chloroplast status to the nuclei of leaf and root cells. By using different approaches we want to elucidate the roles of sugars as possible mobile signals and also the involvement of the spliceosome and the RNA polymerase II as possible targets and/or effectors of these light signaling pathways. Genome-wide analysis of dehydration-responsive alternative splicing in Physcomitrella patens Hsiang-Wen CHEN, Chueh-Ju Shih, Yu-Rong Chen, Wen-Dar Lin, and Shih-Long Tu Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan Abstract Being a major environmental stress, drought dramatically affects plant growth and development and causes significant loss in crop production globally. Plant dehydration responses are complicated molecular procedures that inquire to clarify by genome-wide analysis. Transcriptional regulation of plant genes which responses to drought stress was well studied already. However, whether water deficit affects pre-mRNA splicing remains an open question. We analyzed the transcriptomic data of Physcomitrella patens, a droughttolerant moss, by high-throughput mRNA sequencing (RNA-seq) to evaluate alternative splicing changes under dehydration condition. A large amount of alternative splicing events including intron retention (IR), exon skipping (ES) and alternative donor/acceptor sites (AltDA) are responsive to 2- and 8-hr dehydration treatments, respectively. Gene ontology analysis showed that enriched functions of genes with dehydration-responsive IR are highly correlated with ribosome, photosystems and chloroplast. Enriched functions of genes with notable AltDA are mainly classified into photosystems and membranes. We also validated the RNA-seq data for drought-related genes by quantitative RT-PCR. The results indicate that alternative splicing of regulatory genes involved in drought stress responses are also altered rapidly after dehydration treatment. Data from motif search suggest a UUUA repetitive cis element may participate in splicing regulation for plant drought response. Our current data suggest that plants respond to water deficit by globally and rapidly adjusting alternative splicing of both metabolic and regulatory genes. Physcomitrella phytochromes regulate pre-mRNA splicing in response to light Bou-Yun LIN 1,2,3, Chueh Ju Shih1,2,3 and Shih-Long Tu1,2,4 1Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan, 2 Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, 3Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan 4Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan. Abstract Plant growth and development are mainly under the light regulation. There are many photoreceptors fine-tuning gene expression to control photomorphogenic responses. Recently, existing data showed alternative splicing is also regulated by phytochromes in Arabidopsis. In our previous study, we have confirmed that various alternative splicing events such as exon skipping, intron retention (IR) and alternative donor-acceptor splicing site occur after red light irradiation in Physcomitrella patens. There are at least seven phytochromes (PpPHY1, PpPHY2, PpPHY3, PpPHY4, and PpPHY5a-c) expressed in P.patens, which are believed to function differently in response to light. Among these phytochromes, PpPHY1 and PpPHY4 were functionally identified as phyA- and phyB-type phytochrome respectively. In this study, we are especially interested in the responses of different type of phytochromes in regulating alternative splicing after red light irradiation. We analyzed the mRNA sequencing data of PpPHY1-deficient and PpPHY4-deficient mutants and compared to the wild type in order to identify the phytochrome-dependent alternative splicing events. We found there are 612 IR events and 570 IR events under the control of PpPHY1 and PpPHY4, respectively. Functional enrichment analysis indicates both PpPHY1 and PpPHY4 can regulate IR of genes involving in fatty acid biosynthetic processing and translation processing. The results of the genome-wide analysis between wild type plants and phytochrome-deficient mutants hint us the role of phytochromes in AS. In conclusion, we may provide more actions for those photoreceptors in regulation of pre-mRNA splicing beyond other gene regulations previously discovered. Dissection of genetic interactions between regulatory splicing factors and SnRK1.1 in sugar signaling pathways in Arabidopsis thaliana Dóra SZAKONYI, Raquel Fonseca Carvalho, Elena Baena-González and Paula Duque Instituto Gulbenkian de Ciência, Oeiras, Portugal Abstract Plants need to adjust to environmental changes in order to grow and propagate. One of the pivotal mechanisms for survival is sensing nutrient availability and energy levels within the cells. Free carbohydrates such as fructose and glucose act as signaling molecules and can modulate gene expression profiles and developmental programs of the organism via intricate molecular pathways. SnRK1 protein kinases are involved in a prominent energy homeostasis pathway that is activated by starvation and sugar depletion [1]. Two splicing factors have unexpectedly been implicated as regulators of SnRK1.1 protein stability. The plant-specific SR45 is a serine/arginine-rich (SR)-like protein that has been shown to function in splicing [2]. The PLEIOTROPIC REGULATORY LOCUS1 (PRL1) gene encodes a WD40 protein and is a central component of the spliceosome-activating NineTeen Complex (NTC) [3]. Both sr45-1 and prl1-1 mutants show pleiotropic phenotypes including hypersensitivity to high sugar conditions. In this study, we introduced a SnRK1.1 overexpression construct and a snrk1.1 loss-of function allele into sr45-1 and prl1-1 T-DNA mutant lines and studied sugar response phenotypes on plant medium supplemented with 3% and 4% glucose under light and dark conditions. In our experiments, SnRK1.1 overexpression considerably enhanced sugar sensitivity of sr45-1 and prl1-1 mutants, supporting the observations that SnRK1.1 protein degradation was impaired in these mutants. However, the double mutants behaved differently. Deletion of SnRK1.1 partly rescued the enhanced sugar sensitivity of sr45-1, while prl1-1 remained unchanged. Further genetic and molecular studies will be carried out to unravel the regulatory mechanisms driven by SR45 and PRL1 in sugar stress signaling. References [1] Crozet, P., et al., Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases. Front Plant Sci, 2014. 5: p. 190.[2] Ali, G.S., et al., Regulation of plant developmental processes by a novel splicing factor. PLoS One, 2007. 2(5): p. e471.[3] Palma, K., et al., Regulation of plant innate immunity by three proteins in a complex conserved across the plant and animal kingdoms. Genes Dev, 2007. 