Abstract book - Post-transcriptional Gene Expression Regulation in

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Organizers, Patronage, and Sponsors
Wielkopolska Centre for Advanced Technologies (WCAT)
and its mission
Wielkopolska is among the leading academic centres in Poland, harbouring a great R&D potential.
The essence of the comprehensive knowledge transfer model, generated and pursued in the city of Poznań,
the capital of Wielkopolska region, is building an effective relationship between invention, pursued at
universities and research institutes, and innovation, developed at the Poznań Science and Technology Park
of Adam Mickiewicz University Foundation, by creating all elements necessary for the effective transfer
of knowledge, especially Polish scientific and technological achievements, to business practice.
At the core of the model is the Wielkopolska Centre for Advanced Technologies (WCAT) in Poznań,
a multi-disciplinary institution focused on design and characterisation of new materials and biomaterials
of multiple applications.
The main aim of the foundation of Wielkopolska Centre for Advanced Technologies– Materials and
Biomaterials (WCAT) in Poznan, is to create a multidisciplinary research centre in the field of high-tech
materials, biomaterials and nanomaterials based on recent achievements in related fields of chemistry,
chemical technologies, physics, biotechnology, biology, medicine, pharmacy and agriculture sciences.
WCAT brings together the best specialists of natural and engineering sciences and is an infrastructural
venture of the Poznań scientific community. The Centre is a consortium of five universities: the Adam
Mickiewicz University (AMU), which is the project coordinator, Poznań University of Technology,
Poznań University of Life Sciences, Poznań University of Medical Sciences and Poznań University of
Economics; four institutes of the Polish Academy of Sciences: the Institute of Bioorganic Chemistry, Plant
Genetics, Human Genetics, and Molecular Physics; Institute of Natural Fibres and Medicinal Plants; and
the Poznań Science and Technology Park of the Adam Mickiewicz University Foundation and City
of Poznań. The project is based on the research know-how and credibility of leading scientists, working in
the key institutes of the regions.
The objective of the multidisciplinary activity of the centre is to develop original methods for synthesis
of chemicals, biochemicals and agrochemicals, called fine chemicals, and a new generation of biomaterials
and nanomaterials and their precursors, designed in cooperation between the chemists, physicochemists
and biochemists. These research activity will be followed by the development of advanced technologies
and biotechnologies for the production of these fine (bio)chemicals and precursors of materials to be used
in optoelectronics, ceramics, medicine, pharmacy, agriculture and other fields of high-tech industry.
Another objective of WCAT is to create a technological basis for a number of applications for bioorganic
chemistry and biotechnology in healthcare (e.g. molecular and cellular therapies and medical diagnostics)
as well as applications in agricultural engineering and in the food industry (e.g. DNA tests in plant and
animal production, biodegradable packaging and etc.).
The special mission of WCAT is to develop multidisciplinary projects involving fundamental research
in the material sciences but simultaneously leading to advanced technologies and/or products subsequently
implemented in incubators of the innovative firms localized in Poznań Science and Technology Park, and
other industrial and technological parks, and innovative small and medium firms as well as by
international industry.The start-up companies and innovative SMEs will be at the heart of WCAT mission.
To reach the complex goals mentioned
above, we committed to ensure permanent cooperation between all units of WCAT i.e.
Centre of Chemical Technology and Nanotechnology, Centre of Industrial Biotechnology
with a Greenhouse, Centre of Medical Biotechnology with an Animal House, Centre of
Material Sciences with a Regional Laboratory
of Unique Equipment, functioning as one research organism.
As emphasized, the Regional Laboratory of
Unique Equipment should be of service to all
other units of WCAT. The Laboratory has
highly specialized equipment which will be
available for use by the scientific community and small and medium enterprises from the region as well as
R&D centres of Polish and international (European) companies. The Service and Technical Facilities with
the Technology Transfer Centre will ensure efficient collaboration among all parts of WCAT.
The vision of WCAT is to include existing organizations (universities, research institutes, and sciencetechnology park) to act like one independent entity, which will generate synergies by combining the work
of the best scientists, as an independent institute modelled on the Fraunhofer Society (most experienced of
R&D Centres in Europe with 60-70 years tradition).
The WCAT project is co-financed (85%) by the European Regional Development Fund under
the Operational Programme Innovative Economy 2007-2013 with total budget 63 million EUR.
--------------------------------------------- w w w .w c a t . p l ----------------------------------------Wielkopolska Centre for Advanced Technologies, Umultowska 89c, 61-614 Poznan, Poland
Faculty of Biology
Faculty of Biology
Dean – Prof. Bogdan Jackowiak
The Faculty of Biology is one of the fifteen faculties of Adam Mickiewicz University (AMU).
It achieved its present institutional form in 1984, but natural sciences were always present at AMU
since the very origin of the University in 1919. Today, the Faculty is organized into four institutes:
Anthropology, Experimental Biology, Molecular Biology and Biotechnology, and Environmental
Biology. These are further divided into 27 departments and five laboratories. In addition, there are
also six central laboratories, four of which host core facilities of the Faculty (isotopic lab, electron and
confocal microscopy lab, molecular biology techniques lab, and phytotron facility), one that groups
all the natural collections, and one that specializes in educational issues. At present, the Faculty
comprises more than 300 employees with roughly 200 academic staff. Its strength is built around ca.
40 full professors and 40 AMU professors; 1400 students, studying various full-time or extramural
study programs, together with 150 PhD students, complement the whole community. The Faculty is
seated in Collegium Biologicum, part of the state-funded state-of-the-art AMU Morasko campus
located on the outskirts of Poznań.
The Faculty of Biology AMU offers an outstanding research environment coupled with world-class
state-of-the art infrastructure. The Faculty staff has been engaged in EU FP6/FP7 funded research
projects, such as AEROTOP, MONALISA, FUNGEN, CONTRASTRESS, EURASNET, EVOLGEN, HIALINE;
the Marie Curie Actions. Moreover our research projects were supported by the Norwegian financial
mechanism, European Structural Funds, Ministry of Science and Higher Education, Ministry of
Agriculture and Rural Development, National Science Centre and Foundation for Polish Science.
In May 2014 “Poznań RNA Centre” formed by the Faculty of Biology AMU in Poznań together with
the Institute of Bioorganic Chemistry, Polish Academy of Sciences received a status of Leading
National Research Centre – first of only two such institutions in biological sciences in Poland.
In August 2014 we open recruitment for students wishing to acquire M.Sc. in Biotechnology. This
program continues the successful study program established within a framework of the UNIKAT
project co-funded by the European Union within the European Social Fund. For more details see –
www.unikat.amu.edu.pl. These studies emphasize molecular biology, genetics, gene therapy and
systems biology approaches, and aims to give practical training and advanced knowledge in
bioeconomy and biotechnology legislation.
Umultowska 89, Collegium Biologicum, 61-614 Poznan
tel. +48 61 829 55 52, fax +48 61 829 56 36
[email protected]
www.biologia.amu.edu.pl.........................................................................................................
INSTITUTE OF BIOORGANIC CHEMISTRY
POLISH ACADEMY OF SCIENCES
Director – Prof. Marek Figlerowicz
The Institute of Bioorganic Chemistry Polish Academy of Sciences (IBCH PAS) was
established over 25 years ago. The process of IBCH PAS formation dates back to 1969
when the Department of Stereochemistry of Natural Products was brought into being at
the Institute of Organic Chemistry PAS. In 1980, the Department of Stereochemistry of
Natural Products was transformed into an independent entity - Department of Bioorganic
Chemistry PAS. In 1988, the latter was finally converted into the Institute of Bioorganic
Chemistry Polish Academy of Sciences. Today, together with the affiliated Poznan
Supercomputing and Networking Center, the IBCH PAS has more than 460 staff
members, including 100 research scientists (33 Professors). In addition, about 80 Ph.D.
students are currently involved in the research projects conducted at IBCH PAS.
The scientific portfolio of the Institute has many dimensions: synthesis and structure of
natural products, in particular nucleic acids and their components; biochemistry,
molecular and structural biology of model biological systems, genetic engineering,
genomics and bioinformatics. IBCH PAS is authorized to confer the degree of doctor and
habiliated doctor in chemistry and biochemistry.
The Institute is organized into 10 research departments and 6 core laboratories. Within
the structure of the Institute, there are also other crucial units associated: the PAS
Poznan Science Center, Scientific Publishers, Guest Rooms, and Library. In the latest
years, the Institute in collaboration with the Poznan University of Technology have
created a European Center for Bioinformatics and Genomics, a unique unit in Poland.
Z. Noskowskiego 12/14, 61-704 Poznań, Poland
tel: (+48) 061-852-85-03, 061-852-89-19
fax: (+48) 061-852-05-32
e-mail: [email protected]
www.ibch.poznan.pl
Institute of Molecular Biology and Biotechnology
The Institute of Molecular Biology and Biotechnology (IMBB) at the Faculty of Biology of Adam Mickiewicz
University in Poznań, Poland was created in 1992. The Institute’s mission is to perform outstanding
research to reveal functions of the cell and organism at the molecular level. Organisms belonging to all
domains of life are studied in IMBB: bacteria, animals, fungi, and plants. Studies encompass basic research
on gene expression regulation, epigenetics and other aspects of chromatin structure, microRNA,
alternative splicing, molecular mechanisms of translation regulation, molecular basis of energy conversion
as well as understanding of renal disease, viral infection, atherosclerosis and myotonic dystrophy at both
the molecular and clinical levels. In addition, the IMBB scientists are also involved in translational science
developing assays for diagnostic and prognostic applications in various human diseases, identifying and
characterizing novel therapeutic targets, searching for small molecule inhibitors of RNA-protein interactions
that can be used as drugs, and developing therapies involving antisense oligomers to threat myotonic
dystrophy. In IMBB the molecular basis of plant response to various environmental cues is also intensively
studied. This part of IMBB research is strongly connected with agrobiotechnological tasks to obtain crop
plants that are more adaptable to changing climate conditions. Bioinformatics is recognized as a
fundamental component of modern biology. For the past several years, IMBB has invested numerous
resources for the development of this dynamic field. Computational biology at IMBB is focused on genome
analysis and gene discovery, comparative and evolutionary genomics, and on general mechanisms of
evolution. Moreover, studies on the relationship between the sequence, structure and function of proteins
and RNAs, as well as the modelling of RNA-protein structures, are also conducted. An important goal of in
silico studies performed at IMBB is the discovery of new drugs that is carried out in close collaboration with
molecular biologists and clinicians. One of the most important activities of IMBB bioinformaticians is the
development of advanced algorithms to support biotechnological research. A strong connection between
computational analyses and bench experiments is a trademark of IMBB.
Scientific cooperation is stimulated and supported by international research projects within the limits of
bilateral agreements and programs of the European Union and the organization of the international
workshops and conferences.
Our PhD program is designed to prepare young scientists for successful careers in research, teaching, and
industry throughout the world. If you are interested in applying please contact directly any of the IMBB
group leaders or contact the IMBB secretariat ([email protected]).
For more information see the IMBB webpage http://ibmib.amu.edu.pl/
Umultowska 89, Collegium Biologicum, 61-614 Poznan tel. +48 61 829 59 50, fax +48 61 829 56 36
[email protected]
www.ibmib.amu.edu.pl
Poznań RNA Research Centre
Leading National Research Centre - KNOW
In 2011, a new Act on Higher Education created a possibility to establish the most prestigious status
of KNOW – Leading National Research Centre. This status, assigned through competition, can only be
given to institutions carrying out scientific research at the highest level and providing PhD studies of
the highest quality. Till now 6 KNOW centres have been named in exact sciences (mathematics,
chemistry, physics) and medical sciences. In 2014, 4 new KNOW centres have been named in natural
sciences, and in agriculture, forestry and veterinary sciences. On May 15th, The Poznań RNA Research
Centre consisting of the Faculty of Biology, Adam Mickiewicz University in Poznań and the Institute of
Bioorganic Chemistry, Polish Academy of Sciences, received KNOW status in biological sciences for 5
years. This status is closely related with additional state funds aimed at improving the quality of
science and education. Up to 50 million Polish zlotys will be transferred from the Polish Ministry of
Science and Higher Education to the Poznań RNA Research Centre. The money will support various
aspects of RNA research from basic research on RNA metabolism, through more applied studies on
RNA viruses and the role of RNA in cancer and inherited diseases and their therapies, up to the
development of bioinformatics tools allowing advanced structure-function studies.
Four new research groups will be created. Moreover, the Centre will open a new international school
for PhD students. For young scientists from other scientific institutions a special fund will be created
to provide financial support for training in RNA research techniques. All information about the
recruitment of new group leaders as well as details on our international PhD program will be
announced soon on the newly created Poznań RNA Centre webpage. All young scientists are
welcome to apply!
Umultowska 89, Collegium Biologicum, 61-614 Poznan
tel. +48 61 829 55 52, fax +48 61 829 56 36
[email protected]
www.biologia.amu.edu.pl
Z. Noskowskiego str. 12/14, 61-704 Poznan
tel. +48 61 852 85 03, fax +48 61 852 05 32
[email protected]
www.ibch.poznan.pl
Honorary Patronage
RYSZARD GROBELNY
PROF. BRONISLAW MARCINIAK
PROF. BOGDAN JACKOWIAK
PROF. EWA LOJKOWSKA
PROF. ANDRZEJ K. KONONOWICZ
Mayor of the City of Poznan, Poland
Rector of the Adam Mickiewicz University in Poznan, Poland
Dean of the Faculty of Biology, Adam Mickiewicz University
in Poznan, Poznan, Poland
President of the Polish Society of Experimental Plant Biology
former President of the Polish Society of Experimental Plant
Biology
Organizing Committee
Session 1.
ANDREA BARTA
JOHN BROWN
Regulation of pre-mRNA processing
Medical University of Vienna, Vienna, Austria
University of Dundee at the James Hutton Institute and the
James Hutton Institute, Dundee, Scotland, United Kingdom
Session 2.
JOANNA KUFEL
RNA turnover and surveillance
Warsaw University, Warsaw, Poland
Session 3.
MARTIN CRESPI
Long non-coding RNAs
Centre National de la Recherche Scientifique (CNRS),
Gif-sur-Yvette, France
Session 4.
ARTUR JARMOLOWSKI
ZOFIA SZWEYKOWSKA-KULINSKA
Biogenesis and function of small RNAs
Adam Mickiewicz University in Poznan, Poznan, Poland
Adam Mickiewicz University in Poznan, Poznan, Poland
Local Organizers
TOMASZ BIELECKI
IWONA KANONIK-JEDRZEJAK
ANDRZEJ PACAK
MARCIN PIECZYNSKI
HALINA PIETRYKOWSKA
ALEKSANDRA SWIDA-BARTECZKA
Adam Mickiewicz University in Poznan, Poland
Post-transcriptional Gene Expression Regulation in Plants
Poznań, June 30th – July 2nd 2014
Contents
Session 1. Regulation of pre-mRNA processing
Chair: MARCELO YANOVSKY, DOROTHEE STEIGER
Session 2. RNA turnover and surveillance
Chair: JOANNA KUFEL
Session 3. Long non-coding RNAs
Chair: MARTIN CRESPI
Session 4. Biogenesis and function of small RNAs
Chair: ANDRZEJ WIERZBICKI
Poster session
Contributions are printed as delivered by authors without substantial modifications
Editors of abstract section:
HALINA PIETRYKOWSKA, MARCIN PIECZYNSKI, ANDRZEJ PACAK, ALEKSANDRA SWIDA-BARTECZKA
BioTechnologia
vol. 95(1) C pp. 37-53 C 2014
Session 1
Lecture 1.1
Genome-wide analysis reveals photoreceptors regulate
alternative splicing in plants
HSHIN-PING WU, YI-SHIN SU, YU-RONG CHEN, HSIU-CHEN CHEN,
CHIA-CHEN WU, WEN-DAR LIN, SHIH-LONG TU
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Light is one of the most important factors influencing plant growth and development. Changes of light condition
affect many developmental programs throughout the life cycle of plants. Light-sensing photoreceptors play major
roles in regulating photomorphogenic changes via signal transduction and gene regulation. Although various levels
of gene expression are modulated by light, regulation at the pre-mRNA splicing step is less discussed. We performed
mRNA sequencing to analyze transcriptome changes during light exposure in Physcomitrella patens . In addition to
transcriptional regulation, light induces intensive alternative splicing. Light-responsive intron retention preferentially
occurred in transcripts for photosynthesis and translation in Physcomitrella , which reveals that light-mediated splicing regulation has transcript specificity. Many splicing-related and light signaling gene transcripts were alternatively
spliced in responding to light changes. Moreover, intron retention was rapidly induced by light but misregulated in
phytochrome-deficient and -knockout mutants, suggesting the involvement of photoreceptors in splicing regulation.
We also identified an exonic splicing element that potentially functions in the light-regulated intron retention regions.
In summary, our results support that during photomorphogenesis, alternative splicing is rapidly fine-tuned by light
to re-organize certain metabolic processes and modulate photomorphogenic gene regulation. Photoreceptors primarily participate in regulation of alternative splicing.
38
Session 1
Lecture 1.2
Polypyrimidine tract binding proteins
– regulators of alternative splicing & development
in Arabidopsis
C. RUHL, D. LAMBERT, A. WACHTER
Center for Plant Molecular Biology, University of Tubingen, Tubingen, Germany
Alternative splicing (AS) allows the formation of distinct transcript variants from one type of precursor mRNA
and affects, according to recent studies, more than 60% of all multi-exon genes in Arabidopsis . Despite its prevalence, information on the regulatory factors constituting the splicing code and the functional implications of AS in
plants is scarce. We have identified polypyrimidine tract binding proteins (PTBs), members of the group of heterogeneous ribonucleoprotein (hnRNP) proteins, as major regulators of AS in Arabidopsis. PTB-mediated AS control
is linked to diverse biological processes, including seed germination and flowering regulation. PTB knockdown also
results in a serrated leaf phenotype, stunted growth, and elevated drought resistance. Our current work aims, on
one hand, at providing molecular links between these processes and PTB-mediated AS regulation. On the other hand,
the interaction between plant PTB proteins and their target RNAs is further characterized. Our findings will help
to decipher the plant splicing regulatory code and are expected to provide novel insight into the versatile functions
of AS in plant biology.
39
Short talk 1.1
The SR45 splicing factor, involved in sugar
and ABA signaling, targets alternative splicing
of several Arabidopsis genes
and modulates SnRK1.1 levels
R.F. CARVALHO1, C.G. SIMPSON2, P. CROZET1, I.C. R. BARBOSA1,
J.W. BROWN2,3, E. BAENA-GONZÁLEZ1, P. DUQUE1
1
2
3
Instituto Gulbenkian de Ciência, Oeiras, Portugal
Cell and Molecular Sciences, the James Hutton Institute, Dundee, Scotland, United Kingdom
Plant Sciences Division, University of Dundee at the James Hutton Institute, Dundee, Scotland, United Kingdom
Alternative splicing is a versatile means of regulating gene expression and generating proteome diversity likely
to be crucial in plant responses to external and endogenous cues. Higher plants are able to transduce sugar signals
in order to adjust developmental programs, maintain energy homeostasis, and achieve stress tolerance. Glucose,
being a carbon and energy source, also plays an important regulatory role as a central signaling molecule. The first
enzyme in glycolysis, hexokinase (HXK), is the conserved glucose sensor in a wide range of organisms including
plants, where both HXK-dependent and HXK-independent pathways appear to coexist. Furthermore, an Arabidopsis
thaliana protein kinase, SnRK1.1, has been described as a sugar sensor and central integrator of a transcription
network for stress and energy signaling. We previously reported that SR45, a plant-specific SR-related splicing factor,
negatively regulates glucose signaling via downregulation of the ABA pathway during early seedling development
in Arabidopsis . Surprisingly, we found that disruption of HXK does not supress the glucose phenotypes of the sr45-1
mutant, indicating a mechanism independent of this sensor. However, glucose-treated sr45-1 leaves contain significantly higher levels of the SnRK1.1 protein kinase due to lower degradation rates of this protein in the mutant.
Importantly, the sr45-1 mutation causes changes in alternative splicing of the 5PTase13 gene, encoding an inositol
polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1.1 in vitro thus
providing a mechanistic link between SR45’s function and the modulation of SnRK1.1 levels in response to glucose.
We used a high-resolution RT-PCR alternative splicing panel to identify additional SR45 endogenous targets. To
pinpoint direct targets of this splicing factor, we performed electrophoretic mobility shift assays to test the binding
of recombinant SR45 to the 5PTase13 mRNA as well as to transcripts of selected genes showing substantial
alternative splicing pattern changes in the RT-PCR panel. Finally, using a protoplast transient expression system,
we investigated whether the two isoforms of the 5PTase13 protein have a differential effect in the stability of the
SnRK1.1 protein kinase.
40
Session 1
Short talk 1.2
Critical roles of snRNP biogenesis
for spliceosome formation
in plant regeneration
MISATO OHTANI
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan
RIKEN Center for Sustainable Resource Science, Japan
Pre-mRNA splicing is a critical process in gene expression in eukaryotic cells. This molecular process is executed
by the spliceosome, which contains UsnRNPs (U small ribonucleoprotein particles) comprised of a specific kind of
UsnRNA (uridine-rich small nuclear RNA) and its tightly associated proteins as core factors. Arabidopsis mutants
srd2 (shoot redifferentiation defective2 ) and rid1 (root initiation defective 1 ) were shown to have severe defects
in hypocotyl dedifferentiation and de novo meristem formation in tissue culture under high temperature conditions.
SRD2 encodes an activator of snRNA transcription, and in the srd2 mutant, snRNAs were significantly decreased
under the restrictive temperature. RID1 encodes a DEAH-box RNA helicase similar to yeast pre-mRNA splicing
factor Prp22. Transient expression analysis using intron-containing reporter genes and RT-PCR analysis on alternative splicing events showed that pre-mRNA splicing was affected by the srd2 and rid1 mutations during hypocotyl
dedifferentiation. Interestingly, artificial introduction of RID1 could not recover the growth phenotype of yeast prp22
mutant, indicating that RID1 is not replaceable to Prp22 but has a different function from Prp22 in pre-mRNA splicing at the molecular level; the fact that RID1-YFP was preferentially localized in the nucleolus, a place of snRNP
biogenesis, suggested that RID1 would contribute to snRNP biogenesis and consequently spliceosome formation
in the nucleolus. The results collectively suggest that robust levels of pre-mRNA splicing, which might be regulated
through snRNP biogenesis, are critical for both of cell dedifferentiation and de novo meristem formation, elementary
steps of plant regeneration.
41
Lecture 1.3
Decipher the RNA structural code:
a transformative platform
reveals novel regulatory features
YILIANG DING
Department of Cell and Developmental Biology, John Innes Centre, Norwich, United Kingdom
RNA structure plays critical roles in regulating various post-transcription events involved in translation, splicing,
and polyadenylation. However, determining RNA structure in vivo has been very challenging, particularly for low
abundance RNAs. In most cases RNA structures are based on in vitro synthesized RNAs or in silico predictions. Also
lack of genome-wide in vivo RNA structural data limits our understanding of how RNAs fold and regulate gene expression globally in vivo. Here, we established a transformative platform to probe in vivo RNA structures at both the
genome wide scale and in targeted individual cases. The genome wide study reveals native RNA structural features
that relate to numerous biological processes including translation initiation and efficiency, alternative polyadenylation, and alternative splicing. The targeted individual RNA structure study has achieved attomolar (10!17) sensitivity,
which allows the analysis of very low abundance mRNAs in vivo. These novel and powerful methods for the investigation of RNA structure-function relationships in plants and should be applicable to any organism.
42
Session 1
Lecture 1.4
Exitron splicing, a new type
of alternative splicing event
shaping the eukaryotic proteome
Y. MARQUEZ1, M. HOPFLER1,2, Z. AYATOLLAHI1, A. BARTA1, M. KALYNA1,3
1
2
3
Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria
Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Germany
Department of Applied Genetics and Cell Biology, BOKU – University of Natural Resources and Life Sciences, Austria
Alternative splicing is a key mechanism for increasing transcriptome and proteome diversity. Here, we identify
exitron splicing as a novel type of alternative splicing event in plants and humans. Exitrons (exonic introns) are internal regions of coding exons that can be removed by the spliceosome. Intriguingly, intronless genes can be also
alternatively spliced via exitron usage, providing the first evidence of splicing in these genes. We show that exitron
splicing is regulated in a tissue-specific manner, in response to stress and by splicing factors. Exitron-encoded sequences contain protein domains interacting with different molecules and are enriched in disordered regions and
post-translational modifications; consequently their splicing impacts proteome dynamics and remodeling. We propose
a “splicing memory” mechanism for the origin of exitrons whereby exitrons are derived from ancestral coding exons
through a history of intron loss and maintenance of vestigial splicing regulatory elements that drives exitron evolution.
43
Short talk 1.3
Unraveling post-transcriptional networks:
Analysis of RNA-protein interactions with RNA
immunoprecipitation
K. MEYER, T. KOSTER, D. STAIGER
Molecular Cell Physiology, Faculty for Biology and Institute for Genome Research and Systems Biology,
Bielefeld University, Bielefeld, Germany
RNA-BINDING PROTEINS (RBPs) control the fate of mRNAs including splicing, 3N end formation, nuclear
export and decay through dynamic interaction with cis -regulatory motifs, thereby forming messenger ribonucleoprotein complexes (mRNPs). The knowledge on post-transcriptional networks controlled by RBPs, especially in
plants, is limited, not least due to the experimental challenge in defining RNA targets of candidate posttranscriptional regulators. We established a straightforward protocol for in vivo RNA immunoprecipitation (RIP) to
isolate messenger ribonucleoprotein particles containing RBPs and associated RNAs in Arabidopsis (Köster and
Staiger, 2014). Here, an At GRP7 (Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7)-GFP fusion
protein is precipitated via GFP-Trap beads from whole cell extracts. The associated RNAs are isolated and analyzed
by quantitative Real Time PCR or high-throughput sequencing. Using this method, we showed that AtGRP7 regulates
alternative splicing of downstream targets by direct in vivo binding. Additionally, RIP experiments showed that
At GRP7 interacts with microRNA precursors, suggesting a role of At GRP7 in miRNA maturation. Future approaches
will focus on the adaptation of iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation; König
et al., 2011) to the plant system to get a genome wide insight into the post-transcriptional networks controlled by
At GRP7 and to identify the precise interaction sites.
44
Session 1
Short talk 1.4
Deciphering the functions of P-class PPR proteins
by combining bioinformatic and experimental methods
SHENG LIU1, WENJUAN WU2, K.E. HOWELL1, J. MELONEK1,
H. RUWE3, C. SCHMITZ-LINNEWEBER3, JIRONG HUANG2, I. SMALL1
1
ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Australia
2
3
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology,
Chinese Academy of Sciences, Shanghai, China
Molecular Genetics, Institute of Biology, Faculty of Life Sciences, Humboldt University of Berlin, Berlin, Germany
Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA binding proteins located in organelles that
are involved in broad range of post-transcriptional RNA maturation processes. The plant PPR proteins are grouped
into P and PLS classes which generally fulfil distinct functions. The majority of the PLS-class PPR proteins function
as editing factors. P-class PPR proteins act in defining 5N and 3N RNA termini, splicing, and promoting or blocking
initiation of translation. Thus P-type PPR proteins play key roles in determining the fate (protection or degradation)
of their target RNAs. The recently developed “PPR code” suggests that it might be possible to predict the RNA target sequences of a particular PPR protein. Verifying editing site predictions is technically straightforward, but verification of binding sites of P-class PPR proteins, where no modification of the target RNA ensues, has been challenging. Comparing small RNA libraries between wild-type and mutants lacking specific P-type PPR proteins gives
a rapid and effective way of identifying probable binding sites through analysis of putative “PPR footprints” – RNA
remnants protected from degradation by a PPR protein. Here we present an example where a combination of bioinformatic and experimental methods have led to elucidation of the function of the SOT1 (suppressor of thylakoid
formation 1) protein in maturation of the chloroplast 23S rRNA.
45
Short talk 1.5
Spliceosome disassembly factor NTR1
is involved in transcriptional pausing
at alternative exons in Arabidopsis
Y. GUO 1, J. DOLATA2, G. BRZYZEK1, A. JARMOLOWSKI2, S. SWIEZEWSKI1
1
Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
2
Department of Gene Expression, Institute of Molecular Biology and Biotechnology,
Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
The interconnection between transcription and splicing is a subject of intense study in many different organisms.
We have identified an Arabidopsis homologue of NTR1 (AtNTR1), a conserved spliceosomal disassembly factor, as
a protein required for co-transcriptional pausing at alternative splice sites. We report that AtNTR1 is required for
the correct expression and splicing of DOG1, a regulator of seed dormancy. Analyses of DOG1 and other genes
splicing defects, identification of NTR1 interactors and AtNTR1 co-immunolocalisation with PolII have shown that
in addition to a well-conserved function in splicing, AtNTR1 plays a role in transcription elongation at alternative
exons. In agreement with the altered elongation rate in atntr1, we demonstrate that the majority of splicing defects
caused by the lack of AtNTR1 are opposite to changes observed in the TFIIS mutant, in which endonucleolytic
cleavage by PolII is blocked. In addition, atntr1 shows decreased PolII occupancy at the majority of the alternatively
misspliced exons and introns tested. We provide evidence that the elongation defects observed in atntr1 are not an
indirect effect of splicing defects. We conclude that AtNTR1 is required for localised transcriptional pausing at the
affected alternatively spliced exons and introns.
46
Session 1
Lecture 1.5
Alternative splicing controls translation
of a novel Arabidopsis transporter
to promote plant zinc tolerance
E. REMY1, T.R. CABRITO2, R.A. BATISTA1, M.A.M. HUSSEIN2,
M.C. TEIXEIRA2, A. ATHANASIADIS1, I. SA-CORREIA2, P. DUQUE1
1
2
Instituto Gulbenkian de Ciencia, Oeiras, Portugal
Institute for Biotechnology and Bioengineering (IBB), Instituto Superior Tecnico, University of Lisbon, Lisbon, Portugal
Root vacuolar sequestration is one of the best-conserved plant strategies to cope with heavy metal toxicity.
We have found that zinc (Zn) tolerance in Arabidopsis thaliana requires the action of a novel membrane transporter
belonging to the major facilitator superfamily (MFS). ZIF2 (ZINC-INDUCED FACILITATOR 2) localizes primarily
at the tonoplast of root cortical cells and is a functional transporter able to mediate Zn efflux when heterologously
expressed in yeast. In Arabidopsis, loss of ZIF2 function exacerbates plant sensitivity to excess Zn, while its overexpression enhances Zn tolerance. Zn content analyses indicate that, although not affecting the overall plant Zn
status, ZIF2 activity influences Zn tissue partitioning, driving root immobilization of the metal and thereby preventing its translocation to the shoot. The ZIF2 gene is Zn-induced and an intron retention event in its 5N UTR generates two splice variants (ZIF2.1 and ZIF2.2 ) encoding the same protein. Importantly, high Zn favors production
of the longer ZIF2.2 transcript, which compared to ZIF2.1 confers greater Zn tolerance to transgenic plants by promoting higher root Zn immobilization. We demonstrate that the retained intron in the ZIF2 5’ UTR enhances translation in a Zn-responsive manner, markedly promoting ZIF2 protein expression under excess Zn. Moreover, Zn
regulation of translation driven by the ZIF2.2 5N UTR depends largely on a predicted stable stem loop immediately
upstream of the start codon that is lost in ZIF2.1. Taken together, our findings show that alternative splicing controls the levels of a Zn-responsive mRNA variant of the ZIF2 transporter to regulate plant tolerance to the metal ion.
