International Conference

Chemistry and Biology of Phytohormones and Related Substances 2019

Luhačovice, Czech Republic, May 19 – 21, 2019

BOOK OF ABSTRACTS

Organized by Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany.

http://old.rustreg.upol.cz/zseminar/2019/

OLOMOUC 2019

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SUNDAY MAY 19

ANDREA LUTEROVÁ SOFTWARE FOR METABOLOMIC ANALYSIS LYDIA UGENA IDENTIFICATION OF BIOLOGICALLY ACTIVE SUBSTANCES FROM DIFFERENT TYPES OF BIOSTIMULANTS IN THE INTERACTION WITH THE MODEL PLANT ARABIDOPSIS THALIANA KLÁRA SUPÍKOVÁ BIOSYNTHESIS AND STRUCTURAL DIVERSITY OF PHENOLIC ACIDS ON OAT TOMÁŠ HEGER SEARCHING FOR NEW SODIUM-POTASSIUM ATPASE INHIBITORS VERONIKA GÓROVÁ JUNIOR PD PROJECT: BRASSINOSTEROIDS AND THEIR NEUROPROTECTIVE ACTIVITY VOL. 2 GABRIEL GONZALES NEURODEGENERATION IN LGR: THE REALM OF NEUROPROTECTION MAGDALÉNA BRYKSOVÁ ANTI-SENESCENCE ACTIVITY OF NEWLY PREPARED N9-SUBSTITUTED BAP DERIVATIVES MARTIN HONIG ROLE OF CYTOKININS IN PROGRAMMED CELL DEATH IN PLANTS ONDŘEJ KOVÁČ THE FUTURE OF MODULAR APPROACH IN NATURAL PRODUCT SYNTHESIS VLASTA MATUŠKOVÁ BIOLOGICAL ACTIVITY OF NOVEL PURINE NUCLEOSIDES ASTA ZUKAUSKAITE NEW ANTI-AUXIN WITH DIFFERENT ACTIVITY IN ROOTS AND SHOOTS ŠÁRKA SLAMENCOVÁ PREPARATION AND BIOLOGICAL ACTIVITY OF NOVEL N6 SUBSTITUTED PURINE RIBOSIDES WITH POTENTIAL ANTITUMOR EFFECT IVAN PETŘÍK INNOVATIVE, EFFECTIVE, ROBUST, FAST AND CHEAP PURIFICATION OF PHYTOHORMONES USING DISPERSIVE SOLID PHASE EXTRACTION JAKUB HRDLIČKA METHOD DEVELOPMENT FOR KARRIKININ ANALYSIS KRISTÝNA FLOKOVÁ IMPROVED STRATEGY TO STRIGOLACTONE DETERMINATION FROM COMPLEX SAMPLE MATRICES BABRORA PAŘÍZKOVÁ IP & OEIP – A PERFECT COUPLE TO REGULATE PLANT DEVELOPMENT WITH HIGH SPATIAL RESOLUTION

MONDAY MAY 20

DENISA HENDRYCHOVÁ HISTONE H3 SERINE-57 PHOSPHORYLATION PROMOTES RESPONSES TO DNA REPLICATION STRESS HANA DOSTÁLOVÁ BTK INHIBITORS: TO LYMPHOMAS AND BEYOND MARKÉTA KOVALOVÁ TRANSCRIPTIONAL CDKS: THE WAY TO GO? MIROSLAV PEŘINA BIOLOGICAL DIFFERENCES AMONG THE CDK4/6 INHIBITORS VLASTIMIL TICHÝ HSF1 INTERACTION NETWORK UNDER STRESS CONDITIONS KAMIRÁN ÁRON HAMOW (FY WATERS) APPLICATION OF UNISPRAY™, A NOVEL ION SOURCE FOR THE MEASUREMENT OF PHYTOHORMONES AND HORMONE-LIKE COMPOUNDS HANA SKOUPILOVÁ THE CYTOTOXIC EFFECT OF NEWLY SYNTHESIZED FERROCENES AGAINST CERVICAL CARCINOMA CELLS AND ITS COMBINATION WITH RADIOTHERAPY OLIVER ŠIMONČÍK THE HEAT SHOCK RESPONSE REGULATION IN CANCER CELLS LUKÁŠ UHRÍK ANALYSIS OF INTERACTION BETWEEN CD20 AND NEWLY DEVELOPED MONOCLONAL ANTIBODY USING HYDROGEN-DEUTERIUM EXCHANGE MASS SPECTROMETRY PETR VOŇKA MOLECULAR DOCKING AND STRUCTURE FUNCTION RELATIONSHIPS TO IDENTIFY NOVEL ANTI OESTROGENS JITKA ZROSTLÍKOVÁ (FY HPST) O KROK DOPŘEDU V LC/MS QTOF TECHNOLOGII: AGILENT 6546

TUESDAY MAY 21

ADÉLA HÝLOVÁ STUDY OF MOLECULAR MECHANISMS AND BIOLOGICAL ACTIVITY OF STRIGOLACTONES ALBA ESTEBAN HERNANDIZ AFFORDABLE FIELD PHENOTYPING, BASIC CONCEPTS FOR BEGINNERS KLÁRA PTOŠKOVÁ GIBBERELLIN SIGNALLING IN RESPONSE TO ABIOTIC STRESS IN WHEAT PETRA JIROUTOVÁ BRASSINOSTEROIDS IN THE CONTEXT OF BIOSYNTHESIS AND CROSSTALK SARA RAGGI THE ROLE OF CUTICLE DURING APICAL HOOK DEVELOPMENT JAKUB HAJNÝ CELL SURFACE RECEPTOR COMPLEX FOR PIN POLARITY AND AUXIN-MEDIATED DEVELOPMENT TOMÁŠ VLČKO BARLEY ITPK GENES PARTICIPATE IN ABIOTIC STRESS RESPONSE DAVID KOPEČNÝ NOVEL UREA AND AROMATIC CYTOKININS AS INHIBITORS OF CYTOKININ OXIDASE/DEHYDROGENASE

SCIENTIFIC PROGRAM

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BTK INHIBITORS: TO LYMPHOMAS AND BEYOND

Hana Dostálová

Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27,

78371 Olomouc,Czech republic Bruton’s tyrosine kinase (BTK) is a kinase expressed in B-lymphocytes where it is involved in B-cell receptor signalling cascade. This pathway facilitates crucial signals for cell’s proliferation and survival which makes BTK an attractive target in B-cell malignancies. Ibrutinib is a first-in-class BTK inhibitor approved by the FDA. It was followed by acalabrutinib and a large number of other compounds, still in clinical trials. Although new BTK inhibitors possess higher selectivity than ibrutinib, a wide range of off-target kinases is affected by BTK-targeted drugs. However, these off-targets may turn into an advantage as shown in numerous studies investigating repurposing of ibrutinib and its followers. HER receptors are a kinase family which is impaired by some of these drugs. This work aimed to examine the potential use of selected BTK inhibitors in prostate cancer (PCa) and hepatocellular cancer (HCC) cell models. Data point to the fact that BTK inhibitors may act as inhibitors of HER receptors in both PCa and HCC cell lines. Downregulated phosphorylated forms of HER receptors were observed primarily after 16 hours treatment with 10 µM compounds ibrutinib, acalabrutinib, and zanubrutinib. Nonetheless, the cytotoxicity effect of these drugs was rather weak in both lymphomas and solid cancer cell lines, with IC50 values ranging from 15 to 61 µM, or even over 67 µM. Project is supported from Palacky University (IGA_PrF_2019_013) and Czech health research council (17-31834A).