21(12): p. 1484-93. Computational Identification of miRNA Targets Using Olive EST Library Nehir ÖZDEMIR ÖZGENTURK*, Zehra Ömeroglu Ulu*, Salih Ulu*, Dilek Koptekin**, Cemal Ün** *Yildiz Technical University, Department of Molecular Biology and Genetics, Davutpasa Kampus, Esenler Istanbul, Turkey **Ege University, Department of Biology, Bornova, Izmir, Turkey Abstract Olive (Olea europaea L.) which is a species in the family Oleaceae has major agricultural importance in the Mediterranean region as the source of olive oil. ESTs (Expressed Sequence Tags) are fragments which are obtained the results of sequencing of cDNA clones and are very important data for genomics studies. They have become a powerful tool discovery of new genes and microRNA (miRNA).miRNAs are a family of approximate 16~25 nt noncoding single-stranded RNAs that represent post-transcriptional regulators. In plants miRNAs play important roles post-transcriptional gene regulation by targeting mRNAs for cleavage or repressing translation. The use of computational homology based search for ESTs is suitable towards the discovery of miRNAs. In this study EST library that was constructed from Olive fruit and leaves and analysed with Phred/Phrap/Consed softwares was used. Duplicates of the functional plant miRNAs in miRBase database (Release 21, June 2014) and high-quality 3734 ESTs identified and removed by CD-HIT454. Non-redundant 4624 functional plant miRNA and non-redundant 3416 ESTs aligned by UEA sRNA workbench software. The sequences that have the criteria of secondary structure will be determined with UEA sRNA workbench program as olive target miRNAs. We expect this study will contribute to plant miRNA researches. The Arabidopsis SR protein SCL30a, conferring salt stress tolerance to germinating seeds, regulates the expression_'splicing of genes involved in photosynthesis, retrotransposition and stress response Dale N. RICHARDSON, Tiago M.D. Cruz and Paula Duque Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal Abstract Despite the influx of new evidence in the animal world suggesting myriad roles for SR genes/proteins in the regulation of gene expression — including in transcription, alternative splicing, RNA surveillance or gene and transposon silencing — and the emerging roles in plant development and responses to biotic or abiotic stresses, the functional evidence linking these alternative splicing regulators to plant stress tolerance is scarce. As SR genes typically have critically important roles in transcription and splicing, it is expected that their mis-regulation will have several downstream consequences on gene expression and alternative splicing, which in turn may affect several biological processes, including the response to environmental stress. To better understand the roles of SR genes in the abiotic stress response in plants, we have performed RNA-seq on an Arabidopsis SCL30a knockout mutant and overexpressor line during seed germination under high salt (200mM) concentrations. At the phenotypic level, despite the absence of developmental or morphological defects during the vegetative phase, we observed in these distinct genotypes alterations in seed size and dormancy as well as in the response to salt stress during germination, which were dependent on a functional ABA pathway. Concomitant with these changes, we observed a small set of differentially expressed genes (DEGs), 94 in the scl30a-1 mutant and 19 in the overexpressor, with the majority of these DEGs being down regulated in the mutant (93%) and overexpressor (90%). Functional annotation clustering of the 94 mutant DEGs using DAVID suggests a role for SCL30a in regulating the expression of genes involved in photosynthesis, defense response and response to hormones. Interestingly, we found that SCL30a may also play a role in regulating transposable elements, which we have validated at the expression level using quantitative RT-PCR (RT-qPCR). Although no significant global differences in alternative splicing in the scl30a-1 mutant, overexpressor or wild-type genotypes were detected, we observed that SCL30a changes isoform expression of a restricted set of genes involved in response to abiotic/biotic stimulus, stress and transport. Lastly, among this restricted set of genes, several appear to be related to the germination and ABA sensitivity phenotypes observed in the scl30a-1 mutant and to the enhanced tolerance to salt stress in the SCL30a-1 overexpressor. We are currently in the process of experimentally validating these differentially expressed isoforms using RT-qPCR. Posters - Session 2- The impact of mRNA decay factors on the response to biotic stress in Arabidopsis thaliana Anna GOLISZ, Monika Stepien, Joanna Kufel Faculty of Biology, Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland Abstract Effective execution of many cellular processes largely depends on the efficient regulation of gene expression by RNA metabolic pathways, particularly maturation and decay of mRNAs, not only under normal conditions but also under stress. Also in plants defects in RNA processing and degradation cause developmental problems, impaired hormonal signaling and altered resistance to abiotic and biotic stress. Irregularities in plant response to pathogen infection have been observed for a number of mutants in major RNA quality control, such as NMD, or RNA interference pathways, but the mechanisms underlying these effects are not clear. For example in Arabidopsis thaliana response to biotic stress is regulated by major NMD factors UPF1, UPF3 and SMG7. To assess the impact of other RNA metabolic factors on plant immunity we have analyzed mRNA levels of key pathogenesis markers, including PR1, PR5, NPR1 and NPR3, PDF1.2 and HEL/PR4, as well as WRKY transcription factors, which activate the immune response, in selected mutants with defects in RNA processing and degradation following infection by Pseudomonas syringae pv. tomato DC3000. The expression of analyzed genes was visibly altered in lsm1, xrn4 and dcp5 mutants defective in 5’ mRNA decapping and mRNA decay. This observation is consistent with changes in a number of mRNAs encoding pathogenesis-related factors seen in lsm1 transcriptome. Similarly to the effects in upf and smg7 mutants, lsm1, xrn4 and dcp5 plants showed elevated basal level of major pathogenesis factors PR1, PR5, NPR1, and PDF1.2, suggesting constitutive activation of the response to pathogen. In addition, induction of some transcripts following infection was visibly increased or had altered kinetics in the mutants. These results suggest that mRNA decay may contribute to plan immune response via regulation mRNA stability of key pathogenesis factors Importance of AtUPF1 phosphorylation in plant NMD Aleksandra SULKOWSKA, Paweł J. Sikorski, Izabela Wawer, Joanna Kufel Institute of Genetics and Biotechnology, University of Warsaw, Poland Abstract Nonsense-Mediated mRNA Decay (NMD) is an evolutionary conserved process related to the control of gene expression. This mechanism prevents the production of potentially harmful proteins by eliminating aberrant mRNA transcripts carrying premature termination codons (PTC). Phosphorylation of the key factor UPF1 is a critical step for NMD in mammals and C. elegans. Phospho-UPF1 is bound by 14-3-3-like proteins (SMG5-7 heterodimer and/or SMG6), which trigger mRNA decay and dephosphorylation of UPF1. Despite intense research in recent years, the understanding of NMD mechanisms in plants is still incomplete.We have shown previously that the N- and C-terminal domains of AtUPF1 are phosphorylated, act redundantly during NMD and form the binding platform for AtSMG7. Notably, three of the phosphorylated residues in the N-terminal domain of AtUPF1 were demonstrated to be important for NMD competence.To clarify the role of AtUPF1 phosphorylation in plant NMD, we have analyzed co-localization of specific AtUPF1 and AtSMG7 mutant variants in Arabidopsis protoplasts using confocal microscopy. We have also performed fluorescence resonance energy transfer–fluorescence lifetime imaging assays to investigate the importance of identified AtUPF1 phosphorylation sites for the interaction with AtSMG7. URT1-mediated uridylation determines the extent of messenger RNA deadenylation in Arabidopsis thaliana Hélène SCHEER, Hélène Zuber and Dominique Gagliardi Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, Strasbourg 67000, France. Abstract The pervasive 3’uridylation of mRNAs is emerging as a major process controlling mRNA stability. In Arabidopsis thaliana