47
Lecture 1.6
A role for LSM genes
in the regulation of circadian rhythms
in plant and human cells
S.P. SANTANGELO, M.J. YANOVSKY
Instituto de Investigaciones Bioquimicas de Buenos Aires, Fundacion Instituto Leloir-CONICET, Buenos Aires, Argentina
Circadian clocks allow organisms to time biological processes to the most appropriate phases of the day and year.
We are interested in deciphering the regulatory networks that control clock function in plants, since this knowledge
could be used to manipulate flowering time, a key factor influencing crop productivity. There is increasing evidence
that propper regulation of clock function involves alterations in alternative splicing (AS) of clock genes, but little is
known about the mechanisms linking AS and the clock. We have recently shown that defects in PROTEIN ARGININE METHYL TRANSFERASE 5, which transfers methyl groups to arginine residues present in Sm and LSm
spliceosomal proteins, impair circadian rhythms in Arabidopsis. Here we show that some LSM genes, encoding core
components of the spliceosomal U6 SnRNP complex, play a regulatory role in the control of circadian rhythms in
plants and mammals. We found the circadian clock regulates expression of LSM5 in Arabidopsis plants and several
LSM genes in mouse SCN. Further, mutations in LSM5 or LSM4 genes in Arabidopsis, or down-regulation of
LSM3, LSM5 or LSM7 expression in human cells, lengthens circadian period. Changes in expression and alternative
splicing of some core-clock genes were identified in Arabidopsis lsm5 mutants, but the precise molecular mechanism
causing period lengthening remains to be identified. Genome-wide expression analysis of either a weak lsm5 or
a strong lsm4 mutant allele in Arabidopsis revealed larger effects on alternative compared to constitutive splicing.
Remarkably, no statistically significant defects were observed in the majority of all introns evaluated using RNA-seq
in the strong lsm4 mutant allele used in this study. These findings support the idea that some LSM genes play regulatory rather than constitutive roles in RNA processing, and that clock regulation of LSM gene expression is one
mechanism integrating transcriptional and post-transcriptional regulatory layers within plant and mammalian circadian networks.
48
Session 1
Lecture 1.7
Alternative splicing of conserved
alternative exons and splice
sites as a mechanism
for gene expression regulation in plants
J.W.S. BROWN1,2, M. SPENSLEY1, C. CALIXTO1, C.G. SIMPSON2
1
Division of Plant Sciences, College of Life Sciences, University of Dundee at the James Hutton Institute, Dundee, United Kingdom
2
Cell and Molecular Sciences, The James Hutton Institute, Dundee, Scotland, United Kingdom
Relatively few examples of conserved alternative splicing in plants are known to date. Analysis of the link between alternative splicing (AS) and nonsense-mediated decay (NMD) using a high resolution RT-PCR system identified NMD-sensitive transcripts that contain PTCs located within alternatively spliced exons (Kalyna et al., 2012).
Many of these alternative exons (AE) were conserved among different plant species. We therefore performed a genome-wide analysis of alternative exons in Arabidopsis to identify conserved AEs focussing on PTC+ AEs using
annotated transcripts from TAIR and assembled transcripts from our AS discovery RNA-seq analysis (Marquez et al.,
2012). In addition, an unbiased computational screen for conserved regions within introns identified novel, conserved alternative exons and other alternative splicing events. Multiple sequence alignments have been generated and
manually curated. We are currently validating the conserved AS events in both Arabidopsis and barley and are
examining their AS under stress conditions. Extensive alternative splicing regulates Arabidopsis clock genes through
dynamic changes in AS transcripts when plants are transferred to low temperatures (James et al., 2012). AS events
in AtLHY and AtPRR7, in particular, are temperature-dependent and generate altered levels of productive mRNAs
through AS/NMD. To examine whether clock genes have conserved modes of regulation in other plant species, we
identified 21 barley core clock and clock-associated genes, 60% of which are true Arabidopsis orthologues. We have
identified AS in some of these genes, including HvLHY and HvPpd-H1 (orthologue of AtPRR7 ) and demonstrate
temperature-specific AS. Although there is little conservation of AS events themselves or sequence conservation
around splicing events, regulation of expression by AS at low temperatures may be functionally conserved. This novel
layer of fine clock control observed in two different species, a model plant and a crop species, might help our understanding of plant adaptation in different environments and ultimately may offer a new range of targets for plant improvement.
49
Lecture 1.8
Global analysis of targets of a splicing regulator
in Arabidopsis : Implications in stress responses
D. XING1, YAJUN WANG1, M. HAMILTON2, A. BEN-HUR2, A.S.N. REDDY1
1
Department of Biology, Program in Molecular Plant Biology, Colorado State University, Fort Collins, Colorado, USA
2
Computer Science Department, Colorado State University, Fort Collins, Colorado, USA
Alternative splicing (AS) of pre-mRNAs has been implicated in modulating several developmental and stress responses in plants. Regulation of alternative splicing is a complex process involving many RNA binding regulatory proteins that modulate splice site choice. Serine/arginine-rich (SR) and SR-like RNA binding proteins are key regulators
of both constitutive and alternative splicing of nuclear pre-mRNAs as well as other aspects of RNA metabolism
in eukaryotes. SR45, one of the SR-like proteins that is similar to human RNPS1 in its RNA recognition motif and
in its domain organization, has been shown to regulate AS of pre-mRNAs of SR genes and multiple developmental
and stress responses. To gain mechanistic understanding of AS regulation by SR and SR-like proteins in plants it is
necessary to i) identify RNAs associated with these proteins, ii) map cis -elements necessary for this association, and
iii) determine the effect of this association on regulated pre-mRNA splicing. However, no global studies have been
performed thus far to identify RNAs associated with any of the plant RNA binding proteins. To address this, we
performed the first global study to identify SR45 associated RNAs (SARs) in the transcriptome of Arabidopsis seedlings using RNA-immunoprecipitation (RIP) followed by high throughput Illumina sequencing (RIP-seq). The SR45
knockout mutant line (sr45 ) expressing SR45-GFP fusion was used for RIP analysis using a GFP antibody and
a transgenic line expressing GFP alone was used as a control. The efficacy of RIP was monitored by measuring the
enrichment of two previously known targets of SR45 using quantitative RT-PCR (qRT-PCR). Analyses of the RIP-seq
data has revealed that there are >3000 SARs in seedlings. In independently performed RIP assays, the association
of 22 out of 23 randomly selected SAR targets was further verified, confirming the validity of our RIP results. Further
analyses of these SARs uncovered some unexpected roles of SR45 in RNA metabolism. Analysis of SRAs for potential
RNA motifs has led to identification of four significantly over-represented RNA motifs in specific locations relative
to splice sites. Two of these are similar to known splicing regulatory motifs whereas the other two are novel. Interestingly, all four motifs identified in SARs were also found in a recent study we performed for discovery of putative
splicing regulatory motifs that are conserved across plant species. Finally, the SARs are significantly enriched
in transcripts involved in stress responses, confirming the roles of SR45 in regulating stress responsive genes at
the post-transcriptional level.
50
Session 1
Short talk 1.6
A chloroplast retrograde signal regulates nuclear
alternative splicing
E. PETRILLO1, M.A. GODOY HERZ1, A. FUCHS2, D. REIFER2, J. FULLER3, M.J. YANOVSKY4,
C. SIMPSON3, J.W.S. BROWN3,5, A. BARTA2, M. KALYNA2, A.R. KORNBLIHTT1
1
Laboratorio de Fisiologia y Biologia Molecular, Departamento de Fisiologia, Biologia Molecular y Celular,
IFIBYNE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
2
3
Max F. Perutz Laboratories, Medical University of Vienna, Vienna, Austria
Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, Scotland
4
5
Fundacion Instituto Leloir, IIBBA-CONICET, Buenos Aires, Argentina
Division of Plant Sciences, University of Dundee at The James Hutton Institute, Invergowrie, Dundee, Scotland
Light is a source of energy and also a regulator of plant physiological adaptations. We 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 signalling molecule travels through the plant. Using
photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of
plastoquinones initiates a chloroplast retrograde signalling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.
51
Short talk 1.7
The competition between splicing
and microRNA processing machineries
in the biogenesis of miRNAs located
within the introns of protein-coding genes
K. SKORUPA, M. BARCISZEWSKA-PACAK, L. SOBKOWIAK,
D. BIELEWICZ, A. JARMOLOWSKI, Z. SZWEYKOWSKA-KULINSKA
Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poland
MicroRNAs are small non-coding RNAs of about 21 nt in length, which act by regulating expression of many genes in the cell. The target mRNAs are recognized by miRNAs in a sequence-specific manner and subsequently cleaved or blocked by inhibiting their translation. In plants, miRNAs are encoded mostly by independent transcription
units, but it has been also reported that 11 plant miRNAs are embedded within introns of other genes. Our further
bioinformatic analyses revealed the existence of the new additional 18 intronic microRNAs. 26 of them are located
within protein-coding genes and the 5N RLM RACE experiments confirmed that their transcription starts from hostgene promoters. Is there a crosstalk between spliceosome and miRNA microprocessor machineries during plant intronic microRNA biogenesis? By now three possible mechanisms of plant intronic pre-miRNA processing were
proposed: 1) pre-splicing, 2) co-splicing or 3) post-splicing intronic microRNA cleavage. Yet none of them is experimentally confirmed. To outline the role of spliceosome and plant microprocessor complex during intronic miRNA
maturation we selected A. thaliana miR402 located within the first intron of protein-coding host-gene – At1g77230.
It was previously reported that expression level of the miR402 may vary in response to abiotic stresses. We found
upregulation of mature miR402 level in heat-stress conditions and its accumulation was correlated with the inhibition
of splicing of miR402-carrying intron. Additionally, within this intron we identified several alternative proximal polyadenylation sites using 3N RACE PCR analysis. Their activation was strongly associated with splicing inhibition and
consequently with mature miR402 accumulation after heat-stress treatment in comparison to normal conditions. To
evaluate the exact role of splicing machinery during intronic miR402 processing we decided to generate constructs
containing miR402 host-gene under control of the 35S promoter and transiently expressed them in N. benthamiana
leaves. Identified conservative and alternative splice sites of miR402-bearing intron within host-gene sequence were
mutated in all possible combinations. The strong accumulation of the mature miRNA was observed in each construct
carrying mutated constitutive 5NSS. Real-time PCR results confirmed splicing inhibition after the constitutive 5’SS
inactivation, which was in correlation with the activation of proximal polyadenylation sites within the first intron of
the host-gene. These observations supported our results obtained for miR402 biogenesis regulation in A. thaliana
upon heat-stress treatment. Performed experiments confirmed the strong competition between spliceosomal
U1snRNP and plant microRNA biogenesis machinery in the case of miR402 processing: efficient splicing of miRNAcarrying intron results in lower accumulation of mature miR402. This is opposite to the results obtained for exonic
miRNAs followed by an intron. We also revealed there may be some additional players, like polyadenylation machinery, involved in plant intronic microRNA biogenesis pathway. Our results show new ways of plant MIR genes expression regulation and consequently regulation of their target genes. Funding: This work was supported by the
National Science Center (Grant no. 2012/05/N/NZ2/00955) and The Dean of the Faculty of Biology AMU (Grant
no. GDWB-03/2013).
52
Session 1
Short talk 1.8
Genome-wide analysis of heat-sensitive
alternative splicing
in Physcomitrella patens
CHIUNG-YUN CHANG1,2,3, WEN-DAR LIN1, SHIH-LONG TU1,2,4
1
2
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan
3
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan
4
Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan
As sessile organisms, plant growth and development are often influenced by temperature fluctuations of the environment. To respond to elevated temperature, the universal heat shock response (HSR) is rapidly activated for
plants to prevent damage and enhance tolerance. Different levels of gene regulation are also modulated in the cell.
Alternative splicing (AS) is well-known to increase transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns among all kingdoms, there is still less information about
whether temperature changes affect the dynamics of AS. Here we used heat shock (HS) treatments at nondamaging
temperature and mRNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis
identified significant amount of novel AS events in the moss protonema. Nearly 50% of genes are alternatively
spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site
and exon skipping are induced, indicating differential regulation of AS in response to heat. Transcripts undergoing
heat-sensitive IR are mostly involved in specific functions, which suggest that plants regulate AS with transcript
specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory
cis element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in Physcomitrella
and Arabidopsis reveals heat regulation for AS evolved early during land colonization of green plants. Our results
support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to enhance
gene regulation and reorganize metabolic processes.
53
Short talk 1.9
CBC and SERRATE cooperate
in alternative splicing regulation
of Arabidopsis thaliana gene transcripts
K.D. RACZYNSKA1, A. STEPIEN1, D. KIERZKOWSKI2, C.G. SIMPSON3,
J.W.S. BROWN3,4, Z. SZWEYKOWSKA-KULINSKA1, A. JARMOLOWSKI1
1
2
4
Department of Molecular and Cellular Biology, Institute of Molecular Biology and Biotechnology,
3
Two subunits of the Arabidopsis thaliana nuclear cap-binding protein complex (AtCBC), AtCBP20 and AtCBP80,
and the SERRATE protein (AtSE) have overlapping function in both miRNA biogenesis and pre-mRNA splicing. Recently, we have shown that AtCBC and AtSE can also regulate alternative splicing (AS) in Arabidopsis thaliana. Using
an RT–PCR alternative splicing panel we analyzed alternative splicing events in selected Arabidopsis genes, encoding mainly transcription factors, splicing factors and stress-related proteins. The analyzed events included alternative 5N and 3N splice site selection, exon skipping and intron retention. Splicing profiles were determined in wild
type plants, and the T-DNA insertion mutants: cbp20, cbp80(abh1), se-1 and the cbp20/80 double mutant. In the
cases that showed significant changes in AS, the cbc mutants preferentially affect alternative splicing events located
closest to the cap: within the first intron of the transcript, and particularly at the 5N splice site. Most of these changes
were common in all three cbp mutants suggesting that AtCBC is directly involved in the regulation of these alternative splicing events. Moreover, as we observed more changes in the cbp80(abh1) and cbp20/80 mutants than in
the cbp20 mutant, we concluded that AtCBP80 plays a more significant role in alternative splicing than AtCBP20,
probably being a platform for interactions with other splicing factors. Interestingly, many changes observed in the
cbp mutants were common to those observed in the se-1 mutant. Consequently, AtSE influences alternative splicing
in a similar way to AtCBC – preferentially affecting selection of the 5N splice site of first introns. However, the changes observed in se-1 did not correspond with the changes observed in other mutants of plant miRNA biogenesis
pathway, hyl1-2 and dcl1-7, suggesting that the role of SERRATE in alternative splicing regulation is distinct from
its role in miRNA biogenesis. In conclusion, the AtSE protein acts in cooperation with AtCBC in alternative splicing
regulation of some transcripts in plants. Indeed, using BiFC and pull down techniques we showed that both AtCBP20
and AtCBP80 co-localize and directly interact with AtSE. Possibly, AtCBC and AtSE specify the binding of U1 snRNP
to the 5N splice site on the first intron of some pre-mRNAs. To confirm AtCBC/AtSE/U1 snRNP communication we
will compare the distribution of AtCBC, AtSE and components of U1 snRNP throughout the fractions after sizefractionation of protein extract on continuous glycerol gradient.
BioTechnologia
vol. 95(1) C pp. 55-62 C 2014
Session 2
Lecture 2.1
Linking mRNA methylation and 3N end processing
to development
G. SIMPSON
Division of Plant Sciences, College of Life Sciences, University of Dundee, Scotland, United Kingdom
Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Scotland, United Kingdom
Genetic screens for mutations affecting Arabidopsis thaliana flowering time have recurrently identified proteins
that affect mRNA 3N end formation. For example, my lab revealed that the RNA binding protein encoded by FPA (one
of the original late flowering mutants identified by Maarten Koornneef) controls poly(A) site choice and transcription
termination. We have recently used transcriptome-wide single molecule RNA sequencing to assess how widespread
the impact of FPA on 3N end formation is and in doing so, linked FPA to specific sub-sets of gene targets and to
processes other than flowering time control. In order to understand the mechanism by which FPA controls RNA 3N
end formation we have now developed an in vivo interaction proteomics procedure, which identifies closely associated proteins in living plant cells. Interestingly, this proteomics approach led us to study mRNA methylation. I will
present our latest findings that link mRNA methylation and 3N end formation to development.
56
Session 2
Lecture 2.2
Unique features of the plant RNA exosome
N. SIKORSKA, H. LANGE, D. GAGLIARDI
Institut de Biologie Moleculaire des Plantes, Centre National de la Recherche Scientifique (CNRS),
Universite de Strasbourg, Strasbourg, France
RNA exosomes are present in both nuclear and cytoplasmic compartments of all eukaryotic cells and participate
in 3N-5N processing, surveillance and turnover of both coding and non-coding RNAs. Exosomes are multimeric complexes composed of a nine-subunit core complex (Exo9) which interact with RNases and activators/adapters such
as RNA helicases, RNA binding proteins and non-canonical poly(A) polymerases. The structural organization of the
core exosome (Exo9) is conserved across eucaryotes: six subunits related to bacterial RNase PH form a ring-like
structure, that is capped by three subunits with RNA binding domains. Despite this common organization, Exo9 from
plants differs remarkably from other eukaryotes. In both yeast and humans, Exo9 has lost its catalytic activity and
both endo- and exoribonuclease activities are conferred by associated RNases. By contrast, we show that Exo9 purified from Arabidopsis thaliana has a phosphorolytic activity. AtExo9 has also unique catalytic features as compared
to other phosphorolytic enzymes such as the bacterial RNase PH, PNPases or the archaeal exosomes. Interestingly,
all residues critical for the phosphorolytic activity of AtExo9 are conserved in the green lineage, suggesting an important function in plants. Another interesting feature of plant RNA exosomes is the complexity of associated co-factors. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. Our data indicate that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and
with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the
nucleoplasm. The major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products
whereas HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates.
These substrates include snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts
produced from pseudogenes and intergenic regions. Interestingly, HEN2 is conserved throughout green algae,
mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast
to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively.
57
Short talk 2.1
N 6 adenosine methylation of mRNA
– an expanding role
Z. BODI, N. ARCHER, C. PEPLOE, L. GRIFFIN-BOOTH, D. KING, G. KAHKA, R. FRAY
School of Biosciences, University of Nottingham, Nottingham, United Kingdom
N 6-methyladenosine (m6A) is a ubiquitous base modification found internally in the mRNA of most Eukaryotes.
Levels of methylation equivalent to at least 50% of transcripts carrying m6A are common, and most of this methylation appears to be associated with the 3N ends of transcripts. In Arabidopsis thaliana, all tissues contain m6A but
levels vary between tissues and developmental stages. The methylase itself (MTA) as well as other components of
the methylase complex responsible for m6A formation are essential during embryonic development, and a reduction
in m6A levels during later growth stages gives rise to plants with altered growth patterns, changed cell identities and
reduced apical dominance. A related methylase (MTB) may also be part of this complex but may have a dispensible
function in certain vegetative tissues. The global analysis of gene expression from reduced m6A plants show a dramatic increase in transcript abundance for messages indicative of various stress responses, and there is a preferential distribution of m6A with polysome-associated transcripts under certain stress conditions.
58
Session 2
Short talk 2.2
Functional specialization
of Arabidopsis poly(A) polymerases
in relation to flowering time and to abiotic stress
H. CZESNICK, C. KAPPEL, M. LENHARD
Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany
Polyadenylation of pre-mRNAs is a critical process in eukaryotic gene expression. The Arabidopsis genome encodes three nuclear poly(A) polymerases. Despite their ubiquitous expression pattern at all developmental stages
and their general enzymatic function of polyadenylating mRNAs, the three poly(A) polymerases show differentiation
of function, as assessed by their mutant phenotypes (Vi et al., 2013). One knockdown leads to an early flowering
phenotype and specific growth defects in the plant, while knocking out the other two poly(A) polymerases causes
a delay in flowering time. To unravel the function of poly(A) polymerases in the regulatory network controlling the
onset of flowering, I am using several approaches. An analysis of the transcriptome of mutant seedlings and flowers
by RNAseq revealed profound changes at the transcriptional level in poly(A) polymerase (paps ) mutants. Crosses
between paps mutants and flowering time mutants are being carried out. To reveal the molecular mechanism underlying the paps mutant flowering time phenotype, the poly(A) tail lengths and expression levels of RNAs involved
in flowering time control are being examined under different growth conditions. Recently, the paps mutants have
been revealed to exhibit opposite responses to a set of diverse stress treatments. To broaden our understanding of
the importance of polyadenylation in the plant reaction to stress, I am particularly analysing the paps mutant response to oxidative stress. Interestingly, a different polyadenylation factor mutant has been shown to be resistant to
oxidative stress before (Zhang et al., 2008). Stress-related genes are being examined for changes in poly(A) tail
lengths in the paps mutants. The detailed analysis of the differential paps mutant phenotypes adds important evidence to the novel concept of gene expression regulation based on poly(A) polymerase-specific mRNA polyadenylation control.
59
Lecture 2.3
Mechanism and evolution of the autoregulatory circuit
that controls the expression
of eukaryotic release factor 1 (eRF1) in plants
T. NYIKO, L. SZABADKAI, Z. KERENYI, A. AUBER, D. SILHAVY
Plant RNA Biology laboratory, Agricultural Biotechnology Institute, National Agricultural Research and Innovation Centre, Godollo, Hungary
When a translating ribosome reaches a stop codon three things, normal translation termination, translational
readthrough (RT) or Nonsense-mediated mRNA decay (NMD) can happen. The outcome depends on the stop context
and the level of the eRF1 and eRF3 termination factors. At normal stop context, the eRF1 binds to the stop codon
and terminates translation. RT occurs when the stop context does not favor the binding of eRF1 to the stop. Instead
the stop codon is bound by a near cognate tRNA and elongation is continued till the next in-frame stop codon. If the
3NUTR contains NMD cis elements (unusually long 3NUTR or an intron in the 3NUTR), the translation will be slow
and NMD is induced. NMD does not interfere with the peptide release but trigger the rapid decay of the target
mRNA. In bacteria, the expression of the RF2 termination factor, which functions similarly to eRF1, is strictly controlled by an autoregulatory circuit. High RF2 level leads to early termination of RF2 translation and to the accumulation of an inactive, truncated protein, while low RF2 level stimulates the expression of the active termination factor.
Although several lines of evidence support that eRF1 expression is tightly regulated in eukaryotes, the mechanism
of eRF1 control is not known. We found that in plants, eRF1 level plays a critical role in all three termination codon
related events, it intensifies normal termination, reduces the frequency of RT and stimulates NMD. We show that
in plants an elegant regulatory system has evolved that controls the eRF1 level via RT and NMD, thereby ensuring
the balance among normal termination, RT and NMD.
60
Session 2
Short talk 2.3
Alternative splicing and nonsense-mediated decay
in a null mutant of UPF1 in Arabidopsis
C. SIMPSON1, J. GLOGGNITZER2, D. LEWANDOWSKA1,3, K. RIHA2, J.W.S. BROWN1,3
1
2
3
Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria.
We previously showed that around 18% of genes in Arabidopsis potentially undergo AS/NMD using a high resolution RT-PCR platform and mutants in the NMD protein genes, UPF1 and UPF3 (Kalyna et al., 2012). The upf1-5
and upf3-1 mutants are viable but the upf1-3 mutant is seedling lethal. Disruption of the UPF1 gene in upf1-3 causes
changes in gene expression and AS profiles, increased salicylic acid levels and expression of PR genes giving a hypersensitive defence response (HR) (Riehs-Kearnan et al., 2012) suggesting that one function of NMD is regulation of
plant defence responses. By combining a mutation in the PAD4 gene involved in the salicylic acid pathogen signalling pathway, the lethality of upf1-3 has been overcome (Riehs-Kearnan et al., 2012). The upf1-3 pad4 double mutant
now allows the real extent of NMD on transcripts to be determined in a null background. We have analysed alternative splicing/NMD in the upf1-3 pad4 mutant and a related double mutant, smg7-1 pad4, by high resolution
RT-PCR using primers targeted to known NMD-sensitive transcripts to investigate the effect of the mutants on NMD.
We found large significant changes in the level of NMD-sensitive alternatively spliced products in the mutants and
particularly in the upf1-3 pad4 mutant background. The increases were often far greater than observed in the weaker
upf1-5 and upf3-1 mutant alleles tested previously suggesting that substantial proportions of transcripts from some
genes are degraded and effectively turned over by NMD. We also identified novel NMD-sensitive transcripts and
further demonstrated that many transcripts with unspliced introns are not turned over by NMD. We are currently
examining whether stabilised NMD-sensitive transcripts in the mutants are translated using a novel plant SILAC
method (Lewandowska et al., 2013).
61
Short talk 2.4
Analysis of the stress triggered mRNA decay process
in Arabidopsis thaliana
R. MERRET1,2, J. DESCOMBIN1,2, YU-TING JUAN3, J.-J. FAVORY1,2, M.-C. CARPENTIER1,2,
C. CHAPARRO1,2, YEE-YUNG CHARNG3, J.-M. DERAGON1,2, C. BOUSQUET-ANTONELLI1,2
1
Laboratoire Genome et Developpement des Plantes, Centre National de la Recherche Scientifique, Perpignan, France
2
Laboratoire Genome et Developpement des Plantes, Universite de Perpignan Via Domitia, Perpignan, France
3
Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan
To survive adverse and ever changing environmental conditions, an organism must be able to adapt. It has long
been established that the cellular reaction to stress includes the upregulation of genes coding for specific stress-responsive factors. We demonstrate in the present study that during the early steps of the heat stress response, 25%
of Arabidopsis seedlings transcriptome is targeted for rapid degradation. Our findings demonstrate that this process
is catalysed from 5N to 3N by the cytoplasmic exoribonuclease XRN4 which function is seemingly reprogrammed by
the heat-sensing pathway. The bulk of mRNAs subject to heat-dependent degradation is likely to include both the
ribosome-released and polysome associated polyadenylated pools. The co-translational decay process is facilitated
at least in part by LARP1, a heat specific cofactor of XRN4 required for its targeting to polysomes. Commensurate
with their respective involvement at the molecular level, LARP1 and XRN4 are necessary for the thermotolerance
of plants to long exposure to moderately high temperature with xrn4 null mutants being almost unable to survive.
These findings provide for the first time mechanistic insights regarding a massive stress-induced post-transcriptional
downregulation and outline a potentially crucial pathway for plant survival and acclimation to heat stress.
62
Session 2
Short talk 2.5
Insights into translational regulation
of thermal stress response of Arabidopsis thaliana
by ribosomal profiling
R. LUKOSZEK1,*, P. FEIST1, Z. IGNATOVA1
1
Department of Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany
* current address: Institute of Biology, Molecular Genetics, Humboldt University of Berlin, Berlin, Germany
Translation provides an additional level to regulate expression of the protein coding genes. It allows for a higher
plasticity of plant organisms to response to environmental conditions or internal development signals. Ribosome
profiling coupled with RNA sequencing enables assessing in depth the regulation at the post transcriptional and
translational level. It combines the power of global measurements of steady state levels of transcripts with the sequencing of the ribosome-protected fragments of mRNA, which in turn is informative on which mRNAs are being
actively translated and on the dynamics of this process. Here we apply ribosome profiling in order to assess transcriptional and translational response of plants growing in the sub-lethal heat stress, compared to optimal growth
conditions. The interest on studies on the influence of increased temperature effect on plants is rising because of
the global warming effect: the vegetation zones are shifting pole wards and more and more areas are already experiencing temperatures which are suboptimal or even detrimental to the plant growth and development. Our data
indicates that heat stress in plants leads to global downregulation of the translation processes, however not all of
the genes seem to be affected. We identified groups of genes whose biosynthesis rates are not affected by stressregulated downregulation of translation; they show even higher synthesis rate compared to optimal growth conditions. Most of these proteins are known to be involved in the stress response, however we identified several previously uncharacterized protein which can potentially participate in these processes. Our data add another twist to
the discussion whether non-coding RNAs are translated or not. Upon heat stress, some non-coding RNAs are upregulated on transcriptional level, however most of them, with few exceptions, were not detected in the ribosome
protected fragments datasets. In summary, our data shows a detailed view on the heat stress response in plants, both
on transcriptional and translational levels and provide numerous interesting points, which will be followed in our
ongoing research.
BioTechnologia
vol. 95(1) C pp. 63-72 C 2014
Session 3
Lecture 3.1
Genome-wide characterization
of non-coding transcription
by RNA Polymerase V
G. BOHMDORFER, M.J. ROWLEY, A.T. WIERZBICKI
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA
RNA-mediated transcriptional silencing prevents deleterious effects of transposon activity and controls the expression of protein-coding genes. It involves not only small interfering RNA (siRNA) but also long non-coding RNA
(lncRNA). In Arabidopsis thaliana this lncRNA is produced by a specialized RNA Polymerase V (Pol V). The mechanism by which lncRNA is involved in the establishment of repressive chromatin states remains mostly unknown. We
show evidence that lncRNA serves as a binding scaffold for several RNA-binding proteins. We further show that these
proteins require both lncRNA and siRNA to recognize their genomic targets and are recruited to lncRNA in a stepwise manner. Finally, we show that genome-wide identification of lncRNAs produced by Pol V further supports their
central role as scaffolds for protein binding and provides insights into the molecular mechanism of RNA-mediated
transcriptional silencing.
64
Session 3
Lecture 3.2
Dynamic changes of non-coding transcriptome
of Arabidopsis during development
HUI-WEN WU, JUN LIU, HUAN WANG, NAM-HAI CHUA
Laboratory of Plant Molecular Biology, Rockefeller University, New York, New York, USA
Recent research on long non-coding RNAs (lncRNAs) has expanded our understanding of gene transcription
regulation and the generation of cellular complexity. Depending on their genomic origins, lncRNAs can be transcribed from intergenic or intragenic regions or from introns of protein-coding genes. In Arabidopsis, we have identified
13,230 intergenic transcripts of which 6,480 can be classified as long intergenic non-coding RNAs (lincRNAs). Expression of 2,708 lincRNAs was detected by RNA sequencing experiments. Transcriptome profiling revealed that
the majority of these lincRNAs are expressed at a level between those of mRNAs and precursors of miRNAs. A subset of lincRNA genes shows organ-specific expression, whereas many others are responsive to biotic and/or abiotic
stresses (Liu et al, 2012). Next, we systematically identified long non-coding natural antisense transcripts (lncNATs),
which are transcribed from the opposite DNA strand of coding or non-coding genes. We found a total of 37,238 senseantisense transcript pairs and 70% of annotated mRNAs to be associated with antisense transcripts in Arabidopsis.
