GENOMICS AND PROTEOMICS
OF PLANT SYSTEMS
www.ceitec.eu
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Research Programme
Genomics and Proteomics
of Plant Systems
Programme Coordinator: Prof. RNDr. Jiří Fajkus, CSc.
Characteristics of the Research Programme
The research programme is focussed on understanding of evolution-based developmental
strategies of plant systems to promote their applications in next-generation technologies and
medicine.
Overall Goal
Plants present a unique experimental system due to their enormous genome plasticity and
the naturally totipotent character of their cells, which makes it possible to regenerate the whole
organism from a single cell easily and without ethical problems. The developmental outcomes
of molecular manipulations can thus be studied at the level of whole organisms.
1. Advanced Nanotechnologies
and Microtechnologies
7. Molecular Veterinary
Medicine
6. Brain and Mind
Research
2. Advanced Materials
3. Structural Biology
5. Molecular Medicine
4. Genomics and Proteomics of Plant Systems
CEITEC
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Research Directions
zGenome, karyotype and chromosome evolution; the role of repetitive DNA in genome dynamics;
the structure, evolution and maintenance of telomeres and their role in chromosome stability and
plant speciation; epigenetic regulations
zMolecular mechanisms governing hormonal regulations and their role in plant development and
stress response; developmental outputs of subcellular protein trafficking and cell polarity will be
established
zMetabolic profiling approaches for understanding and exploitation of plant secondary metabolites; using bacterial metabolomics as a model for systems biology; metabolite biomarkers for
diagnostics; the develop ment of a miniaturised drug metabolism system based on capillary
electrophoresis (CE)
zTo provide access to state-of-the-art technologies based on shared resources and highly trained
staff in proteomics; the development of novel techniques for the separation and analysis of nucleic
acids, proteins, small bioactive molecules & drugs and their complexes based on electrophoretic
and microfluidic systems, electrochemical and optical methods and nanotechnologies
Research Groups | Research Group Leaders
RG-4-1 | Bioanalytical Instrumentation | František Foret
RG-4-2 | Plant Cytogenomics | Martin A. Lysák
RG-4-3 | Functional Genomics and Proteomics of Plants | Jan Hejátko
RG-4-4 | Hormonal Crosstalk in Plant Development | Eva Benková (TBC)
RG-4-5 | Metabolomics | Zdeněk Glatz
RG-4-6 | Core Facility - Proteomics | Zbyněk Zdráhal
RG-4-7 | Developmental and Cell Biology of Plants | Jiří Friml (TBC)
RG-4-8 | Chromatin Molecular Complexes | Jiří Fajkus
RG-4-9 | Developmental and Production Biology - Omics Approaches | Břetislav Brzobohatý
Work Packages
WP-4-1 | Comparative cytogenomics of plant families with contrasting genome structure
WP-4-2 | Structure, function and evolution of telomeres and their role in chromosome stability
WP-4-3 | Identification of the molecular factors and specific developmental processes underlying
hormonal regulations in plants
WP-4-4 | Developmental role of subcellular dynamics and polarity of auxin transport components
WP-4-5 | Hormonal control of plant growth correlations and responses to environmental cues
WP-4-6 | Metabolomic tools used in the development of novel drugs; an analysis of plant secondary
metabolism pathways as a source of novel bioactive compounds
WP-4-7 | New instrumental approaches to bioanalysis
Core Facility: Proteomics
Head: Doc. RNDr. Zbyněk Zdráhal, Dr.
Main Activity
Mass spectrometry – based proteomics. Activities of the Core Facility cover all steps of proteomic
analysis – protein isolation, separation of protein mixtures, protein characterisation by mass spectrometry and bioinformatic data processing.
Unique Features
The Core Facility provides academic community and other subjects with access to advanced proteomic
technologies based on shared resources and highly trained staff. The concentration of expensive
instrumentation and know-how results in fast responses to the demands of the research community
and the effective utilization of resources.
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CEITEC
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1.1. Bioanalytical Instrumentation
Research Group Leader : Ing. František Foret, CSc.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
z Capillary separations
z Mass spectrometry coupling
z Miniaturisation
z Single cell analysis
Main objectives
z Development of novel techniques for the separation and analysis of nucleic acids, proteins, small bio active
molecules & drugs and their complexes based on electrophoretic and microfluidic systems, electrochemical
and optical methods and nanotechnologies.
CONTENT OF RESEARCH
Exploring new ways for achieving enhancements in resolution, sensitivity and selectivity of analyses. Utilisation
of theoretical and instrumental approaches for development and applications of microfluidics, nanotechnologies
and novel chemistries. Research of novel techniques for the separation and analysis of nucleic acids, proteins,
small bioactive molecules & drugs and their complexes using electrophoretic and microfluidic systems coupled
with electrochemical, optical and mass spectrometric methods.
z Microseparation systems for the analysis of biomolecules will be developed utilising combination of microfluidic
chips and structured nanoparticles including quantum dots, magnetically activated tags; enzymatic and nanoparticle modified monolithic supports.
z New detection principles, based on surface signal enhancement (e.g. surface plasmon, Raman, fluorescence,
electrochemistry) will be combined with the newly developed separations. These technologies and instrumentation will be developed for joint applications with other investigators of the CEITEC centre.
z Technologies for microanalyses of proteins and their glycosylation will be focused on the combination of electrochemistry, microfludic biosensors and mass spectrometry for analysis of limited sample quantities. The ultimate
goal of single-cell analysis will be aimed at potential application in cancer detection.
KEY RESEARCH EQUIPMENT
Planned research infrastructure
Technology Units
zBioanalytics
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Current research infrastructure
The current research infrastructure includes instrumentation for mass spectrometry and the separation of biomolecules.