we have previously shown that the URidylylTransferase URT1 adds uridylyl residues at the 3’ end of mRNAs. This uridylation protects deadenylated mRNA from further deadenylation and from 3’ to 5’ degradation by exoribonucleases (Sement et al., 2013).To better investigate the mechanisms and roles played by URT1mediated uridylation, we complemented an urt1-1 mutant line by expressing a tagged URT1 fusion protein and analysed the uridylation status of two model transcripts. As expected, we observed an increase level of uridylation in complemented lines compared to the urt1 mutant. In agreement with previously published results, the increase level of U-tailing correlates with a decrease in the number of excessively deadenylated mRNAs. This observation confirms that uridylation protects the 3’ end from further deadenylation and degradation. Interestingly, the sizes of both uridylated and non uridylated poly(A) tails significantly increase in URT1 overexpressing line as compared to WT. This result indicates that URT1 could antagonize the deadenylation step. Data also show that URT1 overexpression restores but does not lead to a dramatic increase in uridylation level suggesting that deadenylation remains a rate limiting step in URT1 mediated uridylation. Taken together our data reveal a dynamic equilibrium between deadenylation and uridylation of mRNAs. References Sement, F.M., Ferrier, E., Zuber, H., Merret, R., Alioua, M., Deragon, J.-M., Bousquet-Antonelli, C., Lange, H.,and Gagliardi, D. (2013). Uridylation prevents 3’ trimming of oligoadenylated mRNAs. Nucleic Acids Res. 41,7115– 7127. Functional and genetic analysis identify a role for Arabidopsis ARGONAUTE 5 in anti-viral RNA silencing Chantal Brosseau and Peter MOFFETT Département de Biologie, Université de Sherbrooke, Sherbrooke, Québec, J1K 2R1, Canada Abstract RNA silencing functions as an anti-viral defense through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. In turn, plant viruses have evolved strategies to counteract this defence mechanism, including the expression of suppressors of RNA silencing. Potato virus X (PVX) does not systemically infect Arabidopsis thaliana Col-0, but is able to do so effectively in mutants lacking at least two of the four Arabidopsis DCL proteins. PVX can also infect Arabidopsis ago2 mutants, albeit less effectively than double DCL mutants, suggesting that additional AGO proteins may mediate anti-viral defenses. Here we show, using functional assays, that all Arabidopsis AGO proteins have the potential to target PVX lacking its viral suppressor of RNA silencing (VSR), P25, but that only AGO2 and AGO5 are able to target wild-type PVX. However, P25 directly affects only a small subset of AGO proteins, and we present evidence indicating that its protective effect is mediated by precluding AGO proteins from accessing viral RNA, as well as by directly inhibiting the RNA silencing machinery. In agreement with functional assays, we show that Potexvirus infection induces AGO5 expression and that both AGO2 and AGO5 are required for full restriction of PVX infection in systemic tissues of Arabidopsis. 5’ mRNA cap surveillance by AtDXO1 in Arabidopsis thaliana Alesandra KWAŚNIK*, Agnieska Gozdek, Michal Krzysztón, Joanna Kufel Intitute of Genetics and Biotechnology, University of Warsaw, Poland Abstract Correct maturation of mRNA 5’ end constitutes an important step in the regulation of gene expression and is therefore subjected to surveillance mechanisms, which detect and degrade potentially dysfunctional transcripts containing aberrant 5’ end structures. Yeast and human members of the RAI1/DXO1 protein family were previously shown to participate in these processes as they exhibit phosphodiesterase (PPE) activity to remove incomplete, unmethylated caps and may additionally act as pyrophosphohydrolases (PPH) or 5’-3’ exoribonucleases (5’EXO) towards uncapped mRNAs containing 5’ end triphosphates or monophosphates [1, 2, 3]. Here we provide an insight into the biochemical activity and physiological significance of the RAI1/DXO1 homologue from Arabidopsis thaliana.In addition to strong active site amino acid sequence conservation to other RAI1/DXO1 proteins, AtDXO1 contains a unique N-terminal unstructured domain that probably affects its biochemical properties. Series of in vitro decapping and degradation assays on specific RNA substrates revealed three distinct enzymatic activities (PPE, PPH and 53EXO) of purified AtDXO1, which were severely affected by the presence of the N-terminal domain. To establish the role of AtDXO1 in A. thaliana RNA metabolism in vivo we have selected two TDNA insertion mutant lines, which exhibit severe growth inhibition and sterility. RNASeq data and analysis of selected mRNAs shown significant transcriptome changes as well as altered mRNA cap methylation status in dxo1 plants.We envisage that AtDXO1 with its three simultaneous enzymatic activities, similarly to the human counterpart, plays a major role in mRNA cap surveillance in plants by decapping and subsequent degradation of various aberrant and probably also normal transcripts. References Chang JH, Jiao X, Chiba K, Oh C-S, Martin CE, Kiledjian M, Tong L (2012). Dxo1 is a new type of eukaryotic enzyme with both decapping and 5′-3′ exoribonuclease activity. Nat Struct Mol Biol. 19: 1011-1017.Jiao X, Xiang S, Oh C-S, Martin CE, Tong L, Kiledjian M (2010). Identification of a quality-control mechanism for mRNA 5′-end capping. Nature. 467: 608-611.Jiao X, Ho Chang J, Kilic T, Tong L, Kiledjian M (2013). A mammalian premRNA 5′ end capping quality control mechanism and an unexpected link of capping to pre-mRNA processing. Mol Cell. 50: 1–12. *In oral short talk too, session 2 Posters - Session 3- Biological function of antisense RNAs that are synthesized by RNA-dependent RNA polymerases under abiotic stress Akihiro MATSUI 1, Kei Iida2,5, Katsushi Yamaguchi3, Maho Tanaka1, Junko Ishida1, Taeko Morosawa1, Shuji Shigenobu3, Kazuo Shinozaki4, Tetsuro Toyoda5, and Motoaki Seki1,6,7 1 Plant Genomic Network Research Team, RIKEN CSRS2 Medical Research Support Center, Graduate School of Medicine, Kyoto University3 NIBB Core Research Facilities, National Institute for Basic Biology4 Gene Discovery Research Group, RIKEN CSRS5 RIKEN ACCC6 Kihara Institute for Biological Research, Yokohama City University7 CREST JST Abstract Arabidopsis whole transcriptome analysis under drought, cold, high-salinity stress and ABA treatment conditions, using tiling array, showed that approximately 6,000 novel transcripts existed on the antisense strand of the AGI code genes and they were induced under abiotic stress. Forward genetic analysis revealed that accumulation of RD29A antisense RNA was reduced by half in RNA-dependent RNA polymerase triple mutant (rdr1/2/6). It is known that RDRs are required for double stranded RNA synthesis in siRNA generation process. RNase protection and RNA decay analysis showed that the RD29A antisense RNA is involved in formation of double-stranded RNA and promoted degradation of RD29A mRNA. Microarray and RNA-seq analysis revealed that most of RDR1/2/6-dependent antisense RNA loci was not overlapped with the siRNAgenerated regions. In addition, rdr1/2/6 mutants showed arrested root growth at recovery stage after drought stress, while dcl2/3/4 mutants did not show this phenotype. These results indicate that the antisense RNAs have biological function independently of the siRNA pathway in abiotic stress response. 5’-3’ exoribonuclease AtXRN3 functions in genome-wide control of Arabidopsis transcriptome. Michał KRZYSZTON 1, Monika ZAKRZEWSKA-PLACZEK 1, Geoff Barton2, Nick Schurch2, Alexander Sherstnev2, Gordon Simpson3 and Joanna Kufel1 1. Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland; 2. Division of Computational Biology, University of Dundee, Dundee, United Kingdom; 3. Division of Plant Sciences, University of Dundee, Dundee, United Kingdom and The James Hutton Institute, Dundee, United Kingdom Abstract Non-coding RNAs produced by pervasive transcription are considered increasingly important for regulation of gene expression and maintaining genome stability and integrity in different organisms from bacteria to humans. Our findings bring new insights to the study of these phenomena in plants, by revealing the role of the nuclear 5’-3’ exoribonuclease AtXRN3 in the regulation of ncRNA abundance in Arabidopsis thaliana.The Arabidopsis genome encodes three XRN proteins, orthologs of the yeast 5’-to-3’ exoribonuclease, Rat1/Xrn2: nuclear AtXRN2 and AtXRN3, and cytoplasmic AtXRN4, that function in different RNA surveillance pathways. The cytoplasmic AtXRN4 participates in the decay of specific transcripts and degrades 3’ products of miRNA-mediated mRNA cleavage in RNAi, whereas the nuclear AtXRN2/3 act as endogenous RNA silencing suppressors and function in prerRNA processing and degradation of polyadenylated rRNA precursors. Our recent studies demonstrate a widespread accumulation of lncRNAs, named XATs (XRN3-associated transcripts), corresponding to intergenic regions located directly downstream of coding genes in xrn3 mutants. These lncRNAs are produced by PolII and often contain large fragments of coding regions, and poly(A) tails, but mostly lack the 5’ cap structure. Interestingly, the sequences corresponding to coding genes, located within XATs, are often effectively spliced. All properties of XATs suggest that they result from defective transcription termination of PolII and indicate direct function of AtXRN3 exoribonuclease in that process probably through the torpedo mechanism. Most importantly, down-regulation of AtXRN3 leads to changes in mRNA levels of many genes, which appears to be related to transcription of XATs. Considering that the loss of AtXRN3 is lethal this regulation of gene expression by non-coding transcripts represents a physiologically significant example, especially that XAT molecules can accumulate in the cell under certain conditions. Posters - Session 4- Proteome analysis of the early heat-stress response leading to plant death or survival in Arabidopsis. Fernández-Calvino Lourdes1*, Echevarría-Zomeño Sira1*, Castro-Sanz Ana B.1, López Juan A.2, Vázquez Jesús 2 and CASTELLANO M. Mar 1 1Centre for Plant Genomics and Biotechnology, INIA-UPM, 28223, Pozuelo de Alarcón, Madrid, Spain2Spanish National Centre for Cardiovascular Research. Instituto de Salud Carlos III, 28029, Madrid, Spain*Co-first author Abstract One of the consequences of climate change is the global warming. The increasing temperatures are a great problem for agriculture, since heat stress is one of the abiotic stresses that most affects crops. On the other hand, plants have evolved to deal with environmental changes that range from extreme cold in winter to suffocating temperatures in summer. The way they manage to survive to these extreme situations lies in the fact that along the year there is a gradual variation of the temperatures that allows plants to acclimate to the incoming conditions, which otherwise would be lethal. In this way, the study of the molecular mechanisms involved in plant acclimation is crucial for the future of agriculture.Acclimation involves an extensive molecular reprogramming that successfully promotes thermotolerance to a severe heat challenge. On the other hand, non-acclimated plants also trigger a molecular response against severe heat shocks to avoid death, but they fail in their purpose. In this sense, the overlapping degree between both responses and the specific players that determine whether the plant will survive or not are currently unknown. The response of plants to heat stress has been examined profusely for years at the transcriptional level, and the lack of correlation with the translation regulation has pushed researchers to analyse proteome changes during this process. Proteomics methodology provides a more direct assessment of the actual proteins represented in the plant transcriptome during the heat stress response but, to date, proteomic studies have only analysed heat challenges without considering the effective acquisition of plant thermotolerance and leaving the response during the recovery period almost completely unknown.For this reason, we have used Arabidopsis as a model plant to carry out an iTRAQ comparative proteomic analysis during acclimation and during the early stages of the plant response to a severe heat stress that lead seedlings either to survival or death. Our analysis shows that only a small number of proteins are coordinately changed under the analysed heat treatments. In addition, our data demonstrate that there are diverse degrees of overlapping to the different heat regimes that include groups of proteins with specialised functions. Furthermore, proteins associated to specific treatments were also identified. Analysis of T-DNA insertion mutants in genes identified in this analysis demonstrates its capacity to uncover new regulators of the heat response. The spatial regulation of gene expression in response to hypoxic stress Janusz NIEDOJADLO Department of Cell Biology, Laboratory of Confocal and Electron Microscopy, Faculty of Biology and Environment Protection, Nicolaus Copernicus University in Torun, Poland Abstract Excess of water or flooding have negative impact on agricultural yields. Flooding of land plants causing hypoxia is a result of the change in levels of three gases, O2, CO2 and ethylene, and their large reduction in diffusion in water relative to air. Decline in O2 availability results in a reduction in the energy produced by the plant, because of the shift from aerobic respiration to anaerobic, which is less efficient in ATP synthesis. We have observed an increased amount of poly(A) RNA in the nucleus of Lupinus luteus roots cells in the subsequent hours during hypoxia treatment. In the cytoplasm, single granules were found. The poly(A) RNA in cytoplasmic granules isn’t translated during hypoxia because not colocalized with rRNA. Measurements of the quantity of active RNA polymerase II and the distribution of SR proteins revealed a strong decrease of transcription under following hours of hypoxia. It can be concluded, that the observed increase of poly(A) RNA in the cell nucleus is a result of the strong retention. After 3h reoxygenation transcription level increased and in the cytoplasm increased significantly homogenous pool of poly(A) RNA were observed. This suggests that accumulated mRNAs in nucleus and cytoplasmic granules can be quickly used after the disappearance of stress. Previously the presence of poly(A) RNA in Cajal bodies (CB) was observed by our group in few plant species (Niedojadło et al. 2014). More percentage of the total nuclear pool of poly(A) RNA were found in the CBs in the subsequent hours of hypoxia. This finding suggests the possibility that Cajal bodies contribute to the retention of mRNA in the cell nucleus. Then we localized transcripts of genes with different level of expression and translational efficiency in the Arabidopsis thaliana roots under hypoxia. We found increasing amount of alcohol dehydrogenase1 (ADH1) mRNAs (core hypoxia response gene) in the cytoplasm hypoxia treatment roots. Then methionine sulfoxide reductase and U1 snRNP 70K