NEW STRATEGIES TO STRIGOLACTONE DETERMINATION IN COMPLEX SAMPLE MATRICES

Kristýna Floková1,2, Mahdere Z. Shimels3, Beatriz Andreo Jimenez3, Yanting Wang2, Miroslav

Strnad1 and Harro J. Bouwmeester2

1Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27, 78371 Olomouc,Czech republic 2Plant Hormone Biology, Swammerdam Institute for Life Sciences, Sciencepark 904,

1098 XH Amsterdam, The Netherlands 3Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

Strigolactones (SLs), chemically sesquiterpene lactones, are the most recently described group of plant hormones. These root-exuded compounds can be recognized by parasitic plants as a host plant signal resulting in enormous negative impact on yield of agriculturally important crops. Detailed insight to biosynthesis and metabolism of SLs is hampered by their limited stability and extremely low quantities in complicated plant/soil matrix. Conventional approaches to SL determination by mass spectrometry-based techniques require large initial volumes of root exudate wash (≥ 500 ml), eventually gram-amounts of plant material followed by multi-step and low specific purification procedures. We present highly sensitive and validated UHPLC-MS/MS method for simultaneous profiling of ten selected SL compounds. In order to minimize strong

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matrix effect, sample size of root exudate extract and root tissue was reduced to 10 ml and 100 mg, respectively. The combination of rapid extraction with water miscible aprotic solvents and single-step purification using macroporous polymer-based sorbents with both hydrophilic and lipophilic retention characteristics simplified current sample preparation procedures, notably improved stability and extraction recovery of selected SLs (≥ 85%). A new ion source, UniSpray (Waters), was employed as the interface of mass spectrometer to improve the signal of SL analytes, lacking ionizable residues in the structure. The novel atmospheric pressure ionization showed the average intensity gain of factor 4.5 compared to electrospray (ESI). Supported by grant CZ.02.2.69/0.0/0.0/16_027/0008482 and the European Research Council (ERC Advanced grant CHEMCOMRHIZO, 670211)

JUNIOR PD PROJECT: BRASSINOSTEROIDS AND THEIR NEUROPROTECTIVE ACTIVITY VOL. 2

Veronika Górová1, Gabriel Gonzalez2

1Department of Biochemistry, Palacký University in Olomouc, Šlechtitelů 27, 78371 Olomouc, Czech

Republic,2Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27, 78371 Olomouc,Czech republic

One of the major groups of civilization diseases make neurodegenerative diseases. On one hand they really differ in symptoms but on the other hand all of them are related on a sub cellular level. This work was focused on Parkinson’s disease, which idiopathic form can be caused by excitotoxicity. This pathological process contributes to the degeneration of neurons since it damages neuron cells by oxidative stress together with the overstimulation of neurotransmitter – L-glu. Glutamate can cause pathology in some ways – one of them is by binding to NMDA and AMPA receptors those are overactivated by that and can cause the entrance of calcium ions to the cytoplasm in fairly high levels. By that action endoplasmic reticulum stress and activation of enzymes are caused and can mean devastation of organelles. Another possible way leading to the excitotoxic process represents the antiporter cystine/glutamate, that regulates intra- and extracellular glutamate concentrations. The uptake of cystine is blocked by the excessive concentration of glutamate and by that glutathion cannot be synthetized. Its lack decreases the ability of cells to eliminate free radicals and due to the overproduction of ROS the oxidative stress can be induced. In case of the inhibition of excitotoxicity by the tested substances, we can consider them active in terms of the antineurodegenerative properties. In Junior PD project the basic screening of brassinosteroids, their synthetic derivatives and polyphenolic substances was done on the glutamated-induced model of excitotoxicity. As in vitro model the differentiated cells of neuroblastoma cell line SH-SY5Y was used. Consecutively two substances those were considered the most active were analyzed in partial studies. The scale of damaged cells was evaluated by the intercalating probe propidium iodide that marks greatly damaged cells. Results obtained by this method were comparable to the results obtained from the commercial LDH cytotoxicity assay kit. Moreover substances were analyzed in terms of the decrease of the oxidative stress by the fluorescent probe dihydroethidium. Based on the experimental data of activities of substances the model of the possible active site was made. The flexible alignment was done with two substances those were the most active and were also really structurally similar

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to each other. Values of similarity and energy of ligands were compared with the 9 new derivatives of brassinosteroids to find other substances with a possibly higher anti-excitotoxic activity.

CELL SURFACE RECEPTOR COMPLEX FOR TISSUE POLARITY AND AUXIN-MEDIATED DEVELOPMENT

Jakub Hajný, Tomáš Prát, Bert de Rybel, Youssef Belkhadir, Jiří Friml

Auxin is unique among plant hormones due to its directional cell-to-cell transport mediated by the polarly localized PIN auxin efflux transporters at the plasma membrane. Auxin has an interesting ability to change the polarity of PINs and thus, control the direction of its own flow. Auxin feedback on its polar transport is crucial for all developmental stages of plants. Our main goal is to elucidate the molecular mechanism of auxin-mediated PIN repolarization. This can improve our understanding of how polarity is perceived and maintained within a tissue and clarify how cells communicate to synchronically change polarity of PIN proteins to create transporting channels of auxin.

APPLICATION OF UNISPRAY™, A NOVEL ION SOURCE FOR THE MEASUREMENT OF PHYTOHORMONES AND HORMONE-LIKE COMPOUNDS

Kamirán Áron Hamow1,2

1Department of Plant Physiology, Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Brunszvik st. 2., 2462 Martonvásár, Hungary 2Department of Zoology, Plant Protection Institute, Centre

for Agricultural Research, Hungarian Academy of Sciences, Herman Ottó st. 15., 1022 Budapest, Hungary

hamow.kamiran@agrar.mta.hu tel: +3622569527

Measurement of plant hormones is a challenging analytical application because of the low concentration of target analytes and the complexity and diversity of matrices. Due to recent advances in mass spectrometry LC-ESI-MS/MS has become the preferred method of choice for this task. However, matrix effects derived ion suppression is still a major problem even in the case of novel ESI-QQQ instruments featuring low detection limits. Therefore, excessive sample preparation with an emphasis on cleanup procedures is needed, as well as the use of stable isotope labelled standards is often addressed. This approach generally results in expensive and low throughput methods. Another approach for countering matrix effects is the dilution of extracts; although, in order to keep the low limits of quantification, the enhancement in sensitivity and signal-to-noise ratio is a must. For this purpose, a novel ionisation technique, UniSpray™ combined with the capabilities of UHPLC and modern MS/MS instruments can fill the gap to allow for the so-called dilute-and-shoot methods, which enable the fast, robust, and cost effective way to quantitate plant hormones and hormone-like compounds around ppb level (ABA, SA, JA, IAA) in plants. In this study, a comparison of ESI and UniSpray™ was carried out for a wide set of analytes including phytohormones and pesticides among others. Application

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of UniSpray™ resulted in enhanced ionisation efficiency and response factors, in the improvement of S/N ratios and robustness – the latter one especially for labile compounds. The optimised method was successfully applied for different green leaf matrices and plant root extracts for hundreds of samples. In order to meet not only academic research oriented calls, the analytical method performance was evaluated according to SANTE/11813/2017. This work was supported by GINOP-2.3.3-15-2016-00018.