These lncNATs could be reproducibly detected by different technical platforms. Moreover, we observed spatial and
developmental-specific light effects on 626 concordant and 766 discordant NAT pairs. Genes for a large number of
the light-responsive NAT pairs are associated with histone modification peaks, and histone acetylation is dynamically
correlated with light-responsive expression changes of NATs (Wang et al., 2014). We have presented our data described above in a plant long non-coding RNA database (PLncDB), which also includes a list of epigenetic modifications and small RNA datasets (Jin et al., 2013). This database will be updated periodically for the research community. We have examined the function of several intronic ncRNAs in Arabidopsis. AGAMOUS (AG) is known to
specify the development of the third and fourth whorl of wild type (WT) flowers. The AG genomic locus is marked
by dispersed histone H3 lysine 27 trimethylations (H3K27me3) which are absent in mutants deficient in CURLY
LEAF (CLF), the catalytic component of the polycomb repressive complex (PRC) 2. Clf mutants express AG transcripts in leaves and roots because of de-repression. We show that the AG second intron encodes several intronic noncoding RNAs (incRNAs), one which is important for CLF-mediated repression. Genetic interaction analysis demonstrated that the AG-incRNA associated with CLF to repress AG. Our results suggest that AG-incRNA is able to
recruit CLF to the AG second intron to repress AG in leaf tissues through H3K27me3-mediated repression. The mechanism of AG-incRNA-mediated gene repression may serves as a general model for investigating tissue-specific
expression of MADS-box genes in Arabidopsis flower development.
65
Short talk 3.1
Transcription, replication and recombination
of genomic and subgenomic RNAs in brome mosaic virus,
a model (+) strand RNA virus
J. SZTUBA-SOLINSKA1, A. DZIANOTT1, R. WIERZCHOSLAWSKI1, J. HORN2, J.J. BUJARSKI1, 3
1
Plant Molecular Biology Center and the Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, USA
2
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, USA
3
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
The process of transcription from the brome mosaic virus (BMV) RNA3 segment generates two subgenomic (sg)
RNAs, sgRNA3a and sgRNA4. By altering the elements of the intercistronic sg promoter (sgp) sequence that is responsible for sgRNA4, we observed both debilitating and stimulating effects on homologous RNA3-RNA3 recombination. This suggested that although linked, the mechanisms of RNA-RNA recombination and transcription follow
separate molecular processes during BMV RNA replication. Other studies demonstrated that sequence alterations
in the coat protein (CP)-binding cis-acting RNA motifs affected crossover frequencies between BMV RNA3 and
sgRNA3a. This implied the role of CP in at least some of the BMV RNA recombination events by either facilitating
RNA-RNA template interaction or by creating the obstacles for the viral replicase. Altogether, our results demonstrate a functional link of such viral functions as RNA replication, RNA transcription and RNA recombination not only
in BMV, but likely in other RNA viruses.
66
Session 3
Short talk 3.2
Non-coding transcription
by alternative RNA polymerases dynamically regulates
an auxin-driven chromatin loop
F. ARIEL1, T. JEGU2, D. LATRASSE2, N. ROMERO-BARRIOS1, A. CHRIST1, M. BENHAMED2, M. CRESPI1
1
Institut des Sciences du Vegetal (ISV), Centre National de la Recherche Scientifique (CNRS), Gif-sur-Yvette, France
2
Institut de Biologie des Plantes, Universite Paris-Sud XI, Orsay, France
The eukaryotic epigenome is shaped by the genome topology in three-dimensional space. Dynamic reversible
variations in the epigenome structure direct the transcriptional responses to developmental cues. However, little
is known about the control of epigenome dynamics. Here, we show that the Arabidopsis thaliana long intergenic noncoding RNA (lincRNA) APOLO is transcribed by RNA polymerase II (Pol II) and V (Pol V) complexes in response
to auxin, a phytohormone controlling numerous facets of plant development. APOLO transcription facilitates the
formation of an oscillating chromatin loop encompassing the promoter of its neighboring gene PID (or PINOID ),
a key regulator of auxin polar transport. Chromatin and RNA Immuno-precipitation (ChIP and RIP), together with
Chromatin Isolation by RNA Purification (ChIRP) and Chromatin Conformation Capture (3C) served to decipher the
ncRNA-mediated mechanisms controlling the chromatin loop opening and closing in response to auxin, modulating
PID promoter activity. Components of the plant Polycomb Repressive Complexes as well as the transcriptional gene
silencing and DNA demethylation machineries contribute to fine-tune chromatin loop dynamics. Altering APOLO
expression or its 24nt siRNA-dependent DNA methylation affects loop formation and, consequently, PID expression.
Hence, the active transcription of a lincRNA by alternative RNA polymerase complexes influences local chromatin
topology and the expression of a neighboring locus, leading to far-reaching consequences on a variety of developmental outputs. Acknowledgements: FA is an EMBO postdoc fellow; NRB and TJ are Paris-Sud University PhD
fellows. This work was funded by LABEX Saclay Plant Sciences.
67
Lecture 3.3
Novel antisense RNA regulation functions
in plant abiotic stress responses
AKIHIRO MATSUI1, KEI IIDA2,5, KATSUSHI YAMAGUCHI3, MAHO TANAKA1, JUNKO ISHIDA1, TAEKO MOROSAWA1,
SHUJI SHIGENOBU3, KAZUO SHINOZAKI4, TETSURO TOYODA5, MOTOAKI SEKI1,6,7
1
Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Japan
2
Medical Research Support Center, Graduate School of Medicine, Kyoto University, Kyoto, Japan
3
Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Japan
5
Integrated Database Unit, RIKEN Advanced Center for Computing and Communication (ACCC), Wako, Japan
6
7
NIBB Core Research Facilities, National Institute for Basic Biology, Okazaki, Japan
4
Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology (JST), Kawaguchi, Japan
Arabidopsis whole genome transcriptome analysis under drought, cold, high-salinity stress and ABA treatment
conditions, using tiling array, showed that approximately 6,000 novel non-codding transcripts existed in the antisense
strand of the AGI code genes and their expression was induced under abiotic stress (Matsui et al. (2008) Plant Cell
Physiol.). To study the biogenesis mechanism of the antisense RNAs, accumulation of RD29A antisense RNA was
analyzed, using RNA-dependent RNA polymerase (RDR) mutant series. Finally, we found that rdr1/2/6 mutation
decreased accumulation of RD29A antisense RNA. And rdr1/2/6 mutants showed arrest of root growth after drought
stress, suggesting that the antisense RNAs have novel biological function in response to abiotic stress. RNase
protection and RNA decay analyses showed that RD29A antisense RNA formed double stranded RNA and promoted
degradation of RD29A mRNA. However, RNA-seq analysis did not detect siRNA accumulation in this locus. We
propose a novel RNA regulation mechanism that RDR-mediated synthesis of antisense RNAs functions in the
turnover of sense mRNAs under the stress conditions in plants.
68
Session 3
Lecture 3.4
Global analysis of RNA-protein interaction sites
and RNA secondary structure
in the Arabidopsis nucleus
S.J. GOSAI1,#, S.W. FOLEY1,2,#, DONGXUE WANG3, I.M. SILVERMAN1,2, A.D.L. NELSON4,
N. SELAMOGLU1, M.A. BEILSTEIN4, F. DALDAL1, R.B. DEAL3, B.D. GREGORY1,2
1
2
Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA
Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania USA
3
4
Department of Biology, Emory University, Atlanta, Georgia, USA
School of Plant Sciences, University of Arizona, Tucson, Arizona, USA
#
equal contributions
At the heart of post-transcriptional regulatory pathways in eukaryotes are cis - and trans-acting features and factors including RNA secondary structure as well as RNA-binding proteins (RBPs) and their binding sites on target
RNAs. However, to date a comprehensive view of the structural and RBP interaction landscape of RNAs in the
nucleus has not been accomplished for any organism. Here, we use our novel protein interaction profile sequencing
(PIP-seq) approach on mixed nuclei from Arabidopsis seedlings to globally profile both RNA secondary structure
and RNA-protein interaction sites in this cellular compartment. From our analysis, we reveal opposing patterns of
secondary structure and RBP binding levels throughout nascent messenger RNAs (mRNAs). These patterns underlie
the regulation of alternative splicing and polyadenylation. We also uncover a class of protein bound, highly conserved,
nuclear, long non-coding RNAs (lncRNAs), some of which interact with chromatin. Finally, we uncover a large collection of protein bound sequence motifs, and identify their interacting proteins and structural contexts. In total, we
provide the first simultaneous, transcriptome-wide view of RNA secondary structure and RNA-protein interaction
sites in a plant nucleus.
69
Short talk 3.3
The exosome and its cofactors contribute
to multiple steps of 18S rRNA maturation
P.J. SIKORSKI1,2, D. GAGLIARDI2, J. KUFEL1, H. LANGE2
1
2
Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland
Institut de Biologie Moleculaire des Plantes du Centre National de la Recherche Scientifique, Universite de Strasbourg, Strasbourg, France
Maturation of 18S rRNA from the polycistronic rRNA precursor involves a number of conserved endonucleolytic
cleavages. Recent studies in yeasts and human have now revealed that exoribonucleolytic degradation by RRP6, a cofactor of the nuclear exosome, can also contribute to processing or degradation of 18S precursors. Here, we investigate the impact of the exosome and its cofactors on 18S rRNA maturation in Arabidopsis thaliana. We show that
plants deficient in the core exosome, the exoribonuclease RRP44, or the nucleolar RNA helicase AtMTR4 accumulate different forms of both 3N- and 5N- extended 18S rRNA precursors. Downregulation of the core exosome or
RRP44 is associated with accumulation of a 2.5 kb 18S precursor spanning from P to A3 processing sites. In rrp41
mutants, these P-A3 precursors are frequently polyadenylated with long poly-A tails up to 200 nucleotides. By contrast, loss of the RNA helicase AtMTR4 results in decreased levels of P-A3 and to increased levels of 5N shortened
PN- A3 fragments, which have no or only short oligo(A) tails. Preliminary results indicate that more than one poly(A)
polymerase is involved in the addition of poly(A) tails to rRNA precursors prior to their degradation by the exosome.
Moreover, loss of the exoribonuclease RRP6L2 is specifically associated with the accumulation of precursors that
have mature 5N ends but carry a 3N extension of 20 nucleotides (18S-A2), indicating that 18S precursors undergo exoribonucleolytic trimming by RRP6L2. Interestingly, the 18S-A2 fragments that accumulate in rrp6l2 mutants often
carry short oligo(U) tails and can be detected in the cytoplasm. Taken together, our data suggest that in addition
to conserved endonucleolytic cleavages, 3N-5N exoribonucleolytic degradation by the exosome and its cofactors contribute to different steps of 18S maturation in plants.
70
Session 3
Short talk 3.4
Genome-wide annotation
of functional regulatory elements
in plant snoRNA/scaRNA genes
GE QU1, K, KRUSZKA2, Z. SZWEYKOWSKA-KULINSKA2, M. ECHEVERRIA3, W. KARLOWSKI1
1
Laboratory of Computational Genomics / Bioinformatics Laboratory, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
2
Department of Gene Expression, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
3
Universite de Perpignan Via Domitia, Perpignan, France
The small nucleolar RNAs (snoRNA) and the related scaRNAs represent an abundant class of small trans-acting
RNAs with very diverse and important functions in all eukaryotes. The snoRNAs fall into two classes that direct
modification of ribosomal RNAs (rRNA): the C/D snoRNAs that direct 2N-O-ribose methylation and H/ACA snoRNAs
that direct pseudouridylation - these modifications are essential to produce functional ribosomes. The major targets
of scaRNAs are the spliceosomal snRNAs where introduced modifications are essential for splicing. The snoRNAs
and related scaRNAs have additional targets – for example they direct methylation of a brain specific mRNA in vertebrates. Moreover, many so-called ‘orphan’ snoRNAs, for which no target could be predicted, have been identified
in all species, suggesting that these may have additional functions. Several hundreds of genes encoding snoRNAs,
scaRNAs and orphan snoRNAs have been identified already in Arabidopsis and rice. The studies show that while
the structure of snoRNAs and scaRNAs is relatively conserved, the plant snoRNA genes exhibit unique genomic organization which is rarely found in other eukaryotes. We are interested in the factors controlling snoRNA and scaRNA
biogenesis in plants. A preliminary analysis of snoRNA gene promoters in Arabidopsis revealed that all polycistronic
snoRNA genes encoding snoRNAs targeting rRNA modifications have conserved promoter motifs (Telo-box and
GCCCR-elements). Moreover, these elements are also found in the promoter of genes encoding ribosomal proteins
and other proteins controlling ribosome biogenesis. Most interestingly, we found that scaRNAs genes have also
a distinct type of conserved elements. This altogether may suggest that the snoRNA/scaRNA gene promoter contains
elements which, in addition to basic transcriptional regulation, play a significant role in the snoRNA or scaRNA function and localization. The main object of the project is to address the question of the role of the promoter of
snoRNA/scaRNA genes by combining in silico and laboratory experimental approaches.
71
Short talk 3.5
Novel players in plant stress response:
micropeptides and/or long non-coding RNAs?
P. TAVORMINA, J. NEUKERMANS, C. VOS, B.P.A. CAMMUE, B. DE CONINCK
Centre of Microbial and Plant Genetics, KU Leuven, Belgium
Department of Plant Systems Biology, VIB, Belgium
Plants as sessile organisms have to resist abiotic and biotic stresses without the option to escape. To cope with
theses stresses, plants rely on a complex array of fine-tuned networks. Plant peptides are part of these networks and
are known to exert their functions directly as antimicrobial peptides or indirectly via interaction with signaling cascades. However, the diversity and the total amount of plant peptides is strongly underestimated. As a consequence,
recent genome annotations of the model plant Arabidopsis thaliana are far from complete. Moreover, peptides that
are produced de novo from a small open reading frame (sORF, <100 aa), without the involvement of a precursor,
are rarely being studied in plants. In other eukaryotes, such sORF-encoded peptides are being studied more intensively as important bioactive molecules, but still appear to be an unexplored part of the peptidome. However, big
controversy arises in this research domain because transcripts (>200 bp) containing no ORFs longer than 100 aa
are being considered as long non-coding RNAs (lncRNAs). The need emerges to study the dual character of these
transcripts as functional small peptides ‘micropeptides’ and/or as RNA. In the present study we aimed at i) identifying novel stress-induced genes, and ii) unravelling the biological function of the gene products, as peptide(s)
and/or as RNA, in the stress response of Arabidopsis thaliana. We therefore performed a tiling array-based transcriptome study on leaf material under reactive oxygen species (ROS)-inducing biotic and abiotic stress conditions.
As such we identified 195 and 176 transcriptional active regions (TARs) that were differentially expressed after infection with the fungal pathogen Botrytis cinerea and the herbicide paraquat, respectively (De Coninck et al., J. Exp.
Bot. 2013). Functional analysis of these TARs is performed at two levels. At one hand, we study novel peptides that
are possibly encoded by small ORFs in these TARs, more specifically via a peptidomics approach. At the other hand,
after determining the exact transcripts of several TARs by rapid amplification of cDNA ends (RACE), we also study
the TARs as potential long non-coding RNAs (lncRNAs). Further characterization of the identified TARs was done
via phenotypical analyses on a selection of corresponding knock-out mutants. For example, Tar1 was more susceptible to infection with the fungal pathogen Fusarium oxysporum. Tar2 showed interveinal chlorosis and was also
more susceptible to infection with Fusarium oxysporum. In conclusion, these TARs function in the stress response
of plants. It is currently being assessed by complementation assays whether this involvement is mediated via peptide(s) and/or RNA.
72
Session 3
Short talk 3.6
Long non-coding RNA modulates alternative
splicing regulators in Arabidopsis
F. BARDOU1, F. ARIEL1, C.G. SIMPSON2, N. ROMERO-BARRIOS1,
S. BALZERGUE3, J.W.S. BROWN2,4, M. CRESPI1
1
2
Institut des Sciences du Végétal, CNRS, Saclay Plant Sciences, Gif-sur-Yvette, France
Cell and Molecular Sciences, the James Hutton Institute, Invergowrie, Dundee, Scotland, United Kingdom
3
Genomique Fonctionnelle d'Arabidopsis, Unite de Recherche en Genomique Vegetale,
UMR INRA 1165 – Universite d'Evry Val d'Essonne – ERL CNRS 8196, Evry, France
4
Plant Sciences Division, College of Life Sciences, University of Dundee at the James Hutton Institute, Invergowrie,
Dundee, Scotland, United Kingdom
Alternative splicing (AS) of pre-mRNA represents a major mechanism that underlies increased transcriptome
and proteome complexity. Here we show that the nuclear speckle RNA-binding protein (NSR) and the alternative
splicing competitor long non-coding RNA (ASCO-lncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change
in AS patterns of many genes, a response largely dependent on NSRs. RNA-immunoprecipitation assays demonstrate
that AtNSRs not only interact with their alternatively-spliced mRNA targets but also with the ASCO-RNA in vivo.
The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. The expression of ASCO-RNA in
Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, lncRNA can hijack nuclear
AS regulators to modulate AS patterns during development.
BioTechnologia
vol. 95(1) C pp. 73-82 C 2014
Session 4
Lecture 4.1
Insights from global analysis
of miRNAs and target RNAs
P.J. GREEN1-4, DONG-HOON JEONG1,2, S.R. THATCHER1,5, S. PARK1,2, S.A. SCHMIDT1,2
J. ZHAI1,2,6, B.C. MEYERS1,2, K.R. FRANKE 1,4, M. ACCERBI1,2, E. DE PAOLI1,7
1
2
3
Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA
Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, USA
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA
4
5
6
Department of Biology, University of Delaware, Newark, Delaware, USA
DuPont Agricultural Biotechnology, Experimental Station, Wilmington, Delaware, USA
Department of Molecular, Cellular and Developmental Biology, University of California, Los Angeles, California, USA
7
Department of Agriculture and Environmental Sciences, University of Udine, Udine, Italy
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that control development, stress
responses and other processes. They most often function by guiding cleavage of mRNA targets and some inhibit
translation. The deep sequencing of miRNAs and their cleaved targets continues to lead to the discovery of new
miRNAs and new regulation such as regulation in response to cold, heat, drought, submergence, and metal stress
and many examples of tissue or organ preferential expression (Jeong et al., Plant Cell 23: 4185; Plant Phys. 162:
1225; Genome Biol. 14: R145). Among new cases of miRNA regulation are distinct miRNA family members exhibiting differential accumulation in different organs that impact target selection and cleavage. Genome-wide target
cleavage data from Parallel Analysis of RNA Ends (PARE) has proved instrumental in these studies and can be even
more powerful when interpreted with the emerging diversity of miRNA variants, ARGONAUTE immunoprecipitation
data, and small RNA expression data. Examples from model plants and beyond that have led to new insights about
particular miRNAs will be highlighted. Some of these derive from an analysis of miRNAs in five floral organs and six
panicles stages, which was augmented with PARE data from anther and pistil. Most of the previously reported rice
miRNAs were identified in this study as well as tens of new miRNAs using a computational pipeline. About 20% of
the miRNAs were preferentially expressed in a specific floral organ, with half being anther preferential. More than
170 miRNAs were differentially expressed during panicle development including some miRNA variants that showed
differential expression patterns in different stages. In addition to enhancing our understanding of miRNAs and their
targets, these studies are providing rich data resources for future work. Funded by the NSF, DOE and USDA.
74
Session 4
Lecture 4.2
Processing of plant microRNA precursors
J. PALATNIK
Institute of Molecular and Cell Biology of Rosario (IBR), School of Biochemistry and Pharmacy, University of Rosario, Rosario, Argentina
MicroRNAs (miRNAs) are transcribed as larger transcripts that contain fold-back structures that are processed
by RNAse III complexes. In both plants and animals, the precursor contains spatial clues that determine the position
of the miRNA along its sequence. However, at a difference with their animal counterparts, plant precursors are very
heterogeneous in size and shape. They are processed completely in the nucleus by a complex formed by DICERLIKE1 (DCL1) and accessory proteins such as HYL1 and SERRATE. In many precursors a 15 nucleotide lower stem
below the miRNA is essential for its processing. This structural determinant present in many, but not all plant
miRNAs, is recognized by the processing machinery to produce a first cleavage at the base of the precursor, while
a second cut below the loop releases the miRNA. In contrast, the biogenesis of miR319 and miR319 proceeds
through a loop-to-base direction. DCL1 produces first a cleavage at the loop of the precursor and continues towards
the base of the precursor, while the mature miRNAs are finally release between the third and fourth cleavage sites.
To bring insights into the biogenesis of miRNAs from a genome-wide perspective in Arabidopsis thaliana, we designed a strategy to detect precursor intermediates using high-throughput sequencing. Using this strategy we were able
to map the cleavage sites for most Arabidopsis miRNA precursors. We found a good correlation between the cuts
in the precursors and the miRNA profile obtained by deep-sequencing small RNAs. The results indicate that miRNAs
are released by at least four different mechanisms starting from either end of the precursors. A genome-wide view
of miRNA processing in Arabidopsis will be presented.
75
Short talk 4.1
Functional analysis
of SERRATE interacting proteins
S. LAUBINGER
Center for Plant Molecular Biology (ZMBP), University of Tubingen, Tubingen, Germany
Plant microRNAs (miRNAs) regulate diverse aspects of plant development including hormone responses, floral
development and phyllotaxy. Mature miRNAs associate with ARGONAUTE (AGO) proteins to bind and regulate
target mRNAs. Mature miRNAs are released from longer primary-miRNAs (pri-miRNA) by the RNAseIII-like enzyme
DICER-LIKE 1 (DCL1). Additional RNA-binding proteins including SERRATE (SE), HYPONASTIC LEAVES1
(HYL1), TOUGH (TGH) and the CAP BINDING COMPLEX (CBC) facilitate efficient and precise processing of primiRNA transcripts. SE and the CBC are of particular interest for us, because they fulfill an additional function in premessenger RNA (pre-mRNA) splicing. In order to identify partners of SE/CBC that assist the complex in miRNA
processing and/or splicing, we screened yeast cDNA libraries for potential interactors. Our results suggest that
SE/CBC interact with a wide range of scaffolding proteins and regulatory proteins, including RNA-binding proteins
and transcript_ion factors. Interestingly, we also identified components of the U1 snRNP, suggesting that SE/CBC
regulate splicing by a direct physical interaction with the spliceosome. Further genetic and molecular analyses will
be presented.
76
Session 4
Short talk 4.2
The crosstalk between microRNA biogenesis
and splicing machineries in plants
A. STEPIEN1, D. KIERZKOWSKI2, K.D. RACZYNSKA1, M. TAUBE1,3,
Z. SZWEYKOWSKA-KULINSKA1, A. JARMOLOWSKI1
1
Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
2
Department of Comparative Development and Genetics, Max Planck Institute for Plant Breeding Research, Koln, Germany
3
Department of Macromolecular Physics, Adam Mickiewicz University in Poznan, Poland
MicroRNAs (miRNAs) are small non-coding RNAs of about 21 nt in length, which take part in a wide variety of
physiological and cellular processes. They act by regulating gene expression by cleaving or inhibiting translation of
target mRNAs. In plants, miRNAs are encoded mostly by independent transcription units or, less frequently, within
introns of protein-coding genes. In addition, the miRNA genes that are transcribed into long non-coding transcripts,
may contain more than one pre-miRNA in both, introns and/or exons. Plant miRNA biogenesis, which is still not fully
understood process, occurs inside the cell nucleus, and is performed by a complex comprising at least five different
proteins: DCL1 (an RNase III ribonuclease), HYL1 (a dsRNA-binding domain containing protein), SERRATE (SE)
(a zinc-finger-domain protein) and CBC (a nuclear cap-binding protein complex) that is composed of two subunits,
CBP20 and CBP80. DCL1 provides nucleolytic activity of the miRNA processing complex, and other factors involved
in miRNA maturation are required for the efficient and correct excision of miRNAs from plant pri-miRNAs. Interestingly, SE and CBC are also involved in pre-mRNA splicing. It is unclear, however, how this dual function of SE
and CBC is fulfilled. Moreover, we have already shown that splicing of pri-miRNA 163 influences biogenesis of mature miRNA 163. Thus, correlation between splicing and miRNA maturation machineries seems to be obvious. We
decided to characterize interactions between SERRATE, CBC complex and spliceosome in plants. In the case of the
latter machinery we chose U1snRNP, that recognizes the 5N splice site, as a potential candidate for crosstalk studied.
First, we performed experiments on the direct interactions between CBP20, CBP80 and the SERRATE protein using
Bimolecular Fluorescence Complementation (BiFC) and in vitro pull down assays. The CBP20/SE and CBP80/SE
complexes seem to be localized exclusively in the nucleus of Arabidopsis thaliana cells where they are mainly accumulated in specific subnuclear bodies. Moreover, in vitro analyses have shown stronger interactions of overexpressed in E. coli MBP-SERRATE with the whole CBC complex, obtained by in vitro translation, in comparison to
the single interaction between SE and CBP20 or SE and CBP80. Furthermore, we tested interactions of SERRATE
with all ten A. thaliana specific U1snRNP proteins by yeast two-hybrid system followed by pull down assay. We found
five binding partners of SE among them, which were PRP39a, PRP39b, PRP40a, PRP40b and LUC7rl. We also
performed experiments on the subcellular localization of these five proteins and SERRATE in A.thaliana cells. All
U1snRNP binding partners co-localize with SE in the cell nucleus. Taking together, we proved the connection between proteins from miRNA biogenesis machinery and spliceosome which functionality is under further studies. This
work was funded by Polish National Science Center (UMO-2012/05/N/NZ2/00880).
77
Lecture 4.3
Post-transcriptional regulation
of auxin signaling homeostasis
D. WINDELS, F. VAZQUEZ
Botanisches Institut, Universitat Basel, Basel, Switzerland
RNA silencing encompasses a wide set of recently discovered RNA-dependent regulatory mechanisms that act
as a major bandmaster to coordinate the expression, protection, stability, and inheritance of eukaryotic genomes.
Our work has recently provided pioneer insights into the regulation of auxin signaling homeostasis and its impact
on plant development. We showed that, during leaf development, the expression of TIR1/AFB2 AUXIN RECEPTOR
(TAAR ) genes and are regulated by the miRNA miR393 and by a specialized secondary siRNA network, which we
termed siTAARs, to fine-tune auxin signaling homeostasis. We have also now identified an additional layer in the
regulation of TAAR transcripts that involves RNA decay, and that appears as a major regulator of auxin signaling
homeostasis and plant development. I will present these most recent data and highlight the future directions of our
work that aims to understand the biological role of the simultaneous regulation of TAARs by RNA silencing and RNA
decay.
78
Session 4
Short talk 4.3
Regulation of pri-miRNA processing
by an hnRNP-like protein
T. KOSTER1, K. MEYER1, C. WEINHOLDT2, L.M. SMITH3, M. LUMMER1,
C. SPETH4, 5, I. GROSSE2, D. WEIGEL3, D. STAIGER1
1
2
Molecular Cell Physiology, Bielefeld University, Germany
Institute of Computer Science, Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany
3
4
Max Planck Institute for Developmental Biology, Tuebingen, Germany
Center for Plant Molecular Biology, University of Tuebingen, Tuebingen, Germany
5
Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany
The hnRNP-like RNA-binding protein At GRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) regulates pre-mRNA splicing. Small RNA-seq was used to show that At GRP7 also affects the miRNome. Overexpression
of At GRP7 caused a significant reduction of at least 2-fold for around 30 miRNAs. Among those, reduced levels of
miRNA398 were accompanied by an increased level of its targets COPPER ZINC SUPEROXIDE DISMUTASE 1
(CSD1), CSD2, and the COPPER CHAPERONE CCS. Furthermore, reduced accumulation of miR390, an initiator
of trans-acting small interfering RNA (ta-siRNA) formation, led to lower TAS3 ta-siRNA levels and increased mRNA
expression of the target AUXIN RESPONSE FACTOR4. An accumulation of several primary transcripts at the expense of the mature miRNAs suggested that At GRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that At GRP7 interacts with several pri-miRNAs in vivo. Mutation of an arginine residue in the RNA
recognition motif abrogated in vivo binding and the effect on miRNA and pri-miR levels, indicating that the RNAbinding activity of At GRP7 is required for these functions. Moreover, we show that At GRP7 affects alternative splicing of pri-miRNAs. Thus, At GRP7 is an hnRNP-like protein with a role in processing of specific pri-miRNAs in addition to its role in pre-mRNA splicing.
79
Short talk 4.4
Salt stress reveals a new role of AGO1
in the miRNA biogenesis pathway at both,
transcriptional and post-transcriptional levels
J. DOLATA1, M. BAJCZYK1, D. BIELEWICZ1, K. NIEDOJADLO2,
W. WALCZAK1, Z. SZWEYKOWSKA-KULINSKA1, A. JARMOLOWSKI1
1
2
Department of Cell Biology, Faculty of Biology and Environment Protection, Nicolaus Copernicus University, Torun, Poland
MicroRNAs (miRNAs) are short (21-24 nt) RNA molecules that control gene expression at the post-transcriptional level, by cleavage of mRNA targets or by inhibiting their translation. These small molecules are important in plant
development and responses to different biotic and abiotic stresses. Recently we developed a high-throughput platform, mirEX, to simultaneously examine of all known A. thaliana miRNA precursors (pri-miRNAs) in different developmental stages, using Real Time PCR. We applied the mirEX panel to test the influence of high salt stress conditions on MIR genes expression in A. thaliana T87 cells. Our data clearly indicate that salt stress has a great influence on the expression pattern of A. thaliana MIR genes: the profile of 40% of all pri-miRNAs was significantly changed under salt stress conditions. Many of them (63pri-miRNAs) were down-regulated in comparison to control conditions. There was also a large group of up-regulated miRNA precursors (53pri-miRNAs). Data obtained from the
qPCR and northern blot analyses shows that the majority of pri-miRNA and miRNA follows in the same direction
(both are up or down regulated). under stress conditions. These results indicate that the mentioned above MIR
genes are transcriptionally regulated. Selected examples were confirmed using ChIP (Chromatin Immunoprecipitation) technique and GUS system. We also observed following profiles of MIR gene expression regulation upon salt
stress: the level of pri-miRNA was down-regulated but the level of the mature miRNA was increased (e.g. MIR 161,
MIR 173). Such MIR gene expression profiles indicate post-transcriptional regulation of miRNAs biogenesis. In further experiments we have shown that miR161 and miR173 are more stable in salt stress condition and that AGO1
is responsible for this phenomenon. Additionally, we found that AGO1 is present not only in cytoplasm but also in
nuclei. Using ChIP approach we have shown that AGO1 co-localizes on MIR 161 and MIR 173 genes and its level is
increased under stress conditions. Parallely, we observed that RNA Pol II drops off during the transcription of these
MIR genes. Our results indicate that AGO1 function is not only limited to the last step of miRNA pathway but has
also important role in co-transcriptional regulation of MIR gene expression. This project was supported by Foundation for Polish Science (MPD/2010/7).