Ñ
2011, K. Klepárník, Institute of Analytical Chemistry AS CR, D. Valík, Masaryk Memorial Cancer Institute, I. Míšek, Institute of Animal
Physiology and Genetics.
zÑSingle-cell analysis by mass spectrometry (GAP206/11/2377), Czech Science Foundation, 2011-2013, K. Klepárník, Institute of Analytical
Chemistry AS CR.
zÑNew methods of analysis of proteins and their glycosylation in cancer - combination of electrochemistry, microfludic biosensors
and mass spectrometry (P301/11/2055), Czech Science Foundation, 2011-2014, F. Foret, Institute of Analytical Chemistry AS CR,
E. Paleček, Institute of Biophysics AS CR.
zÑIntegrated bioanalytical technologies for microanalyses and diagnostics with laser induced fluorescence and mass spectrometry
coupling (LC06023), Ministry of Education, Youth and Sports, 2006-2011, Z. Glatz, Masaryk University, L. Fajkusová, University
Hospital Brno, F. Foret, Institute of Analytical Chemistry AS CR.
zÑNanoparticle modified monolithic supports for bioanalysis (2SGA2721), South Moravian Programme for Distinguished Researchers
MAIN PROJECTS
z Nanotechnology in functional diagnostics of apoptotic and tumor cells (GA203/08/1680), Czech Science Foundation, 2008-
(SoMoPro), 2011-2013, F. Foret, Institute of Analytical Chemistry AS CR.
Ñ
SELECTED PUBLICATIONS
Ñ
zÑ
zÑ
zÑ
z LISKOVA, M., VORACOVA, I., KLEPARNIK, K., HEZINOVA, V., PRIKRYL, J., FORET, F. Conjugation reactions in the preparations of quantum
z
dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis. Anal. Bioanal. Chem. 2011, 400, p. 369379.
SMEJKAL, P., SZEKRENYES, A., RYVOLOVA, M., FORET, F., GUTTMAN, A., BEK, F., MACKA, M. Chip-based CE for rapid separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans. Electrophoresis. 2010, 31, p. 3783–3786.
JUSKOVA, P., OSTATNA, V., PALECEK, E., FORET, F. Fabrication and Characterization of Solid Mercury Amalgam Electrodes for Protein
Analysis. Anal. Chem. 2010, 82, p. 2690-2695.
TOMAS, R., KOVAL, M., FORET, F. Coupling of hydrodynamically closed large bore capillary isotachophoresis with electrospray
mass spectrometry. J.Chromatogr. A. 2010, 1217, p. 4144–4149.
RYVOLOVÁ, M., PREISLER, J., FORET, F., HAUSER, P., C., KRASENSKY, P., PAULL, B., MACKA, M. Combined Contactless Conductometric,
Photometric, and Fluorimetric Single Point Detector for Capillary Separation Methods. Anal. Chem. 2010, 82, p. 129–135.
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1.2. Plant Cytogenomics
Research Group Leader : Doc. Mgr. Martin A. Lysák, Ph.D.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
z Karyotype and genome evolution in plants
zChromosome rearrangements in speciation
zWhole-genome duplications (polyploidy)
zChromosome structure
z Evolution of repetitive DNA
zComparative and evolutionary phylogenomics
zMolecular phylogenetics
Main objectives
zInvestigation of the evolution of chromosome complements (karyotypes) in land plants.
zUnderstanding the role of chromosome repatterning and whole-genome duplicati on events in genome
evolution and speciation.
zAnalysis of chromosome and genome collinearity using methods of comparative molecular cytogenetics
and sequence genomics.
CONTENT OF RESEARCH
The overarching objective of the Plant Cytogenomics group is to document, analyse and compare genome
structure across the plant kingdom at different complexity levels:
1.
2.
3.
4.
DNA level (genome size, repetitive elements),
chromosomal level (karyotype evolution, chromosome collinearity),
whole-genome level (genome collinearity, polyploidy),
species level (molecular phylogenetic frameworks, paleobiogeography).
The primary focus of the research group is comparative cytogenomics in species and plant groups with contrasting genome features and well-documented phylogenetic frameworks. The prime target between 2011
and 2015 will be the mustard family (crucifers, Brassicaceae); possibilities of cytogenomic research in other
angiosperm groups furnished with genomic resources and tools will be explored.
The research will concentrate on understanding (1) the role of chromosome rearrangements in reproductive
isolation and speciation, (2) the impact of whole-genome duplication events on genome structure and cladogenesis, and (3) the evolutionary dynamics and chromosome organisation of repetitive elements.
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KEY RESEARCH EQUIPMENT
Planned research infrastructure
Technology Units
zPlant cytogenomics
Current research infrastructure
Current infrastructure includes equipment for molecular cytogenetics, standard laboratory equipment for
molecular biology and plant cultivation.
Ñ
2007- 2009, M. Lysák, Masaryk University, J. Macas, Biology Centre AS CR, Ilia J. Leitch, Royal Botanic Gardens.
zÑChromosome evolution in crucifers (Brassicaceae) revealed by comparative chromosome painting (IAA601630902),
Academy of Sciences of the Czech Republic, 2009-2012, M. Lysák, Masaryk University.
zÑGenome evolution in Cardamine allopolyploids of contrasting phylogenetic age (GAP501/10/1014), Czech Science Foundation,
2010-2012, M. Lysák, Masaryk University, K. Mummenhoff, Universität Osnabrück, K. Marhold, Charles University in Prague.
zÑMolecular basis of cell and tissue regulations (MSM0021622415), Ministry of Education, Youth and Sports, 2005-2011, J. Fajkus,
MAIN PROJECTS
z Molecular and cytogenetic analysis of the giant genomes of Fritillaria (Liliaceae) (GA521/07/0284), Czech Science Foundation,
Masaryk University.