AFFORDABLE FIELD PHENOTYPING, BASIC CONCEPTS FOR BEGINNERS

Alba Esteban Hernandiz

Department of Chemical Biology and Genetics, Palacký University and Institute of Experimental Botany ASCR,

Šlechtitelů 27, 78371 Olomouc, Czech Republic Phenotyping activities are an essential tool for crops breeding and genetic improvement programs. In fact, Field Based Phenotyping (FBP) is the only approach able to provide an accurate description of the crops trait expression in field conditions. It is commonly assumed that these activities are expensive since they are based on the use of high technological devices; however nowadays there are affordable sensors able to deliver reliable and robust information, such as the conventional RGB cameras followed by a proper image analysis. There are many researches done in this topic and the methodologies differ in the type of selected camera, the chosen “vehicle”, as well as in the sort of extracted information and the workflow for the extraction. It is important to understand the process to obtain information from the images and how is linked to the crop traits. Despite the fact that number of FBP publications has increased exponentially in the recent years there are not many articles with “new user friendly” explanations. These publications are highly multidisciplinary, for this reason to understand it, is necessary to have a strong background on mathematics, statistics and image processing techniques. Thus, this work describes in an easy and understandable the workflow, and the most common mathematical methods and algorithms, as well as, summarizes the most used information derived from RGB images.

QUANTIFICATION OF KARRIKINS USING UHPLC-MS/MS

Jakub Hrdlička1,2, Tomáš Gucký1, Kristýna Floková2,3, Manoj Kulkarni4, Shubhpriya Gupta4, Ondřej Novák1,2, Harro Bouwmeester3, Johannes van Staden4, Karel Doležal1,2

1Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký

University, Faculty of Science, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 2Department of Chemical Biology and Genetics, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic 3Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, Netherlands 4Research Centre for Plant Growth

and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa

Karrikins (KARs), chemically butenolide derivatives, are plant growth regulators that promote seed germination and the subsequent growth and development of seedlings of many plant species

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in extremely low concentration (Light et al., 2009). In nature they are generated and released by combustion of plant material and promote the restoration of ecosystems after wildfires or bushfires (Flematti et al., 2015). Smoke waters (SW, artificially prepared as a saturated extract of all substances in smoke produced by burning plants) could be used as an affordable biostimulant in agriculture and horticulture (Light and Van Staden, 2004). Despite the importance of knowing the optimal concentration of KARs in smoke water for both research and practical applications, no method suitable for monitoring karrikinins in biological matrices have been developed and published so far. Therefore, we employed a new analytical approach for quantification of KARs using ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). Due to the separation by reverse phase and quantification by multiple reaction monitoring we developed, validated and applied a fast, specific and sensitive method which will give us the possibility to characterize these new interesting class of biostimulants in more detail. This work was supported by the Erasmus+, the Ministry of Education, Youth and Sport of the Czech Republic, ERDF project "Plants as a tool for sustainable global development" (No. CZ.02.1.01/0.0/0.0/16_019/0000827) as well as by the Internal Grant Agency of Palacký University (IGA_PrF_2019_018).

STUDY OF MOLECULAR MECHANISMS AND BIOLOGICAL ACTIVITY OF STRIGOLACTONES

Adéla Hýlová, Tomáš Pospíšil and Lukáš Spíchal

Department of chemical biology and genetics, Centre of the Region Haná for Biotechnological and Agricultural

Research, Palacký University Olomouc, Slechtitelu 241/27, 78371 Olomouc Strigolactones have been recently discovered as signalling molecules and plant hormones. Apart from other functions in plants, they are active as germination stimulants for seeds of parasitic weeds. The life cycle of parasitic species Striga, Orobanche and Phelipanche spp. is well adapted to their host plants, which are crops, e.g. sorghum, maize, soy, sunflower, rice or tomato. The host plants produce SLs in root exudates into soil, where the seed germination is induced. The host provides the growing parasite with nutrients, which causes massive damage of crops and loss in yield. The seeds are tiny and remain viable in soil up to 20 years. These parasites are widespread in southern Asia, subtropical African areas and Mediterranean part of Europe. SLs could be used for suicidal seed germination – a method using germination stimulation of seeds of parasitic weeds, which causes death in the absence of the host. The synthesis of natural SLs is complex and expensive. Therefore, their structural analogues with less complicated synthesis were designed. This work focuses on germination assays, their optimization and wide testing of new biologically active SLs. Supported by grant from The Ministry of Education, Youth and Sport of the Czech Republic via ERDF project “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827) and the Internal Grant Agency of Palacký University (IGA_PrF_2019_018).

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BRASSINOSTEROIDS IN THE CONTEXT OF BIOSYNTHESIS AND CROSSTALK

Petra Jiroutová1, Jana Oklešťková1, Nemanja Vukasinovic3, Michal Karady2, Jenny Russinova3, Miroslav Strnad1

1Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký University, Faculty of Science, Šlechtitelů 27, 78371 Olomouc, Czech Republic. 2Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University & Institute of ExperimentalBotany, Academy of

Sciences of the Czech Republic,Šlechtitelů 27, CZ‐78371, Olomouc, Czech Republic 3Center for Plant Systems Biology, VIB, 9052 Ghent, Belgium

Brassinosteroids (BRs) are a group of steroidal plant hormones involved in a variety of physiological processes in plants. BR mutants are characteristic for their dwarf phenotype with curled, dark-green leaf rosette, short internodes, and male sterility. The first and most active BR – brassinolide (BL), was isolated in 1979 from pollen of Brassica napus. Since then, a significant amount of work about their biosynthesis, signaling, biological activity or practical application, especially in agriculture, have been reported. In Arabidopsis the biosynthesis of BRs starts with a conversion of campesterol to campestanol and then continues to brassinolide via two parallel pathways - the early and the late C-6 oxidation pathways. An early C-22 oxidation branch called “campestanol independent pathway” has also been described. This branch is linked to the later part of the late C-6 oxidation pathway. By using BR enzymatic mutant lines of Arabidopsis and various BR precursors, we were studying structure–activity relationship of biosynthetic precursors of brassinolide to discover which structural motif is important for BR activity. Various cross-talk mechanisms between BRs and other hormones are suggested or have been characterized. In this work we investigated interaction between BL and ethylene and direct ethylene precursor - 1-aminocyclopropane-1-carboxylic acid (ACC). We observed that level of the interaction between these hormones is different in Arabidopsis roots and shoots respectively. This work was supported by IGA of Palacký University (IGA_PrF_2019_020) and GAČR (19-00973S). UREA-DERIVED AND AROMATIC CYTOKININS AS INHIBITORS OF CYTOKININ

OXIDASE/DEHYDROGENASE

David Kopečný1, Jaroslav Nisler2,3, Radka Končitíková1, Václav Mik4, Pierre Briozzo5, David Zalabák6, Solange Moréra7, Miroslav Strnad2, Karel Doležal2,4

1Department of Protein Biochemistry and Proteomics, CRH, Faculty of Science, Palacký University, Olomouc, Czech Republic 2Laboratory of Growth Regulators, CRH, Institute of Experimental Botany, AS CR & Palacký

University, Olomouc, Czech Republic 3University of Chemistry and Technology in Prague, Faculty of Food and Biochemical Technology, Department of Chemistry of Natural Compounds, Prague, Czech Republic. 4Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty

of Science, Palacký University, Olomouc 783 71, Czech Republic 5Institut Jean-Pierre Bourgin, INRA, AgroParisTech, Université Paris-Saclay, Versailles, France 6Department of Molecular Biology, CRH, Faculty of Science, Palacký University, Olomouc, Czech Republic 7Institute for Integrative Biology of the Cell, CNRS-CEA-