80
Session 4
Short talk 4.5
Parallel evolution of two microRNAs controlling
AGL17-like MADS-box genes
in distantly related flowering plants
L. GRAMZOW, D. LOBBES, G. THEIßEN
Friedrich Schiller University, Jena, Germany
Even though hundreds of microRNA (miRNA) families have been found in plants so far their origin remained
mostly unclear. A number of hypotheses on the origin of new plant miRNA families have been brought forward over
the years. These hypotheses include the origin of miRNA families by inverted duplication of their target genes, from
inverted-repeat containing transposable elements, transcription of genomic regions containing inverted repeats, and
by duplication of existing miRNA families. Nevertheless, elucidating the origin for certain miRNA families proved
difficult, especially for older families. Hence, our knowledge on the origin and evolution of miRNA families is still
limited. Here, we exemplarily study the origin of the two miRNAs miR444 and miR824. Both miRNAs post-transcriptionally regulate AGL17-like MADS-box genes encoding transcription factors which have important functions in
the nitrate-signaling pathway amongst others. However, both miRNAs do not share any significant sequence similarity and seem to be restricted to non-overlapping plant phyla. The miR444 has so far been discovered in several
species belonging to the grass family Poaceae, and in Musa accuminata, a species of wild banana (from the family
of Musaceae, order Zingiberales) which are all monocot plants. In contrast, the miR824 has only been detected in
the eudicot species from the crucifer family Brassicaceae, such as the major model plant Arabidopsis thaliana, so
far. We use data obtained from large-scale transcriptome projects, sequencing efforts and Northern blot hybridization
to elucidate when and how the two miRNAs miR444 and miR824 originated and started to regulate the same family
of target genes. Our analyes indicate, that both miRNAs may have originated by an inverted duplication of their
target genes. Our data thus reveal the intriguing case of the independent origin and parallel evolution of two miRNAs
targeting a highly conserved class of developmental control genes in distantly related clades of flowering plants.
81
Short talk 4.6
AtERI has suppressor function
on PTGS via changing the population of small RNAs
I. JUPIN1, G. HONIG2, G. REUTER2, M. KUHLMANN3
1
Laboratoire de Virologie Moleculaire, CNRS-Univ Paris Diderot, Institut Jacques Monod, Cell Biology Department, Paris, France
2
Institute of Genetics/Developmental Genetics, Martin-Luther University Halle-Wittenberg, Halle, Germany
3
Research group Abiotic Stress Genomic, IPK Gatersleben, Gatersleben, Stadt Seeland, Germany
Interdisziplinares Zentrum fur Nutzpflanzenforschung (IZN), Martin-Luther-Universitat Halle-Wittenberg, Halle (Saale), Germany
In all known eukaryotic organisms short interfering RNAs (siRNAs) are the effector molecules of gene silencing.
The presence of siRNAs, homologous to the affected gene is a typical hallmark for post-transcriptional gene silencing
(PTGS). In plants the mechanism of silencing is very well understood and the most important factors are known.
Although in other organisms siRNA degrading enzymes are already described, up to now no investigation was aiming
to the degradation pathway of siRNAs. The function of the Arabidopsis thaliana enhancer of RNAi (ERI) homolog
from C. elegans in PTGS was analyzed in planta. Therefore plants with ERI loss-of-function mutation and plants over
expressing AtERI (Pro35S::ERI ) were studied. Plants with modulated level of AtERI show an increased accumulation of the virus load after infection with turnip yellow mosaic virus (TYMV). Utilizing a 2xGUS reporter silencing
system for analysis of PTGS frequency, an increase of silencing frequency was found in the ERI loss-of-function
mutant. Molecular analysis of siRNAs by small RNA sequencing and Northern hybridization revealed an ERI related
change of a specific class of siRNAs in the 5Nregion of the silenced GUS reporter gene. Ectopic overexpression of
AtERI result in an increase in biomass after backcrosses to wild type plants resembling the heterosis effect.
82
Session 4
Short talk 4.7
Small RNAs in chloroplasts and mitochondria represent
footprints of RNA-binding proteins
H. RUWE1, YUJIAO QU1, K. HOWELL2, C. SCHMITZ-LINNEWEBER1, I. SMALL2
1
2
Molecular Genetics, Institute of Biology, Faculty of Life Sciences, Humboldt University of Berlin, Berlin, Germany
Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Australia
Regulatory small RNAs like miRNAs and siRNAs are well studied in plants. Using high-throughput sequencing
small RNAs are usually sequenced from size fractionated total RNA. About 20% of sequence reads have an organellar
origin and did not get a lot of attention so far. We investigated small RNAs from a wide variety of plant species that
map to the chloroplast and mitochondrial genome. We found about 100 of such small RNAs that show a strong bias
towards localization in intergenic regions. We mapped transcript 5N- and 3N-ends and found that small RNAs and
transcript ends coincide in most of the investigated cases. Whereas in chloroplasts, most 5N- and 3N-ends of mRNAs
overlap with small RNAs, in mitochondria small RNAs are found to map mostly to 3N-ends of messages. Several
mRNA ends have been reported to be dependent on RNA-binding proteins and we find small RNAs with sequences
that have been described as binding sites for RNA-binding proteins. Investigating “knock-outs” for RNA binding
proteins we show that processed mRNAs and small RNAs are missing in these mutants. This finding indicates that
small RNAs might represent naturally occurring footprints of RNA-binding proteins. Many of the known RNA-binding
proteins that affect RNA stability in chloroplasts belong to the class of pentatricopeptide repeat proteins (PPRproteins). This class of proteins has strong sequence specificity and members of this class bind with high affinity
(Kd in the picomolar range). Given the high specificity and affinity, it is speculated that these proteins act as protein
caps preventing RNA degradation by exonucleases (Pfalz J. et al., 2009). We show that sequencing of small RNAs
in mutants of PPR proteins allows the identification of their target sites. We show that some of these targets are
in transcripts antisense to messenger RNAs, indicating that PPR proteins act in the metabolism of chloroplast sense
and antisense RNAs.
BioTechnologia
vol. 95(1) C pp. 83-127 C 2014
Poster session
Poster 1
Blue-light-activated phototropin2 trafficking
from the cytoplasm to Golgi/post-Golgi vesicles
C. AGGARWAL1,2, A.K. BANAS2, A. KASPROWICZ-MALUSKI1, H. GABRYS2
1
2
Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
Phototropins are plasma membrane localized UVA/blue-light photoreceptors which mediate phototropism,
inhibition of primary hypocotyl elongation, leaf positioning, chloroplast movements and stomatal opening. Blue light
irradiation activates the C-terminal serine/threonine kinase domain of phototropin which autophosphorylates the
receptor. Arabidopsis thaliana encodes two phototropins, PHOT1 and PHOT2. In response to blue light PHOT1
moves from the plasma membrane into the cytosol and PHOT2 translocates to the Golgi complex. Our work show
the molecular mechanism and route of blue-light-induced PHOT2 trafficking. It is shown that AtPHOT2 behaves in
a similar manner when expressed transiently under 35S or its native promoter. The PHOT2-kinase domain but not
blue light-mediated autophosphorylation is required for the receptor translocation. Using co-localization and western
blotting, the receptor was shown to move from the cytoplasm to the Golgi complex, and then to the post-Golgi
structures. The results were confirmed by brefeldin A (an inhibitor of the secretory pathway) which disrupted
PHOT2 trafficking. An association was observed between PHOT2 and the light chain2 of CLATHRIN via bimolecular
fluorescence complementation. The fluorescence was observed at the plasma membrane. The results were confirmed using co-immunoprecipitation. However, tyrphostin23, (an inhibitor of CLATHRIN-mediated endocytosis) and
wortmannin (a suppressor of receptor endocytosis) were not able to block PHOT2 trafficking indicating no involvement of receptor endocytosis in the formation of PHOT2 punctuate-structures. Protein turnover studies indicated
that the receptor was continuously degraded in both darkness and blue light. The degradation of PHOT2 proceeded
via a different transport route than translocation to the Golgi complex.
Poster 2
Deep: non-reference based method for functional sRNA
identification and first bioinformatics analysis of
liverwort Pellia endiviifolia microtranscriptome
S. ALABA1, P. PISZCZALKA2, Z. SZWEYKOWSKA-KULINSKA1,2, W. KARLOWSKI1
1
2
Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poland
Small RNA (sRNA) are short 18-26nt sequences generated by RNA endonucleases and functionally associated
with Argonaute protein family members to regulate gene expression level via complementarity. Recently, thanks to
decreasing Next Generation Sequencing costs, sRNA world started to grow rapidly. Nevertheless, discovery of novel
84
Poster session
sRNA classes is mainly restricted to single sRNA types from model organisms with known genomic sequences. This
is a result of two major disadvantages of currently used sRNA identification methods: 1) focus on best described
sRNA class – microRNA (miRNA) only and 2) usage of mandatory reference sequence. To overcome known software
limitations we have developed Deep – a novel non-reference based method for functional sRNA discovery. Our approach is based on three criteria focused on features related to sRNA processing: 1) 5N-end cleavage accuracy,
2) sequence length, and 3) abundance. Small RNA clusters built from sRNAs are encoded as position-specific feature
profiles that allows sequence-free identification of novel short RNAs. Resulting sequence lists created by grouping
similar profiles and ranked by sRNA abundance, allow simple sRNA candidate selection. For learning step, algorithm
requires as input only raw sRNA data and any user-specifed known sRNA sequences. Small RNA data from Pellia
endiviifolia – a liverwort representing a group of first land plants was selected to test our approach. No genomic
sequence is available for P. endiviifolia. Five samples of P. endiviifolia sRNA data from male and female thalli with
or without sex organs, were analyzed. First, all sequences homologous to plant miRNA were identified representing
311 miRNA families with up to 2 substitutions. Northern hybridization performed on selected candidates confirmed
its presence in Pellia thalli. Small RNAs annotated as plant miRNA without substitutions were then used for algorithm learning step. By applying the new approach we were able to select 69 positive candidates representing putative novel liverwort specific short RNAs. Using northern hybridization, 41 of them were confirmed as stable sRNA
products. For further validation of 41 candidate sequences we performed transcriptome and degradome sequencing.
Genome Walking technique confirmed 10 miRNA genes identified in silico. Analysis of degradome data revealed
13 mRNA targets cleaved precisely at 9-11 position of sRNA:mRNA duplex. One out of 41 sRNA candidates represented Pellia specific miR* sequence of miR408 family which expression was experimentally confirmed.
Poster 3
Novel proteins interacting with the SERRATE protein
M. BAJCZYK, Z. SZWEYKOWSKA-KULINSKA, A. JARMOLOWSKI
Adam Mickiewicz University, Poznan, Poland
In plants the SERRATE protein (SE) is involved in two important pathways of RNA metabolism: microRNA biogenesis and pre-mRNA splicing. Originally, SE was characterized as a protein involved in microRNA biogenesis,
where together with DCL1 (DICER-LIKE 1) and HYL1 (HYPONASTIC LEAVES 1) form a core of the plant microprocessor. In this complex SE influences the cleavage accuracy of pri-miRNAs by DCL1, interacting directly with
DCL1 and some other miRNA biogenesis factors. Interestingly, the Arabidopsis se null mutant is embryonic lethal
what proves the key role of SE in plant development and growth. SE has been also found to be involved in splicing
of pre-mRNA. Moreover, its role in splicing is connected with interaction of SE with both subunits of the nuclear
cap-binding complex (CBC): CBP20 and CBP80. Thus, CBC and SE have dual functions: in pre-mRNA splicing as well
as in processing of pri-miRNA. We suggest that SE can be a link in the crosstalk between biogenesis of miRNA, primiRNA splicing and transcription of MIR genes. In order to understand better such key role of SE, we decided to
search for proteins that interact with the Arabidopsis SERRATE protein. To this end, we constructed the Arabidopsis thaliana transgenic line, in which in the genetic background of the se-1 mutant the FLAG-tagged version of
the SE gene was integrated into the genome. Next, we carried out immunoprecipitation against the FLAG epitop
of the expressed fusion protein to find SE protein partners using mass spectrometry. First of all, we optimized the
method. Using this protocol we performed co-immunoprecipitation experiments which gave us a list of potential SE
interactors. Surprisingly, a big set of proteins interacting with SE are factors involved in transcription and chro-
Poster session
85
matine structure remodeling. Our data suggest that a zinc finger protein SERRATE can be involved in the regulation
of transcription carried out by RNAPII as well as RNA-directed DNA methylation. Currently, we are confirming the
interactions using the yeast two hybrid system and microscopic methods (BiFC and FRET).
Poster 4
Comparison of expression LlFCA, LlFY and LlFLD genes from
the flowering autonomous pathway of Lupinus luteus
M. BANACH, W. WOJCIECHOWSKI, J. KESY, P. GLAZINSKA, E. WILMOWICZ,
A. KUCKO, K. MARCINIAK, J. KOPCEWICZ, A. TRETYN
Chair of Plant Physiology and Biotechnology, Faculty of Biology and Environment Protection,
Nicolaus Copernicus University, Torun, Poland
Centre for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Torun, Poland
The flowering time is one of the factors providing reproductive success. At least four genetically defined pathways have been identified that control flowering: vernalization, photoperiod, gibberellin or hormonal and autonomous
pathways. All pathways control of flowering cooperate regulation of key flowering genes which name ‘integrator
genes’. The autonomous pathway includes at least 7 of flowering induction genes. All these genes are negative
regulators of FLOWERING LOCUS C (FLC ) – main inhibitor of flowering induction. RNA BINDING/FLOWERING
TIME CONTROL PROTEIN FCA ALPHA (FCA) is a nuclear protein mediates the floral transition. This protein
contains two conserve domains RNA recognition motifs (RRM) and a WW domain. The RRM domain is involved in
selection of polyadenylation site, chromatin silencing of single and low copy genes, interaction with small interfering
RNA-directed DNA methylation pathway for regulating common targets. FCA participates in post-transcriptional FLC
mRNA modifications. FCA pre-mRNA is alternatively spliced. FCA gene has several alternative versions of different
length mRNAs in various tissues it follows that activity of FCA protein is connection with kind of mRNA used for
translation. FCA plays important roles in RNA 3 processing and transcription termination, thus limiting intergenic
transcription. FLOWERING TIME CONTROL PROTEIN (FY) plays dual roles in FLC regulation. FY is an RNA 3N
end-processing factor. FY interacts with FCA (FY/FCA) by two proline-rich (PPLPP) motifs in the C-terminus end
of FY. FY/FCA interaction shares in the autoregulation of FCA expression and the selection polyadenylation site in
the FLC pre-mRNA. FCA/FY is the key factor in regulating the RNA processing machinery. In likely model, FY acts
in conjunction with FCA to repress FLC, but also has an FLC-promoting activity that is FCA independent.
FLOWERING LOCUS D (FLD) regulate FLC expression by chromatin remodeling. FLD regulates FLC by preventing hyperacetylation of the locus. FLD is histone demethylase. In likely model, FLD might participate in the
deacetylation of FLC chromatin as a component of a histone deacetylase (HDAC) complex. In this study, expression
of FCA, FY and FLD gene was quantitative used real time PCR technique. Lupinus luteus were cultivated in
a growth chamber at a temperature of 22 ±1EC under long day conditions. The research materials were vegetative
organs. Before collected plants were sprayed aqueous solutions of auxin, abscisic acid or gibberellins. Control plants
were sprayed water. Plants were collected after one hour after sprayed. Preliminary results obtained here will enable
us to determine FCA, FY and FLD expression pattern in vegetative organs of L. luteus cultivars – the agricultural
valuable species in Poland. It will also facilitate to characterize the role of these genes in the regulation of development of L. luteus crops in different growth conditions. Acknowledgements: The work was supported by the MultiYear Programme of the Polish Ministry of Agriculture and Rural Development, No. 149/2011 and the National
Science Centre (Poland) grants No 2011/01/B/NZ9/03819.
86
Poster session
Poster 5
RNAi as a tool for a functional analysis of ABC transporters
in the model legume plant Medicago truncatula
J. BANASIAK1, W. BIALA2, K. JARZYNIAK2, M. JASINSKI1,2
1
2
Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Poznan, Poland
Nowadays genomic projects provide researches with numerous genes with unknown functions. To solve this
problem the strategy called reverse genetic has been established. It is based on phenotype analysis of plants in which
expression of a particular gene is switched off. This approach exploits mutagenesis or gene silencing. The latter can
be exemplified by RNA interference (RNAi) triggered through double-stranded RNA (dsRNA) which inhibits gene
expression in a sequence-specific manner. This post-transcriptional gene silencing (PTGS) is still the method of
choice to gain information about function of any chosen target gene. Especially when mutants collection of a given
plant is scarce or knockouts are lethal. We have used PTGS in the functional studies of ABC (ATP-binding cassette)
transporters in the model legume plant Medicago truncatula. Transgenic hairy roots and root cultures with silenced
MtABCG10 have been obtained by Agrobacterium rhizogenes- mediated RNAi. To suppress MtABCG10 expression,
a 139 bp fragment from the coding region was introduced into the pK7GWIWG2(II)-p35S::DsRED binary vector.
Presence of DsRED marker allowed for a quick and efficient inspection of the chimeric nature of A. rhizogenes transformed roots and selection of material for proper phenotyping. By usage of this construct we were able to silenced
MtABCG10 expression what was confirmed at the mRNA and protein level by Real-Time PCR and Western Blot
respectively. Selected transgenic hairy root clones have been used for metabolomic analysis and biological assays
(pathogen infection and nodulation efficiency).
Poster 6
Novel Arabidopsis thaliana miRNAs responsive
to different abiotic stress conditions
M. BARCISZEWSKA-PACAK, K. SKORUPA, D. BIELEWICZ,
J. DOLATA, A. JARMOLOWSKI, Z. SZWEYKOWSKA-KULINSKA
MicroRNAs are key regulators of eukaryotic gene expression via post-transcriptional inhibition and cleavage.
They play crucial roles in plant development and response to various abiotic and biotic stresses. However, the expression of MIR genes themselves is a subject of careful control. A high throughput real-time PCR platform (mirEX)
has been developed to discriminate nearly all individual known primary miRNA precursors (pri-miRNAs) and to analyze reliably their individual expression patterns (Bielewicz et al. 2012, http://comgen.pl/mirex). In progressive
drought experiment, the stress was applied to Arabidopsis plants at 1.10 growth stage (Boyes et al. 2001) by water
withholding, and continued until the soil moisture level reached 30% field capacity (FC) (2 days before wilting) and
20% FC (wilting stage), both for different plant batches. The experiment was monitored by leaf relative water content
(LRWC) measurements. 14 day-old Arabidopsis seedlings were subjected independently to 24h salinity, sulfur deficiency, copper deficiency, copper excess, cadmium excess and 12h heat stresses. The polyA+ RNA isolated from
plants subjected to 30% and 20% FC drought conditions and the other abiotic stresses was analyzed by mirEX
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Poster session
platform for pri-miRNA and by Northern hybridizations for mature miRNA expression profiles, respectively. The
analyses revealed altogether 33 known and novel mature miRNAs responsive to different abiotic stresses. With
regard to the applied 20%FC and 30%FC drought stresses and salinity stress 13 new mature drought-responsive
miRNAs and 9 new salinity-responsive miRNAs were identified. The progressive drought analysis showed also different responsiveness of pri- and mature miRNAs in the time course. For metal toxicity stresses the responsiveness
of 25 miRNAs was shown and can be compared to the data known only from metal toxicity experiments done in rice,
Medicago truncatula and Brassica napus. For Arabidopsis sulfur deficiency responsive miRNAs, new data have been
obtained for 8 novel and 9 generally known stress responsive miRNAs, what can be compared to Brassica napus
published data. The changes of the mature miRNA levels were analyzed for correlation with the changes of their
cognate precursors in the applied abiotic stresses. Using available data for single gene miRNA family members that
were detected as new Arabidopsis stress responsive miRNAs, we noted potential 7 transcriptional (correlated priand mature miRNAs changes) and 9 post-transcriptional (uncorrelated pri- and mature miRNAs changes) miRNA
genes expression regulations. For drought stress 2 transcriptional and 3 post-transcriptional gene expression regulations can be postulated, meanwhile only 3 transcriptional gene regulations are potential under salt stress. Under
copper as well as cadmium stresses, out of 6 analyzed 5 post-transcriptional gene regulations can occur. The work
was supported by the NCN Harmonia funding scheme UMO-2012/04/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”.
Poster 7
Translational regulation during root developmental
adaptation to phosphate starvation
J. BAZIN1,2, M. CRESPI2, J. BAILEY-SERRES1
1
Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, California, USA
2
Institut des Sciences du Vegetal (ISV), Centre National de la Recherche Scientifique (CNRS), Saclay Plant Sciences,
Gif-sur-Yvette, France
The sessile lifestyle of plants requires them to adapt their growth and development to environmental variation.
This implies a tight spatiotemporal regulation of gene expression to control developmental responses. Translational
control, the regulation of the initiation, elongation or termination of ribosomes on an mRNA, provides cells additional
mechanisms to rapidly control gene expression in a reversible manner, in response to environmental and developmental cues. It is known that non-coding RNA (ncRNA) can modify spatiotemporal gene expression patterns, and
play a key role in developmental plasticity. Their role in the regulation of initiation of mRNA translation have been
described in animals and hinted in plants, where mechanistic details are limited. Improving tolerance to nutrient
deficiency is a major goal of agricultural research. Improvement of inorganic phosphate (Pi) starvation has been the
target of classical breeding and genetic engineering. ncRNAs (i.e. miRNA399) have been shown to regulate root
development during Pi starvation in roots of the model plant Arabidopsis thaliana. The three objectives of this project are to a) analyze the role of translational control at the genomic level in response to Pi starvation using state-ofthe-art methods that resolve ribosome activity to the codon level; b) to assess the role of diverse classes of ncRNA
in translational regulation in response to Pi starvation; and c) to delve into the mechanistic role of selected ncRNAs
in translational regulation during Pi starvation. Experimental approaches used to fulfill these objectives will be
presented. This will include the use of tissue-specific ribosome footprint sequencing and mRNA-seq of Arabidopsis
roots cells to study translation regulation.
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Poster session
Poster 8
mirEX2: a new version of RT-qPCR and sRNA NGS platform
for comparative exploration of plant miRNA expression data
D. BIELEWICZ1, J. DOLATA1, K. KRUSZKA1, A. ZIELEZINSKI2, S. ALABA2,
A. PACAK1, A. SWIDA-BARTECZKA1, K. SKORUPA1, A. STEPIEN1, P. PISZCZALKA1,
H. PIETRYKOWSKA1, A. PIASECKA1, A. JARMOLOWSKI1, W.M. KARLOWSKI2, Z SZWEYKOWSKA-KULINSKA1,2
1
2
mirEX is a comprehensive RT-qPCR platform for comparative analysis of microRNA expression data. In the new
version of mirEX we have combined data from RT-qPCR-based gene expression profiles of pri-miRNAs and data from
NGS-based expression profiles of mature miRNAs from three plant species: Arabidopsis thaliana, Hordeum vulgare,
and liverworth Pellia endiviifolia. Currently, the mirEX2 integrates information about the expression profile of all
(299) Arabidopsis thaliana pri-miRNAs in thirteen different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, for two-week-old seedlings we analyzed the expression level of pri-miRNA for
three microRNA biogenesis mutants (hyl1-2, se-1, and cbp20xcbp80 ) an for three-week-old plant we performed analyses of pri-miRNAs for four mutants (dcl1-7, hyl1-2, se-1, and cbp20xcbp80 ). Pri-miRNA analyses for barley were
carried out for five developmental stages: 1-,2-,3-,6-week-old plants and from 68-day-old plants when kernels reach
milk ripeness. Pri-miRNA expression analyses for dioecious Pellia were performed for female thalli producing archegonia, male thalli producing antherida or for female or male thalli without sex organs. One of the main new features
of the mirEX2 database is integration of information about expression level of mature microRNAs. NGS data will
cover two developmental stages in Arabidopsis thaliana. There will be data from wild type, hyl1-2, se-1, cbp20xcbp80
from two-week old seedlings and data from 35-day-old wild type plants. For Hordeum vulgare deep sequencing was
carried out for all (5) developmental stages and for Pellia endiviifolia NGS sequencing was performed separately for
female and male thalli with or without sex organs. The possibility of correlation of pri-miRNAs expression level with
expression level of mature miRNAs gives users more detailed information of microRNA gene expression regulation.
All data are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface.
A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and
dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows
a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a onestep solution for microRNA information in Arabidopsis, Hordeum and Pellia. A web-based mirEX interface can be
accessed at http://bioinfo.amu.edu.pl/mirex.
Poster 9
ARP4 – the busiest protein in the plant nucleus?
T. BIELUSZEWSKI, L. GALGANSKI, M. ABRAM, A. BIELUSZEWSKA, P. ZIOLKOWSKI, J. SADOWSKI
Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poland
ARP4 belongs to the actin related protein (ARP) family which is represented by cytoplasmic and nuclear proteins
in all eukaryotes. Nuclear ARPs are subunits of several protein complexes responsible for chromatin remodeling and
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histone acetylation. Among nuclear ARPs, ARP4 is the most evolutionary conserved and occurs in the greatest variety of complexes. In human, ARP4 is a subunit of the chromatin remodeling complexes SWI/SNF, INO80 and
SRCAP as well as the Tip60-p400 complex which has a dual role of a H2A-H2A.Z histone-exchange complex and a histone H4 acetyltransferase. Our results suggest that ARP4 is a subunit of at least seven different chromatin modifying
complexes in the model plant Arabidopsis thaliana, including plant analogs of SWI/SNF and SWR1 complexes which
are known as important regulators of transcription in plant development. Here we focus on two less well studied complexes, INO80 and NuA4 and their possible roles in Arabidopsis.
Poster 10
AtNTR1 is required for histone methyltransferase activity
G. BRZYZEK, Y. GUO, S. SWIEZEWSKI
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
In higher eukaryotes messenger RNA splicing is required for gene expression as most of the nuclear-encoded
genes harbor multiple introns. Splicing of pre-mRNA is carried out by spliceosome, a multi-megadalton ribonucleoprotein complex in a complex multistep process. Most of splicing is believed to take place co-transcriptionally and
splicing has been shown to feed back on transcription. NTR1 is one of splicosomal accessory factors, highly conserved within all eukaryotes, required for disassembly of splicing complex and completion of the splicosomal cycle. We
have analyzed the AtNTR1 interacting proteins using a complementing tagged version of AtNTR1. One of the interactors was a putative histone methyltransferase. We have confirmed this interaction by Y2H and BiFC assay. In vitro
biochemical assay with purified putative methyltransferase failed to show biochemical activity. However when this
putative methyl transferase was combined with purified AtNTR1 we could observe a specific methyltrasferase activity
towards H3K36. Our data suggest a direct link between splicing and histone posttranslational modification that could
potentially provide a mechanistic explanation for spliceosome mediated feedback on chromatin.
Poster 11
An evidence for reversible DNA methylation changes
in drought stress-responsive genes in barley
K. CHWIALKOWSKA, I. SZAREJKO, M. KWASNIEWSKI
Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland
Plants are constantly challenged by biotic and abiotic stresses and, thus, have developed a number of strategies
allowing for a rapid adaptation to unfavorable environmental conditions. DNA methylation is one of the epigenetic
phenomena that play a crucial role in regulation of gene expression in response to environmental stimuli. In the
presented study, the effect of drought stress on DNA methylation level, pattern and its correlation with gene expression in Hordeum vulgare L. were assessed. The global evaluation of changes in the level and pattern of cytosine
methylation under drought stress was carried out using methylation sensitive amplification polymorphism (MSAP)
technique on leaf and root samples of plants exposed to drought stress, after re-watering and unstressed controls.
Moreover, we modified the MSAP method by replacing of the conventional separation of MSAP amplicons on polyacrylamide gels with their direct sequencing using Next Generation Sequencing methods and automated data analysis (MSAP-Seq). The identified DNA methylation changes in specific genomic loci were confirmed using methylation-sensitive restriction enzyme digestion coupled with quantitative PCR (MSRE-qPCR). Our study revealed that
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the overall methylation level within 5N-CCGG-N3 sequences in barley genome was at a high and comparable level
in both leaves and roots (about 70%). Drought stress induced global-wide changes of DNA methylation pattern in
barley genome and most of them consisted of new methylation profiles. There were more methylation alterations
induced by drought in leaves than in roots and most of them went back to their initial status after re-watering. Detailed MSAP-Seq identification of loci undergoing DNA methylation changes revealed that most of new methylation
profiles were established in genes, whereas demethylation occurred mainly within repeated sequences, especially
in regions of transposable elements. Establishment of new methylation profiles under drought was observed among
genes involved in basic metabolic processes such as oxido-reductive processes, chromatin condensation regulation,
DNA repair, translation, RNA metabolism, as well as in stress-responsive genes, for example: response to jasmonic
acid, pathogen resistance, anthocyanin biosynthesis and signal transduction. Interestingly, genic demethylation of
cytosine was identified within similar group of stress-related genes, which suggests that stress triggers dynamic
methylation/demetylation events preferentially in stress-responsive genes. Single-loci analysis using MSRE-qPCR
confirmed that a large fraction of identified genomic loci was subjected to methylation changes under drought and,
in addition, revealed that they all went back to their initial level under re-watering. Simultaneous gene expression
profiling with RT-qPCR indicated that most of these genes underwent transcriptional down-regulation under drought
and also returned to the basic expression level after recovery from water deficit. Thus, our results demonstrate
a strong correlation of DNA methylation changes and gene expression modulation under drought and indicate their
coupled reversibility.
Poster 12
Wheat (Triticum aestivum L.) gibberellins biosynthesis genes
expression alteration during growth regulator application
K. DUDZIAK, M. ZAPALSKA, J. LESNIOWSKA-NOWAK, M. NOWAK, K. KOWALCZYK
Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Lublin, Poland
Gibberellins (GAs) are plant hormones which mediate plant growth and development. Production of biologically
active GAs is a complex process involving number of enzymes. Two of them: oxidases GA20 and GA3 play crucial
role in bioactive GAs biosynthesis and have been tested in this study. Additionally, level of bioactive gibberellins is
also regulated by GA2 oxidase, which transform bioactive gibberellins into biologically inactive forms. Last studies
indicate that some of cereals dwarfing genes encode modified enzymes of gibberellins biosynthesis pathway. The
purpose of our study was determination of the influence of plant growth regulator application on transcription
alteration of three main gibberellins biosynthesis pathway genes (GA20OX, GA3OX and GA2OX ) in common wheat
(Triticum aestivum L.) Bezostaya 1 isogenic lines containing RHT-B1b and RHT12 dwarfing genes. As a control
form tall isogenic line without dwarfing genes was used. In presented study seven-day-old seedlings were treated with
etephon in concentration suggested for commercial application. After three days plant material was harvested, and
total genomic RNA was extracted by means of Trizol reagent method. For determination of analyzed dwarfing genes
transcript level in plant tissue qPCR method based on SYBR Green dye was applied. For quantitative PCR cDNA
obtained from reverse transcription of total RNA was used as template. For amplification reaction the sequence
specific primers for selected dwarfing genes developed previously were used. Obtained results showed, that etephone application caused alteration of analyzed genes expression in tested wheat lines. After growth regulator treatment the level of expression for GA20OX gene was lower for both dwarfing genes containing lines, whereas for tall
line an increase of transcript level was noticed. The level of GA3OX gene expression decreased in response to etephone activity in all analyzed lines. For GA2OX gene, responsible for biosynthesis of enzyme involved in degradation
of bioactive gibberellins forms, increase of transcript level in all three analyzed lines was observed. Obtained results
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Poster session
confirm, that changes in gibberellins biosynthesis pathway genes expression is a part of complex plant response to
application of growth regulation. Etephone treatment caused decrease of expression in genes encoding enzymes
responsible for bioactive gibberellins biosynthesis (GA20OX, GA3OX ). In the same time the level of transcription
of GA2OX gene increase, what suggests intensification of bioactive gibberellins degradation process in tissue.
Presented results were obtained as a part of the project #N N310 774140 granted by Polish National Science Centre.