protein mRNAs were localised.Concluding, our results indicate the important role of the spatial organization of gene expression in response to hypoxia stress. References Niedojadło et al. (2014) PLoS ONE 9(11):e111780. Distribution of poly(A) RNA in sperm cells of Arabidopsis thaliana and Hyacinthus orientalis Katarzyna NIEDOJADLO, Janusz Niedojadło Department of Cell Biology, Faculty of Biology and Environmental Protection, Nicolaus Copernicus University in Toruń, Poland Abstract The mature pollen grain of flowering plants represents a unique structure with a key function in sexual plant reproduction. Upon pollination of a receptive stigma pollen grains hydrate and germinate to produce pollen tubes. Pollen tube formed by a vegetative cell transports of the two male gametes through the pistil tissue to the embryo sac where double fertilization takes place. Depending on the timing of generative cell mitosis, two sperm cell may be formed during pollen development in the anther (tricellular pollen grain) or during pollen tube growth (bicellular pollen grains). The male gametophyte of Arabidopsis thaliana is a tricellular pollen grain and Hyacinthus orientalis is a bicellular pollen grain. Thus in hyacinth the grain must be culture in order to grow pollen tubes, trigger mitosis and obtain sperm cells.In the recent years, the genome-wide analysis of the male gametophyte transcriptome in A. thaliana suggest that sperm cells possess a transcriptome with the unique composition also with sperm cell-specific transcripts. The aim of our study was to determine in situ the spatial and temporal distribution of poly(A) RNA in A. thaliana and H. orientalis sperm cells. The observations indicated two patterns of poly(A) RNA distribution in sperm cell of A. thaliana in pollen grain which differed from that in germinating pollen tube. Directly after anthesis poly(A) RNA was accumulated in the clusters in the cytoplasm whereas after rehydratation was homogenously distributed throughout in the whole cytoplasm. A physical relationship between the sperm cells and the vegetative nucleus (termed male germ unit-MGU) was visible. During growth the pollen tube the high accumulation of mature transcripts in the cytoplasm was still localized. In hyacinth, after sperm cells formation, MGU was observed and in such an organized structure the poly(A) RNA in both of the sperm cells was localized in the form of numerous granules. Our previous studies in hyacinth revealed the presence of AGO1 and AGO4 proteins in sperm cells. Therefore, we suggest that shortly before fertilization in male gametes the degradation of mRNA by small RNA pathways takes place. It is also discussion whether sperm cells transfer paternal transcripts during fertilization. Moreover, we know that the egg cell of H. orientalis does not accumulate large amounts poly(A) RNA and fertilization induces the chromatin remodeling and the activation of the zygote and endosperm genome. The research was supported by the National Science Centre (NCN) grant 2011/03/D/NZ3/00603 Translatome profiling in germinating sunflower seeds reveals post-transcriptional regulation of dormancy Elodie LAYAT1, Juliette Leymarie1, Hayat El-Maarouf-Bouteau1, José Caius², Nicolas Langlade3, and Christophe Bailly1 1UMR 7622, Sorbonne Universit_es, UPMC Univ Paris 06, 75005 Paris, France ²UMR 1165, INRA URGV, 91000 Evry, France 3Laboratoire Interactions Plantes Microorganismes, INRA, 31326 Castanet Tolosan, France Abstract (Text) : In higher plants, regulation of gene expression results from the combination of transcription and translation and it is well known that the abundance of a transcript does not necessarily reflect its translation. Selective translation of messengers permits rapid and efficient adaptation of cell signaling in response to environmental stresses and to developmental transitions. Seeds of many species are dormant at harvest and thus unable to germinate, even in favourable environmental conditions, but dry storage (after-ripening) allows seeds to become nondormant and able to germinate. We raised up the hypothesis that germination completion and dormancy alleviation would more rely on the ability of the cells to translate a subset of mRNA templates than on transcriptional activity. Thus we compared the dynamics of mRNA recruitment to polysomes in dormant and non-dormant sunflower seeds during their imbibition. Association of transcripts with polysomes reached a maximum after 15 h of seed imbibition when 194 transcripts were specifically translated in non-dormant embryos but only 47 in dormant embryos. Analysis of the polysome associated transcripts by microarrays revealed that germination completion was associated with selective translation. The proteins that were selectively translated in non-dormant embryos appeared as very pertinent for germination and ranged mainly in RNA metabolism, regulation of transcription, posttranslational modifications of proteins, cell wall modification or amino acid metabolism. Our data allow revisiting the actual concept of a transcriptomic regulation of germination and emphasize that only a small number of messengers can be considered as essential for germination. References Layat et al., 2014 New phytologist Investigation of translational regulation and molecular mechanisms involved during the NB-LRR-mediated defense response in plants Mathias COHEN1, Louis-Valentin Meteigner1, Mohamed El-Oirdi1, Teura Barff1, Daniel Garneau1, Ji Zhou2, and Peter Moffett1 1Département de Biologie, Université de Sherbrooke, 2500 Boulevard de l’université, Sherbrooke, Québec, Canada, J1K 2R1 2The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom Abstract One major layer of plant immunity is conferred through NB-LRR proteins, the products of Resistance genes. These proteins recognize specific proteins encoded by pathogens and activate multiple signaling pathways. Translational repression of viral mRNAs upon NB-LRR response activation has already been described. However, the molecular pathways by which these viral RNAs are degraded by the plant cell remain to be fully understood. In many cases, translational control occurs through the formation of cytosolic foci called Processing Bodies (PBs), where the translationally repressed RNAs are directed. PBs are dynamic and reversible structures composed by numerous proteins ensuring the decapping, the degradation or the storage of the viral RNAs. Using molecular imaging techniques and transient transformation of fusion PB proteins in Nicotiana benthamiana, we are studying the biogenesis and composition of PBs after the triggering of the NB-LRR defense response. With the use of a PB quantification assay, we find that NB-LRR activation induces a robust increase in the number of cellular PBs. In addition to translational repression of viral RNAs, transcriptional reprogramming of endogenous genes has also been previously described. However, little is known about post-transcriptional gene regulation of this response, such as translational control of endogenous genes. Thus we used Translating Ribosome Affinity Purification followed by RNAseq (TRAPseq) as a mean to evaluate translational regulation of host mRNAs upon NB-LRR signaling activation, in Arabidopsis thaliana. Translatome analysis revealed extensive translational regulation of hundreds of genes, and provided for several novel candidate genes implicated in NB-LRR-mediated resistance. Finally, as a proof of concept, we show that the knocking out or down of certain of these candidates can either inhibit or enhance NB-LRR-mediated resistance. Use ribosome profiling to discover