Univ. Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France Homeostasis of cytokinins is regulated by a flavoenzyme family of cytokinin oxidases/dehydrogenases (CKO/CKX) that irreversibly oxidize this plant hormone. Thidiazuron

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(N-phenyl- N′-1,2,3-thiadiazol-5-yl urea, TDZ) and N-(2-chloro-pyridin-4-yl)-N´-phenylurea (CPPU) are well-known CKO inhibitors, which were used in many previous studies. Also aromatic cytokinins such as 6-benzylaminopurine or kinetin are very poor substrates and rather behave as inhibitors. These compounds compete with substrates for the binding site above the isoalloxazine plane of FAD cofactor of CKO. CKO inhibitors might increase the lifetime of endogenous cytokinins and affect different cytokinin functions, thereby having possible positive effects on seed filling, delayed senescence and stress tolerance toward biotic and abiotic stresses and thus improving crop yield. Several maize CKO isoforms including ZmCKO1, ZmCKO4a, ZmCKO5 and ZmCKO8 were used to study the inhibitory strength of new inhibitors as well as their binding mode by X-ray crystallography. Diffraction data were collected at the synchrotron SOLEIL (Saint-Aubin, France) at Proxima 1 and Proxima 2 beamlines. We identified several urea derivatives with IC50 values in low nanomolar range and solved crystal structure complexes up to 1.7 Å resolution using ZmCKO8 and ZmCKO4a. Fluorescently labelled cytokinins derived from 6-benzylaminopurine or kinetin were also analyzed and several compounds exhibit IC50 values in low micromolar range. Their binding was analyzed using ZmCKO8 up to 2.1 Å resolution. The strongest inhibitors represent attractive candidates for in planta trials. This work was supported by grant no. 18-07563S from the Czech Science Foundation, grant LO1204 from the National Program of Sustainability I and grant CZ.02.2.69/0.0/0.0/16_027/0008482 by the Ministry of Education, Youth and Sports, Czech Republic.

TRANSCRIPTIONAL CDKS: THE WAY TO GO?

Markéta Kovalová, Radek Jorda and Vladimír Kryštof

Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27, 78371 Olomouc,Czech republic

Many cancers, including hematologic malignancies, are highly dependent on oncogenes with a short half-life at both mRNA and protein level. These oncogenes codes, for example, cell cycle regulators and anti-apoptotic proteins like Bcl-2, Mcl-1, and XIAP. They are continuously transcribed and their expression is sensitive to transcriptional disruption. Thus, targeting general transcriptional machinery is a promising therapeutic window. The transcriptional cyclin-dependent kinases (tCDKs) plays an essential role in the basal transcriptional machinery and also are a druggable target. Even though that transcriptional inhibitors globally affect transcription, they are selective to transcript wish rapid turnover. This contributes to the specificity of inhibitors to cancer cells over non-transformed cells. Several selective inhibitors of tCDKs were discovered and some of them are in clinical trials. During our research, we will test new compounds that are potential inhibitors of tCDKs. Compounds will be tested on selectivity towards tCDK and their effect on gene expression and protein levels of oncogenes from MYC and Bcl-2 families. Project is supported from Palacky University (IGA_PrF_2019_013) and Czech science foundation (19-09086S).

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METABOLOMIC ANALYSIS FOR HIGH DENSITY MS DATA INTEGRATION OF POSITIVE AND NEGATIVE MODE

Andrea Luterová, Tomas Furst, Miroslav Strnad and Jiří Grúz

Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27,

78371 Olomouc,Czech republic Metabolomic analysis typically performed by LC-MS, is used for systematical identification and quantification of metabolites from a biological sample. The measurement proceeds in positive or negative modes, which provides the complete information about molecules obtained from a biological sample. Integration of positive and negative modes is basic requirement for further effective data processing and structural identification. This necessary step for database creation is based on the knowledge of the pseudo-molecular ion for each group of ions originate from a single metabolite – the friendly ions. The resulting dataset obtained from positive and negative mode contains rows with information from both modes and identifier of molecular ions, which makes it possible to find all the molecules that belong to each other. Another file is created after integration - the MVA file. This file contains only one molecule describing the group of ions originate from single metabolite. By this way, the highly correlated variables, those friendly ions, are removed. This is a basic requirement for further multivariate analysis, such as Principal Cluster Analysis or Hierarchical Cluster Analysis. IP & OEIP – A PERFECT COUPLE TO REGULATE PLANT DEVELOPMENT WITH

GREAT SPECIFICITY

Barbora Pařízková1,2, Ioanna Antoniadi3, Michal Karady2, David J. Poxson4, Daniel T. Simon4, Ivan Petřík1,2, Miroslav Strnad1, Ondřej Novák1, Karel Doležal1,2, Karin Ljung3

1Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 2Department of Chemical Biology and Genetics,

Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 3Umeå Plant Science Centre, Department of Forest Genetics and

Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden 4Laboratory of Organic Electronics, Department of Science and Technology, Linköping University, 601 74 Norrköping, Sweden

Cytokinins are plant hormones playing crucial roles in plant development. They are involved in many critical physiological processes, including cell division and differentiation, regulation of activity of apical meristems, senescence delaying or regulating the root system architecture (RSA). Since the modulation of plant growth lies on dynamic interactions between phytohormones, it is of a high interest to develop precise hormone application methods with high spatial resolution and over selected developmental frames. Here we demonstrate an emerging electrophoretic drug delivery technology, the organic electronic ion pump (OEIP), for the flow-free and highly specific transport of isoprenoid-type cytokinin isopentenyladenin (iP) in vivo to intact plant seedlings. Using RSA as a model system, we estimated the resolution of cytokinin delivery to be less than 1 mm, affecting only the OEIP-treated LR, with no effect of neighbouring roots. Moreover, our data demonstrate that while specifically targeting one LR primordia with an OEIP device, the influence of cytokinin in LR development may differ in different stages of LR

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development. Taken together, OEIP based technologies represent a novel and tuneable iP treatment method, which can contribute in unravelling the functions of cytokinin in different developmental processes with great specificity.

BIOLOGICAL DIFFERENCES AMONG THE CDK4/6 INHIBITORS

Miroslav Peřina1, Radek Jorda1

1Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27, 78371 Olomouc,Czech republic

CDK4 and CDK6 are cyclin-dependent kinases that control the transition between the G1 and S phases of the cell cycle, which are typically deregulated and overactive in cancer cells [1]. Three inhibitors of CDK4/6 (Palbociclib, Ribociclib, and Abemaciclib) have been approved for the treatment of breast cancer and several others CDK4/6i (e.g. ON123300, Trilaciclib, Lerociclib, AMG925) entered clinical trials [2, 3]. All drugs mentioned above share similar structural motifs and are reported to have similar mechanisms of action (mostly CDK4/6 inhibition) although recent in vitro data pointed at some distinctions. Selectivity studies clearly confirmed differences in potential off-target kinases [4, 5, 6]. Our aim is to study differences in CDK4/6i at cellular level and show important features that could be therapeutically (dis)advantageous for the potential therapy and to improve the knowledge leading to the possibility of choice of ideal CDK4/6i as a drug. CDK4/6i usually arrest proliferation of cells in G1 phase of the cell cycle without significant induction of the cell death during the short treatment. Our data showed that some CDK4/6 inhibitors cause G2/M block of the cell cycle after 24 h treatment, importantly also in Rb-negative cells. Further, we observed considerable differences between CDK4/6i in acute myeloid leukemia cell lines, namely EOL-1 and MV4-11 cells bearing oncogenic PDGFR and FLT3-ITD mutation, respectively. We found, that while phosphorylation of STAT proteins in studied cells were significantly reduced after the short treatment with Trilaciclib and ON123300, no effect was observed in the cells treated with Palbociclib, Abemaciclib or Ribociclib.