Poster 13
New tools to monitor PBs and SGs dynamic in response
to heat stress in Arabidopsis
J.-J. FAVORY1,2, J. DESCOMBIN1,2, J.-M. DERAGON1,2, C. BOUSQUET-ANTONELLI2,1
1
2
Laboratoire Genome et Developpement des Plantes, Universite de Perpignan Via Domitia, Perpignan, France
Laboratoire Genome et Developpement des Plantes, Centre National de la Recherche Scientifique, Perpignan, France
For each living organism it is of prime importance to be able to adapt to changes of environmental conditions.
For plants, as sessile organisms, the fine tuning of the gene expression in an ever changing environment is even
more vital. Post-transcriptional regulations play a crucial role in gene expression control. In animals two types of
cytosolic ribonucleoprotein (RNP) structures have been described in response to translation repression: stress granules (SGs) and processing bodies (PBs). SGs are dynamic aggregates of untranslated mRNAs in association with
translation initiation factors and PBs are RNA protein aggregates containing untranslated mRNAs associated with
the mRNA decapping and 5N- decay machineries. Presently, only few components involved in PBs and SGs formation
are identified in plants. In addition, recent studies showed that some key factors are common to plants and animals
and can thus be used to identify more partners, as well as makers of these granules. These aggregates are visible
as cytoplasmic foci which can be followed using fluorescent microscopy approaches. To study the dynamics of these
RNP granules in Arabidopsis, stable transgenic lines expressing YFP and RFP tagged version of AtDCP1 and AtPAB2
under control of their endogenous promoters were selected to study PBs and SGs respectively. Our experiments
conducted in young seedlings root tips indicate that both structures response to selected stresses with different
dynamics. These first studies show that these selected lines provide useful cytosolic RNP granules dynamic reporter
lines and stress sensors in Arabidopsis. Our team’s main interest focuses on the understanding of the reprogramming of translation and cytoplasmic mRNA stability in response to heat stress. We are in particular aiming at
understanding the dynamics of SG and PB formation and dissociation in various heat stress regimes. We will present
our latest data about the dynamics of PB and SG along a short-term acquired thermotolerance (SAT) process.
Poster 14
Post-transcriptional regulation of an Arabidopsis protein
involved in the heat stress response
N. FERNANDEZ BAUTISTA, L. FERNANDEZ-CALVINO, A. MUNOZ, M.M. CASTELLANO
Centre for Plant Biotechnology and Genomics (CBGP) INIA-UPM, Pozuelo de Alarcon, Madrid, Spain
High temperature is one of the most deleterious environmental factors affecting plants. It seriously constrains
plant development and growth, not only by reducing crop yield and quality, but also by limiting the arable land area
and the use of varieties to crop. Therefore, elucidating the heat stress response is an important goal to understand
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how plants can adapt to this physical parameter. During the last years our lab has developed different genomic approaches, analyzing transcriptional and translational changes during the survival process of plants to the increase
of temperatures. This research has allowed identifying a group of proteins which functions in heat stress response
have not been studied before in plants. One of these proteins is the protein 3P. The transcriptional changes in the
3P gene during the heat shock treatment has been studied and compared with other already known proteins involved
in heat stress response. These analyses show that its expression is highly increased during the heat stress, but actively degraded under normal conditions. The 3P protein is localized in the cytoplasm under non stress conditions
and at the early stages during the heat stress response. However, it changes its localization to cytoplasmic isolated
foci and to the nucleus later on under stress conditions. All these results suggest that 3P is a highly regulated protein
whose function during the plant response to heat stress is currently been evaluated.
Poster 15
Changes in the chromatin state and gene transcription
in response to salinity stress in T87 Arabidopsis thaliana cells
A. FOGTMAN1, A. KWIATKOWSKA3, A. PALUSINSKI2, R. IWANICKA-NOWICKA1,2, A. PACEK2, A. MACIOSZEK4,
B. WILCZYNSKI4, M. KOTLINSKI1,2, J. DOLATA5, A. JARMOLOWSKI5, A. JERZMANOWSKI1,2, M. KOBLOWSKA1,2
1
Corelab, Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland
2
Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, Warsaw, Poland
3
4
5
Department of Botany, University of Rzeszow, Rzeszow, Poland
Institute of Informatics, Faculty of Mathematics, Informatics and Mechanics, University of Warsaw, Warsaw, Poland
Chromatin is a nucleoprotein complex encompassing DNA and core histones in the nucleus of an eukaryotic cell.
Stability of chromatin structure is crucial for the proper regulation of all nuclear DNA-templated processes – transcription, replication, repair and recombination. Emerging studies show that various cell signaling pathways trigger
changes in the chromatin state and thus incoming signals can regulate gene expression through chromatin reorganization. Plants as sessile organisms developed diverse mechanisms allowing for quick response and adaptation to
abiotic stress conditions. One of the first levels of the plant cell response to stress is induction of transcription of
different gene classes. Defining how changing environmental conditions influence the chromatin state is essential
for understanding regulation of plant gene transcription critical for improvement the resistance of cultivated plant
to environmental stress. We studied in parallel the quick changes at both levels: transcription and chromatin
structure in response to salt stress. As a model we chose T87 Arabidopsis thaliana cell line grown in suspension.
Analysis of transcriptional changes was performed on Affymetrix GeneChip ATH1 microarrays. We carried out
microarray experiments of treated with 250 mM NaCl T87 cells in 6 time points (0N, 20N, 40N, 60N, 80N and 100N).
A number of known (COR15a, DREB2A ) and unknown genes were identified to have changed transcription levels
in response to salinity, which was confirmed by qPCR analysis. Our transcriptomic analysis identified new genes
potentially crucial for plant adaptation to salinity stress. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) with the use of antibodies against characteristic for transcriptional regulation core histone
modifications revealed global changes in chromatin state under salt stress conditions, which correlated with changed
expression of early responsive (ER) genes. Our results show the relations between cell signaling, chromatin state
and gene regulation in response to environmental stress in plants.
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Poster 16
Functional characterization of Arabidopsis LUC7 proteins,
components of the U1 snRNP
M. DE FRANCISCO AMORIM, S. LAUBINGER
Center for Plant Molecular Biology (ZMBP), University of Tubingen, Tubingen,Germany
and Chemical Genomics Centre (CGC), Max Planck Institute of Molecular Physiology, Dortmund, Germany
Splicing, which is the removal of intronic regions from mRNAs transcripts followed by the exons joining, is an
essential RNA processing step in all eukaryotes. Splicing is carried out by a macromolecular complex, the spliceosome, which consists of 5 so-called small nuclear ribonucleoproteins particules (snRNP) sub-complexes (for instance,
U1, U2, U4, U5 and U6 snRNP for the major spliceosome). Each snRNP is assembled of a common core set of proteins (SM or LSM proteins), snRNP specific proteins and specific small nuclear RNA (snRNA). Splicing of introns
proximal to the cap, which is a covalent modification attached to polymerase II derived transcripts, is assisted by
additional proteins, among them the nuclear cap-binding complex (CBC) and SERRATE (SE). The CBC is an heterodimeric complex formed by CBP20 and ABH1/CBP80 that serves as a platform for binding of different proteins
dictating the RNA fate. The CBC directly interacts with the zinc-finger protein SE. This interaction is not only required for efficient splicing, but also important for pri-miRNA processing and possibly other RNA processing events.
While the role of SE/CBC in the miRNA biogenesis has been extensively studied, little is known about how this complex regulates splicing. In a yeast two-hybrid screen using SE as bait, we identified several components of the spliceosome as potential SE interactors. These findings suggest that SE/CBC physically interact with the spliceosome to
facilitate splicing of cap-proximal introns. The main focus of this work is a protein called LUC7, which is part of the
U1 snRNP and is encoded by a small gene family (LUC7A, LUC7B and LUC7RL) in Arabidopsis. While a mutation
in LUC7A has been reported to impair fertilization in plants, we were able to identify hypomorphic alleles with only
slightly compromised functions. A hypomorphic luc7 triple mutant exhibited severe developmental defects: it is
dwarf, flowers late and display loss of the apical dominance. Complementation analysis revealed that all the three
LUC7 proteins act redundantly to control plant development. Splicing analyses, localization studies and proteinprotein interaction data will be discussed.
Poster 17
The new RNAi approaches as tools in functional analysis
of genes in polypolid cereals – introduction to research
S. GASPARIS1, A. NADOLSKA-ORCZYK1, W. ORCZYK 2
1
Department of Functional Genomics, Plant Breeding and Acclimatization Institute – National Research Institute, Radzikow, Blonie, Poland
2
Department of Genetic Engineering, Plant Breeding and Acclimatization Institute – National Research Institute, Radzikow, Blonie, Poland
In the last decade a significant progress has been made in studying molecular mechanism of RNA interference
(RNAi). RNAi is now routinely used in model plant species to characterize gene function or to alter the phenotype.
Because of some limitations this approach has not been commonly used in case of polyploid species, however it has
the same great research potential. The recent studies performed by our team have proved the high effectiveness
of post-transcriptional gene silencing (PTGS) in polyploid cereals. This method was successfully used for silencing
the expression of genes encoding important agronomic and quality traits i.e. puroindoline genes in wheat and HvCKX
genes in barley. Here we present the background of our new research in which another two RNAi pathways have
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been studied – transcriptional gene silencing (TGS) and silencing by artificial microRNA (amiRNA). Both methods
were applied for silencing of puroindoline genes PINa and PINb in allohexaploid wheat and amiRNAs were also used
for silencing of secaloindoline genes in triticale – the orthologs of wheat puroindolines. Grain hardness determined
by puroindoline genes is one of the most important technological trait in wheat. In the first bioinformatical approach
the proper RNAi constructs were designed for TGS and amiRNA experiments. For TGS, the silencing cassettes of
hpRNA type were constructed. Promoters of PINa and PINb genes were sequenced and analyzed to find the fragments containing regulatory elements. These fragments were cloned into RNAi vector pMCG161 in a sense and
antisense orientation. amiRNAs for PINa and PINb genes were designed using special algorithm. 21 nt amiRNA
fragments were then inserted into precursor miRNA from wheat – Tae-miR164. This construct was cloned into overexpression vector pBract214. Finally, the TGS and amiRNA constructs were used for Agrobacterium -mediated
transformation of wheat and triticale and transgenic plants were obtained. In the next part of the research the molecular and phenotypic analysis will be performed to determine the effectiveness and preciseness of gene silencing
with RNAi pathways used. We believe that the results of our studies will be helpful in choosing the most suitable and
efficient method for either functional analysis of agronomically important genes or for genetic engineering of polyploid cereals. This research is supported by the National Science Center grant UMO-2011/03/B/NZ9/01383.
Poster 18
Identification and analysis of miRNA precursors
in yellow lupine based on the cDNA sequences submitted
in Sequence Read Archives NCBI
P. GLAZINSKA1,2, M. GRZECA1,2, W. WOJCIECHOWSKI1,2, E. WILMOWICZ1,2,
K. MARCINIAK1,2, J. KESY1,2, J. KOPCEWICZ1
1
Chair of Plant Physiology and Biotechnology, Faculty of Biology and Environment Protection, Nicolaus Copernicus University, Torun, Poland
2
Yellow lupin (Lupinus luteus L.) is an important legume crop characterized by high content of seed proteins. The
crucial stage for lupine productivity, the formation and development of flowers and seeds, is often associated with
the abscission of flowers. Plant miRNAs are approximately 21-nt-long small regulatory RNAs that recognize their
mRNA targets based on imperfect sequence complementarity, thereby suppress expression of the target gene by
guiding degradation and/or translational repression of the cognate mRNA target, which are involved in the regulation
of plants growth and development. MiRNAs are produced from either their own genes or from introns and then
processed from single-stranded precursors that form hairpin structures, with the miRNAs residing in one arm of the
stems. In contrary to mature sequences, much less evolutionary conserved precursors of miRNA hinder the
identification of pre-miRNA homologous in other plant species. Using the fact that, plant pri-miRNAs are primarily
transcribed by RNA polymerase II to produce mRNA-like structure with 5N-end cap and 3N- end polyA tails, we
decided to identify miRNA precursors using data from 454 sequencing cDNA Lupinus luteus L. libraries submitted
in Sequence Read Archives NCBI (SRP014198). In the present study, we have used seven miRNAs sequences (from
miRBase) that play important role in generative development of another plant species. As the result, we have found
that SRA data base contains homology sequences to five known miRNAs of seven used in the study. Part of them
form hairpin structures and contain the mature miRNA sequence in one arm of the stems. Identified putative
precursors are very similar to soybean and Medicago with minor difference in the hairpin structure outside of the
highly conserved miRNA/miRNA* region. The occurrence of the identified pre-miRNA in yellow lupine have been
confirmed by PCR and sequencing. This is the first step to study the putative miRNAs involving in flower morphogenesis and abscission in Lupinus luteus L.
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Poster 19
mRNA decay factors contribute to ABA singaling
A. GOLISZ, I. WAWER, A. SULKOWSKA, J. KUFEL
Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland
Sm-like (Lsm) proteins have been identified in all organisms and function in RNA metabolism. They form two
distinct heptameric complexes: the nuclear Lsm2-8 complex is a core component of the U6 snRNP and is involved
in mRNA splicing and the cytoplasmic Lsm1-7 is engaged in 5N-3N mRNA degradation. We have recently confirmed
that similar LSM8 and LSM1 complexes exist also in Arabidopsis and are involved in mRNA splicing and decay,
respectively. Interestingly, a mutant in the SAD1/LSM5 gene, was shown to be hypersensitive to the plant hormone
abscisic acid (ABA), salt and drought, and displays altered expression of some stress related genes, whereas lsm4
plants are hypersensitive to salt and ABA. To further investigate the link between RNA decay and plant hormone
response we have analyzed Arabidopsis lsm1 mutant, which similarly to sad1 shows growth sensitivity to ABA. As
lsm1 transcriptome profiles revealed accumulation of some mRNAs encoding core components of ABA signaling
we have tested mRNA level for chosen genes, including PYL/PYR/RCAR ABA receptors, PP2C protein phosphatases
and SnRK2 kinases. Notably, PYR1 and PYL5 transcripts were upregulated, with PYR1 mRNA stabilized in lsm1
plants, which shows that it is a direct subtrate of the LSM1 decay pathway. Consistently, the activity of ABA-dependent SnRK2 kinases were enhanced, and mRNA level of some ABA-induced and SnRK2-regulated PP2C phosphatases were upregulated in this mutant. mRNAs changed in cytoplasmic lsm mutants partially overlap with substrates
of the cytoplasmic 5N-3N exonuclease AtXRN4, the decapping enzyme AtDCP2 as well as NMD effectors AtUPF1 and
AtUPF2. In addition, mutants in these factors are sensitive to ABA. We have therefore tested the level of ABA
receptor mRNAs and it transpired that in upf1 and dcp5 mutants, but not in xrn4, PYR1 and PYL5 behave alike as
in lsm1 plants. Also, SnRK2 kinase activity is increased in the absence of AtUPF1. These results indicate that LSM1
complex, and possibly other components of the cytoplasmic mRNA decay pathway, contribute to the regulation of
stress- and hormone-related processes via their role in mRNA metabolism.
Poster 20
Alternative splicing events in two maize lines
under herbicide stress conditions
J. GRACZ1, A. TYCZEWSKA1, A. HOFFA2, A. ZMIENKO1, A. SWIERCZ1,2, J. BLAZEWICZ1,2, T. TWARDOWSKI1
1
2
Institute of Computing Science, Poznan University of Technology, Poznan, Poland
Plants, as sessile organisms, must adapt their growth and metabolic style to a changing environment. Splicing
is one of the mechanisms which play an important role in plant adaptation and is an additional element of fitness benefit adjusted to the limited capacity of genome size. Studies of splicing and its role in diverse aspect of cell biology,
pathology and stress response, has remained undescribed for many plant species, including maize. Through the
mechanism of alternative splicing, exons from primary transcripts (pre-mRNA) with multiple introns may undergo
ligation in many different ways generating multiple proteins from single gene. This process can affect mRNA stability
and translation efficiency as well as activity, cellular localization, regulation and stability of coding protein. For better
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characterization of alternative splicing role in plant herbicide stress response, we sequenced transcriptomes of two
maize breed lines – sensitive and tolerant to herbicide RoundUp. We used Illumina next-generation sequencer
Genome Analyzer IIx and we conducted pair-end sequencing. As a result we obtained 35 to 76 mln 50nt reads per
sample. Using bioinformatics tools such as BowTie, TopHat, Cufflinks, Cuffdiff and CummRbund we managed to
identify between sensitive and tolerant maize line. We also managed to identify different types of splicing events with
java script. Funding acknowledgement: Ministry of Science and Higher Education 3098/B/P01/2010/39; National
Science Center DEC-2011/01/N/NZ9/02900
Poster 21
Transcriptional gene silencing induced by VIGS-BSMV system
in rye for functional analysis of ScBx1
J. GROSZYK1, M. RAKOCZY-TROJANOWSKA2, W. ORCZYK1
1
2
Department of Plant Genetics, Breeding, and Biotechnology, Warsaw University of Life Sciences, Warsaw, Poland
Benzoxazinoids (BXs) are defensive compounds of secondary metabolism that have been found in several species
of Poaceae, including the major agricultural crops maize (Zea mays ), wheat (Triticum aestivum ) and rye (Secale cereale) and wild barley (Hordeum lechleri ). The compounds are considered as important factors involved in allelophatic interactions and biotic and abiotic stress tolerance. The most important BX synthesized in rye is DIBOA
(2,4-dihydroxy-1,4-benzoxazinon-3-one). The compound is the final product of benzoxazinoid biosynthetic pathway
catalyzed by enzymes encoded by Bx1 ÷ Bx5 genes. The goal of this project was to experimentally verify the biological function of ScBx1 gene encoding indole-3-glycerol phosphate lyase, probably the first specific enzyme of
BIBOA biosynthesis in rye. The experimental VIGS (virus induced gene silencing) system was selected for functional
analysis of this gene. The genomic sequence of the analyzed ScBx1 gene including the sequence of promoter region
was obtained as a result of this project. Selected fragments of promoter regions were cloned into cDNA of modified
BSMV (Barley stripe mosaic virus ) β and γ units. The resultant plasmids pT7_BSMV: α, pT7_BSMV: β_prom-ScBx1
and pT7_BSMV: γ_prom-ScBx1 were used as the templates for in vitro transcription. The mixture of α, β_ promScBx1 and γ_prom-ScBx1 transcripts were used for inoculation of rye seedlings cultivar Konto F1. Leaves with
symptoms of BSMV infection were collected 14, 21 and 99 days post inoculation (dpi) and used for: i) analysis of
ScBx1 transcript level, ii) detection of CG methylation in promoter region selected and cloned into VIGS vectors
and iii) analysis of total amount of DIBOA. The analysis of gene expression and CG methylation of target region was
compared with control plants i.e. the plants inoculated with the ‘empty’ BSMV: α, BSMV: β and BSMV: γ RNA
mixture. ScBx1 transcript level in experimental plants ranged from 0.03 to 0.09 14dpi, from 0.14 to 1.33 21dpi and
from 0.02 to 0.67 99dpi compared with transcript level in control plants. CG methylation of target ScBx1 promoter
in plants with lowered expression was from 1.31% to 1.96% 14 dpi, from 3.06% to 34.87% 21 dpi and from 12.16%
to 33.92% 99 dpi. CG methylation of corresponding region in control plants was 1.55% and it was similar in all six
control plants in three (14, 21, 99 dpi) experimental time-points. The results indicate that VIGS-BSMV system can
be used as efficient experimental tool for directed CG methylation of selected genomic DNA regions. We conclude
the observed lowered transcript level was the result of methylation-induced transcriptional silencing. Total DIBOA
content and its correlation with ScBx1 transcript level will be investigated. The research has been financed by The
National Centre for Research and Development grant nr PBS1/A8/12/2012.
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Poster 22
Evidence for alternative splicing mechanism for meadow
fescue (Festuca pratensis) and perennial ryegrass
(Lolium perenne) RcaA gene
B. JURCZYK, M. RAPACZ
Department of Plant Physiology, Faculty of Agriculture and Economics, University of Agriculture in Krakow, Cracow, Poland
RuBisCO ACTIVASE (RCA) catalyzes the activation of RuBisCO. In several plant species two RCA isoforms are
evidenced as an effect of alternative splicing of pre-mRNA. On the other hand two isoforms may be also products
of separate genes. The aim of the study was to confirm, that two isoform of RCA in Lolium perenne and Festuca pratensis are products of alternative splicing of RCAa gene. 3N RACE (rapid amplification of cDNA ends) has been performed for meadow fescue and perennial ryegrass RCAa. In both species PCR products of about 1200 bp was obtained and sequenced after isolation from agarose gels. As confirmed by ClustalW alignment, this fragment represented
the majority of the coding sequence of RCAa. A very high homology in coding sequence has been observed between
Lolium perenne and Festuca pratensis. PCR primers flanking the orthologous gene region, where splicing has been
observed in barley, were designed. Two PCR products differing 48 bp in length were evidenced. Both products was
isolated from agarose gel and sequenced to confirm that they represent two splicing variants of RCAa. The insertion
contains an early stop codon, in the same position as observed in barley RCAa2 mRNA. The presence of two RCA
isoforms was additionally confirmed by Western Blot.
Poster 23
Apple miRNAs and their role in fire blight resistance
E. KAJA1, T. MCNELLIS2, M. SZCZESNIAK1, I. MAKALOWSKA1
1
Laboratory of Bioinformatics, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
2
Department of Plant Pathology, Penn State University, Pennsylvania, USA
Micro RNAs (miRNAs) are small, single stranded RNA molecules, which are involved in post-transcriptional gene
silencing in plant and animal cells. To date, it has been reported that plant miRNAs, by targeting many regulatory
genes, play an important role in such processes as: plant development, hormone signaling or biotic and abiotic stress
response. Although, many interesting facts have already been discovered about miRNA nature and way of action,
those molecules are still surprising and not fully understood. In this research we characterized miRNAs, which are
specific for Gala apple scions grafted on four different rootstocks: B.9, G.30, M.27 and M.111, presenting diverse
fire blight resistance. Our previous studies (Jensen et al., 2009) showed, that those rootstocks also induce a different
gene expression pattern in the apple scion as well as they determine tree size. Although the mechanism of this regulation is not known yet, we suggest that miRNAs might play a crucial role in it. In order to identify miRNA species,
as well as their expression levels in selected trees, SOLiD sequencing of small RNAs has been performed. All the
reads have been mapped to the apple genome (http://www.rosaceae.org/projects/apple_genome) and searched for
conserved and apple-specific miRNAs. Performed analyses allowed us to extend the apple miRNA repertoire by 38
conserved and 78 novel, apple specific, miRNA as well as verify 143 miRNAs from previous studies. We confirmed
five of new miRNAs using qPCR or RT-PCR. We also identified miRNAs with significantly changed expression among
analyzed rootstocks. In addition, we searched for potential miRNA targets using psRNATarget focusing on transcripts with significantly higher expression in fire blight resistant trees.
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Poster session
Poster 24
Links between F-box proteins and ARGONAUTE1 N domain
S. KAUSIKA, P. BRODERSEN
School of Biology, University of Copenhagen, Copenhagen, Denmark
ARGONAUTE (AGO) proteins are the core components of RNA induced silencing complex (RISC) that cause
post-transcriptional gene silencing (PTGS) guided by small RNAs. Four domains, the N, PAZ, MID and Piwi are common to all AGOs, and have key functions in small RNA binding and target mRNA repression. Our interest is in the
function of the poorly understood N domain. It has been shown that membrane association of AGO is important for
function and our previous data shows that a mutation in the N domain of Arabidopsis AGO1 alleviates membrane
association. In this study, we used the N domain of AGO1 as bait in a yeast two-hybrid screen to identify interactors.
We found two F-box proteins and a ubiquitin-like protein as candidates. F-box proteins are core components of the
Skp1-Cullin1-Fbox (SCF)-type E3 ubiquitin ligases that recognize specific target substrates and catalyze their ubiquitination. The two AGO1-interacting F-box proteins require two distinct set of amino acids in AGO1, potentially identifying important interaction sites in AGO1. Point mutants in amino acid residues predicted to be surface-exposed at
these sites led to severe phenotypes, suggesting that these sites are crucial for AGO1 function in planta. Knockout
of either F-box protein led to appreciable increases in AGO1 protein levels, suggesting that the F-box proteins could
be involved in of AGO1 in regulated proteolysis of AGO1 in planta.
Poster 25
Can we design PPR proteins to bind user-defined RNA targets?
P. KINDGREN, A. YAP, I. SMALL
Plant Energy Biology, Australian Research Council Centre of Excellence, University of Western Australia, Crawley, Australia
Pentatricopeptide repeat (PPR) proteins bind RNA and determine a wide range of RNA processing events required to prepare plant organellar RNA for translation, such as stability, splicing and editing. PPR motifs are capable
of sequence-specific interaction with the target RNA via interactions involving 2-3 amino acids in each motif that
recognise individual nucleotides in the RNA target. The aim of this project is to obtain the knowledge needed to
design and construct proteins capable of binding user-defined RNA sequences. By systematically mutating the RNAinteracting amino acids in CLB19, which edits rpoA and clpP in plastids, and the nucleotides in its target RNAs, we
have confirmed the exact binding sites and the residues that determine target specificity. We have shown that it is
possible to predictably alter the binding preferences of RNA editing factors in vitro and in vivo. To investigate if it
is possible to make more drastic changes, synthetic proteins have been created where the PPR tract from another
editing factor, YS1, have replaced the PPR tract of CLB19. The specific role of individual PPR motifs along a tandem
PPR tract and their involvement in RNA recognition will be discussed.
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Poster session
Poster 26
The role of small RNAs in the tomato root response to cyst
nematode infection
M.D. KOTER1, M. SWIECICKA1, A. PACAK2, M. FILIPECKI1
1
2
Department of Plant Genetics Breeding and Biotechnology, Warsaw University of Life Sciences, Warsaw, Poland
Plant cyst nematodes (PCN) infect roots and induce formation of multinuclear syncytium, a specialized structure
becoming a sole food source for developing larvae and adults. The formation of syncytium is accompanied with active
suppression of defense response as well as substantial reprogramming of development and metabolism of incorporated cells. Such process are likely to engage different mechanisms of gene expression regulation including those mediated by small RNAs. Since down-regulation of genes upon plant parasitic nematode infection is a common phenomenon concerning a third part of regulated genes we decided to purify and sequence the sRNA fraction of infected
root transcriptome. The tomato roots were grown in vitro and infected with PCN. Root fragments with syncytia were
collected and RNA was isolated and fractionated. The indexed sRNA libraries were sequenced using Illumina MiSeq
genome sequencer. Resulting sequences were analyzed using UEA sWorbench 3.1 and potential target genes were
identified using psRNATarget server. The results show many changes in composition of sRNA profiles. 54 known
miRNAs have been found with 6 of them manifesting over 2-fold induction/suppression as compared to control samples. Their predicted target genes in tomato genome are transcription factors (GRAS, MYB, NAM), proteins involved
in signal transduction (glucose/ribitol dehydrogenase, phosphatidate cytidyltransferase) and LRR receptor-like serine/threonine-protein kinase. Substantial portion of target candidates are likely to participate in other stress responses. Several miRNA homologues identified, such as mir1446, mir164 and mir399 are up-regulated in infected tissues
showing the potential mechanism of plant response suppression by cyst nematodes.
Poster 27
Heat stress-regulated microRNAs in barley
K. KRUSZKA1, A. PACAK1, A. SWIDA-BARTECZKA1, P. NUC1,
S. ALABA2, W. KARLOWSKI2, A. JARMOLOWSKI1, Z. SZWEYKOWSKA-KULINSKA1,2
1
2
Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
Heat stress is one of the major abiotic factors that can induce severe plant damages leading to a decrease in crop
plant productivity. Despite barley being a cereal of great economic importance, little data is available concerning its
thermotolerance mechanisms. Here we investigated miRNAs involved in heat stress response in barley. Using
northern hybridization we found out that four mature miRNAs: miR160a, 166a, 167h and 5157a were up-regulated
under heat stress in barley. Our studies also revealed that the level of their pri-miRNAs was affected under the heat
stress conditions. Surprisingly, the splicing of the intron-containing precursors pri-miR160a and pri-miR5175a was
induced by heat, suggesting the post-trancriptional regulation of miRNA precursor processing. Furthermore, we
experimentally identified conserved (HD-Zip transcription factors and auxin response factors) as well as novel target
genes (HOX9, ACC oxidase and Nek5-like kinase ) of the heat-responsive barley miRNAs using the degradome
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Poster session
analysis and 5NRACE approach. The observed induction of the mature miRNAs expression was correlated with the
down-regulation of the expression level of their corresponding target genes. The identified target genes of barley
heat-regulated miRNAs are involved in the regulation of a leaf morphology and polarity, shoot morphology, flower
development, adventitious root formation, microtubule function and ethylene biosynthesis. These morphological and
physiological features were often reported to be affected under heat stress conditions leading to a significant
decrease in plant height and biomass reduction or the arrest of the root elongation and branching that frequently
resulted in a decrease of crop yield. Our findings showed that barley miRNAs, together with target genes function
in a complex regulatory network that barley plants developed to cope with stressful conditions. This work was
supported by POLAPGEN-BD project no. UDA.POIG.01.03.01-00-101/08 “Biotechnological tools for breeding cereals
with increased resistance to drought”, subject 20: “The role of microRNA in regulation of mechanisms leading to
drought adaptation in plants”, executed within Innovative Economy Programme 2007-2013, subject “Biological
progress in agriculture and environment protection”.
Poster 28
Transcriptomic and proteomic study of seeds priming
and post-priming germination emphasize the importance
of mRNA translation regulation and post-translational
processing in priming-induced improvement
of seeds germination
S. KUBALA1, M. QUINET2, L. WOJTYLA1, A. KOSMALA3, S. LUTTS2, M. GARNCZARSKA1
1
2
3
Department of Plant Physiology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
Groupe de Recherche en Physiologie Vegetale (GRPV), Earth and Life Institute – Agronomy (ELI-A),
Universite catholique de Louvain, Louvain-la-Neuve, Belgium
Department of Environmental Stress Biology, Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland
Osmopriming is a pre-sowing treatment that exposes seeds to a low external water potential that allows partial
hydration but prevents germination. Priming improves seed germination performance as well as stress tolerance
of germinating seeds and seedlings. In this work, rape (Brassica napus L.) seeds were osmoprimed with !1.2 MPa
polyethylene glycol (PEG 6000) for 7 days. A global expression profiling method was used to compare transcriptomic
and proteomic data for osmoprimed seeds at the crucial phases of priming procedure (soaking, drying), whole
priming process and subsequent germination. Total number of 952 genes and 75 proteins were affected during the
main phases of priming and post-priming germination. In general, more genes were up-regulated during osmopriming
treatment and post-priming germination. Regarding the different phases of priming, most of genes down-regulated
during PEG soaking were affected in opposite way during drying. There were more proteins which abundance decreased in response to PEG soaking and seed drying while most of the proteins analysed during complete osmopriming and post-priming germination showed an increase of their abundance during these processes. Progress towards
germination in primed seeds was associated with an increase in protein synthesis potential, post-translational processing capacity and targeted proteolysis. Higher expression of genes involved in regulation of transcription, water
transport, cell wall modification, cytoskeletal organization and cell division was also linked to the advanced germination of primed seeds. Moreover, improved germination of primed seeds was associated with higher genes expression and abundance of proteins involved in the management of oxidative stress during post-priming germination. The
differences between transcriptome and proteome data set (the match between genes and proteins was limited to
only 12 gene-protein pairs) reinforce the importance of the regulation of mRNA translation and post-translational
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Poster session
processing during priming and post-priming germination. This weak correspondence between mRNA levels and
protein abundance is due to the existence of complex post-transcriptional processes such as transcript de/stabilization, translation, posttranslational modifications and protein degradation which determine and modulate the
quality and quantity of expressed proteins. This work was supported by grant no. 2011/03/B/NZ9/00068 from the
National Science Centre given to MG. SK obtained financial support for the best PhD students in PO KL 8.2.2.
program editions: 2011/2012 and 2012/2013 and from the National Science Center DEC-2013/08/T/NZ9/01019.