Ñ
of Brassicaceae. Trends Plant Science. 2011, 16, p. 108-116.
zÑSCHUBERT, I., LYSAK, M., A. Interpretation of karyotype evolution should consider chromosome structural constraints. Trends
Genet. 2011, 27, p. 207-216.
zÑMANDAKOVA, T., JOLY, S., KRZYWINSKI, M., MUMMENHOFF, K., LYSAK, M., A. Fast diploidization in close mesopolyploid relatives
of Arabidopsis. Plant Cell. 2010, 22, p. 2277-2290.
zÑMANDAKOVA, T., LYSAK, M., A. Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae). Plant Cell.
2008, 20, p. 2559-2570.
zÑLYSAK, M., A., BERR, A., PECINKA, A., SCHMIDT, R., MCBREEN, K., SCHUBERT, I. Mechanisms of chromosome number reduction in
SELECTED PUBLICATIONS
z FRANZKE, A., LYSAK, M., A., AL-SHEHBAZ, I., A., KOCH, M., A., MUMMENHOFF, K. Cabbage family affairs: the evolutionary history
Arabidopsis thaliana and related Brassicaceae species. Proc Natl Acad Sci USA. 2006, 103, p. 5224-5229.
CEITEC
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1.3. Functional Genomics
and Proteomics of Plants
Research Group Leader : RNDr. Jan Hejátko, Ph.D.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
zRole of cytokinins in the vascular tissue and root apical meristem formation and development
zInteraction of cytokinin and other hormones, particularly auxin
zInteraction of cytokinins with light
zStructural basis of multistep phosphorelay signaling in plants
zPlant proteome and phosphoproteome response to cytokinins
zEmploying the knowledge of molecular mechanisms of multistep phosphorelay-based signalling in molecular breeding
zProduction of bioactive compounds in plant production systems
Main objectives
zDetermination of molecular mechanisms governing hormonal regulations and their functions in plant
development.
CONTENT OF RESEARCH
The research group is interested in the hormonal regulation of plant development and underlying molecular
mechanisms with particular emphasis on the understanding of cytokinin signalling, action and interaction
with other plant growth regulators.
Plant cells are well known for their tremendous developmental plasticity. Plant hormones, particularly auxins
and cytokinins were found to be major regulators of intrinsic developmental programs associated with changes
of differentiation status of plant cells and tissues. That allows de novo formation of entire plants from virtually
all types of specialised plant tissues. Identification of basic molecular principles involved in the regulation
of plant cell division and differentiation will provide developmental model useful in the comparative biology
approaches and identification of corresponding regulatory and developmental events in animal and particularly human cell systems.
In the group of Functional Genomics and Proteomics of Plants, we are interested in the molecular mechanisms
underlying the regulation of plant development by plant hormones cytokinins (CKs) and their interaction with
other plant hormones, e.g. auxin. In our studies we employ comprehensive approaches including forward and
reverse genetics, proteomics, protein biochemistry, protein structure analysis and bioinformatics to recognise
the principles of complex molecular events involved in the cytokinin signal transduction and action.
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We are particularly interested in study of following problems:
1. Interaction of auxin and cytokinins in the processes of de novo organogenesis, regulation of root meristem patterning and vascular tissue development. We are interested in the identification of molecular targets
acting downstream the CK signalling pathways and the role of gene regulatory networks constituting CKdependent developmental circuits.
2. Elucidating molecular determinants of specificity in multistep phosphorelay (MSP), with special emphasis
on the role of MSP in CK signalling. Particularly we are interested in the structural analysis of intracellular
receiver domains of sensory histidine kinases that, as we have found, specifically interact with downstream
signaling His-containing phosphotransfer proteins, determining thus specificity in plant MSP pathways.
3. Hormonal regulations of plant proteome, particularly the study of CK-dependent proteome changes with
the aim of identifying novel regulatory targets of CK-mediated regulations of plant development and elucidating molecular mechanisms underlying interaction of CK with other plant growth regulators, e.g. ethylene.
4. Development and application of novel approaches, e.g. immunomodulation (production of scFv fragments
recognising both cytokinins and CK signalling proteins in vivo) to modulate CK-dependent regulation of plant
development. Extending our fundamental research and use of its results in applied science and development
of novel strategies useful in e.g. molecular breeding or using of plant systems for biotherapeutics production.
KEY RESEARCH EQUIPMENT
Planned research infrastructure
Technology Units
zMolecular biology, genomics and proteomics of plants
Current research infrastructure
Equipment for cultivation of plants under tightly regulated conditions (fytotrons) and under defined light
quality and quantity. Expertise and high-end instrumentation in advanced microscopy techniques that
include high-end confocal microscope equipped with 'white ' laser, highly sensitive hybrid detectors and
module for fluorescence life-time imaging (FLIM) and fluorescence correlation spectroscopy (FCS). Image
analysis software allowing highly sensitive and specific fluorescence imaging in living cells via fluorescence
intensity decay shape analysis microscopy (FIDSAM). High-throughput system for automated microscopy
(Olympus 'slide' ) and horizontal confocal macroscope (modified Nikon AZ-C1 system), allowing detailed,
real-time fluorescent protein localisation and dynamics in vivo.