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1. Malumbres, M.; Barbacid, M. Curr Opin Genet Dev. 2007 1, 17. 2. Knudsen, E. S.; Witkiewicz, A. K. Trends Cancer 2017 3, 39. 3. Otto, T.; Sicinski, P. Nat.Rev.Cancer 2017 17, 93. 4. Klaeger, S. et al., Science 2017 358. 5. Chen, P. et al., Mol.Cancer Ther. 2016 15, 2273. 6. Jorda R. et al., J Med Chem. 2018 Oct 25;61(20):9105-9120.

INNOVATIVE, EFFECTIVE, ROBUST, FAST AND CHEAP PURIFICATION OF PHYTOHORMONES USING DISPERSIVE SOLID PHASE EXTRACTION

Ivan Petřík1,2, Anna Valníčková1, Karin Ljung3, Miroslav Strnad1, Ondřej Novák1,3

1Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký

University, Faculty of Science, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 2Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 3Umeå Plant Science Centre,

Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83 Umeå, Sweden

Cytokinins and auxins are naturally occurring plant growth regulators. They play an important role in controlling growth and developmental processes in plants. Similarly to other phytohormones, their concentrations in plant tissues are usually very low (pmol per gram of fresh weight). Therefore, their identification and quantification are based on highly sensitive analytical approaches, such as an ultra-high performance liquid chromatography coupled with a tandem mass spectrometry (LC-MS/MS). Moreover, sample preparation, especially removal of salts and isolation of analytes from a complex plant matrix, is the most critical procedure to achieve high quality data. For many years, the purification of cytokinins and auxins has been based on a well-established solid phase extraction (SPE). However, a dispersive SPE (dSPE) has been introduced a few years ago as an effective and robust approach for isolation of a wide range of analytes. In the contrast with conventional SPEs, this technique uses free solid phase particles dispersed in a

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liquid crude sample extract. We tested dSPE as an innovative, fast and cheap approach to purifying selected phytohormonal groups (cytokinins and auxins). Our work has been mainly focused on investigation of the main parameters contributing to the extraction efficiency compared to the conventional SPE technology. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (European Regional Development Fund-Project ‘Centre for Experimental Plant Biology’ no. CZ.02.1.01/0.0/0.0/16_019/0000738) and the Internal Grant Agency of Palacký University (IGA_PrF_2019_020).

MACHINE READABLE DATA

Michal Polák

Department of Chemical Biology and Genetics, Faculty of Science, Palacky University Olomouc In today’s academic research, there is growing effort to automate some parts of research process. Especially in biological experiments, where experimental data are generated with calibrated devices and standardized protocols. Huge data volume is generated with drones or phenotyping chambers and this is a situation, in which machine learning models can be extremely efficient and useful. Machine learning is the scientific study of algorithms and statistical models that computer systems use to effectively perform a specific task without using explicit instructions, relying on patterns and inference instead. But application of some smart machine learning method is a just tip of the iceberg. Before that, you need to collect data in a consistent way and store it effectively. The goal is to have machine readable and FAIR data (findability, accessibility, interoperability, reusability). It is a long way to reach this goal. You need to define and build data infrastructure which suits well to your experiments. And this issue, I will describe in my presentation.

GIBBERELLIN SIGNALLING IN RESPONSE TO ABIOTIC STRESS IN WHEAT

Klára Ptošková1, Peter Hedden1,2

1Laboratory of Growth Regulators, Czech Academy of Sciences, Institute of Experimental Botany & Palacký University, Šlechtitelů 27, 783 71 Olomouc, Czech Republic 2Rothamsted Research, Harpenden, Hertfordshire AL5

2JQ, United Kingdom Wheat is one of the most important sources of nutrition for humanity globally, but yields can be severely limited by abiotic stress, particularly drought. This work is focused on the role of gibberellin (GA) signalling in the response of wheat seedlings to water limitation. The effect of water restriction on shoot and root growth and on the levels of stress factors and hormones in these organs was determined in soil-grown wheat seedlings. The expression level of genes involved in the GA-biosynthetic and signal transduction pathways was examined in well-watered and stressed leaves and roots by qRT-PCR and RNAseq. GA acts by promoting the degradation of the growth-suppressing DELLA proteins, which accumulate when GA concentrations are low, as can occur under stress conditions. They act by regulating gene expression via interaction with different transcription factors (TFs). The wheat DELLA protein RHT-1 was shown in yeast 2-hybrid assays to interact with PHYTOCHROME INTERACTING FACTOR-LIKE (PIL) TFs,

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which are involved in light-regulated gene expression. As the rice PIL1 has been shown to regulate stem height in response to drought, mutants of the wheat orthologue TaPIL1 and the closely related TaPIL3 were generated from a TILLING population to determine their response to drought. The interaction between RHT1 and PILs is being further tested by bimolecular fluorescence complementation. Future experiments will include determining the response of wheat mutants with altered GA signalling to water limitation.

THE ROLE OF THE PLANT CUTICLE DURING APICAL HOOK DEVELOPMENT

Sara Raggi, Sijia Liu, Stéphanie Robert

Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden

In order to protect the apical meristem from mechanical damage while emerging from the soil, dicotyledonous plants develop a transient structure at the upper end of the hypocotyl named apical hook. Apical hook development involves differential growth in a well-defined time frame, including formation, maintenance and opening phases. Among the cellular signaling mechanisms involved in the regulation of apical hook development, those involving phyto-hormones such as auxin, ethylene and gibberellins are certainly the best characterized. However, the structural modifications implicated in this process are not clearly understood yet. In a search for new players in the modulation of hook development, we found that three cuticle-related mutants, defective in cuticular ridges (dcr), cytochrome p450, family 77, subfamily a, polypeptide 4 (cyp77a4) and lipid transport protein 2 (ltp2), display defective apical hook development. The cuticle is a lipidic layer mainly composed of cutin and waxes, which is deposited outside the cell wall of epidermal cells in aerial organs and on the root cap during early seedling development and lateral root emergence. The mutants selected in our screening are involved in the biosynthesis and transport of cutin precursors: DCR encodes an acyltransferase indispensable for normal cutin formation, CYP77A4 encodes an enzyme able to catalyze fatty acid epoxidation and LTP2 encodes for a lipid transport proteins that maintains the integrity of the cell wall-cuticle continuum in etiolated Arabidopsis hypocotyls. Our data suggest that cutin is important for proper apical hook maintenance during hypocotyl elongation, however, the mechanisms underlying its role remain unclear. Interestingly, the dcr mutant has been previously reported to be resistant to the inhibitory effect of cytokinin on etiolated hypocotyl elongation. In addition, the application of cytokinins promotes a sustained maintenance phase in wild type apical hook while the impaired hook maintenance in either dcr or cyp77a4 can not be rescued by cytokinins. Further characterization of these cuticle-defective mutants will pave the way to a better understanding of the role of the cuticle and cytokinins in apical hook development. Supported by grant the Olle Engkvist Byggmästare Foundation (Sara Raggi) Kempestiftelserna (Sijia Liu) Vetenskapsrådet VR 2016-00768 (Stéphanie Robert)

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THE CYTOTOXIC EFFECT OF FERROCENE DERIVATIVES IN COMBINATION WITH RADIOTHERAPY AGAINST CERVICAL CARCINOMA CELLS