SK is a scholarship holder of The Adam Mickiewicz University Foundation in Poznan in 2013/2014.
Poster 29
Participation of genes encoding biosynthesis
and metabolism compound of jasmonates in flower
morphogenesis of Ipomoea nil
A. KUCKO1, 2, E. WILMOWICZ1,2, K. FRANKOWSKI1, K. MARCINIAK1,2, J. KESY1,2, J. KOPCEWICZ1
1
2
Chair of Plant Physiology and Biotechnology, Nicolaus Copernicus University, Torun, Poland
Centre for modern interdisciplinary technologies, Nicolaus Copernicus University, Torun, Poland
Jasmonates, with representative jasmonic acid (JA), are lipid-derived phytohormones with diverse functions, ranging from the initiation of biotic and abiotic stress responses, to the regulation of plant growth and development.
It has been shown that bioactivity of jasmonates is not restricted to the free acid, but also some of its precursors and
derivatives are activators of plant responses. JA biosynthesis originates from LIPOXYGENASE (LOX) - catalyzed
oxidation of polyunsaturated fatty acids in chloroplast, which are subsequently converted by ALLENE OXIDE SYNTHASE (AOS) and ALLENE OXIDE CYCLASE (AOC) to 12-oxophytodienoic acid. Cyclopentanone ring structure
of jasmonates is established by OPDA REDUCTASE (OPDR) activity in peroxisome. Cytosol is the compartment
of JA biochemical diversification, e.g. formation of predominant amino acid conjugate with isoleucine (JA-Ile) catalyzed by JAR (JASMONATE RESISTANT) or MeJA (JA-methyl ester) synthesis performed by JMT (JA CARBOXYL
METHYLTRANSFERASE), which has been recognized as highly active in bioassays for JA activity. In this study we
reported the expression profile of enzymes involved in biosynthesis (InLOX, InAOS, InOPR3 ) and metabolism
(InJMT, InJAR ) of JA in vegetative (roots, hypocotyls, cotyledons, apexes and leaves) and generative (stamens, pistils
and petals) organs of Ipomoea nil. Experiments have been performed by Real-Time PCR (qRT-PCR) with genespecific primers and UPL probes, with actin as a reference endogenous control. InLOX transcript accumulation was
greater in generative than in vegetative tissues. There was no significant difference between InAOS and InOPR3.
The highest transcripts level of these genes in hypocotyls and petals was observed. The activity of genes involved
in metabolism of JA in cotyledons, hypocotyls and leaves was similar. InJMT strongly accumulated in petals, whereas
InJAR in pistils, almost at all stages of their development. The level of mRNA InLOX and InOPR3 increased during
pistils and stamens development. We suggest that that differential expression of JAs biosynthesis and metabolism
genes during vegetative and generative development, allows plants to maintain specific JAs homeostasis, necessary
for proper plant development.
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Poster session
Poster 30
Model expansion: gene expression under binary metal
treatment, hydroponics vs. peat pots
A. KUTROWSKA, A. MALECKA, A. PIECHALAK, B. TOMASZEWSKA
Department of Biochemistry, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
Trace metals present in the environment in excess cause adverse effects on plant yield and biomass quality. Impact of the elements depends on the soil pH, its redox state, organic content, ion capacity and many other factors.
Despite this, most of the fundamental research on metal treatment performed in the laboratories is limited to the
semi-ideal environment created with the use of hydroponics or perlite foundation. This approach, although burdened
with many disadvantages, presents unique opportunity to isolate and closely examine interesting phenomena. In our
study we aimed to enrich the standard model of metal uptake by examining the simultaneous treatments with binary
metal combinations in a hydroponic culture (with Hoagland solution). We then performed additional experiments
replicating our approach but on the commercially available peat pots instead of hydroponic medium. Our model involved the use of 3-weeks-old seedlings of Indian mustard (Brassica juncea ), one of the known metal hyperaccumulators. Careful examination of different metal treatments resulted in the following final metal concentrations: for individual treatments of Cu2+, Zn2+, Cd2+, Pb2+ – 50 μM of each metal in hydroponics and 100 μM for the peat pots; for
binary combinations (CuZn, CuPb, CuCd, CdPb, ZnCd, ZnPb) – 25 μM of each metal in hydroponics and 50 μM of
each metal for the peat pots. Double concentration of metals was used for the peat pots due to the lowered metal
mobility in this medium. Additionally, plants showed similar levels of stress (measured by the antioxidant enzyme
activity and reactive oxygen generation, among other) on these separate mediums, when treated with selected concentrations. For the gene expression analysis, we decided to measure the short response to metal stress, thus limiting ourselves to following time points: 0N, 4N, 8N hours of metal treatment for roots and 0N, 8N, 24N hours for the
aboveground tissues. Relative levels of transcription for genes encoding superoxide dismutase (Mn-SOD, Cu,ZnSOD ), gamma-glutamylcysteine synthetase (γ-ECS ) and glutathione reductase (GR ) were determined with the use
of RT-PCR and qPCR. Generally, gene expression was induced mostly by the binary combinations. Genes encoding
antioxidant genes (SOD) responded to a lower extent to metals than the genes encoding detoxicative enzymes (ECS,
GR). We observed different patterns of gene expression on hydroponics and peat pots, suggesting that though the
plant stress symptoms on the higher level (RFT, antioxidants) were similar for these media, gene expression levels
could not have been easily translated. This work was partially supported by NCN grant no 3811/ B/P01/2010/38.
Poster 31
In vitro biochemical characterization
of Arabidopsis thaliana DXO1 protein
A. KWASNIK, K. STEPNIAK, A. GOZDEK, J. KUFEL
Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland
The correct formation of mRNA 5N end is a crucial component of the regulation of gene expression and is therefore subjected to surveillance mechanisms that detect molecules containing aberrant 5N end structures and target
these dysfunctional transcripts for degradation. Yeast and human members of the RAI1/DXO1 family were recently
shown to participate in these processes as they exhibit phosphodiesterase (PPE) activity to remove incomplete,
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Poster session
unmethylated caps and may additionally act as pirophosphohydrolase (PPH) or 5N-3N exoribonuclease towards uncapped mRNAs containing 5N end triphosphates and monophosphates, respectively. Here we describe the in vitro biochemical activity of a putative RAI1/DXO1 homologue from Arabidopsis thaliana. This protein displays high conservation of active site amino acid sequence with other RAI1/DXO1 proteins, but it additionally contains unique
N-terminal unstructured domain that may affect its biochemical properties. The fact that Arabidopsis dxo1 insertion
mutant lines show severe growth inhibition, sterility and several defects in molecular phenotypes underlines the
importance of this protein for RNA metabolism in plants. We performed a series of in vitro assays using purified
AtDXO1 and its catalytically inactive or N-terminally truncated variants to test for the PPE, PPH and 5N-3N exoribonuclease activities towards oligoribonucleotide substrates containing methylated or unmetylated cap, as well as triphosphate or monophosphate group at their 5N ends. These experiments demonstrated that AtDXO1 exhibited all
three activities, with enzymatic properties somewhat different than those of human DXO. Our data suggest that
AtDXO1 activities are probably employed to remove abnormal cap structures and degrade improperly capped or
uncapped RNA molecules.
Poster 32
Calcium-dependent phosphoproteins
– a multiprotein hub for stress signal transduction
A. LUDWIKOW1, F. MITULA1, M. TAJDEL1, A. CIESLA1,2, L.H. MISZTAL 1, J. SADOWSKI 1,2
1
Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
2
Institute of Plant Genetics of the Polish Academy of Sciences, Poznan, Poland
Calcium-dependent protein kinases (CPKs) comprise a large family of structurally conserved serine/threonine
kinases involved multiple biological processes. A key challenge in CPK research is to understand the structural basis
for activation of CPK. Here, we implemented advanced proteomic approaches to identify and analyze the droughtinduced CPKs in Hordeum vulgare. To facilitate the analysis we raised antibodies against active site of kinase domain
and used them to immunoprecipitate protein complexes containing CPKs from drought tolerant and drought sensitive barley genotypes. Subsequent LC-MS/MS analysis allowed identification of two putative barley CPKs involved
in regulation of drought stress response in stress tolerant genotype. Interestingly, identified putative barley CPK
shows substantial similarity to Arabidopsis CPK17 and CPK34. To further investigate the function of CPKs an initial
insight into cellular localization was achieved. In the Arabidopsis and barley protoplasts HvCPK fused to the GFP
reporter was targeted to the plasma membrane localization. To test mechanism of HvCPKs activation we purified
recombinant HvCPKs and analyzed kinase activity using in vitro and in gel assay methods. Together, we show evidence for the role of barley CPKs in the regulation of drought stress response.
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Poster session
Poster 33
High-throughput sequencing identification of novel
and conserved miRNAs in the Brassica oleracea leaves
A. LUKASIK1, H. PIETRYKOWSKA2, L. PACZEK1,3, Z. SZWEYKOWSKA-KULINSKA2, P. ZIELENKIEWICZ1,4
1
Institute of Biophysics and Biochemistry, Polish Academy of Sciences, Warsaw, Poland
2
3
Department of Immunology, Transplant Medicine and Internal Medicine, Medical University of Warsaw, Warsaw, Poland
4
Department of Plant Molecular Biology, Institute of Experimental Plant Biology and Biotechnology,
University of Warsaw, Warsaw, Poland
Plant microRNAs are short (-21 nt) non-coding molecules that regulate gene expression by targeting the mRNA
cleavage or protein translation inhibition. In this manner, they play many important roles in the cells of living organisms. One of the plant species in which the entire set of miRNAs has not been yet completely identified is Brassica
oleracea var. capitata (cabbage). For this reason and for the economic and nutritional importance of this food crop,
high-throughput small RNAs sequencing has been performed to discover the novel and conserved miRNAs in mature
cabbage leaves. In this study, raw reads generated from three small RNA libraries were bioinformatically processed
and further analyzed to select sequences homologous to known B. oleracea and other plant miRNAs. As a result of
this analysis, 261 conserved miRNAs (belonging to 62 families) have been discovered. MIR169, MIR167 and MIR166
were the largest miRNA families, while the highest abundance molecules were miR167, miR166, miR168c and
miR157a. Among the generated sequencing reads, miRNAs* were also found, such as the miR162c*, miR160a* and
miR157a*. The unannotated tags were used in the prediction and evaluation of novel miRNAs, which resulted in the
26 potential miRNAs proposal. The expressions of 13 selected miRNAs were analyzed by northern blot hybridization.
The target prediction and annotation for identified miRNAs were performed, according to which discovered molecules may target mRNAs encoding several potential proteins – e.g., transcription factors, polypeptides that regulate
hormone stimuli and abiotic stress response, and molecules participating in transport and cell communication.
Additionally, KEGG maps analysis suggested that the miRNAs in cabbage are involved in important processing pathways, including glycolysis, glycerolipid metabolism, flavonoid biosynthesis and oxidative phosphorylation. Conclusively, for the first time, the large set of miRNAs was identified in mature cabbage leaves. Potential targets designation
for these miRNAs may suggest their essential role in many plants primary biological processes. Presented study not
only supplements the knowledge about B. oleracea miRNAs, but additionally it may be used in other research concerning the improvement of the cabbage cultivation.
Poster 34
smRNAome sequencing to identify conserved
and novel microRNAs in Stevia rebaudiana Bertoni
VIBHA MANDHAN, KASHMIR SINGH
Department of Biotechnology, Panjab University, Chandigarh, India
MicroRNAs (miRNAs) constitute a family of small RNA (sRNA) species that regulates the gene expression and
plays an important role in plant development, metabolism, signal transduction and stress response. Extensive studies
of miRNAs have been performed in different plants such as Arabidopsis thaliana, Oryza sativa and volume of the
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Poster session
miRNA database, mirBASE has been increasing on day to day basis. Stevia rebaudiana Bertoni is an important perennial herb which accumulates high concentrations of diterpene steviol glycosides which contributes to its high indexed
sweetening property with no calorific value. Several studies has been carried out on understanding molecular mechanism of biosynthesis of these glycosides, however, information about miRNAs has been lacking in S. rebaudiana.
Deep sequencing of small RNAs combined with transcriptomic data is a powerful tool for identifying conserved and
novel miRNAs irrespective of availability of genome sequence data.To indentify miRNAs in S. rebaudiana, sRNA library was constructed and sequenced using Illumina genome analyzer II. A total of 30,472,534 reads representing 2509190
distinct sequences were obtained from sRNA library. Based on sequence similarity, we identified 100 miRNAs belonging
to 34 highly conserved families. Also, we identified 12 novel miRNAs whose precursors were potentially generated
from stevia EST and nucleotide sequences. All novel sequences have not been earlier described in other plant species. Putative target genes were predicted for most conserved and novel miRNAs. The predicted targets are mainly
mRNA encoding enzymes regulating essential plant metabolic and signaling pathways. This study led to the identification of 34 highly conserved miRNA families and 12 novel potential miRNAs indicating that specific miRNAs exist
in stevia species.Our results provided information on stevia miRNAs and their targets building a foundation for future
studies to understand their roles in key stevia traits.
Poster 35
LlGAMyb gene expression profile in generative organs
of yellow lupine (Lupinus luteus L.)
K. MARCINIAK, P. GLAZINSKA, E. WILMOWICZ, A. KUCKO, W. WOJCIECHOWSKI, M. BANACH, J. KOPCEWICZ
GAMyb, a positive regulator involved in the GA signalling pathway, has been known to act as an important downstream component in the degradation of DELLA proteins. GAMyb gene was first identified in barley (Hordeum vulgare ) aleurone cells, where its expression is upregulated by GA treatment. The synthesized protein can bind specifically to GA-response elements in promoter regions of an α-amylase gene and many other GA-regulated genes, for
example SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 ) and LFY (LEAFY ).Therefore, the
GAMyb has been demonstrated to play an important role in such processes as seed and flower development. In the
first step of our work, the GAMyb homologue in yellow lupine (Lupinus luteus L.) cultivar Taper was identified (Marciniak et al., 2013). As a consequence, the transcriptional activity of LlGAMyb at different development stages of
inflorescence, flowers and pods was determined using RT-qPCR technique. During the growth of the whole inflorescence (8 phases), a decrease of mRNA level by about five times between the first and last phase was observed.
In turn, in the six individual flower whorls of fully mature inflorescence were no significant differences in the content
of the LlGAMyb transcripts. At the later stage of flowers withering and pods forming, rapid increase in the transcriptional activity of investigated gene was recorded, especially in the two upper whorls of all six. In plants with fully
mature pods in each whorl (3-6), we observed a similar level of gene expression as in plants with fully developed flowers. During single flower development slight upward tendency of mRNA accumulation was observed, whereas
during single pod development (from 1 to 6 seeds) was downward tendency. The results indicate the specific expression pattern of the studied gene in different generative organs of Lupinus luteus L. Due to the highest level of transcriptional activity in the examined variants, the LlGAMyb gene may be involved mainly in the early stage of pod
development. It should be added, that presented results are an introduction to the extensive research that in the
near future will determine the precise mechanism of the flower and pod development. This in turn can lead to improved yields of yellow lupine. This work was supported by the Ministry of Agriculture and Rural Development of
Poland Grant No 149/2011.
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Poster session
Poster 36
Natural variation of transposon silencing
in Arabidopsis thaliana is caused by transposition
and regulation by MUR1
T. MEYER, I. BAURLE
The silencing of DNA transposons and retroelements is essential for all eukaryotic organisms to maintain genome stability and integrity and suppress their mutagenic potential. Otherwise active mobile elements can destroy
functional genes by insertion, alter gene expression or cause a rapid growth of the genome size. A large number of
interacting pathways, including DNA methylation, small RNA-based silencing and histone modifications have evolved
to ensure transposon silencing. While it is accepted that transposon activity over millions of years has contributed
to the evolution of species, it is currently unclear how much variation in transposon activity exists at a smaller timescale, for example on an intraspecific level. As a model case for this question, we have chosen to study the DNA
transposon AtMu1 of Arabidopsis thaliana, which is characterized by an over 100fold expression difference between
two studied ecotypes. Using eQTL mapping we were able to identify the genomic region responsible for this large
expression difference. A single trans-QTL on the upper arm of chromosome 1, narrowed to a 100 kb interval by fine
mapping, is responsible for the variation. Within this interval, we identified a new copy of AtMu1 inserted in the
3NUTR of a protein coding gene. Furthermore, in the mapped region a previously uncharacterized gene (MUR1 )
was found, which has a role in AtMu1 activation. Taken together, our work identifies the genetic basis for natural
variation in the silencing of a mutagenic DNA transposon and it also demonstrates the existence of host factors that
positively regulate transposon expression.
Poster 37
Novel algorithm for designing plant artificial miRNA
A. MICKIEWICZ1, A. RYBARCZYK1,2, A. HOJKA-OSINSKA1,
P. JACKOWIAK1, J. BLAZEWICZ1,2, M. FIGLEROWICZ1,2
1
2
MicroRNAs (miRNA) are small non-coding RNAs found in most eukaryotic organisms. They are involved in posttranscriptional control of gene expression in a sequence specific manner. The miRNAs are generated from endogenous transcripts synthesized mainly by RNA polymerase II. These precursors contain stem-loop structures, which
harbor a ca. 21-nucleotide long miRNA. There are differences in the miRNA biogenesis and mode of function in
plants and animals. Animal miRNA precursors are about 80 nt and two different proteins are involved in their processing. The first step occurs in the nucleus and the second yielding miRNA/miRNA* duplex occurs in the cytoplasm.
Plant miRNA precursors are significantly longer, up to 600 nt, display structural diversity and are digested to
miRNA/miRNA* by a nuclear DICER-LIKE 1 protein. Both in animals and plants, the miRNA (guide strand) is loaded
onto the ARGONAUTE protein component of the RNA-induced silencing complex while the miRNA* is destroyed.
The miRNA then directs the cleavage of mRNA or represses translation. Animal miRNAs harbor “seed” region: 2-8
nucleotides of 5 ends complementary to ca. 7 nt of target. Plant miRNAs usually display perfect or near-perfect pai-
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107
ring with their mRNA target sites. In animals, miRNA target sites are located mostly within 3Nuntranslated regions
whereas plant miRNAs bind both to the coding and untranslated regions. The main concept of the artificial miRNA
(amiRNA) is to design a 21 nt RNA molecule that is able to regulate the target gene expression and is incorporated
into the backbone that forms a fold similar to miRNA precursor structures. At present, there are few bioinformatics
tools capable of designing amiRNA. One of the mostly used tools is Web MicroRNA Designer (WMD3), which is the
only one dedicated to plants. Recently developed tools, miR-Synth or AmiRzyn, are designed preferentially for human
amiRNA. In this work we present a new approach to design plant amiRNA. Our method is based on the analysis of
the decomposed free energy profiles of known miRNA/miRNA* and miRNA/target interactions. These thermodynamic profiles serve as templates for amiRNA design. The major advantage of AmiRNA Designer, as compared to other
tools, is a possibility to introduce mismatches within miRNA/miRNA* and amiRNA/target duplexes. This allows designing the regulatory RNA for targets whose expression cannot be regulated by amiRNA obtained with the currently
available tools. The use of artificial miRNA is a promising technique for functional genetic studies, cultivable plant
improvement, as well as for plant antiviral protection. Thus, it is very probable that this algorithm will turn out to
be powerful and broadly applied. Acknowledgments: This work was partially financed by the Polish Ministry of Science and Higher Education grant number IP2012 014972, to PJ, and by grant No. 2012/05/B/ST6/03026 from the
National Science Centre, Poland.
Poster 38
RNApathwaysDB – a database of RNA maturation and decay
K. MILANOWSKA1,2, K. MIKOLAJCZAK2, A. LUKASIK2, M. SKORUPSKI2,
Z. BALCER2, M.A. MIKA1, K.M. ROTHER1,2, J.M. BUJNICKI1,2
1
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology in Warsaw, Warsaw, Poland
2
Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
Many RNA molecules undergo complex maturation, involving e.g. excision from the primary transcripts, posttranscriptional modification, splicing, and polyadenylation. The level of mature RNAs in the cell is controlled by degradation, which proceeds via many different reactions including, but not limited to endo- and exonucleolytic cleavage. The systematization of data about RNA metabolic pathways and enzymes taking part in RNA maturation and
degradation is essential for the full understanding these processes. RNApathwaysDB (RNA pathways database) is
the first database of metabolic pathways involving RNA as the substrate. It presents information about reactions and
enzymes (proteins, RNA molecules or complexes) that take part in RNA processing. The database provides also links
to other databases and literature information. The current dataset is limited to the maturation and degradation of
tRNA, rRNA and mRNA, and describes pathways in three model organisms: Escherichia coli, Saccharomyces
cerevisiae and Homo sapiens. Other RNAs, enzymes and pathways and data for other organisms will be successively
added in the future. The database can be queried with keywords or by the name of a pathway, a reaction, an enzymatic complex, a protein or an RNA molecule. Amino acid sequences of protein enzymes involved in pathways included in RNApathwaysDB can be compared to a user-defined query sequence with a BLAST utility. Options for data
presentation include graphs of pathways and tabular forms with enzymes and literature data. Structures of macromolecular complexes are presented as “potato models” using DrawBioPath – a new javascript tool. The contents of
RNApathwaysDB can be accessed through the World Wide Web at http://genesilico.pl/rnapathwaysdb.
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Poster session
Poster 39
Effects of sugars as endogenous signals
and Fusarium oxysporum on expression level of isoflavonoids
biosynthesis pathway genes in yellow lupine
I. MORKUNAS1, M. FORMELA1, D. NAROZNA2, W. NOWAK3
1
2
3
Department of Plant Physiology, Poznan University of Life Sciences, Poznan, Poland
Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Poznan, Poland
Laboratory of Molecular Biology Techniques, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
Sugars not only function as substrates for growth of sink tissues, which are dependent on the import of carbohydrates, but also affect sugar-sensing systems that initiate changes in genes expression. These genes encode proteins that function in many metabolic pathways and developmental programs. Therefore, sugar-induced signaling
pathways will interact with many other signaling pathways to form regulatory webs that allow the integrated response
to changing environmental conditions, including invasion of pathogens. The aim of the present study was to examine
effects of sucrose and monosaccharides (glucose and fructose) as endogenous signals, and a hemibiotrophic fungus
Fusarium oxysporum f.sp. lupini on the expression of genes of flavonoid biosynthetic pathway. Real-time PCR analyses of the level of mRNA encoding enzymes involved in the synthesis of isoflavones, revealed post-infection accumulation of mRNA in embryo axes of yellow lupine cv. Juno. In embryo axes infected with F. oxysporum cultured
in vitro on the medium with sucrose, glucose and fructose (+Si, +Gi, +Fi) the level of mRNA for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS) was higher
than in non-infected axes (+Sn, +Gn, +Fn) in the period from 0 to 96 h. Moreover, up to 72 h after inoculation
in inoculated axes with a sugar deficit (-Si) a higher level of mRNA encoding PAL, CHS, CHI and IFS was observed
post infection in relation to non-inoculated axes (-Sn). At the next time point after inoculation, i.e. at 96 h in infected
axes cultured at carbohydrate deficit (-Si) a very strong reduction was recorded in the mRNA level, while at 96 h
in infected axes with a high level of carbohydrates (+Si, +Gi and +Fi) the level of mRNA encoding the above
mentioned enzymes was highest. It needs to be stressed that a very high post-infection level of mRNA was recorded
for enzymes of the specific isoflavone synthesis pathway, i.e. chalcone synthase (CHS) and isoflavone synthase (IFS)
in embryo axes inoculated with F. oxysporum, being much higher than for PAL and CHI. These results suggested
that the sensing of carbohydrate levels and the response to pathogen can be interrelated at some level. This study
was supported by the Polish Ministry of Science and Higher Education (MNiSW, grant no. N N303 414437).
Poster 40
RNAi in practice – PTGS silencing in functional studies
of agronomically important cereal genes
A. NADOLSKA-ORCZYK1, S. GASPARIS1, W. ZALEWSKI1, Y. YANUSHEVSKA2, W. ORCZYK2
1
2
Biotechnology offers a range of tools for functional analysis and for improvement of selected traits such as yield,
product quality, resistance to biotic and tolerance to environmental stresses. One of the most promising experimental tools, especially useful for research on allopolyploid cereals, is RNAi-based gene silencing. We proved its
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Poster session
applicability by silencing three groups of genes: PINa / PINb in wheat and their orthologs in triticale and, in other
line of experiments, HvCKX1 / HvCKX2 and HvGSK in barley. The PIN genes determine grain hardness in wheat,
which is important technological trait. Silencing of the genes in wheat resulted in reduction of the transcript exceeded 90% what caused significant reduction or lack of both puroindoline proteins and increased grain hardness
up to the level of T. turgidum var. durum cultivar, which lacks of the genes (Gasparis et al., 2011). Unexpectedly
silencing of SINa and SINb genes in triticale however resulted in significant reduction of transcripts and secaloindoline proteins, did not prove their role in grain hardness (Gasparis et al., 2013). The second group of silenced genes belongs to the HvCKX family of barley. They encode cytokinin dehydrogenase enzymes (CKX), which regulate
cytokinin level in different tissues of developing plants. The detailed functions of the genes are not known. We have
already documented that silencing of the HvCKX1, which expression was the highest in the roots and developing
spikes of wild plants, decreased cytokinin dehydrogenase level in these tissues. This leaded to higher plant productivity expressed as the yield, the number of seeds per plant and the 1000 grain weight and greater mass of the roots
(Zalewski et al., 2010). Silencing of HvCKX2 (Zalewski et al. 2012), which expression was the highest in the developing spikes as well as in the young and fully developed leaves of wild plants, decreased transcript and CKX level
in these tissues. There was a positive correlation between the low level of transcript in spikes 7 DAP and the higher
plant productivity. The data of productivity of modified lines up to T3 generation will be presented. Current project,
and unpublished results, is focused on identification and functional analysis of putative barley homologs of
brassinosteroid regulators OsGSK1 in rice and BIN2 / AtSK2 in Arabidopsis. Depressing transcript level of the
tested gene to 0.09 – 0.22 of the transcript in control plants revealed the clear correlation with elevated salt tolerance of the seedlings. The research has been financed by grants: N302 013 31/1517, 620/N-COST/09/2010, UMO2011/03/B/NZ9/01383.
Poster 41
Retention of poly(A) RNA in the cell nucleus
of roots subjected to the hypoxia treatment
J. NIEDOJADLO, B. KALICH, E. KUBICKA
Flooding of agricultural lands is one of the biggest problems of modern agriculture, because it causes the reduction in yields. The reason for that is the reduced availability of oxygen (hypoxia) for the root system of plants. In this
report, distribution and quantity of active RNA polymerase II, poly (A), SR and SUMO proteins were investigated
in Lupinus luteus root cell nuclei under natural and stress conditions. The stress was induced by flooding the seedlings in water, which imitates the conditions during the flood. After 3 and 6 h of hypoxia treatment, a significant
increase in the level of poly (A) in the cell nuclei was observed. In the cytoplasm, stress granule-like poly (A) RNA
clusters were also noticed. Since the presence of poly(A) RNA in Cajal bodies (CBs) (Smolinski and Kolowerzo 2012)
has recently been demonstrated, the signal level in these structures was also measured. It appeared that in the hypoxia-treated cells, the growth of poly (A) in CBs is even higher than in the whole nucleus. Measurements of the
quantity of active RNA polymerase II and distribution of SR proteins revealed a strong termination of transcription
during consecutive hours of hypoxia treatment. It can be concluded, that the observed increase in poly (A) is a result
of the strong retention in the of nucleus, including Cajal bodies, and not transcription process intensification. One
also checked, whether poly(A) RNA accumulated in the nucleus, includes RNA coding proteins. Recently, a relationship between SUMO1proteins accumulation and the retention of poly (A) RNA in the nucleus under two abiotic
stress treatments, heat and ethanol treatment respectively, has been shown (Muthuswamy and Meier 2011). Our
studies have demonstrated changes in the nuclear distribution of SUMO1 protein in control cells and hypoxia-treated
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Poster session
cells. In addition, during the initial period of hypoxia, SUMO1 protein was accumulated in Cajal bodies. Our preliminary results indicate retention of poly (A) RNA as an important mechanism of gene expression transcribed via
RNA polymerase II in hypoxia-treated cells. The obtained results also suggest the involvement of Cajal bodies in the
retention of poly (A) RNA in plant cells.
Poster 42
Barley microRNA 444.1 expression is regulated by alternative
splicing and affects barley tillering upon heat stress
A. PACAK1, K. KRUSZKA1, A. SWIDA-BARTECZKA1,
W. KARLOWSKI2, A. JARMOLOWSKI1, Z. SZWEYKOWSKA-KULINSKA1,2
1
2
Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
MicroRNAs are key molecules regulating gene expression. Barley microRNAs are encoded by the genes with
diverse organization, representing mostly independent transcriptional units with or without introns. The intron
– containing miRNA transcripts undergo complex splicing events to generate various spliced isoforms. There are
three genes encoding different species of barley microRNA444 (MIR444.1, MIR444.2 and MIR444.3 ). All of them
contain long introns that separate microRNA* from microRNA. Functional pre-miRNA444 can be formed only after
intron removal. MiRNA 444.1, 444.2 and 444.3 differ from each other by one or two nucleotide substitutions. Recently we have tested barley response to different abiotic stresses and found that heat stress induces significantly
expression of miR444. By Northern blotting and small RNA NGS analyses we identified the MIR444.1 as a heatinduced gene. Transcript of MIR 444.1 gene undergoes complex alternative splicing events that generate three RNA
isoforms: i) fully spliced, functional pri-miR444.1 isoform, ii) alternatively spliced non-functional isoform in which
the exon encoding miR444.1* is removed, and iii) non-functional spliced isoform in which the whole region containing exons encoding miR444.1* and miR444.1 together with the intron separating these exons is spliced out. We
found that upon heat stress conditions the general level of pri-miR444.1 transcript as well as the level of the functional, spliced pri-miRNA isoform were significantly elevated compared to the control conditions. We observed also an
interplay between all spliced isoforms of pri-miR444.1 leading to the increase of the functional pri-miRNA 444.1
isoform when compared to the control conditions. Interestingly, the MIR444.1 and its target gene – transcription
factor belonging to the MADS-box type II gene family are encoded within the same locus at the opposite DNA
strands. Barley degradome analysis showed that indeed MADS.1 is cleaved by microRNA444.1. Exons forming spliced MADS-box TF are found almost exclusively within the introns of the MIR444.1 gene. Thus spliced pri-miRNA
miR444.1 isoforms and the MADS-box TF spliced mRNA represent different in the nucleotide sequence molecules
having only a short, about 62 nt long complementary region encompassing binding sites between the miRNA 444.1
and mRNA target site. Accordingly to the elevated level of the mature miR444.1 during the heat stress, we observed
dramatic decrease of its target mRNA. We found that plant growth is affected upon heat-stress. We postulate that
the miR444.1 increase during the plant response to the heat-stress is responsible for barley tillering inhibition via
MADS.1 downregulation. To validate the involvement of MADS.1 an tillering inhibition we use VIGS approach (Virus
Induced Gene Silencing). Using in vitro transcribed BSMV (Barley Stripe Mosaic Virus) RNAs: RNAα, RNAβ and
modified RNAγ-containing MADS.1 fragment in sense orientation, we silence MADS.1 to observe tillering phenotype. The work was supported by the European Regional Development Fund through the Innovative Economy for
Poland 2007-2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD “Biotechnological tools for breeding
cereals with increased resistance to drought”.