MAIN PROJECTS
zÑThe role of cytokinins and auxin interactions in the regulation of root gravitropism in Arabidopsis thaliana (GAP501/11/1150),
Czech Science Foundation, 2011-2013, J. Hejátko, Masaryk University.
zÑImmunomodulation as a functional proteomics tool for cytokinin signaling study in Arabidopsis thaliana (GA521/09/1699),
Czech Science Foundation, 2009-2012, L. Janda, Masaryk University, M. Faldyna, Veterinary Research Institute.
zÑIdentification of molecular components and the mechanism of polar targeting of PIN auxin transport proteins in Arabidopsis thaliana
(IAA601630703), Academy of Sciences of the Czech Republic, 2007-2011, J. Friml, Masaryk University.
zÑStructural basis for the specificity of signal transduction in plants: interaction network of histidine kinase receiver domains
in Arabidopsis (GAP305/11/0756), Czech Science Foundation, 2011-2014, L. Žídek, Masaryk University.
zÑRegulation of morphogenesis of plant cells and organs (LC06034), Ministry of Educaton, Youth and Sports, 2006-2011,
E. Zažímalová, Institute of Experimental Botany AS CR, I. Kašík, Institute of Photonics and Electronics AS CR, Z. Opatrný, Charles
University in Prague, J. Hejátko, Masaryk University, Z. Novotná, Chemical–Technology University in Prague, B. Brzobohatý, Mendel
University in Brno.
CEITEC
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SELECTED PUBLICATIONS
zÑPEKAROVA, B., KLUMPLER, T., TRISKOVA, O., HORAK, J., JANSEN, S., DOPITOVA, R., BORKOVCOVA, P., PAPOUSKOVA, V., NEJEDLA,E.,
SKLENAR, V., MAREK, J., ZIDEK, L., HEJATKO, J., JANDA, L. Dynamic structure and binding specificity of the receiver do main of sensor
histidine kinase CKI1 from Arabidopsis thaliana. Plant Journal. 2011, 67, p. 827-839.
zÑHORAK, J., JANDA, L., PEKAROVA, B., HEJATKO, J. Molecular Determinants of the Signalling Specificity via Phosphorelay Pathways
in Arabidopsis. Current Protein & Peptide Science. 2010, 12, p. 126-136.
zÑHEJATKO, J., RYU, H., KIM, G., T., DOBESOVA, R., CHOI, S., CHOI, S., M., SOUCEK, P., HORAK, J., PEKAROVA, B., PALME, K., BRZOBOHATY, B.,
HWANG, I. The histidine kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 regulate vascular tissue
development in Arabidopsis shoots. Plant Cell. 2009, 21(7), p.2008-2021.
zÑPERNISOVA, M., KLIMA, P., HORAK, J., VALKOVA, M., MALBECK, J., SOUCEK, P., REICHMAN, P., HOYEROVA, K.,DUBOVA, J., FRIML, J.,
ZAZIMALOVA, E., HEJATKO, J. Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux.
Proceedings of the National Academy of Sciences of the USA. 2009,106, p. 3609-3614.
zÑKUDEROVA, A., URBANKOVA, I., VALKOVA, M., MALBECK, J., BRZOBOHATY, B., NEMETHOVA, D., HEJATKO, J. Effects of conditional
IPT -dependent cytokinin overproduction on root architecture of Arabidopsis seed lings. Plant and Cell Physiology. 2008, 49,
p. 570-582.
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1.4. Hormonal Crosstalk
in Plant Development
The research group will start its research activities in 2012.
Research Group Leader : Mgr. Eva Benková, CSc. (TBC)
THEMATIC RESEARCH FOCUS
Research areas
zGrowth and development of plants are regulated by signalling substances such as plant hormones. In plants,
interactions between hormonal pathways represent crucial factors that govern their action. The molecular basis
for hormonal crosstalk is largely unknown.
zResearch group aims to identify the molecular and cellular mechanism(s) underlying crosstalk of hormonal
pathways in organogenesis and other plant developmental processes.
zResearch group uses lateral root formation in Arabidopsis as an ideal experimental model to study mechanisms
of plant hormone action, the molecular basis of their interactions, and the role of these interactions in organogenesis.
Main objectives
1. Convergence of hormonal pathways on transport-dependent auxin distribution upstream of lateral root
formation:
zIdentification of key points in which auxin and other signalling pathways converge during lateral root formation
and the molecular components involved in the process.
2. Role of auxin-cytokinin interaction in lateral root formation:
zCell type-specific transcriptome analysis to investigate molecular events involved in auxin-cytokinin-regulated
lateral root organogenesis
3. Identification of components of hormonal crosstalk by genetic approaches:
z Mutant screens that will specifically target interactions between selected hormonal pathways.
4. Formulation of general models for hormonal regulation of organogenesis:
z The acquired knowledge on molecular networks and their mutual interactions in lateral root organogenesis
will be used to mathematically model these processes and to extrapolate them onto other developmental
situations.
CEITEC
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1.5. Metabolomics
Research Group Leader : Prof. RNDr. Zdeněk Glatz, CSc.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
z Metabolomics
z Metabolism
z
z
z
z
z
z
z
z
Metabolites
Biomarkers
Drug metabolism
Nuclear magnetic resonance
Mass spectrometry
Liquid chromatography
Gas chromatography
Capillary electrophoresis
Main objectives
z Using bacterial metabolomics as a model for systems biology.
z Establishing metabolite biomarkers for diagnostics.
z Development of a miniaturised drug metabolism system based on capillary electrophoresis.
CONTENT OF RESEARCH
Metabolomics is one of the newest “omics” in the field of systems biology. The subject of its study - metabolome
is the complete set of metabolites that are present in cells under particular physiological or developmental
ircumstances. Although the number of metabolites in a given organism is projected to be less than those
of genes and proteins, the metabolome analysis presents a difficult analytical task. It is given not only by the wide
diversity in physico-chemical properties of metabolites but also by the large differences in their abundance.
The main analytical techniques employed for metabolomic studies are based on NMR spectroscopy and
mass spectrometry (MS). Besides the direct injection MS, this technique usually requires pre-separation
of the metabolic components using either gas (GC) or liquid chromatography (LC). In addition to these wellestablished methodologies capillary electrophoresis (CE) even in combination with MS is gaining a position
in this field.
