Download Abstracts - Institute of Plant Sciences Paris

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
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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.