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Poster session
Poster 43
Protein interacting with post-translationally modified
H3 histone in Arabidopsis thaliana
A. PALUSINSKI1, M. KOTLINSKI1,2, T. RUBEL3, D. BUSZEWICZ2,
J. OLEDZKI4, M. DADLEZ4, A. JERZMANOWSKI1,2, M. KOBLOWSKA1,2
1
2
Laboratory of Systems Biology, University of Warsaw, Warsaw, Poland
Laboratory of Plant Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
3
4
Institute of Radioelectronics, Warsaw University of Technology, Warsaw, Poland
Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
The post-translational modifications of histones are among the major mechanisms modulating chromatin
structure. These modifications are mainly located in N-and C-terminal unstructured domains of core histones (called
histone tails), extending beyond the core of nucleosome. The best known modification include methylation of lysine
(K) and arginine (R), acetylation of lysine and phosphorylation of serine (S) and threonine (T). One of the most important functions of these modifications is mediating the recruitment of specific nonhistone proteins to chromatin
loci. However, presence of post-translational histones modifications may function not only by the recruitment of
these proteins, but also act to prevent their binding. Histone-bounding proteins are crucial in the reorganization of
chromatin and regulation of numerous processes occurring at the DNA level. Therefore, understanding the roles
of histone modifications is of primary importance for the study of gene regulation at the chromatin level. Relatively
large numbers of animal proteins that associate with post-translationally modified histones is known. However, data
on such interactions in plants is extremely poor. Our studies aim to identify proteins that bind to modified core histone H3 in Arabidopsis thaliana. To this end, we adapted for the first time for plant material a technique of peptide
pull-down that has been used earlier for analogous experiments in animals. This assay allows to search for protein
partners interacting in vitro with chemically synthesized peptides that are analogous to core histone tails carrying
selected posttranslational modifications. So far, using mass spectrometry technology (Orbitrap Velos or Orbitrap
Elite), we successfully identified several proteins binding to modified core histone in Arabidopsis thaliana.
Poster 44
U12 intron in A. thaliana CBP20 gene is necessary
for correct pre-mRNA splicing and mRNA/protein level
M. PIECZYNSKI1, D. BIELEWICZ1, J. DOLATA1, M. SZCZESNIAK2,
A. WYRZYKOWSKA1, W. KARLOWSKI2, A. JARMOLOWSKI1, Z. SZWEYKOWSKA-KULINSKA1
1
2
Laboratory of Bioinformatics, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
Eucaryotic RNA polymerase II transcripts are characterized by the presence of the cap structure and polyA tail at
their 5N and 3N ends, respectively. Cap binding complex (CBC) is a nuclear complex composed of two CAP-BINDING
PROTEINS: CBP20 and CBP80. It is known that binding of the CBC to the 5N cap is crucial for the proper mRNA maturation and transport. We show that the CBP20 gene structure is highly conserved across land plants from liverwort
higher plants. The gene contains always seven introns with the fourth intron belonging to U12 class. Additionally
the U12 intron divides the gene in two parts: one that encodes the core domain containing RNA recognition motif
(RRM) and the second one that encodes the tail domain containing nuclear localization signal (NLS). In all investi-
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Poster session
gated plants CBP20 genes first four exons coding the core domain have always the same length whereas exons
coding the terminal domain differ considerably in length. To answer the question why the presence and location of
the U12 intron in the CBP20 gene is preserved across all plant species we prepared constructs representing CBP20
mini-genes and its mutated full versions. Mini-gene constructs containing 4th and 5th exons from A. thaliana CBP20
gene and U12 introns derived from different plants and differing in length (from 134nt to 2733nt) were transfected
to tabaccum mesophyll protoplasts and splicing was analysed. Our results show that the longer the U12 intron the
more efficient splicing was observed. Additionally, transcripts splicing analyses of the mini-gene construct containing
a U2 intron in the U12 intron natural position revealed that 37% of mRNAs undergo alternative splicing. Additionally
we prepared five constructs containing full A. thaliana CBP20 gene in which i) the U12 intron was replaced by a U2
one, ii) the U12 intron was removed, iii) exons flanking the U12 intron were exchanged with each other, and iv-v)
the U12 intron was moved to different locations within the gene body. These constructs were introduced into
A. thaliana cbp20 T-DNA insertion mutant. Our results show that transcripts derived from the mutated CBP20 gene
constructs (in which the U12 intron was moved to the other gene locations) generate additional isoforms containing
up to 58% of alternatively 5N spliced U2 intron, which was placed in U12 intron natural position. Simultaneously the
U12 intron in different locations within CBP20 gene body was correct and efficient spliced. Additionally we found
that plants containing mutated CBP20 gene in which the U12 intron was replaced with a U2 one show strong downregulation of the CBP20 mRNA and protein levels. All these data show that the U12 intron in the proper position
in A. thaliana CBP20 genes is necessary for correct pre-mRNA splicing and mRNA/protein level.
Poster 45
The first liverwort microtranscriptome: Liverworts share
molecular traits exclusive for green algae and other land plants
P. PISZCZALKA1, S. ALABA2, H. PIETRYKOWSKA1, P. PLEWKA1,
I. SIEROCKA1, KASHMIR SINGH3, W. KARLOWSKI2, Z. SZWEYKOWSKA-KULINSKA1,2
1
2
3
Department of Biotechnology, Panjab University, Chandigarh, India
MicroRNAs are key regulatory elements of eukaryotic gene expression. These short (18-24 nt) molecules act
post-transcriptionally by sequence-specific guidance of RNA Induced Silencing Complex (RISC) to complementary
mRNAs which results in slicing or translation inhibition of targeted mRNAs. Because of its importance in gene
expression regulation, miRNAs are a field of intensive research and much is known for model plants or plants with
great economic importance. Yet, until now there are no publicly available data on any liverwort microtranscriptome.
Liverworts represent the most the basal group of land invaders and are the evolutionary oldest land plants present
in our days. We applied t high-throughput sequencing technique (SOLEXA, Illumina) and sequenced small RNAs,
transcriptome, and degradome from the dioecious liverwort Pellia endiviifolia species B. 311 miRNA families of conservative miRNA species that are identical to model moss Physcomitrella patens and/or other land plants miRNAs
were identified. Surprisingly, our analysis reve1ed in Pellia endiviifolia the presence of 3 miRNA families identified
exclusively in the green algae C. reinhardtii. We confirmed the presence of selected C. reinhardtii miRNAs by northern hybridization using RNA isolated from axenically grown P. endiviifolia plants. Also, the presence of selected
conservative miRNAs with homologs in higher land plants identified in P. endiviifolia was confirmed by northern
hybridization. With the use of bioinformatic approaches we studied also novel Pellia endiviifolia miRNA candidates
which have not been previously described. Using northern hybridization we evidenced the presence of 41-21 nt long
stable, small RNAs, which represent novel, unique liverwort miRNAs. Analysis of P. endiviifolia transcriptome re-
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Poster session
vealed the presence of at least twenty miRNA putative precursors. Ten of them were verified using experimental
approaches like RACE and genome walking resulting in establishing the gene structure of the first known liverwort
MIR genes and their primary transcripts. Four of identified MIR genes contain one or more introns. Interestingly,
in all intron containing MIR genes the miRNA and its stem-and-loop sequence are located in last exon. Degradome
sequencing revealed mRNA targets for 38 conservative miRNAs and 13 novel miRNAs. Targets for 3 miRNAs
(miR160, miR166, and miR408) were evolutionary conserved while the remaining mRNAs represent new, previously
unknown targets. Our results are the first to characterise liverwort microtranscriptome. This new data will supplement our knowledge and understanding of plant miRNA evolution and represent an interesting example of research
case for other scientists.
Poster 46
Identification of molecular factors involved in biogenesis
of tRNA-derived fragments (tRFs) in plants using
next-generation sequencing
P. PLEWKA1, M. SZYMANSKI2, A. PACAK1, Z. SZWEYKOWSKA-KULINSKA1, A. JARMOLOWSKI1, W. KARLOWSKI2
1
2
Laboratory of Computational Genomics, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland
The mechanism of negative regulation of gene expression by small non-coding RNAs (sRNAs) is among the most
studied subjects in molecular biology. The sRNAs act by silencing of gene expression at both transcriptional and
post-transcriptional levels, and they have been reported to play an essential role in plant growth and development
as well as are recognized as important players in responses to environmental stresses. Besides extensively studied
microRNAs and siRNAs, other small functional RNAs have been identified in various organisms by using highthroughput sequencing techniques. Recent reports indicate that abundant, non-coding RNAs, like rRNA, tRNA and
snoRNA may, aside of their canonical functions, be a source of small regulatory RNAs. Current knowledge about
biogenesis and function of these molecules in plants is poor, however studies on human and bacterial cells point out
at their putative negative regulatory action at the gene expression level, mainly through translation inhibition. Considering a great regulatory potential of small RNAs, we decided to study the biogenesis of small RNAs derived from
tRNAs, called tRFs from tRNA-derived fragments. They represent a newly discovered and interestingly the most
conservative class of small RNAs (they have been identified in all three domains of life). The tRFs of 14-26 nucleotides in length have long been regarded as random byproducts of tRNA biogenesis or degradation process, but nowadays there are more and more evidence proving their stability and putative regulatory capabilities. Results of highthroughput sequencing experiments demonstrate that tRFs are not only generated through specific cleavage pattern
that predominantly favors either the 3N or 5N end but also preferentially maintained in the cells. This asymmetric accumulation and defined length of tRFs molecules reflect characteristic features of microRNAs and siRNAs. What is
more, experiments carried out on animal cells show that these molecules can be generated by the main endonuclease
of miRNA maturation pathway – Dicer or alternatively, by RNase Z and RNase P which are responsible for maturation of the 3N and 5N-end of tRNA molecules, respectively. However, there is no experimental data concerning the
mechanism of generation of tRNA fragments in plants. In order to perform comprehensive, whole-genome studies
on biogenesis of tRFs in Arabidopsis thaliana, we selected almost 50 mutants disrupted in biogenesis and degradation of microRNAs, siRNA and tRNA transcripts, assuming that sRNAs resulting from tRNA cleavages can represent
byproducts of tRNA maturation and/or degradation machinery, or alternatively, they can be processed by the factors
involved in siRNA/microRNA biogenesis. Until now, 22 representative Arabidopsis mutants were subjected to RNA
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Poster session
deep sequencing. Detailed bioinformatic analysis of obtained results has led to identification of almost 6300 unique
sequences that map to 725 annotated genes encoding tRNA molecules and show differential expression in mutants
in comparison to wild type plants. This work was supported by grant from the National Science Center
2011/03/B/NZ2/01416.
Poster 47
Analysis of GASA genes in germinating tomato seeds
Solanum lycopersicum cv. Moneymaker
W.E. PLUSKOTA, J. PERKOWSKA, K. GLOWACKA, M.M. JASTRZEBSKA,
M. PIKULINSKI, P. PUPEL, S.B. OKORSKA, R.J. GORECKI
Department of Plant Physiology, Genetics and Biotechnology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
Gibberellin (GA) stimulated transcript (GAST ) family genes, encoding a group of small cysteine rich proteins,
has been reported in petunia (Petunia hybrida ), potato (Solanum tuberosum ), rice (Oryza sativa ), maize (Zea mays )
and Arabidopsis (Arabidopsis thaliana ). GAST proteins also known as GASA (Arabidopsis ), GASR (rice) or SNAKIN
(potato) proteins contain 18 - 29 amino acid residues, a highly divergent intermediate region with polar amino acid
residues, and a conserved 60 amino acid C-terminal domain, named as GASA domain, containing 12 conserved cysteine residues. Those proteins are involved in plant development, plant responses to biotic and abiotic stress and
hormone crosstalk and redox homeostasis. Microarray data indicates that two member of SlGASA gene family are
expressed in the micropylar region of germinating tomato seeds. Nucleotide sequences of array probes show the
highest similarities with GAST1, first GA-stimulated transcript cloned from tomato and GASA4-like gene from rose,
respectively. Base on the literature some GASA proteins can promote and the others inhibit seed germination.
Alignment of cloned cDNA and genomic nucleotide sequences reveal the structure of SlGASA6, the new member
of GASA gene family from tomato. Predicted amino acids sequences of GASA6 was compared with amino acid
sequences of GASA proteins available in GenBank. The temporal and spatial expressions of both SlGASA (GAST1
and its new homolog GASA6 ) genes in germinating tomato seeds are presented. Changes in transcript level of
mRNA GAST1 and cloned GASA6 as well as expressions of genes involved in biosynthesis of GA in dissected tomato
seeds are shown. Moreover the 5N upstream region of the GASA6 gene was cloned and its activity was studied
in transgenic Arabidopsis thaliana seeds using a green fluorescent protein and/or β-glucuronidase reporter gene.
Additionally the potential cis-regulatory elements contained in 5Nupstream region of SlGASA6 are described.
Poster 48
Transcriptomic insights into SCL30a,
an Arabidopsis SR protein involved in salt stress tolerance
during seed germination
D.N. RICHARDSON, P. DUQUE
Instituto Gulbenkian de Ciencia, Oeiras, Portugal
Despite the influx of new evidence in the animal world suggesting myriad roles for SR genes/proteins in diverse
biological processes such as transcription, alternative splicing, gene and transposon silencing and response to biotic
or abiotic stresses, little functional evidence exists for linking this key post-transcriptional regulatory mechanism
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to plant stress tolerance. As SR genes typically have critically important roles in transcription and splicing, it is expected that their misregulation 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 concentrations. At the phenotypic level, 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 an intact ABA pathway. Aside from
these changes, we observed several differentially expressed genes (1667 in the SCL30a knockout and 434 in the
overexpressor). Among these differentially expressed genes, 35 were associated to the term, “response to abscisic
acid stimulus” in the knockout, whereas 13 were associated thusly in the overexpressor, of which 11 were common
to the 35 in the knockout. Broad level gene ontology analysis using DAVID highlighted several biological processes
correlated with the observed germination phenotypes, such as embryonic development, dormancy process, protein
biogenesis, transport/localization and cell growth. Several differentially expressed genes were also associated with
photosynthesis. Of the differentially expressed genes in the SCL30a knockout, the most up-regulated (22-fold) gene
is a copia-like retrotransposon, leading us to suspect a role for SCL30a in transposon silencing. As SCL30a is a splicing regulator, our next step will be to analyze global changes in alternative splicing in the SCL30a misregulation
lines using these RNA-seq data. Comparative analysis of alternative splicing with the list of differentially expressed
genes should allow for a narrowing down of potential target genes for experimental validation to garner functional
evidence for SCL30a’s role in salt stress tolerance in Arabidopsis.
Poster 49
RNA-seq reveals global differences in gene expression
between male and female gametophytes producing sex organs
in dioecious liverwort Pellia endiviifolia sp B
I. SIEROCKA1, S. ALABA2, W. KARLOWSKI2, Z. SZWEYKOWSKA-KULINSKA1,2
1
2
Laboratory of Computational Genomics, Institute of Molecular Biology and Biotechnology, Faculty of Biology,
Regulation of gene expression plays a pivotal role in controlling all aspects of multicellular organisms development, including sexual reproduction. In flowering plants a number of genes has been identified which control the
transition from vegetative to generative phase of life cycle. Among liverworts, the most basal lineage of bryophytes,
there is almost no data about the genes and mechanisms controlling this transition. This fact puts liverworts in critical evolutionary position to investigate the genetic basis of key innovations which allowed them to survive in demanding terrestrial environment and to give fertile offspring. We have chosen Pellia endiviifolia species B, a dioecious
liverwort from class Jungermaniopsida to profile the differences in transcripts level between different stages of the
male and female thalli development. We applied the next generation sequencing technology to identify genes engaged in the antheridia and archegonia production in P. endiviifolia. RNA-seq was performed using four different developmental stages: the male thalli i) producing or ii) without antheridia, the female thalli iii) producing or iv) without
archegonia. For each library over 40 mln reads were generated which were mapped to the reference de novo transcriptome sequencing data of P. endiviifolia. To select genes with the highest differences in expression between the
male and female thalli producing/not producing sex organs bioinformatics analyses were performed with criterion
log2_fold_change $ 10. As a result 72 Differentially expressed genes (DEGs) were selected. Out of 10 genes up-regulated in sperm-producing male thalli, 8 are also expressed in the vegetative phase of males thalli. In turn, out of 62
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Poster session
up-regulated genes in archegonia-producing female thalli, 46 are also expressed in the vegetative phase of females
growth. To verify the differentially expressed genes selected from the RNA-seq, real-time PCR analysis was performed which validated 9 male and 47 female specifically expressed genes. All verified DEGs were analyzed by blast
search to classify their gene function. The most enriched DEGs belong to RNA or DNA binding protein families,
serine/threonine-protein phosphatases, LRR receptor-like kinases and ubiquitin protein ligases. 24 DEGs showed
no similarity to known proteins or nucleotide sequences, and the lengths of these transcripts reach from -250 to
-600 nt with no putative open reading frames. It cannot be excluded that these transcripts represent non-coding
RNAs or represent partial sequences of untraslated regions of original mRNA molecules what needs to be further
investigated. Our studies provide possibility to learn about the gene expression regulation within the representative
of genus Pellia, which is recognized as the one of the most basal lineage of the simple thalloid liverworts. The obtained results are one of first steps to understand the processes that trigger the development of liverworts from vegetative to generative stage of life. The work was supported by the Foundation for Polish Science, grant number
POMOST/2012-5/7.
Poster 50
Regulation of environmental stress memory in Arabidopsis
through the AGO1-miR156-SPL module
A. STIEF, S. ALTMANN, K. HOFFMANN, B. DATT PANT, W.-R. SCHEIBLE, I. BAEURLE
Max Planck Institute for Molecular Plant Physiology, Potsdam, Germany
Plants have developed coping strategies for reproductive success and survival under stressful conditions. The
responses directly following abiotic stress such as high temperatures have been well characterized. In contrast, the
mechanisms allowing adequate reaction to recurring environmental stress are poorly understood, despite their
importance for organisms in their natural surroundings. We present molecular and physiological evidence for the
involvement of ARGONAUTE1 and the microRNA pathway in the adaptation to recurring heat stress, an active
maintenance of acquired tolerance which we call heat stress memory. We show that besides AGO1, DCL1 and SUO
are required for heat stress memory. Different miRNAs are induced by high temperatures in Arabidopsis seedlings,
and elevated expression is maintained for several days. The miR156 family, and especially isoform miR156h, displays
a particularly interesting expression profile of high induction and long maintenance, and we can show its functional
requirement by manipulating the levels of mature miR156. Depletion of miR156 leads to higher susceptibility towards recurring heat stress, while its overexpression improves memory. Using heat-inducible constructs we can
show that miR156 is only required after the heat stress. Interestingly, increased levels of miR156h clearly prolong
the sustained expression of heat stress memory genes (such as HSFA2, HSA32, and other HSPs ). We furthermore
show that repression of SPL genes, prominent targets of the miR156 family regulating the juvenile-to-adult phase
transition, is required for heat stress memory. We demonstrate that SPL genes are post-transcriptionally downregulated after heat, and this repression is a direct effect of miR156, as miR156 -resistant SPL transcripts are not
downregulated equivalently. Seedlings expressing rSPL transcripts are more susceptible to recurring heat stress,
showing a level of damage similar to that seen in miR156 knockdown lines. Reduced heat stress memory in rSPL
plants corresponds to reduced maintenance of heat stress memory genes, arguing for a role of SPL proteins as
transcriptional repressors of these memory genes. Altogether, our analyses demonstrate a yet unknown, central role
of the miR156-SPL module in the integration of development and environmental stress.
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Poster 51
Different pattern of H2A.Z distribution in Arabidopsis genes
W. SURA1, M. KUS3, L. PAWELOSZEK1, W. KARLOWSKI2, J. SADOWSKI1, P.A. ZIOLKOWSKI1
1
2
Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poland
Laboratory of Computational Genomics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poland
3
The role of histone variant H2A.Z in the control of gene expression has been reported in a wide variety of eukaryotes. Moreover, its amino acid sequence is highly conserved and its presence is essential for viability in many species. Although it is known that H2A.Z-dependent regulation is based on transcription initiation and the variant occupies mainly gene transcription start sites (TSSs), its mode of action is still unclear. Possible models include modification of nucleosome stability or position, as well as recruitment of other factors to TSS. To investigate correlation
between changes in presence of H2A.Z in chromatin and changes in gene transcription levels in Arabidopsis thaliana
we studied its nucleosomal distribution at the whole-genome scale. We performed ChAP-seq (chromatin-affinity purification followed by high-throughput sequencing) and RNA-seq analyses of plants submitted to drought stress in comparison to control plants. Our results indicate that there are several different patterns of H2A.Z distribution along
gene bodies. The most common pattern with distinctive 5N-end peak is typical for constitutive genes, while genes
involved in stress response show more complex distribution of H2A.Z. It may suggest that this histone variant regulates expression of different genes in several ways. While we are still comparing H2A.Z distribution in genes which
are either induced or repressed under drought stress, our initial analysis shows that the H2A.Z pattern is relatively
constant and not subjected to alterations upon transcription. This may indicate that the main mode of H2A.Z action
is based on attraction of additional regulation proteins, likely transcription factors, to regions enriched in H2A.Z-containing nucleosomes along gene bodies.
Poster 52
AtCCR4s degrade the poly(A) tail of GBSS1 mRNA
which is responsible for amylose synthesis
YUYA SUZUKI1, P.J. GREEN2, JUNJI YAMAGUCHI1,3, YUKAKO CHIBA1,3,4
1
2
Graduate School of Life Science, Hokkaido University, Sapporo, Japan
Delaware Biotechnology Institute, University of Delaware, Delaware, USA
3
4
Faculty of Science, Hokkaido University, Sapporo, Japan
Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Japan
Gene expression is tightly regulated during plant growth and development as well as in response to internal or
external environmental changes. Most studies regarding control of gene expression have largely focused on transcriptional regulations. However, steady-state levels of mRNA are in fact determined by the balance between mRNA
synthesis and degradation. Removal of poly(A) tail, deadenylation, is the first and rate-limiting step of mRNA degradation and apparently an effective step not only for controlling mRNA stability, but also for translation in many eukaryotic transcripts. CARBON CATABOLITE REPRESSOR 4 (CCR4) has been identified as a major cytoplasmic deadenylase
in yeast. The Arabidopsis homologs of yeast CCR4, AtCCR4a and AtCCR4b, have been identified by the sequence-based analysis; however their functions and physiological significance for plants remain to be elucidated. In this study,
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we showed that FLAG-tagged AtCCR4a or AtCCR4b exhibited poly(A) specific degrading activity in vitro. Transient
expression analysis using the GFP fusion of AtCCR4a or AtCCR4b indicated that both were localized in P-bodies,
which are specific granules in the cytoplasm consisting of many enzymes involved in mRNA turnover. To understand
the functional significance of AtCCR4a and AtCCR4b in vivo, we took an advantage of reverse genetics strategy. The
double mutant of AtCCR4a and AtCCR4b showed the insensitivity to a high level of sucrose. Levels of sucrose in the
seedlings of the double mutants were reduced, whereas no difference was observed in the glucose level. In addition,
we revealed that the amylose fraction in the double mutants was slightly higher than in the control plants. Furthermore, the poly(A) length of the transcripts encoding GRANULE BOUND STARCH SYNTHASE 1 (GBSS1), which is the
key enzyme for amylose synthesis, was longer in the double mutants. This result indicated that GBSS1 transcript
is the target of AtCCR4a and AtCCR4b. Our results presented here suggest the regulation of amylose contents via
deadenylation of GBSS1 transcripts by AtCCR4a and AtCCR4b.
Poster 53
miR172 is involved in control of somatic embryogenesis
induced in vitro in Arabidopsis
A. SZCZYGIEL-SOMMER, M. GRZYB, K. SZYRAJEW, K. NOWAK, M.D. GAJ
In plants, microRNAs were indicated to play a main role in regulation of many aspects of development and responses to the environment. Thus, the involvement of miRNAs in control of somatic embryogenesis (SE), a developmental process induced in vitro in somatic cells, can be assumed. In support, the dcl1 mutant defective in DICER
LIKE1 (DCL1) activity required for miRNAs biogenesis, was found totally unable for the SE-induction. Moreover,
numerous MIRNA genes were found differentially expressed during SE-induction stage, including MIR172c and
MIR172d to be highly up-regulated (100- and 31-folds, respectively) (Szyrajew, 2012a,b). To further verify the
involvement of miR172 in SE the relation between MIR172 expression level and explant capacity for SE was evaluated. To this end, the embryogenic potential of cultures derived from a transgenic line overexpressing MIR172d
and three insertional mutants (mir172b, mir172c and miR172d ) were analysed. It was found that all the analyzed
genotypes were impaired in capacity for SE and displayed a significantly reduced number of responding explants
and/or somatic embryos produced per explant. In planta, MIR172 genes are involved in regulation of flowering time
and floral organ identity in Arabidopsis and among their targets a small group of APETALA2 (AP2)-like transcription
factor genes including AP2, TOE1, TOE2, TOE3, SNZ and SMZ were indicated (Aukerman and Sakai, 2003). The
present results of qRT-PCR analysis revealed that two of miR172 targets, AP2 and SMZ, are down-regulated during
SE suggesting their role as the negative regulators of SE induction in Arabidopsis. In support, auxin treatment used
to induce SE in cultured explants was found to significantly modulate expression of AP2 and SMZ. In contrast to
AP2 and SMZ, expression profiles of TOE genes in embryogenic culture have not indicated their regulatory relation
with miR172 during SE. However, a significant and auxin-dependent stimulation of TOE1 and TOE2 in SE together
with a significantly decreased embryogenic response of toe1 mutant suggest that these genes can positively control
embryogenic transition in somatic cells of Arabidopsis. Further analysis are needed to reveal other genes of
regulatory pathway operating during SE induction in which miR172 and its targets in planta (AP2, SMZ, TOE1,
TOE2 ) seem to be involved.
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Poster 54
At4g25290 has at least eight different splicing variants
O. SZTATELMAN, J. LABUZ, A.K. BANAS
Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
UVB is known to seriously harm living cells. Beside causing oxidative damage of proteins, lipids and nucleic acids
it can also induce dimer formation between adjacent pyrimidines in a DNA strand. Such photoproducts may be both
mutagenic and cytotoxic. They are removed via highly efficient light repair pathway by photolyases (photoreactivation
repair) and/or relatively inefficient dark repair mechanisms (excision repair pathways). Up to date, three Arabidopsis
genes have been shown to have photolyase activity: i) At1G12370 (PHR1 photolyase1, UVR2 – UV resistance2);
ii) At3G15620 (UVR3 ); iii) At5G24850 (CRY3 cryptochrome3). Two others, PHR2 and At4g25290 have been classified as photolyases based on in silico analysis. The protein encoded by At4g25290 has two domains, a photolyase
at the N-terminus and a hydrolase at the C-terminus. When we used mRNA isolated from different plant organs as
a template for the PCR reaction with At4g25290 specific primers we had found eight splicing variants of this gene.
The steady state mRNA levels of most of these forms were strongly up-regulated by light. Several of the splicing
variants identified contained premature STOP codon and subsequently encoded a shorter protein having only
a photolyase domain. Interestingly, due to ASIP database (http://www.plantgdb.org/tmp/ASIP/) one of the splicing
forms can have alternative start of translation, located in the alternatively spliced area. Three other forms also
contain long ORFs starting at different position in the alternatively spliced region. In all these cases proteins having
only a hydrolase domain would be produced. Transcripts with premature translation-termination codons may undergo
nonsense mediated mRNA decay (NMD). Thus, we have used Arabidopsis mutants of the NMD pathway (upf1-5,
upf3-1 and upf3-2 ) to address a possible role of alternative splicing in the regulation of At4g25290 mRNA level. No
differences in mRNA levels of one of the splicing variant as well as the pattern of its light up-regulation between wild
type and the mutants have been observed. Acknowledgements: The study was supported by Polish National Science
Centre, a grant no. UMO-2011/03/D/NZ3/00210
Poster 55
Whole genome computational analysis of small RNA fragments
derived from tRNAs in response to abiotic stress
A. THOMPSON1, M. SZYMANSKI1, A. JARMOLOWSKI2, W.M. KARLOWSKI1
1
Laboratory of Computational Genomics / Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology,
2
Department of Gene Expression, Institute of Molecular Biology and Biotechnology,
Small RNA (sRNA) play an important role in regulation of gene expression, and as a consequence are essential
for proper function of living cells. RNA silencing has been recognized for its critical role in development, stress response and housekeeping. Due to increasing amount of data from Next Generation Sequencing, the research community is continuously adding to the ever-expanding pool of newly identified sRNAs that potentially may contribute to
the regulatory landscape of gene expression. One such recently discovered group are fragments derived from transfer RNA (tRNA) genes. Transfer RNAs are well-characterized, conserved components of the translational machinery
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with lengths varying between 73 and 90 nucleotides and having a characteristic secondary and tertiary structure.
Recent results demonstrate that their transcripts undergo processing to form fragments ranging between 19 and
26 nucleotides in length and are known to accumulate during stress conditions. In order to evaluate the regulatory
potential of tRNA fragments we have selected a model plant Arabidopsis thaliana. We performed a genome-wide analysis and predicted approximately one hundred previously unclassified tRNA candidates for a total of seven hundred
and twenty-five tRNA positions. The sequences of tRNA genes derived from the annotations provided by GtRNAdb
and TAIR databases were validated with two criteria: i) the ability to fold into standard tRNA cloverleaf structure ii)
the presence of promoter elements – boxA and boxB. As a means to investigate which factors are responsible for
accumulation of tRNA fragments, we have collected close to seventy datasets from publically available Gene Expression Omnibus (GEO) database. The datasets consisted of wild type samples from different tissues and environmental
conditions. We then analyzed small RNA sequencing data and created expression profiles for precursor tRNA
transcripts. Our results demonstrated that tRNA fragments originate from both precursor and mature tRNAs. Our
recent advances will be presented in the poster. This work was supported by grant from the National Science Center
2011/03/B/NZ2/01416.