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Since the metabolites are downstream of all genome and proteome regulatory structures, they provide
valuable information about the regulatory and catalytic properties of a gene product. In this consequence
there is a growing interest in an application of metabolomics for functional genomics studies, for detection
of metabolic dysfunctions and diseases and for study of drug metabolism.
As a result the metabolomic research will be focused on three key outcomes:
z Comprehensive development of metabolomic analytical tools for purpose of systems biology
z New diagnostic tools based on metabolomic biomarkers discovery for detection of metabolic dysfunctions and diseases
z High throughput analytical tools for drug development processes especially for drug metabolism studies
All above-mentioned analytical techniques should be applied for these research tasks.
KEY RESEARCH EQUIPMENT
Planned research infrastructure
Technology Units
z Metabolomics
Current research infrastructure
The metabolomic laboratoriy is equipped with three capillary electrophoresis (CE) instruments; one of them
is connected to the new Agilent 6320 Ion Trap for mass spectrometry detection. In addition a new analytical HPLC system HP 1200 is available that could also be connectable to the above-mentioned MS system.
The laboratories are further equipped with combined gradient HPLC systems (Shimadzu, ESA, Ecom) with
an autosampler, column oven and diode-array, fluorescence and coularray detectors, UV VIS microtiter
plate spectrophotometer and luminometer.
Ñ
2010-2013, Z. Glatz, Masaryk University.
zÑCapillary electrophoresis as a member of the metabolomic analytical toolbox (GAP206/11/0009), Czech Science Foundation,
2011-2015, Z. Glatz, Masaryk University.
zÑIntegrated bioanalytical technologies for microanalyses and diagnostics with laser induced fluorescence and mass spektrometry
MAIN PROJECTS
z Miniaturized on-line drug-metabolism system based on capillary electrophoresis (GAP206/10/0057), Czech Science Foundation,
coupling (LC06023), Ministry of Education, Youth and Sports, 2006-2011, Z. Glatz, Masaryk University, L. Fajkusová, University Hospital
Brno, F. Foret, Institute of Analytical Chemistry AS CR.
SELECTED PUBLICATIONS
z ZEISBERGEROVA, M., REMINEK, R., MADR, A., et al. On-line drug metabolites generation and their subsequent target analysis
by capillary zone electrophoresis with UV-absorption detection. Electrophoresis. 2010, 31(19), p. 3256-3262.
z MUSILOVA, J., SEDLACEK, V., KUCERA, I., et al. Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted
metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans. Journal of separation science. 2009,
32(14), p. 2416-2420.
z ZEISBERGEROVA, M., ADAMKOVA, A., GLATZ, Z. Integration of on-line protein digestion by trypsin in CZE by means of electrophoretically mediated microanalysis. Electrophoresis. 2009, 30(13), p. 2378-2384.
z KONECNY, J., MICIKOVA, I., REMINEK, R., et al. Application of micellar electrokinetic capillary chromatography for evaluation of inhibitory effects on cytochrome P450 reaction. Journal of chromatography A. 2008, 1189(1-2), p. 274-277.
z ZHANG, J., KONECNY, J., GLATZ, Z., et al. Application of capillary electrophoresis in drug metabolism studies. Current analytical
chemistry. 2007, 3(3), p. 197-217.
CEITEC
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1.6. Core Facility - Proteomics
Research Group Leader : Doc. RNDr. Zbyněk Zdráhal, Dr.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
z
z
z
z
Separation of complex protein mixtures
Characterisation of proteins and their modifications by mass spectrometry
Mass spectrometric techniques for quantification of protein mixtures
MS-based proteomics in general
Main objectives
z To provide access to state-of-the-art technologies based on shared resources and highly trained staff in MSbased proteomics for research groups within CEITEC and outside the project.
z The development of novel techniques for the separation and analysis of proteins using mass spectrometry.
CONTENT OF RESEARCH
Our research interests are focused on the field of MS-based proteomics including isolation of protein samples from various matrices, fractionation and separation of complex protein mixtures by chromatographic and
electrophoretic techniques, and namely on application of mass spectrometric techniques for qualitative and
quantitative characterisation of proteins and their modifications.
At present, we participate on several research projects with diverse topics in area of molecular biology, biomedicine and agriculture. Our roles within these projects represent: development and aplication of MALDI-MS profiling for taxonomic studies of selected bacterial genera and funghi, characterisation of differentially expressed
proteins (e.g. plant proteins after hormonal treatment), characterisation of protein complexes, characterisation
of histone acetylations and assessment of the influence of selected histone deacetylase inhibitors on histone
acetylation state, characterisation of phosphorylation state of selected proteins, characterisation of group
of patatin proteins in different potato cultivars or searching for potentional disease biomarkers in blood plasma.
KEY RESEARCH EQUIPMENT
Planned research infrastructure
Proteomics Core Facility
zCF Proteomics - Mass spectrometry
zCF Proteomics - Protein separation
zCF Proteomics - Sample storage and preparation
z CF Proteomics - Protein separation
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Current research infrastructure
The current infrastructure includes protein isolation, protein separation and mass spectrometry.
MAIN PROJECTS
z Functional characterization of pollen unique storage ribonucleoprotein particles (GAP501/11/1462), Czech Science Foundation,
2011-2014, D. Honys, Institute of Experimental Botany AS CR, Z. Zdráhal, Masaryk University.
z Methods of eradication of production diseases of cattle - bovine viral diarrhoea - mucosal disease and paratuberculosis
(QI101A094), Ministry of Agriculture, 2010-2013, M. Faldyna, Institute of Veterinary Research, Z. Zdráhal, Masaryk University, V. Celer,
University of Veterinary and Pharmaceutical Sciences Brno, J. Kučera, Farmers Union of Czech Pied Cattle.
z Development of diagnostics and treatment of serious heart and vascular diseases using genomic and proteomic approaches
(2B08060), Ministry of Education, Youth and Sports, 2008-2011, L. Fajkusová, University Hospital Brno, T. Freiberger, Cardiovascular
and Transplant Surgery Center, Z. Zdráhal, Masaryk University.
z Molecular basis of cell and tissue regulations (MSM0021622415), Ministry of Education, Youth and Sports, 2005-2011, J. Fajkus,
Masaryk University.