new small peptides in Arabidopsis Jeremie BAZIN1,2, Martin Crespi2, Julia Bailey-Serres1 1 Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 Plant Science, Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, F-91198 Gif-surYvette, France 2 Saclay Abstract The proportion of eukaryotic genomes annotated as protein coding gene regions represent only a small fraction of the total genome. However, unbiased, high throughput transcriptome analysis technologies such as deep sequencing and tilling arrays revealed extensive transcription in intergenic regions. Non-protein coding RNA molecules include structural and housekeeping RNA (ribosomal RNA, tRNA, small nucleolar RNAs, small nuclear RNA), as well as a large and heterogeneous class of regulatory RNAs. In the last past years, studies in plants and animals have drawn attention to lncRNAs (> 200 nt) and shown some lncRNA can have a functional role in the regulation of gene expression. Intriguingly, ribosome footprint sequencing from immunopurifed ribosome of arabidopsis root seedlings revealed a large number of putative long non-coding RNA are associated with ribosome footprints. This data suggested transcript annotated as lncRNA might actually encode for peptides. To answer this question, we applied an analysis pipeline to detect ribosome footprints distribution patterns on lncRNAs, which are characteristic of an active translation. This study allowed use to identified a set of new putative small peptide encoding transcripts in Arabidopsis roots and to show the majority of ribosome-associated lncRNAs do not harbor typical features of active translation. Posters - Session 5- Analysis of small RNAs involved in tracheary element differentiation of plant Tian Tian TAN 1, Hitoshi Endo1, Ryosuke Sano1, Misato Ohtani1, Taku Demura1 1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan Abstract Xylem tissues consist of specialized, water-conducting cells known as tracheary element (TE). One of the characteristics of TE is thick secondary cell wall (SCW), which is major source of plant woody biomass. Thus, the understanding of molecular mechanisms underlying TE formation is important to develop innovative biotechnologies for controlling quantity and quality of woody biomass. Recent progress of molecular biology has demonstrated that small RNAs, such as microRNA, have critical roles in gene expression regulation. However, roles of small RNAs in TE formation have not been well understood; involvement of microRNA 165/166 in vascular tissue development is the single well-known example. In this study, we established an in-vitro TE formation system to study the roles of small RNAs that involved in TE differentiation. In the established in-vitro system, ectopic TEs were formed in cotyledons of Arabidopsis young seedlings by the application of three phytohormones: auxin, cytokinin and brassinosteroid. The ectopic TE possess lignified and patterned SCW, which mimics the characteristics of TE in planta. Further analysis using Arabidopsis mutants and reporter lines revealed that our in-vitro system requires the master regulators of TE formation, VASCULAR-RELATED NAC-DOMAIN proteins. To understand the process of the ectopic TE formation, we obtained the time-course RNA-seq data on both of mRNA and small RNA fractions using Illumina Genome Analyzer II platform. 42 potential miRNA-mRNA target gene candidates were identified including miR165/166 (PHB/PHV: early marker genes for TE formation) and miR858 (MYB63/83: SCW formationrelated genes). In summary, our in vitro system successfully identified novel miRNA-mRNA pairs, suggesting the importance of miRNA-mediated gene regulation in TE formation. DCL2-dependent silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutant results in phloem transport defect and carbohydrate over-accumulation Yu-Yi WU1, Bo-Han Hou1, Wen-Chi Lee1, Shin-Hua Lu1, Chen-Jui Yang1, Hervé Vaucheret2 and Ho-Ming Chen1 1 Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 11529, Taiwan2 Institut Jean-Pierre Bourgin, UMR1318, INRA, 78026 Versailles Cedex, France Abstract Dicer and Dicer-like (DCL) enzymes process double-stranded RNA into small RNAs, which regulate gene expression. Arabidopsis DCL2 plays a role in the silencing of viruses and transgenes, but its activity is mostly obscured by DCL4. In the absence of DCL4, DCL2 ensures silencing activity against viruses and transgenes, but its activity also contributes developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying the DCL2-dependent dcl4 phenotype was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that purple discoloration of dcl4 leaves is associated with carbohydrate over-accumulation and phloem transport defect and depends on the activity of Suppressor of Gene Silencing 3, RNAdependent RNA polymerase 6 and DCL2. An outburst of DCL2-dependent small RNAs deriving from two vasculature expressed genes, SMAX1-Like 4 (SMXL4) and SMXL5, was detected in dcl4 mutants. The smxl4 smxl5 double mutant also showed increased anthocyanin accumulation, starch accumulation and phloem transport defect, similar to dcl4 mutants. These results suggest that when DCL4 function is impaired, DCL2-dependent silencing of SMXL4 and SMXL5 disrupts phloem transport and carbohydrate homeostasis. As DCL4 function is suppressed when Arabidopsis is infected with turnip crinkle virus, these findings indicate new DCL2 function in virus-infected plants in addition to processing viral RNA. Characterization of small RNAs involved in the plant response to parasitic nematodes of genus Meloidogyne. Clémence Medina, Marc Magliano, Martine Da Rocha, Bruno Favery, Pierre Abad, and Stéphanie JAUBERT-POSSAMAI INRA, Univ. Nice Sophia Antipolis, CNRS, UMR 1355-7254 Institut Sophia Agrobiotech, 06900 Sophia Antipolis, France Abstract Plant response to bioagressors involves modifications of gene expression. Recently, microRNAs have been evidenced as crucial regulators of host gene expression during plantspathogen interactions. Root-knot nematodes (RKN) are biotrophic plant parasitic worms that transform plant cells from root vascular cylinder into hypertrophied, multinucleate and highly metabolically active giant feeding cells. Since RKN are able to induce the formation of feeding cells in roots of almost all cultivated plants, they are thought to manipulate essential and conserved plant molecular pathways. Previous transcriptomic analyses evidenced that redifferentiation of root cells into giant feeding cells implies transcriptional reprogramming with a large repression of gene expression. Our study aims to investigate the role of microRNAs in the regulation of transcriptional repression observed during the redifferentiation into feeding cells. Small RNAs from Arabidopsis thaliana roots infected with the RKN model species Meloidogyne incognita were sequenced by SOLID technology. As a control, small RNAs from non infected roots were also sequenced. First, a catalog of microRNA expressed in healthy and infected roots was established. Then, microRNAs that are differentially expressed between healthy and infected roots were then identified by statistical analyses. Preliminary results show that only few microRNAs are differentially expressed in infected roots and statistically relevant. Interestingly some of these microRNAs, such as At-mir398a and At-mir408 are evolutionary conserved in plants and are known as main factors of plant response to biotic and abiotic stresses or of interaction with symbiotic bacteria. Our results suggest that microRNAs are involved in the regulation of gene expression that results in redifferentiation of root