Poster 56
Dicer-like proteins in Medicago truncatula,
a model legume plant
A. TWORAK1, A. URBANOWICZ1, N. KORALEWSKA1, M. POKORNOWSKA1,
A. KURZYNSKA-KOKORNIAK1, J. PODKOWINSKI1, M. FIGLEROWICZ1,2
1
2
The biogenesis of both miRNAs and siRNAs in plants depends on a specific group of ribonucleases known as
DICER-LIKE (DCL) proteins. Based on functional analysis of DCL proteins (DCL1-4) identified in Arabidopsis thaliana, four functional DCL types were distinguished in plants. DCL1-type ribonucleases mainly produce 21 nt long
miRNAs. The products generated by DCL2-, DCL3-, and DCL4-type ribonucleases belong to various classes of
siRNAs. DCL2-type enzymes are involved in the biogenesis of 22 nt long siRNAs from natural antisense transcripsts
(nat-siRNA), DCL3-type ribonucleases produce an abundant class of 24 nt long heterochromatic small interfering
RNAs (hc-siRNA) and DCL4-type proteins are mainly involved in 21 nt long trans-acting siRNAs (ta-siRNA) production. In addition, DCL2-, 3- and 4-type enzymes contribute to the plant defense against a diverse range of pathogens.
In many plants duplications of the genes encoding DCL proteins have been observed. For example, the rice genome
encodes two DCL2- and two DCL3-type proteins whereas in soybean two genes encoding DCL1-, DCL2- and DCL4type ribonucleases were identified. Medicago truncatula is a model legume plant closely related to many economically important cultivable species. In order to increase our knowledge on miRNA and siRNA biogenesis in Medicago
we have screened the current genome assembly available from Medicago truncatula Genome Project in search for
DCL-coding genes. In addition to MtDCL1, 2 and 3 characterized in the previous studies we identified three other
DCL genes: MtDCL4 and two new MtDCL2 homologs. We found that one of the newly identified MtDCL2 genes
codes for a truncated version of DCL2 protein. Using droplet digital PCR (ddPCR) we confirmed the existence of
all MtDCL transcripts in the total RNA fractions extracted from Medicago plants. Additionally, we identified an alternative splicing variant of MtDCL1 mRNA. Translation of the latter may result in the formation of the truncated DCL1
protein. Finally, we determined the expression profiles of the six MtDCL genes in different parts of the plant at various developmental stages. The mRNA abundance for all assayed DCL mRNAs was significantly increased in the
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nodule, compared to root and other plant organs which may suggest the important role of DCL genes in nodule function. This work is co-financed by the European Regional Development Fund through the Operational Programme
Innovative Economy, Innovation grants. This work was partially supported by the European Union Regional Development Fund within the PARENT-BRIDGE Programme of Foundation for Polish Science (Pomost/2011-3/5 to A.K.-K).
Poster 57
Changes in small RNA populations
under herbicide stress in maize
A. TYCZEWSKA1, M. ZYWICKI2, J. GRACZ1, L. HANDSCHUH1,
K. ADAMCZEWSKI3, J. ADAMCZYK3, M. FIGLEROWICZ1, T. TWARDOWSKI1
1
2
Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University in Poznan, Poland
3
Plant Breeding and Acclimatization Institute, National Research Institute, Poznan, Poland
Maize, Zea mays, a plant that originally comes from South America, is currently cultivated in the world on a large
scale. Its great potential is broadly used in food and fodder industry, textile industry but also in bioethanol production. Herbicides, commonly known as weedkillers, are compounds used to destroy or inhibit the growth of plants,
especially weeds. The most popular weedkillers, widely used in the maize fields, are nonselective, which means that
they affect not only weed populations but influence all plants that are growing in the sprayed area. To guarantee their
survival under adverse environmental conditions plants have developed exquisite adjustments to stresses at all levels
(anatomical, morphological, cellular, biochemical and molecular). It has been observed in the fields that there is a variety of phenotypic differences between maize lines in response to herbicide spraying. Therefore our goal is to
identify molecular basis of plant’s increased/decreased resistance to herbicides. First, we chose two maize lines that
differ significantly in susceptibility to herbicide RoundUp, then using Next Generation Sequencing (NGS) we detected differences in small RNA populations. Bioinformatic analyses (miRDeep-P, miREvo and our own programs) were
performed to profile the expression of known and identify new small RNA molecules that are involved in herbicide
stress response, cDNA libraries were mapped to B73 maize genome (assembly AGPv2, March 2009). We identified
over a hundred of small RNA molecules with changed expression profiles in tested lines (treated or not with the
herbicide), nearly 50 of them have never been described before in the literature. Funding acknowledgement: the
work is supported by a grant no. N N310 769040 from Ministry of Science and Higher Education, Poland.
Poster 58
Bioenergetic cross-talk between RNA silencing
and photosynthesis in plants
F.G. VERRET1, N.E. IOANNIDIS2, X. KOTAKIS2, K. KOTZABASIS2, K. KALANTIDIS1,2
1
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece
2
Department of Biology, University of Crete, Heraklion, Greece
RNA silencing is the regulatory control of gene and genome expressions by small (21-25 nt) non-coding RNAs
including short interfering RNAs (siRNAs) and microRNAs (miRNAs). In plants RNA silencing is involved during
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development, genome stability, defence against virus, and responses to abiotic stressors. Conversely RNA silencing
efficiency is affected by environmental factors such as temperature and photoperiod. Using Nicotiana benthamiana
transgenic line exhibiting spontaneous RNA silencing of a GFP transgene we have recently shown that photoadaptation to high light condition increases both the frequency of RNA silencing and the transcript levels of key components of the RNA silencing machinery. Here analysis of the composition and activity of the photosynthetic apparatus
during ongoing PTGS is reported. In vivo photosynthetic measurements using chlorophyll fluorescence based approaches and qPCR analysis of key photosynthetic genes have been carried out in A. thaliana and N. benthamiana
transgenic lines exhibiting PTGS against a GUS and a GFP transgene respectively. Our project aims to assess the
presence of a bioenergetic cross-talk between RNA silencing and photosynthesis, identify its molecular players and
provide a better understanding of the place of the RNA silencing machinery within the plant physiology.
Poster 59
Auxin inhibits flowering of Ipomoea nil through stimulation
of JASMONIC ACID CARBOXYL METHYLTRANSFERASE
expression
E. WILMOWICZ, K. FRANKOWSKI, A. KUCKO, K. MARCINIAK, J. KOPCEWICZ
1
2
Centre for modern interdisciplinary technologies, Nicolaus Copernicus University, Torun, Poland
Studies indicate the great significance of interaction between indole-3-acetic acid (IAA) and jasmonates (JAs)
in the regulation of various physiological processes in plants, e.g. stem cell growth, abscission, secondary abscission
zone formation, tendril coiling and wounding, but still little is known about their interaction in the transition from
vegetative to reproductive phase. In A. thaliana flowers, auxin signalling requires AUXIN RESPONSE FACTOR6
(ARF6) and ARF8, both of which induce the expression of JA biosynthesis genes in filaments. We investigated the
interaction between IAA and JAs in photoperiodic flower induction of short-day plant Ipomoea nil using Real-Time
PCR and GC-MS methods. Also we investigated the effect of IAA on the endogenous jasmonates (JA, MeJA) level
in cotyledons of Ipomoea nil during 16-h long inductive night and the expression of JAs biosynthesis (InLOX2
– LIPOXYGENASE, InAOS – ALLENE OXIDE SYNTHASE, InOPR3 – 12-OXOPHYTODIENOATE REDUCTASE)
and metabolism genes (InJMT – JA CARBOXYL METHYLTRANSFERASE, InJAR – JASMONATE RESISTANT).
We have shown that IAA applied to the cotyledons before the inductive night inhibits flower induction. The level of
endogenous JAMe in cotyledons of I. nil during inductive night was low. Application of IAA just before the inductive
night increased the level of endogenous MeJA in the cotyledons and also stimulated the expression of InJMT. Our
results demonstrate that IAA inhibits flower induction through the stimulation of InJMT and as a consequence
enhancement MeJA content, which is considered as another flowering inhibitor in I. nil.
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Poster 60
Identification and preliminary expression analysis
of LlFPA gene from Lupinus luteus
W. WOJCIECHOWSKI, M. BANACH, J. KESY, P. GLAZINSKA, E. WILMOWICZ,
A. KUCKO, K. MARCINIAK, J. KOPCEWICZ, A. TRETYN
Chair of Plant Physiology and Biotechnology, Faculty of Biology and Environment Protection,
Nicolaus Copernicus University, Torun, Poland
The induction of flowering is one of the most important stages of the development of higher plants. A lot of data
indicates the necessity of cooperation of many factors both exogenous and endogenous to provide an optimum time
of flowering. At least 4 pathways controlling this process (photoperiodic, vernalization, autonomous, and hormonal)
were distinguished with the model long day plant Arabidopsis thaliana. All pathways control of flowering cooperate
regulation of key flowering genes which name integrator genes. The autonomous pathway includes at least 7 of flowering induction genes. The key task of the proteins coded by them is to stop the transcript activity of the FLC
(FLOWERING LOCUS C ) gene flowering inhibitor. The mutants of AP (autonomous pathway) genes showed an
increased level of mRNA FLC. The FLC inactivation occurs on the road of two mechanisms of remodeling chromatin
and mRNA processing of this gene. In these study we identify homologue of FPA in vegetative organs from Lupinus
luteus. Known full-length cDNA sequence shows high identity to the known gene cDNAs described in other legume
species (Glycine max, Medicago truncatula ). Into the predicted amino acid sequence LlFPA evolutionarily conserved
RRM (RNA recognition motifs) domain was found. This suggests that in the lupine as in others species this protein
takes part in alternative cleavage and polyadenylation of RNAs. Examined the transcriptional activity of the gene was
determined in vegetative organs (leaves, petioles roots) of lupine. No significant differences in the expression of LlFPA
were detected. These results may indicate that the protein encoded by this gene is involved in many physiological processes. Acknowledgements: The work was supported by the Multi-Year Programme of the Polish Ministry of Agriculture
and Rural Development, No. 149/2011 and the National Science Centre (Poland) grants No 2011/01/B/NZ9/03819
Poster 61
miR393 controls somatic embryogenesis in Arabidopsis through
regulation of auxin signaling components (TIR1 and AFB2)
A.M. WOJCIK, M.D. GAJ
MicroRNAs (miRNAs) play essential role in regulation of gene expression and their involvement in control of
developmental processes in plants and animals was indicated. In plants, a key regulatory role of miRNAs in morphogenic processes, including zygotic embryogenesis and hormone signaling was documented. Beside development in
planta, miRNAs are also believed to control morphogenic processes induced in somatic cells cultured in vitro, including somatic embryogenesis (SE). In support for this hypothesis, we found the mutant in DICER-LIKE1 (DCL1),
a gene required for miRNAs biogenesis pathway, incapable of SE induced in culture of Arabidopsis explants. Moreover, MIR393A and MIR393B genes involved in auxin signaling, a regulatory pathway expected to be essential for SE
induction, were found up-regulated in SE induction and the respective mutants (miR393a, miR393b and miR393ab )
showed impaired embryogenic potential. In the present study, the involvement of MIR393A and MIR393B genes in
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SE was further confirmed and overexpression of these genes was found to negatively affect culture capacity for SE. To
extend the knowledge on miR393-controlled mechanism of SE induction, four candidate miR393-target genes involved in auxin perception were analyzed, including: AFB1 (AUXIN SIGNALING F-BOX1), AFB2, AFB3 and TIR1
(TRANSPORT INHIBITOR RESPONSE1). The candidate targets are members of the TIR1/AFB2 clade of auxin
receptors (TAARs) in the AFB family of plant F-box proteins. We found insertional mutants in the candidate targets
(tir1-1, afb1-3, afb2-3 and afb3-4 ) significantly defected in embryogenic response induced in vitro. In addition, expression analysis with the use of real time RT-PCR indicated that two of the candidate genes, TIR1 and AFB2, are negatively regulated by miR393 molecules in SE. In contrast to SE-stimulated MIR393 expression, the transcription of
TIR1 and AFB2 was found down-regulated during this process. Expression profiles of AFB1 and AFB3 did not suggest their miR393-controlled involvement in SE. In addition, a spatiotemporal expression pattern of the candidate
miR393-target genes was analyzed in explants undergoing SE induction with the use of GFP reporter lines
(TIR1pro:GFP, AFB1pro:GFP, AFB2pro:GFP and AFB3pro:GFP ). GFP analysis indicated that TIR1 and AFB2 are
highly expressed in explant areas involved in embryogenic transition (cotyledons, proximity of SAM) while expression of AFB1 and AFB3 is localized mainly in explant parts not undergoing SE induction (root, epidermis). Collectively, the results indicated that miR393 molecules control SE induction in Arabidopsis through regulation of TIR1
and AFB2 genes encoding auxin receptors.
Poster 62
Post-transcriptional gene silencing of putative HvGSK leads
to enhanced salt stress tolerance of barley seedlings
Y. YANUSHEVSKA1, A. NADOLSKA-ORCZYK2, W. ORCZYK1
1
2
Brassinosteroids (BRs) are a class of naturally occurring steroidal plant hormones involved in diverse biological
processes. BRs play a key role in plant growth and development and function as crucial regulators of plant tolerance
for biotic and environmental stresses. The goal was to verify that clone AK251287, showing high similarity to
OsGSK1 (Koh i in., 2007), was a putative ortolog of this gene and, similar to OsGSK1, functioned as a negative regulator on BR-dependent signalling pathway and was involved in salt stress tolerance. Transcript assigned as AK251287
was amplified using barley cv. Golden Promise cDNA as a template, cloned and sequenced. Nucleotide and encoded
amino acid sequence confirmed high similarity to OsGSK1 and BIN2/AtSK21 – a gene earlier recognized as negative
regulator of BR signaling. Fragments of ORF, 5N- and 3N-UTRs were cloned into pBRACT207 silencing cassette and
used for barley transformation, via Agrobacterium. The 11 primary transgenic plants (T0) were confirmed for the
presence of T-DNA. Relative expression of the analyzed gene, a putative HvGSK in T1 plants ranged between 0,09
and 0,22 of the expression determined in the control plants. Transgenic T1 plants with silenced expression of
analyzed gene showed better seedling development under salt stress (25 mM NaCl germination and 200 mM NaCl
growth) as well as normal conditions compared with control non-transgenic plants. Biomass of randomly selected
14 days old T1 seedlings compared with control was between 71% and 117% for seedlings grown in normal conditions
and between 67% and 145% for salt stress conditions. Analyzed seedlings represented T1 generation and the
observed range of results might reflect segregation of the silencing cassette in the selected transgenic plants. In the
summary we can state that silencing of the analyzed gene HvGSK resulted in better growth of seedlings in both –
normal and salt stress conditions. Similarity of the nucleotide sequence of the genes (HvGSK and OsGSK1 ) as well
as the similar phenotypes of OsGSK1 -knock-out rice and HvGSK -silenced barley indicate that identified barley gene
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is a homolog of OsGSK1. Due to the expected bigger number of barley genes in GSK3 gene family, further study
is required to establish detailed phylogenetic and functional characteristics of the gene. The work was financed from
National Science Centre grants nr. 718/N-COST/2010/0 and UMO-2011/01/B/NZ9/02387.
Poster 63
Transcriptome surveillance by the nuclear 5N-3N exonuclease
XRN3 in Arabidopsis
M. ZAKRZEWSKA-PLACZEK1, M. KRZYSZTON1, G. BARTON2,
N. SCHURCH2, A. SHERSTNEV2, G. SIMPSON3, J. KUFEL1
1
2
3
Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland
Division of Computational Biology, University of Dundee, Dundee, United Kingdom
Division of Plant Sciences, College of Life Sciences, University of Dundee, Scotland, United Kingdom and
Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Scotland, United Kingdom
Two nuclear 5N-3N exonucleases AtXRN2/3 in Arabidopsis thaliana are homologs of the yeast Xrn2/Rat1, which
is involved in degradation and processing of several classes of nuclear RNAs and in transcription termination of RNA
polymerases I and II. We have recently shown that AtXRN2/3 contribute to polyadenylation-mediated nuclear quality
control of rRNA precursors and excised spacer fragments. Here, we report that several mRNAs and pri-miRNAs are
significantly upregulated in xrn3 mutants, and this is accompanied by a strong accumulation of non-coding RNAs that
originate from these loci. As chromatin immunoprecipitation confirmed increased PolII occupancy in these regions,
these species most likely represent readthrough transcripts that result from termination defects. In addition, our
high-throughput data reveal a prevailing accumulation of lncRNAs corresponding to intergenic regions in xrn3 mutants. These transcripts, named XATs (XRN3-associated transcripts), are produced by PolII and often contain fragments of coding regions and poly(A) tails, but mostly lack the 5N cap structure. Considering that plant XRN2/3 act
as endogenous silencing suppressors, these aberrant RNAs could in principle activate RNAi, however, we were not
able to detect changes in DNA methylation or histone modifications in XAT-generating regions. Although XAT transcripts have an impact on the expression of neighbouring genes, the mechanism of their action is still unclear. We
propose two models for XATs biogenesis. One of them assumes that XATs are readthrough transcripts resulting
from XRN3 involvement in transcription termination. The alternative mechanism considers XATs a result of pervasive transcription of the genome and implicates their rapid removal by AtXRN3. The two pathways are not mutually exclusive and AtXRN3 may be involved in both regulation of transcriptional termination and surveillance of
genome-wide transcription.
Poster 64
Reference Gene Selection for Quantitative Real-time PCR
Normalization in Quercus robur
M. ZAPALSKA, K. DUDZIAK, M. NOWAK, K. KOWALCZYK
Institute of Plant Genetics, Breeding and Biotechnology, University of Life Sciences in Lublin, Lublin, Poland
Gene expression can vary across tissues, developmental stages as well as under different experimental conditions. The relative quantification of gene expression by reverse transcription quantitative PCR (RT-qPCR) requires
reliable internal controls in order to avoid misinterpretation of experimental results. Recent studies confirm that
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Poster session
an universal reference gene does not exist, therefore a selection of most suitable RG for each species or condition
is essential. Quercus robur is a deciduous tree of great ecological and industrial importance. Due to its significance
and scarce knowledge of gene expression in this species, an attempt of RG selection for pedunculate oak was carried
out. In this study, several candidate reference genes were evaluated to determine the most stable internal reference
for quantitative PCR normalization in Quercus robur. Among tested genes were ACT (ACTIN ), EF-1α (ELONGATION-FACTOR-1ALPHA ), GAPDH (GLYCERALDEHYDE-3-PHOPHATE DEHYDROGENASE ), β-TUB (β-TUBULIN ), UBQ (UBIQUITIN ) and 18S rRNA. The transcript abundance of these genes was analyzed in leaves tissue
of 1-year old Quercus robur seedlings subjected to three different CdCl2 concentrations (0 μM, 10 μM and 50 μM).
Total RNA extraction was carried out according to Le Provost et al., (2007) protocol. RNase contamination from
glass and plastic surfaces was removed with the use of RNaseZap ® RNase Decontamination Solution (Ambion, cat.#
AM9780). cDNA was synthesized from 1 μg of total RNA using the SuperScript® VILOTM cDNA Synthesis Kit
(Invitrogen). The Quantitative Real-time PCR analysis was performed with the SYBR Select Master Mix (Applied
Biosystems) chemistry, using gene-specific primers. Specificity of amplification of the transcripts was checked by
the analysis of melting curves and by gel electrophoresis, showing a single PCR amplification product with the expected size for each gene. The gene expression stability was assessed with the NormFinder statistical approach. From
all candidate genes tested, GAPDH and β-TUB were identified as the most stable, while ACT showed the lowest
expression stability. In this study, we have identified and validated reference genes in pedunculate oak that can be
used for quantification of target gene expression in leaves tissues under heavy metal treatment and will be useful
as a starting point for further gene expression studies in this species.
Poster 65
The microRNA transcriptome change
during tuberization in potato
RUNXUAN ZHANG, M. LINEY, D. MARSHALL, G.J. BRYAN, C. HORNYIK
The James Hutton Institute, Invergowrie, Dundee, United Kingdom
Potato is a major global food crop, and is cultivated for its underground storage stems (tubers), rich in starch
and nutrients. Potato is unique among the major crops in tuber formation. It is extremely important to understand
the as yet poorly known molecular events playing roles in development or responses upon biotic or abiotic stresses,
ultimately impacting on breeding of cultivars with improved tuber characteristics (tuber initiation, yield, size distribution, shape etc.). Our aim is to explore the potato microRNA (miRNA) transcriptome to investigate the role of
these small non-coding RNAs in one of the most important potato developmental processes, tuberization. Using this
knowledge we can identify the genes which play a role in the switch which lead to stolon and tuber intiation and tuber
development. Previously, we used small RNA next generation sequencing approach to identify small RNAs and the
recently published potato genome for the prediction of microRNAs (miRNAs). We have developed a pipeline for the
analysis of miRNAs in potato. The prediction of candidate miRNAs is based mainly on plant miRNA characteristics,
such as minimum folding energy of candidate precursors, read distributions and matching patterns of the mature
miRNA and ‘star’ sequences. Conserved and potato specific miRNAs were identified and non-conserved miRNAs
were validated experimentally. Additionally, we predicted targets for all of the miRNAs applying different levels of
stringency using the potato transcriptome data. In our study we used a photoperiod inducible tuberization system
(Solanum tuberosum group Andigena ). This primitive cultivar can tuberize under short days (SD) but do not make
tubers under long day (LD) conditions. During a time course experiment we sampled plants under SD and LD conditions from the induction of tuberization (early time points) until SD plants had small tubers. Using high-throughput
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Poster session
sequencing approach we identified small RNAs in different samples. Both leaf and stolon tissues were used in biological replicates to compare the miRNA transcriptome between SD and LD plant at different time points. Conserved
and potato specific miRNAs were predicted with their targets and miRNA expression was calculated at the different
time points between SD and LD plants. Data will be presented on how the expression levels of miRNAs change during this unique developmental transition. Our findings will ultimately facilitate the molecular biology in potato
through the knowledge of miRNAs and their predicted targets.
Poster 66
Distribution of AGO1, HYL1 and AGO4
in Hyacinthus orientalis L. male gametophytes
K. NIEDOJADLO1, M. KUPIECKA1, A. KOLOWERZO1,2, R. LENARTOWSKI3, J. NIEDOJADLO1
1
2
3
Laboratory of Isotope and Instrumental Analysis of Biology and Environment Protection, Nicolaus Copernicus University, Torun, Poland
Small non-coding RNAs (sRNAs) have emerged as key guide molecules in the regulation of various biological
processes in plants, including developmental transition and patterning, responses to the environment, maintaining
genome stability and defense against viruses and bacteria. Our knowledge of small RNAs biogenesis and their mechanisms of action has dynamically expanded over the past decade, but their functions in the regulation of plant sexual
reproduction are still not fully understood. The present study uses immunofluorescence techniques to examine the
spatial and temporal distribution of the molecules involved in the biogenesis of small non-coding RNAs, i.e. AGO1
(ARGONAUTE 1), HYL1 (HYPONASTIC LEAVES 1) and AGO4 (ARGONAUTE 4), in mature pollen grain and
in vitro growing pollen tubes of Hyacinthus orientalis. AGO1 and HYL1 are specifically associated with the miRNA
pathway. AGO4 is involved in siRNA gene silencing and in RNA-directed DNA methylation. The results have demonstrated a different localization of these antigens in mature pollen grain. Also, changes in the pattern of the labeling
were visible during pollen tube growth in the vegetative and generative nuclei and after division generative cell were
visible in sperm cells in the pollen tube after 8-12 h of growth in medium. Our preliminary study indicates that
in H. orientalis processes including sRNAs take place and play an important role in the regulation of gene
expression in these cells during male gametophyte development and fertilization. The research was supported by
the National Science Centre (NCN) grant 2011/03/D/NZ3/00603
List of participants
AGGARWAL CHHAVI
ALABA SYLWIA
[email protected]
[email protected]
P1
P2 P8 P27 P45 P49
ARIEL FEDERICO
BAJCZYK MATEUSZ
[email protected]
[email protected]
S3.2 S3.6
P3 S4.4
BANACH MARIUSZ
BANAS AGNIESZKA KATARZYNA
BANASIAK JOANNA
[email protected]
[email protected]
[email protected]
P4 P35 P60
P54 P1
P5
BARCISZEWSKA-PACAK MARIA
BARTA ANDREA
BAZIN JEREMIE
BIALA WANDA
BIELEWICZ DAWID
BIELUSZEWSKI TOMASZ
BROWN JOHN
BRZEK ALEKSANDRA
BRZYZEK GRZEGORZ
BUJARSKI JOZEF J.
CARVALHO RAQUEL
CASTELLANO M. MAR
CHANG CHIUNG-YUN
CHWIALKOWSKA KAROLINA
CRESPI MARTIN
CZESNICK HJORDIS
DERAGON JEAN-MARC
DING YILIANG
DOLATA JAKUB
DUDZIAK KAROLINA
DUQUE PAULA
FAVORY JEAN-JACQUES
FERNANDEZ-CALVINO LOURDES
FIGLEROWICZ MAREK
DE FRANCISCO AMORIM MARCELLA
FRAY RUPERT
GAGLIARDI DOMINIQUE
GAJ MAŁGORZATA
GASPARIS SEBASTIAN
GLAZINSKA PAULINA
GOLISZ ANNA
GRACZ JOANNA
GRAMZOW LYDIA
GREEN PAMELA
GREGORY BRIAN
GROSZYK JOLANTA
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
P6 S1.7
L1.4 S1.6
P7
P5
P8 S1.7 S4.4 P6 P44
P9
L1.7 S1.1 S1.6 S1.9 S2.3 S3.6
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected];
[email protected]
[email protected]
[email protected]
[email protected]
P10 S1.5
S3.1
S1.1
P14
S1.8
P11
S3.6 S3.2 P7
S2.2
S2.4 P13
L1.3
S4.4 S1.5 P6 P8 P15 P44
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
P13 S2.4
P14
P37 P56 P57
P16
S2.1
L2.2 S3.3
P53 P61
P17 P40
P18 P4 P35 P60
P19
P20 P57
S4.5
L4.1 P52
L3.4
P21
P12 P64
L1.5 S1.1 P48
GRUPA ANNA
HOJKA-OSINSKA ANNA
HORNYIK CSABA
JACKOWIAK PAULINA
JARMOLOWSKI ARTUR
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
JASINSKI MICHAL
JURCZYK BARBARA
KAJA ELZBIETA
KALYNA MARIA
KAUSIKA SWATHI
KINDGREN PETER
KOBLOWSKA MARTA
KONONOWICZ ANDRZEJ K.
KORALEWSKA NATALIA
KOTER MAREK
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
KRUSZKA KATARZYNA
KUBALA SZYMON
KUCKO AGATA
KUFEL JOANNA
KUHLMANN MARKUS
KURZYNSKA-KOKORNIAK ANNA
KWASNIEWSKI MIROSLAW
KWASNIK ALEKSANDRA
LAUBINGER SASCHA
LABUZ JUSTYNA
LEWANDOWSKA DOMINIKA
LUDWIKOW AGNIESZKA
LUKASIK ANNA
LUKOSZEK RADOSLAW
MACIOSZEK VIOLETTA
MALECKA ARLETA
MARCINIAK KATARZYNA
MARQUEZ ORTIZ YAMILE
MELONEK JOANNA
MERRET REMY
MEYER KATJA
MEYER TINA
MICKIEWICZ AGNIESZKA
MILANOWSKA KAJA
MILCZAREK DOROTA
MORKUNAS IWONA
NADOLSKA-ORCZYK ANNA
NIEDOJADLO JANUSZ
NIEDOJADLO KATARZYNA
NOWAK KATARZYNA
NUC PRZEMYSŁAW
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
P37
P65
P37
S1.5 S1.7 S1.9 S4.2 S4.4 P3 P6 P8 P15
P27 P42 P44 P46 P55
P5
P22
P23
L1.4 S1.6
P24
P25
P15 P43
P56
P26
P27 S3.4 P8 P42
P28
P29 P4 P35 P59 P60
S3.3 P19 P31 P63
S4.6
P56
P11
P31
S4.1 P16
P54
S2.3
P32
P33 P38
S2.5
P30
P35 P4 P18 P29 P59 P60
L1.4
S1.4
S2.4
S1.3 S4.3
P36
P37
P38
P39
P40 P17 P62
P41 P66
P66
P53
P27
OHTANI MISATO
ORCZYK WACLAW
PACAK ANDRZEJ
PALATNIK JAVIER
[email protected]
[email protected]
[email protected]
[email protected]
S1.2
P17 P21 P40 P62
P42 P8 P26 P27 P46
L4.2
PALUSINSKI ANTONI
PETRILLO EZEQUIEL
PIECZYNSKI MARCIN
PIETRYKOWSKA HALINA
PISZCZALKA PAWEL
PLEWKA PATRYCJA
PLUSKOTA WIOLETTA
POKORNOWSKA MARIA
QU GE
RACZYNSKA K. DOROTA
REDDY ANIREDDY
RICHARDSON DALE
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
P43 P15
S1.6
P44
P8 P33 P45
P45 P2 P8
P46 P45
P47
P56
S3.4
S1.9 S4.2
L1.8
P48
RUWE HANNES
SADOWSKI JAN
SEKI MOTOAKI
SIEROCKA IZABELA
SIKORSKI PAWEL
SILHAVY DANIEL
SIMPSON CRAIG
SIMPSON GORDON
SINGH KASHMIR
SKORUPA KATARZYNA
SMYDA PAULINA
SOLTYS-KALINA DOROTA
STAIGER DOROTHEE
STEPIEN AGATA
STEPNIAK KAROLINA
STIEF ANNA
STREITNER CORINNA
SULKOWSKA ALEKSANDRA
SURA WERONIKA
SUZUKI YUYA
SWIEZEWSKI SZYMON
SZAJKO KATARZYNA
SZAKONYI DORA
SZAREJKO IWONA
SZCZYGIEL-SOMMER ALEKSANDRA
SZTATELMAN OLGA
SZWEYKOWSKA-KULINSKA ZOFIA
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
S4.7 S1.4
P9 P32 P51
L3.3
P49 P45
S3.3
L2.3
S2.3 S1.1 L1.7 S1.6 S1.9 S3.6
L2.1 P63
P34 P45
S1.7 P6 P8
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
S4.2 S1.9 P8
P31
P50
TAVORMINA PATRIZIA
THOMPSON AGNIESZKA
TU SHIH-LONG
[email protected]
[email protected]
[email protected]
S4.3 S1.3
P19
P51
P52
S1.5 P10
P11
P53
P54
S1.7 S1.9 S3.4 S4.2 S4.4 P2 P3 P6 P8
P27 P33 P42 P44 P45 P46 P49
S3.5
P55
L1.1 S1.8
TWORAK ALEKSANDER
TYCZEWSKA AGATA
VAZQUEZ FRANCK
VERRET FREDERIC
WACHTER ANDREAS
WIERZBICKI ANDRZEJ
WILMOWICZ EMILIA
WOJCIECHOWSKI WALDEMAR
WOJCIK ANNA
WU HUI-WEN
WYRZYKOWSKA ANNA
YANOVSKY MARCELO
YANUSHEVSKA YULIYA
YIN ZHIMIN
ZAKRZEWSKA-PLACZEK MONIKA
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
P56
P57 P20
L4.3
P58
L1.2
L3.1
P59 P4 P18 P29 P35 P60
P60 P4 P18 P35
P61
L3.2
P44
L1.6 S1.6
P62 P40
ZAPALSKA MAGDALENA
ZIELENKIEWICZ PIOTR
[email protected]
[email protected]
P64 P12
P33
P63

Abstract book - Post-transcriptional Gene Expression Regulation in

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