Hana Skoupilováa, Vladimír Rakb,c, Jindřich Karband, Jiří Pinkase and Roman Hrstkaa

aRECAMO, Masaryk Memorial Cancer Institute, Žlutý kopec 7, 656 53 Brno, Czech Republic; bDepartment of Radiation Oncology, Masaryk Memorial Cancer Institute, Žlutý kopec 7, 656 53 Brno, Czech Republic; cDepartment

of Radiation Oncology, Masaryk University, Faculty of Medicine, Kamenice 5, 625 00 Brno, Czech Republic; dInstitute of Chemical Process Fundamentals of the CAS, v. v. i., Rozvojová 135, 165 02 Prague 6, Czech Republic;

eJ. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Dolejškova 2155/3, 182 23 Prague 8, Czech Republic

Cervical cancer is the the second most frequent female malignancy (1). Despite the availability of preventive screening program and vaccination against most prevalent „high-risk“ human papillomaviruses (HPV), approximately 400 women die due to cervical cancer each year in the Czech Republic. Optimal treatment choice depends on the stage of the disease. Early stages are treated by surgery. More advanced stages are usually treated with a combination of radiotherapy and chemotherapy. Most commonly used chemotherapeutic drug is still cisplatin, which has a good antitumor effect but also serious toxicity (2, 3). This fact led us to the synthesis and testing of new potential anti-cancer organometallic compounds. A series of new structures based on ferrocene moiety was tested in vitro and showed even better efficiency than cisplatin (4, 5).

N+

H

FeN+H

Cl-

Cl-

N

FeN

N

OH

OHFe

NHO

HO 1b 2a 3 Scheme 1. - Most active ferrocenes We used cervical cancer cell lines (SiHa, HeLa) and non-tumor cell lines (HEK293, RPE-1) for our testing. Cytotoxicity of the compounds was screened by MTT assay. Several of the tested compounds altered mitochondrial potential, cell cycle distribution, and induced production of oxygen radicals, which was associated with significant cytotoxicity. These compounds were then tested in combination with ionizing radiation to analyze possible radiosensitizing effect. We used a colony forming assay (CFA), which evaluates ability of cells to create colonies after the treatment and represents a good option for measurement of tumor cells’ potential to cause recurrence of disease. A candidate molecule 1b was identified among tested substances as the most promising, since it increases the sensitivity of cells to ionizing radiation by more than 50% in both cervical cancer cell lines. The results of our work indicate promising enhancement of antitumor activity that some of the tested ferrocenes displayed in combination with radiotherapy, suggesting that the combined effects of ionizing radiation and organometallic compounds could bring new insights for scientific research as well as clinical practice. This project was supported by GACR 17-05838S, MEYS - NPS I - LO1413 and MH CZ - DRO (MMCI, 00209805).

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1. International Agency for Research on Cancer – Cancer Fact Sheets 2012. 2. Sturdza et al. Radiother Oncol. 2016; 120(3): 428-433. 3. Vale et al. J Clin Oncol. 2008; 26(35): 5802-5812. 4. Bartošík et al. Analyst. 2015; 140(17): 5864-5867. 5. Hodík et al. J. Organomet. Chem. 2017; 846: 141-151.

PREPARATION AND BIOLOGICAL ACTIVITY OF NOVEL N6 SUBSTITUTED PURINE RIBOSIDES WITH POTENTIAL ANTITUMOR EFFECT

Šárka Slamencová1, Magdaléna Bryksová1, Jiří Voller2,3 and Karel Doležal1,2

1Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and

Agricultural Research, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic. 2Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27, 78371 Olomouc,

Czech Republic 3Department of Clinical and Molecular Pathology, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Hněvotínská 3, 775 15 Olomouc, Czech

Republic Cytokinin ribosides (structurally N6 substituted adenosines) show usually high activity in cytokinin bioassays, one of the most active compound is for example meta-topolin riboside. On the other hand, some of the ortho-hydroxylated aromatic cytokinin ribosides exhibit considerable cytotoxic effect against various human cell lines. Several of these cytotoxic compounds occur also naturally in planta – ortho-topolin riboside (oTR) riboside or 2-hydroxy-3-methoxybenzylaminopurine riboside (2OH3MeOBAPR) are examples of them. Both compounds express very strong cytotoxicity against cancer cell lines, which can be used in cancer therapy, although the mechanism of 2OH3MeOBAPR action is still unknown. Based on the cytotoxicity of oTR and 2OH3MeOBAPR, six derivatives of oTR with different substituents on the benzene ring were synthesized through condensation of 6-chlorpurine riboside with appropriate benzylamine or through reaction of inosine with appropriate benzylamine. Prepared compounds were then purified and analysed with physico-chemical methods (HPLC-UV/LSD, UHPLC-HR MS/MS, NMR, EA, melting point) The cytotoxicity was tested on cancer (K-567, MCF7) and normal (ARPE-19, HaCaT, BJ) cell lines. Some of the prepared compounds showed high cytotoxicity on cancer cell lines, but unfortunately also on normal cell lines. On the other hand, two of prepared compounds were absolutely nontoxic on tested cell lines. Cytokinin activity of prepared compound was measured with senescence, tobacco callus and Amaranthus bioassays. This work was supported by the Erasmus+, the Ministry of Education, Youth and Sport of the Czech Republic, ERDF project "Plants as a tool for sustainable global development" (No. CZ.02.1.01/0.0/0.0/16_019/0000827) as well as by the Internal Grant Agency of Palacký University (IGA_PrF_2019_018).

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THE HEAT SHOCK RESPONSE REGULATION IN CANCER CELLS

Oliver Šimončík, Petr Müller, Veronika Martinková, Bořivoj Vojtěšek

Masaryk Memorial Cancer Institute, Regional Centre for Applied Molecular Oncology, Brno, Czech Republic Activation of stress response manifested by increased activity of chaperones is a natural compensatory mechanism that accompanies a malignant transformation. Activation of the transcription factor HSF1, which regulates the expression of stress proteins, is then essential for the survival of tumour cells. Although HSF1 and the heat shock response has been extensively studied, key aspects governing their activation in cancer remain a mystery. To study the mechanism of HSF1 activation, we have established several independent methods to characterize its trimerization, conformation and DNA binding. We used cell line models as well as recombinant purified HSF1 to investigate the key mechanisms of HSF1 activation. We have established stable cell lines expressing fluorescent fusion proteins of HSF1 and its homolog HSF2 to study their subcellular localization and chromatin interactions. Our previous results have shown that HSF1 is localized in cell nuclei even under normal conditions. However, isolation of cell nuclei revealed that inactive monomeric HSF1 is soluble and is washed out from the nuclear fraction and therefore appears in the cytoplasmic fraction. In contrast to physiological conditions, stress activates HSF1 trimerization and subsequently HSF1 associates with the nuclear chromatin fraction. We used western blotting to distinguish HSF1 solubility in control and stressed cells in previous reports. We have also developed a rapid and cost-effective method based on fluorescent detection of mCherry-HSF1 that allows reproducible quantification of HSF1 in soluble and insoluble fractions and makes it suitable for reliable screening of HSF1 activators. To study the conformational changes of HSF1 and mechanisms responsible for its trimerization, we have used purified recombinant HSF1. High resolution native electrophoresis was used to quantify HSF1 trimerization induced by macromolecular crowding and/or by increased temperature. The results demonstrate direct activation of HSF1 in response to increased macromolecular crowding and increased temperature. These results indicate that HSF1 works as a primary sensor of proteotoxic stress since mechanisms responsible for stress sensing lies in conformational change leading to its trimerization. Acknowledgement: This project was supported by the GAČR 17-07822S and MEYS - NPS I - LO1413