SELECTED PUBLICATIONS
z SEDO, O., SEDLACEK, I., ZDRAHAL, Z. Sample preparation methods for MALDI-MS profiling of bacteria. Mass Spectrom. Rev. 2011,
30(3), p. 417-434.
z STOPKOVA, P., ZDRAHAL, Z., RYBA, S., SEDO, O., SANDERA, M., STOPKA, P. Novel OBP genes similar to hamster Aphrodisin in the bank
vole, Myodes glareolus. BMC Genomics. 2010, 11(45), 10 pp.
z LOCHMANOVA, G., ZDRAHAL, Z., KONECNA, K., KOUKALOVA, S., MALBECK, J., SOUCEK, P., VALKOVA, M., KIRAN, N., S.,
BRZOBOHATY, B. Cytokinin induced photomorphogenesis in dark-grown Arabidopsis: A proteome analysis. J. Exp. Bot. 2008,
59(13), p. 3705-3719.
z EYER, L., PANTUCEK, R., ZDRAHAL, Z., KONECNA, H., KASPAREK, P., RUZICKOVA, V., HERNYCHOVA, L., PREISLER, J., DOSKAR, J.
Structural protein analysis of the polyvalent staphylococcal bacteriophage 812. Proteomics. 2007, 7(1), p. 64-72.
z BENES, P., MACECKOVA, V., ZDRAHAL, Z., KONECNA, H., ZAHRADNICKOVA, E., SMARDA, J. Role of vimentin in regulation of monocyte/
macrophages. Differentiation. 2006, 74(6), p. 265-276.
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1.7. Developmental and Cell Biology
of Plants
The research group will start its research activities in 2012.
Research Group Leader : Prof. Mgr. Jiří Friml, Ph.D., Dr. rer. nat. (TBC)
THEMATIC RESEARCH FOCUS
Plants as sessile organisms must adapt to ever changing environmental conditions. The adaptations to environmental cues are essential for plant survival and consequently for crop productivity. The adaptation processes rely
on sensing and transduction of environmental signals, integration of various forms of signals and determination
of the final response. Plant hormones are implicated in various aspects of regulation of plant development,
growth and responses to environmental cues. Our work is focused on systematic analysis of environmental and
hormonal signaling in regulating plant growth and development, as well as adaptation to environmental cues.
We focus on how different environmental and endogenous signals are integrated into the subcellular dynamics
and polar localisation of transporters for plant hormone auxin. We expect to get a deeper knowledge on plasticity of plant responses to environmental cues.
Main objectives
1. Plant hormonal signalling for regulation of cell polarity and subcellular dynamics:
zIdentification of key entry points by which auxin and other signalling pathways modulate subcellular dynamics and polar trafficking of PIN auxin transporters
2. Cell polarity and subcellular dynamics in plant cells:
zWe elucidate molecular and cellular mechanisms underlying cell polarity and trafficking processes by combination of genetics and high resolution microscopy approaches.
3. Perception of external signals and their integration into subcellular dynamics and cell polarity:
z Using combination of reverse genetic, chemical genomics, life cell and high-resolution imaging approaches;
we gain new insights into how gravity and light perception is integrated into regulation of subcellular dynamics.
4. Integration of hormonal signalling and subcellular dynamics for multicellular tissue development by mathematical modelling:
z The acquired knowledge on hormonal networks; processes of subcellular dynamics and integration of different
signals will be used to mathematically model these processes and to extrapolate them onto multicellular
developmental situations.
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1.8. Chromatin Molecular Complexes
Research Group Leader : Prof. RNDr. Jiří Fajkus, CSc.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
z Structure, evolution and maintenance of telomeres and their role in chromosome stability and plant
speciation; this includes characterisation of nucleoprotein composition of telomeres and telomerases,
biophysical analysis of interactions between telomere components by quantitative methods, analysis
of structure-function relationships of telomerase subdomains and analysis of alternative (telomeraseindependent) strategies of telomere maintenance
z Epigenetic mechanisms in the regulation of gene expression, chromosome stability and telomere maintenance
z Structure, evolution and function of SMC complexes; characterisation of SMC5-6 complex subunits and
MAGE proteins in vitro and in vivo; their role in DNA repair and chromosome dynamics
Main objectives
z Structure, evolution and maintenance of telomeres and their role in chromosome stability and plant
speciation. Epigenetic regulations.
CONTENT OF RESEARCH
Chromatin is the supramolecular complex of DNA, proteins and other associated molecules (e.g. RNA
species). It is the building material of chromosomes, which can be observed during cell division in their
most condensed state. Chromatin was first discovered in plant cells – as were cells themselves, or genes.
While the nucleotide sequence of the DNA component of chromatin constitutes the genetic material of the cell,
the other chromatin components (and also modifications of bases in DNA itself ) participate in so-called
epigenetic functions. These include spatiotemporal regulation of gene activity and DNA replication, correct
and precise segregation of genetic material to daughter cells, maintenance of chromosome stability, protection of genetic material from damage. Last but not least, chromatin structure compacts several metres of genomic DNA to fit the single cell nucleus of only several microns in diameter, while remaining functional despite
the high degree (10 5-10 6) of compaction.