cells into giant feeding cells in response to RKN infection. Moreover some microRNA-networks appear to be shared by plant response to biotic and abiotic stress. Looking for new genes involved in root growth and development Céline SORIN1 Thomas Blein2, Tracy Plainchamp2, Santosh Satbhai3, Wolfgang Busch3 and Martin Crespi2 1Université Paris Diderot-IPS2, France, 2IPS2, France, 3GMI, Austria, Abstract Roots are essential for water and nutrients acquisition in plants. Root architecture can be modulated by endogenous and environmental factors. In plants, microRNAs (miRNAs) play various developmental and physiological roles. These small RNAs regulate their targets by transcript cleavage and/or inhibition of protein translation and are known as major posttranscriptional regulators of various developmental pathways and stress responses. GWAS was performed in Arabidopsis to identify candidate genes involved in root growth and development (Meijon et al 2014; Slovak et al 2014). Among them SNPs involving miR170 were selected as potentially modulating gravitropic responses in specific growth conditions. To analyse the role of this miRNA in such responses characterization of miR170 overexpressing lines and lines with decreased miR170 has been undertaken. Preliminary results on the impact of miR170 in plant development will be presented. References Meijon M et al 2014 Nat Genet 46:77-81Slovak et al 2014 Plant Cell 26 (6):2390-2403 Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses Maria BARCISZEWSKA-PACAKC 1, Kaja Milanowska1, Katarzyna Knop1, Dawid Bielewicz1, Przemyslaw Nuc1, Patrycja Plewka1, Andrzej Pacak1, Franck Vazquez2, Wojciech Karlowski3, Artur Jarmolowski1, Zofia Szweykowska-Kulinska1 1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland 2Zurich-Basel Plant Science Center, Part of the Swiss Plant Science Web, Botanical Institute, University of Basel, Basel, Switzerland3Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland Abstract Arabidopsis microRNA expression regulation was studied in a wide array of abiotic stresses such as drought, heat, salinity, copper excess/deficiency, cadmium excess, and sulfur deficiency (Barciszewska-Pacak et al., 2015). A home-built RT-qPCR mirEX platform for the amplification of 289 Arabidopsis microRNA transcripts was used to study their response to abiotic stresses. Small RNA sequencing, Northern hybridization, and TaqMan® microRNA assays were performed to study the abundance of mature microRNAs. A broad response on the level of primary miRNAs (pri-miRNAs) was observed. However, stress response at the level of mature microRNAs was rather confined. The data presented show that in most instances, the level of a particular mature miRNA could not be predicted based on the level of its pri-miRNA. This points to an essential role of posttranscriptional regulation of microRNA expression. New Arabidopsis microRNAs responsive to abiotic stresses were discovered. Four microRNAs: miR319a/b, miR319b.2, and miR400 have been found to be responsive to several abiotic stresses and thus can be regarded as general stress-responsive microRNA species. The work was supported by the NCN Harmonia funding scheme UMO2012/04/M/NZ2/00127: “The regulation of Arabidopsis thaliana microRNA genes expression in response to selected abiotic stresses: the role of transcription and splicing factors in microRNA biogenesis”.Keywords: miRNA, pri-miRNA, abiotic stress, gene expression References Barciszewska-Pacak M, Milanowska K, Knop K, Bielewicz D, Nuc P, Plewka P, Pacak AM, Vazquez F, Karlowski W, Jarmolowski A and Szweykowska-Kulinska Z (2015). Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses. Front. Plant Sci. 6:410. doi: 10.3389/fpls.2015.00410 The transcriptomic landscape of the Chlamydomonas chloroplast as revealed by sRNAseq and stranded-RNAseq. Marina CAVAIUOLO, Yves Choquet, Richard Kuras, Francis-André Wollman, Olivier Vallon Institut de Biologie Physico-ChimiqueUMR 7141 Laboratoire de Physiologie Membranaire et Moléculaire du Chloroplaste13, rue Pierre et Marie Curie, F-75005 Paris Abstract Regulation of gene expression is a dynamic process controlled by a number of mechanisms going from RNA synthesis to degradation. In the chloroplast of the green alga Chlamydomonas reinhardtii regulation of transcript accumulation is mainly posttranscriptional and involves gene specific stabilizing factors and ribonucleases that cooperate in maintaining the steady state levels of RNA abundance from mRNA maturation and stabilization to decay [1-3].We used Illumina strand-specific RNA sequencing of long RNA to explore both sense and antisense transcription of the chloroplast genome and small RNA sequencing of short RNAs (10-nt to 40-nt) to estimate RNA decay under transcriptional and translational inhibition.Strand-specific RNA sequencing indicated that the chloroplast genome is heavily transcribed: the higher RNA levels were produced from the sense strand, but a low-abundance generation of antisense RNAs was present. By smallRNAseq, we found highly abundant short RNAs in correspondence to the 5’ends of mRNAs representing footprints of stabilizing factors as previously shown [4-5]. Interestingly, short RNAs mapping to coding sequences originate nearly equally from both strands and mapped to the same regions. When chloroplast transcription was inhibited with rifampicin, antisense short RNAs were disappearing more rapidly compared to the sense. In contrast, when cells were treated with lincomycin, an inhibitor of chloroplast translation initiation, the abundance of antisense short RNAs was increased. This suggests that they are processing products of double-stranded RNA molecules generated in the absence of ribosomes by the pairing of the sense RNA, much more abundant, with an antisense RNA.Our results suggest that antisense RNAs precursors, whether produced as transcriptional noise or as read-through from genes, are rapidly degraded in the cells by a variety of mechanisms. They may have a function in controlling stability or translation of the sense RNA through base pairing. References [1]Stern DB et al. Chloroplast RNA metabolism. Annual Review of Plant Biology (2010); 61, 12555[2] Eberhard S et al. Searching limiting steps in the expression of chloroplast encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. The Plant Journal (2002); 31(2), 149-160[3] Monde RA et al. Processing and degradation of chloroplast mRNA. Biochimie (2000); 82, 573−582.[4] Ruwe H et al. Short non-coding RNA fragments accumulating in chloroplasts. Nucleic Acids Research (2012); 40(7), 3106–3116.[5] Loizeau K et al. Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas. Nucleic Acids Research (2014) 42(5), 3286-3297. Biochemical techniques for the study of long noncoding RNAs Aurélie CHRIST1, Federico Ariel1 and Martin Crespi1 Institute of Plant Sciences Paris-Saclay (IPS2), UMR 9213/UMR1403, CNRS, INRA, Universite Paris-Sud, Universite d’Evry, Universite Paris-Diderot, Sorbonne Paris-Cite, Batiment 630, 91405 Orsay, France 1 Abstract Noncoding RNAs have emerged as major components of the eukaryotic transcriptome. Genome-wide analyses revealed the existence of thousands of long noncoding RNAs (lncRNAs) in several plant species. They are involved in a wide range of regulatory mechanisms impacting on gene expression, including chromatin remodeling, modulation of alternative splicing, fine-tuning of miRNA activity, and the control of mRNA translation or accumulation. In our lab we perform alternative biochemical approaches to decipher the molecular mechanisms involving lncRNAs, such as Chromatin Immunoprecipitation (ChIP), RNA Immunoprecipitation (RIP) and Chromatin Isolation by RNA Purification (ChIRP). This variety of techniques has allowed us to determine DNA and protein partners of lncRNAs. Furthermore, Next Generation Sequencing technologies served to identify genomic regions recognized by chromatin-related lncRNAs. Noncoding transcription shapes genome topology Federico ARIEL1, Teddy Jegu1, Natali Romero-Barrios1, Aurelie Christ1, Moussa Benhamed1,2 and Martin Crespi1 Institute of Plant Sciences Paris-Saclay (IPS2), UMR 9213/UMR1403, CNRS, INRA, Universite Paris-Sud, Universite d’Evry, Universite Paris-Diderot, Sorbonne Paris-Cite, Batiment 630, 91405 Orsay, France 2 Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia 1 Abstract Noncoding RNAs have emerged as major components of the eukaryotic transcriptome. Genomewide analyses revealed the existence of thousands of long noncoding RNAs (lncRNAs) in several plant species. Plant lncRNAs are transcribed by the plant-specific RNA polymerases Pol IV and Pol V, leading to transcriptional gene silencing, as well as by Pol II. They are involved in a wide range of regulatory mechanisms impacting on gene expression, including chromatin remodeling, modulation of alternative splicing, fine-tuning of miRNA activity, and the control of mRNA translation or accumulation. Recently, dual noncoding transcription by alternative RNA polymerases was implicated in epigenetic and chromatin conformation dynamics. Reversible variations in the epigenome shape the genome topology in three-dimensional space structure, directly influencing the transcriptional responses to developmental cues. We have shown that the Arabidopsis long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerases II and V in response to auxin, a phytohormone controlling numerous facets of plant development. This dual APOLO transcription regulates the formation of a chromatin loop encompassing the promoter of its neighboring gene PID, a key regulator of polar auxin transport. Altering APOLO expression affects chromatin loop formation, whereas RNA-dependent DNA methylation, active DNA demethylation, and Polycomb complexes control loop dynamics. This dynamic chromatin topology determines PID expression patterns. Hence, the dual transcription of a lincRNA influences local chromatin topology and directs dynamic auxin-controlled developmental outputs on neighboring genes. Genome-wide identification of APOLO–interacting loci may reveal an intricate mechanism shaping genome topology driven by noncoding transcription, thus allowing the coordination of gene expression. Posters - Session 7- Compartment Markers for Plant Science Hossain, Zakir*, Linn FRANSSON.**, Brown, Christopher M.* *Environmental Proteomics, Sackville, New Brunswick, Canada **Agrisera AB, Box 57, 911 21 Vännäs, Sweden Chloroplasts are ideal hosts for transgenic expression. Introduced sequences undergo homologous recombination to integrate into plastid genomes and are thereafter maternally inherited, minimizing pleiotropic effects and containment risks. Production of heterologously expressed proteins within chloroplasts also limits contamination of the cytosol and cytotoxicity toward plant cells and tissues. Confirmation of chloroplast targeting and assessment of expression levels can be achieved by tracking introduced proteins and measuring their abundance relative to marker proteins. Fractionation of cellular compartments such as cytosol, mitochondria and chloroplasts, and further sub-fractionation into thylakoids, stroma and lumen can be followed by quantitative detection of both introduced and compartment marker proteins. Agrisera and Environmental Proteomics have created a comprehensive set of antibodies for detection and tracking of protein compartment markers in plants and algae. Several of these are fully quantitative, allowing normalization of expression levels of heterologous proteins. We demonstrate the fractionation of chloroplasts from cytosol and other organelles with antibodies toward RbcL (chloroplast stroma), PsbA or PsbD (chloroplast thylakoid), SPS (cytosol), PEPC (cytosol, mesophyll enriched in C4 plants), AOX (mitochondrial), and VDAC (vacuole). We also show the enrichment of Rubisco and PEP carboxylase using quantitative RbcL and and PEPC markers following fractionation of mesophyll and bundle-sheath cells from maize. Plant response to zinc excess Nga NT Nguyen1,2, Zaigham Shahzad1 Mohannad Alsulaiman1, Carinne Alcon1, Pierre Berthomieu1 and Françoise GOSTI1. 1. Biochimie and Physiologie Moléculaire des Plantes, Campus SupAgro-INRA - Bât. 7 - 2 place Pierre Viala 34060 Montpellier cedex 2, FRANCE. 2. present address: Department of Plant Molecular Biology (DBMV), University of Lausanne, UNIL–Sorge, Biophore Building, 1015 Lausanne, Switzerland. Abstract In an extreme case of adaptation to stressful environmental conditions, metal hyperaccumulator plants not only thrive on metal-enriched soils, but also accumulate metals to exceptionally high concentrations in their aerial tissues without suffering toxicity. Knowledge of the mechanisms used by plants to tolerate higher zinc concentrations idrags applications in both phytoremediation particularly considering the replanting of metal polluted areas and biofortification of the plant edible parts that will benefit to animal and human nutrition [1]. The extremophile species Arabidopsis halleri has become a model to study molecular evolutionary mechanisms related to the acquisition of zinc tolerance by comparison to A. thaliana [2]. Molecular genetic and physiological researches identified that over-expression of metal related genes in A. halleri are required for zinc tolerance and hyper-accumulation [3, 4]. Plant Defensins of type 1 (PDF1s) are genes that are also over-expressed in A. halleri [5] and their encoded proteins were discovered to play a role in cellular zinc tolerance [6]. These genes are especially interesting to investigate zinc tolerance mechanism because they are not metal related genes and are actually mostly recognized for their role in plant response to biotic stresses such as pathogen attack [7, 8]. To decipher the contribution of PDF1 over-expression to adaptive zinc tolerance processes, several PDF1s originating from A. halleri were expressed under the control of their own promoter in the non-tolerant A. thaliana species. We will present results highlighting that PDF1 over-expression is not the only requirement to provide plant with zinc tolerance and that several members of the PDF1s family are subjected to different post transcriptional and/or posttranslational mechanisms. In addition, our current investigation indicates that cellular localization of PDF1 promoter activity is also an important factor to consider and that it is also regulated. Taken as a whole, presented work will contribute to understand the mechanisms by which PDF1s specifically play a role in plant zinc tolerance, to the acquisition of this character in metal extremophile species and how it could lead us to improve the direct valuation of plant natural capital for the benefit of a greening economy. References 1. Guerinot, M.L. and D.E. Salt, Plant Physiol, 2001. 125(1): p. 164-7. 2. Roosens, N.H., et al., Trends Plant Sci, 2008. 13(5): p. 208-15. 3. Deinlein, U., et al., Plant Cell, 2012. 24(2): p. 708-23. 4. Hanikenne, M., et al., Nature, 2008. 453(7193): p. 391-5. 5. Talke, I.N., et al., Plant Physiol, 2006. 142(1): p. 148-67. 6. Mirouze, M., et al., Plant J, 2006. 47(3): p. 329-42. 7. Sels, J., et al., Plant Physiol Biochem, 2008. 46(11): p. 941-50. 8. Shahzad, Z., et al, New Phytol, 2013. 200(3): p. 820-33.