BIOSYNTHESIS AND STRUCTURAL DIVERSITY OF PHENOLIC ACIDS IN OAT

Klára Supíková1, Andrea Luterová1, Ron Wever2, Anja Hartmann3, Jiří Pospíšil1, Miroslav Strnad1, Jiří Grúz1

1Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 27,

78371 Olomouc,Czech republic 2Van 't Hoff Institute for Molecular Sciences, Faculty of Science, University of Amsterdam, Netherlands 3Department of Pharmacognosy, University of Innsbruck, Austria

For the study of biosynthesis and structural diversity of phenolic acids feeding experiment was performed. Isotopically labeled benzoic (BA) and trans-cinnamic acids (tCA) were applied to seedlings of oat (Avena sativa) which was selected as a representative of monocotyledonous

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plants. Treated oat was extracted and analyzed by differential LC-MS/MS. Data were processed by in house metabolomic software followed by manual sorting. Altered isotopic profiles of metabolites were searched which resulted in detection of about 100 candidate features. Together 18 metabolites were detected and structurally characterized after that. Most of the tCA-derivatives were predominantly hydroxy/methoxy derivatives and conjugates with hexoses and other substances, whereas hydroxylation of BA did not occur. BA formed conjugates with other substances but exclusively via its carboxyl group. Further tCA-derivatives were not synthesizes from BA and vice versa. Part of the study was analysis of naturally occuring BA or tCA-derived metabolites in oat. There were detected for example conjugates with malate or with hexoses. Suprisingly there were detected group of sulfated phenolic acids. While free BAs or CAs were not detected in oat, these sulfated phenolic acids were detected as free. Some of these sulfated substances are known to occur in seagrass Zostera marina or in algae Dasycladus vermicularis.

HSF1 INTERACTION NETWORK UNDER STRESS CONDITIONS

Vlastimil Tichy, Petr Muller, Veronika Martinkova, Filip Trcka, Michal Durech, Jakub Faktor, Borivoj Vojtesek

Masaryk Memorial Cancer Institute, Regional Centre for Applied Molecular Oncology, Brno, Czech Republic

The eukaryotic heat shock response is an important adaptation mechanism resulting in production of many cytoprotective proteins in the presence of thermal and other stress. One of the major regulators of cellular heat shock response is Heat Shock Factor 1 (HSF1) that plays an important role as a central sensor of proteotoxic stress. Higher temperatures can cause the C-terminal domain of HSF1 to unfold, expose hydrophobic leucine zippers, and result in the generation of DNA-binding HSF1 trimers. Currently, the negative regulation of HSF1 protein by HSP70 / HSP90 chaperones is well described, but the factors involved in HSF1 activation are largely unexplored. For this reason, we have focused our study on identifying proteins that interact with activated HSF1 in response to various stress conditions. For these interaction studies we created human cell lines producing the SBP tagged HSF1 protein, which exhibits a monomeric conformation under normal physiological conditions. To find out the possible common features of protein complexes associated with activated HSF1, we used various stress stimuli including heat shock and Hsp90 or proteasome inhibitors. In pulldown studies, samples were tested at several time intervals to monitor HSF1 activation dynamics, its post-translational modification, and assembly of protein complexes. Due to quantitative proteomics, we have revealed several protein clusters that produce different protein complexes with activated HSF1 trimers. The first and probably the most important group are proteins assembled into the prefoldin-like (R2TP/PFDL) complex and U5 snRNP spliceosome (RUVBL1, RUVBL2, AAR2, EFTUD2, PRPF8 and SNRNP200). In the second group of interaction partners, we can include the nuclear proteins AAA+, such as KIF4A, which interaction is associated with the sumoylation of HSF1. Hsp70 isoforms and their co-chaperones represent the third group of interacting proteins involved in negative feedback control. Our results demonstrate the complexity of spaciotemporal changes in protein interactions during stress exposure and reveal a link between HSF1 activation, chromatin remodelling, and recruitment of pre-mRNA splicing factors.

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The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic; National Programme of Sustainability I (MEYS – NPS I – LO1413) and by the Ministry of Health Development of Research Organization, MH CZ - DRO (MMCI, 00209805). IDENTIFICATION OF BIOLOGICALLY ACTIVE SUBSTANCES FROM DIFFERENT TYPES OF BIOSTIMULANTS IN THE INTERACTION WITH THE MODEL PLANT

ARABIDOPSIS THALIANA

Lydia Ugena1, Goizeder Almagro2, Javier Pozueta-Romero2, Nuria De Diego1, Lukáš Spíchal1, Jiří Grúz3

lydia.ugena@upol.cz

1Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Šlechtitelů 27, Palacký University, Olomouc, CZ-78371, Czech Republic 2Instituto de

Agrobiotecnología (CSIC/Gobierno de Navarra). Avenida Pamplona 123, 31192 Mutilva, Navarra, Spain 3Laboratory of Growth Regulators, The Czech Academy of Sciences, Institute of Experimental Botany & Palacký

University, Šlechtitelů 27, Olomouc CZ-78371, Czech Republic. The interaction between plants and microorganisms occurs in many different ways and different levels 1. Microbes may provide compounds that stimulate plant growth, making the plant more resistant to abiotic or biotic stress or enhancing nutrient uptake. For these reasons, microorganisms are defined as biostimulants 2. The study of the interaction between plant and microorganism that reveals mechanisms of plant reactions is conducted on the level of genome, transcriptome, proteome, and metabolome 3. Metabolomic technique is an important part of research conducted in the direction of breeding new varieties of crop plants tolerant to the affecting stresses and possessing good agronomical features. These studies would permit to define the physiological and biochemical changes in the mechanisms of plant tolerance to stress 3. Arabidopsis thaliana has been exposed to diverse microorganisms with different phylogenia, fungi and bacteria, via flood-inoculation technique 4. Moreover, the interaction has been performed in various concentrations to check the level of change produced in the metabolome. Afterwards, the metabolomic screening performed by UPLC-QTOF-MS/MS has played an important role for the identification of biologically active substances. In this approach, it has been possible to observe a clear difference between the metabolome obtained from the interaction from fungi than bacteria, where fungi interaction provides better positive results. Common candidates found for all treatments are considered as marker stress, while the search of interaction specific candidates is the main goal to go further with the reseach. 1 Schirawski, J. and Perlin, M. H. International journal of molecular sciences, vol.19, no.5, 2018. 2 du Jardin, P. Scientia Horticulturae, vol.196, pp.3–14, 2015. 3 Piasecka, A., Kachlicki, P., and Stobiecki, M. International journal of molecular sciences, vol.20, no.2, 2019. 4 Ishiga, Y., Ishiga, T., Uppalapati, S. R., et al. Plant Methods, vol.7, no.1, pp.32, 2011.