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Our research group addresses the following topics in current chromatin and epigenetic research:
z The structure, evolution and maintenance of telomeres (terminal parts of linear chromosomes) and their
role in chromosome stability and plant speciation. This includes characterisation of nucleoprotein composition of telomeres and telomerases (ribonucleoprotein enzymes performing telomere elongation),
biophysical analysis of interactions between telomere components in vivo and in vitro, including quantitative methods, analysis of structure-function relationships of telomerase subdomains and analysis of alternative (telomerase-independent) strategies of telomere maintenance. We are also interested in the roles
of telomeres and their components in DNA damage signalling and DNA repair.
z Epigenetic mechanisms involved in the regulation of gene expression, chromosome stability and telomere
main tenance. This includes analysis of epigenetic marks (e.g. DNA methylation, covalent modifications
of histones, variant histones, nucleosome positioning and dynamics). Experimental analysis of nucleosome
positioning is supported by computer predictions based on DNA deformability sequence pattern recently
derived by the group of E. N. Trifonov. This pattern represents the cracking of the so-called chromatin code,
which has been searched for since 1980.
z Structure, evolution and function of SMC 5-6 and SMC-like complexes (Structure Maintenance of Chromatin).
Characterisation of SMC5-6 complex subunits, and MAGE (melanoma antigen) proteins in vitro and in vivo.
Association of SMC complex subunits and MAGE proteins with specific chromatin domains (e.g. telomeres,
centromeres, rDNA loci). Roles of SMC and MAGE proteins in DNA repair, transcription and chromosome
dynamics.
KEY RESEARCH EQUIPMENT
Planned research infrastructure
Technology Units
zMolecular biology, genomics and proteomics of plants
zPlant Cytogenomics
Current research infrastructure
The current research infrastructure includes fluorescence microscopy, centrifugation and ultracentrifugation,
preparative and analytical chromatography, electrophoresis, in situ and Southern hybridisation, phosphofluoroimaging, real-time PCR and a documentation system for chemiluminiscence and fluorescence.
Ñ
2009-2011, J. Paleček, Masaryk University.
zÑTelomeres and telomerase: transition from molecular to structural biology approach (IAA500040801), Academy of Sciences
of the Czech Republic, 2008-2012, E. Sýkorová, Institute of Biophysics AS CR, J. Fajkus, Masaryk University.
zÑEpigenetic mechanisms of plant telomeres regulation (GAP501/11/0569), Czech Science Foundation, 2011-2014, M. Fojtová,
Masaryk University.
zÑLoss of specific DNA repeats in response to dysfunction of CAF1 in plants (GAP501/11/0289), Czech Science Foundation, 20112015, J. Fajkus, Masaryk University.
zÑMolecular basis of cell and tissue regulations (MSM0021622415), Ministry of Education, Youth and Sports, 2005-2011, J. Fajkus,
MAIN PROJECTS
z Characterisation of MAGE proteins: cofactors of E3-ubiquitin ligases? (IAA501630902), Academy of Sciences of the Czech Republic,
Masaryk University.
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SELECTED PUBLICATIONS
z MOZGOVA, I., MOKROS, P., FAJKUS, J. Dysfunction of Chromatin Assembly Factor 1 Induces Shortening of Telomeres and Loss
of 45S rDNA in Arabidopsis thaliana. Plant Cell. 2010, 22(8), p. 2768-2780.
z FOJTOVA, M., et al. Telomere maintenance in liquid crystalline chromosomes of dinoflagellates. Chromosoma. 2010, 119(5),
p. 485- 493.
z DVORACKOVA, M., et al. AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows
highly dynamic association with chromatin. Plant Journal. 2010, 61(4), p. 637-649.
z HOFR, C., et al. Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and
kinetics. Biochemical Journal. 2009, 419, p. 221-228.
z RUCKOVA, E., et al. Role of alternative telomere lengthening unmasked in telomerase knock-out mutant plants. Plant Molecular
Biology. 2008, 66(6), p. 637-646.
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1.9. Developmental and Production
Biology - Omics Approaches
Research Group Leader : Prof. RNDr. Břetislav Brzobohatý, CSc.
Contact : [email protected]
THEMATIC RESEARCH FOCUS
Research areas
z Molecular mechanisms underlying:
zhormonal regulation of plant development
zplant adaptation to abiotic and biotic cues
z Molecular markers related to economically important traits
Main objectives
zDetermination of molecular mechanisms governing hormonal regulations and their functions in plant
development and stress-response. Developmental outputs of cell polarity will be established.
CONTENT OF RESEARCH
Hormonal control of plant growth correlations and responses to environmental cues
Throughout their life cycle, plants as sessile organisms depend critically on sensing a number of external
and internal signals that they use to alter their physiology, morphology and development. Plant hormones,
mainly auxin and cytokinin, have been recognised as key players in the control of plant growth correlations, and
an increasing body of evidence implicates their involvement in the responses to environmental cues. We will
investigate the molecular and cellular mechanisms underlying the hormonal modulation of the plant body
architecture, mainly shoot and root branching, under various environmental cues including nutrient and/or
water deficiency. Our pilot experiments indicate that alterations in the hormone level or signalling can compensate for the undesirable effects of the environmental cues on plant growth and morphogenesis. We will
develop and employ reverse genetic tools to get a deeper understanding of the particular roles of a number
of candidate genes implicated in the compensation responses. In addition, green fluorescent protein (GFP)
fusions will be used to monitor promoter activities and protein products of genes reportedly involved
in branching, for example genes involved in the maintenance and differentiation of the axillary meristems
and auxin transport. Based on the knowledge gained in the project we will evaluate the possibilities of crop
engineering for better performance under limited nutrient and water availability.
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Much progress has been made recently in our understanding of cytokinin biosynthesis and signaling, and how
environmental cues interact with these components to modulate plant growth, development and physiology.
Cytokinin probably plays a role in the response to many environmental signals. Physiological studies have correlated the changes in cytokinin levels in response to environmental cues. Genome-wide microarray studies reveal
overlapping transcriptional responses between cytokinin and various environmental inputs. The components
of the cytokinin biosynthetic and signaling pathway are, in turn, transcriptionally altered by environmental
stimuli. While many of the links of cytokinin to various environmental stimuli have been simply correlative, recent
studies using mutants that alter cytokinin biosynthesis or signalling have begun to demonstrate an important
role for cytokinins in these responses. However, because of the wide range of outputs of the cytokinin signalling
pathway, dissecting the role of cytokinin in the response to a particular stress remains challenging. We will employ a number of tools now available to alter cytokinin levels and responsiveness in combination with non targeted
transcriptomic and proteomic analyses to deepen our knowledge of this subject. The investigation of structurefunction relationships in proteins involved in these interactions will provide us with knowledge of their mechanisms of action at the molecular level. We will focus on the interactions of cytokinin and the light, temperature
and drought response pathways. We will also examine the effects of altered cytokinin levels and signalling
on the accumulation of and tolerance to heavy metals. Attention will be paid to cytokinin interactions with other
hormones in these processes. As we further deepen our understanding of the circuitry underlying the input
of cytokinin into the response of the environmental signals, we should be able to engineer these pathways
to produce plants with increased tolerance to abiotic stresses.
KEY RESEARCH EQUIPMENT
Planned research infrastructure
Technology Units
zDevelopmental and production biology
Current research infrastructure
The current research infrastructure of the group includes plant cultivation facilities, microscopy, molecular
biology and biochemistry equipment.
Ñ
Republic, 2007-2010, B. Brzobohatý, Mendel University in Brno, J. Bobáľová, Institute of Analytical Chemistry AS CR, P. Bouchal,
Masaryk University.
zÑThe role of cytokinins and polyamines in heat stress response and thermotolerance in tobacco and Arabidopsis plants
(GA206/09/2062), Czech Science Foundation, 2009-2013, R. Vaňková, Institute of Experimental Botany AS CR, B. Brzobohatý,
Mendel University in Brno.
zÑApplication of conventional and molecular genetic approaches for the development of grain legumes resistant to viral and
fungal pathogens and insect pests (QI91A229), National Agency for Agriculture Research, 2009-2013, M. Griga, Agritec
Plant Research Ltd., D. Kodrík, Biology Centre AS CR, M. Navrátil, Palacký University Olomouc, V. Reinöhl, Mendel University in Brno.
zÑFunctional genomics and proteomics for crop improvement (1M06030), Ministry of Education, Youth and Sports, 2006-2011,
B. Brzobohatý, Mendel University in Brno.
zÑRegulation of morphogenesis of plant cells and organs (LC06034), Ministry of Education, Youth and Sports, 2006-2011,
MAIN PROJECTS
z Proteome dynamics in response to increased cytokinin levels in Arabidopsis (IAA600040701), Academy of Sciences of the Czech
E. Zažímalová, Institute of Experimental Botany AS CR, I. Kašík, Institute of Photonics and Electronics, Z. Opatrný, Charles University
in Prague, J. Hejátko, Masaryk University, Z. Novotná, University of Chemical Technology in Prague, B. Brzobohatý, Mendel University
in Brno.
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SELECTED PUBLICATIONS
z BALLA, J., KALOUSEK, P., REINOHL, V., FRIML, J., PROCHAZKA, S. Competitive canalization of PIN-dependent auxin flow from axillary
buds controls pea bud outgrowth. Plant Journal. 2011, 65(4), p. 571-577.
z CERNY, M., DYCKA, F., BOBALOVA, J., BRZOBOHATY, B. Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana
identified by proteome and phosphoproteome profiling. Journal of Experimental Botany. 2011, 62(3), p. 921-937.
z HEJATKO, J., RYU, H., KIM, G.-T., DOBESOVA, R., CHOI, S., CHOI, S., M., SOUCEK, P., HORAK, J., PEKAROVA, B., PALME, K., BRZOBOHATY, B.,
HWANG, I. The histidine kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 regulate vascular tissue
development in Arabidopsis shoot. Plant Cell. 2009, 21, p. 2008-2021.
z LOCHMANOVA, G., ZDRAHAL, Z., KONECNA, H., KOUKALOVA, S., MALBECK, J., SOUCEK, P., VALKOVA, M., KIRAN, N., S., BRZOBOHATY, B.
Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis. Journal of Experimental Botany. 2008, 59,
p. 3705-3719.
z SAUER, M., BALLA, J., LUSCHNIG, C., WISNIEWSKA, J., REINOHL, V., FRIML, J., BENKOVA, E. Canalization of auxin flow by Aux/IAA-ARFdependent feedback regulation of PIN polarity. Genes and Development. 2006, 20, p. 2902-2911.
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CONTENT
General Overview ....................................................................................................................................................3
Research Groups .....................................................................................................................................................6
1.1. Bioanalytical Instrumentation .....................................................................................................................6
1.2. Plant Cytogenomics ........................................................................................................................................8
1.3. Functional Genomics and Proteomics of Plants ...............................................................................10
1.4. Hormonal Crosstalk in Plant Development .........................................................................................13
1.5. Metabolomics ...............................................................................................................................................14
1.6. Core Facility - Proteomics .........................................................................................................................16
1.7. Developmental and Cell Biology of Plants ..........................................................................................18
1.8. Chromatin Molecular Complexes ...........................................................................................................20
1.9. Developmental and Production Biology - Omics Approaches ....................................................23
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Contacts
CEITEC – Central European Institute of Technology
Žerotínovo nám. 9, 601 77 Brno, Czech Republic
Phone: +420 549 494 981
E-mail: [email protected]
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