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ANALYSIS OF INTERACTION BETWEEN CD20 AND NEWLY DEVELOPED MONOCLONAL ANTIBODY USING HYDROGEN-DEUTERIUM EXCHANGE MASS

SPECTROMETRY

Lukas Uhrik1, Adam Krejci1, Marta Nekulova1, Lenka Hernychova1, Petr Muller1, Ted Hupp2, Borivoj Vojtesek1

1Masaryk Memorial Cancer Institute, Regional Centre for Applied Molecular Oncology, Brno, Czech Republic,

2Institute of Genetics and Molecular Medicine, Edinburgh Cancer Research UK Centre, Edinburgh, UK Lymphomas belong to the most common malignant tumours of dogs and most of the canine lymphomas originate from transformed B-cells. CD20 is a cell surface antigen of B-cells and it has a role in their proliferation and activation. Therapeutic monoclonal antibodies (mAbs) such as Rituximab, Ofatumumab and Ocrelizumab that targets CD20 are successfully used to treat human cancer and autoimmune diseases. CD20 mAbs can act via various mechanisms including direct induction of apoptosis, antibody-dependent cell-mediated toxicity and complement-dependent lysis. Recently, we developed NCD1.2 mAb, which specifically binds canine CD20 and can be used in veterinary medicine as a diagnostic and potentially therapeutic tool [1]. Using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) we analysed interaction between NCD1.2 mAb and CD20 canine peptide. We compared samples of deuterated NCD1.2 with and without CD20 peptide preincubation. The formation of NCD1.2-CD20 peptide complex decreased deuteration of peptides located at the sites of interaction. Using HDX-MS, we found that the residues from CDR1 (complementarity determining region) of light chain, CDR2 and CDR3 of heavy chain are the main mediators of CD20 binding. Increased deuteration levels in NCD1.2 peptides which do not correspond to the interaction interface, shows that the antibody relaxes some regions to enable CDRs to bind antigen more tightly. These findings help us to understand the mechanism how antibody binds its epitope on the antigen and adapts its structure, which could help to developing more effective diagnostic and therapeutic agents in human as well as veterinary medicine. The work was supported by the project MEYS - NPS I - LO1413 [1] Jain, S., Aresu, L., Comazzi, S., Shi, J., Worrall, E., Clayton, J., Humphries, W., Hemmington, S., Davis, P., Murray, E., Limeneh, A., Ball, K., Ruckova, E., Muller, P., Vojtesek, B., Fahraeus, R., Argyle, D. and Hupp, T. (2016). The Development of a Recombinant scFv Monoclonal Antibody Targeting Canine CD20 for Use in Comparative Medicine. PLOS ONE, 11(2), p.e0148366.

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MOLECULAR DOCKING AND STRUCTURE FUNCTION RELATIONSHIPS TO IDENTIFY NOVEL ANTI OESTROGENS

Petr Voňkaa,b, Lucie Rárovác, Václav Bazgierb,d, Karel Berkad,e, Miroslav Kvasnicab, Jana

Oklešťkováb, Eva Kudováf, Miroslav Strnadb, Dalibor Valíka, Roman Hrstkaa,b

aRECAMO, Masaryk Memorial Cancer Institute, Žlutý kopec 7, 656 53 Brno, Czech Republic bLaboratory of Growth Regulators, Institute of Experimental Botany AS CR & Palacký University Olomouc, Šlechtitelů 27, 783 71 Olomouc,

Czech Republic cDepartment of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University Olomouc, Šlechtitelů 27, 783 71 Olomouc, Czech Republic

dDepartment of Physical Chemistry, Faculty of Science, Palacký University Olomouc, třída 17. listopadu 12, 771 46 Olomouc, Czech Republic eRegional Centre of Advanced Technologies and Materials, Department of Physical

Chemistry, Palacký University Olomouc, třída 17. listopadu 1131, 779 00 Olomouc, Czech Republic fInstitute of Organic Chemistry and Biochemistry AS CR, Flemingovo náměstí 2, 166 10 Praha 6, Czech Republic

Oestrogen receptor (ER) is a key biomarker for breast cancer, and ER presence or absence in breast and other cancers influences treatment regimens and patient prognosis. ERs are activated after ligand binding (typically by the steroid, oestradiol), which is accompanied by conformational changes, mainly by phosphorylation and dimerisation. ERs then translocate into the nucleus where they bind EREs (Oestrogen receptor Responsive Elements) and activate transcription of specific genes. Steroids represent a group of chemical compounds with four rings skeleton. A library consisting of approximately 8 000 compounds with described synthetic pathways was used for molecular docking studies to find novel potential ligands for ERs to predict compounds that may show anticancer activity. Compounds potentially interacting with ERs were chosen from the library of steroid compounds by molecular docking analyses. The structure of ER with oestradiol (PDB ID: 1ERE) representing its natural ligand was used for docking. The cytotoxic effect of selected compounds was tested experimentally using MTT (3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide) cell viability test in MCF-7 cells (ER-dependent breast cancer). The impact on ER activity was determined by luciferase reporter test, which we developed for this purpose. Twenty high affinity compounds were chosen from the library by molecular docking. Two compounds, MU-5562 and MU-5611, showed ER inhibitory activity comparable to the clinically used ER inhibitors tamoxifen and fulvestrant. We found that our compounds stabilize ERs like tamoxifen. Determination of luciferase activity showed reduced signals, similar to commercial inhibitors. However, immunochemical analysis revealed decreased AGR2 expression, indicating a different mechanism of action compared to tamoxifen. The inhibitory effect of these compounds on ER is probably caused by the presence of a double bond in their D ring, which protects activation of ERs by decreasing the electron density of the keto group. This configuration blocks the development of hydrogen bonds, which is responsible for conformational changes of the α helix H12 associated with ER activation. The combination of computational and experimental methods represents rapid approach to determine the activity of compounds towards ERs. These data will be helpful not only in research of ER activity and inhibition, but may also be useful for the development of novel drugs for clinical application. This work was supported by the project MEYS-NPS I-LO1413, by MH CZ DRO (MMCI, 00209805), by the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and by the Grant Agency of the Czech Republic (GACR 19 01383S, and GACR 19-02014S).

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NEW ANTI-AUXIN WITH DIFFERENT ACTIVITY IN ROOTS AND SHOOTS

Asta Žukauskaitė1, Kristýna Bieleszová1, Iñigo Saiz-Fernández2, Michaela Sedlářová3, Monika Iškauskienė4, Martin Kubeš1,5, Karolina Dzedulionytė4, Barbora Pařízková1, Iva Pavlović1,

Václav Bazgier6, Thomas Vain7,*, Vida Malinauskienė4, Algirdas Šačkus4, Stéphanie Robert7, Richard Napier5, Miroslav Strnad1, Karel Doležal8, Ondřej Novák1

1Laboratory of Growth Regulators, Faculty of Science, The Czech Academy of Science, Institute of Experimental

Botany & Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic 2Laboratory of Plant Molecular Biology, Institute of Biophysics AS CR, CEITEC – Central European Institute of Technology, Phytophthora Research

Centre, Faculty of Agronomy, Mendel University in Brno, Zemědělská 1, CZ-613 00, Brno, Czech Republic 3Department of Botany, Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic

4Department of Organic Chemistry, Kaunas University of Technology, Radvilėnų pl. 19, LT-50254, Kaunas, Lithuania 5School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK 6Department of Physical

Chemistry, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, 17. listopadu 12, CZ-771 46 Olomouc, Czech Republic 7Department of Forest Genetics and Plant Physiology, Umeå

Plant Science Centre, Swedish University of Agricultural Sciences, SE-90183 Umeå, Sweden 8Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty

of Science, Palacký University, Šlechtitelů 27, CZ-78371 Olomouc, Czech Republic *Present address: DIADE, University of Montpellier, IRD, 911 avenue Agropolis, 34394 Montpellier, France

Auxins are a class of plant hormones influencing nearly every stage of plant life cycle. Anti-auxins, on the contrary, competitively inhibit the actions of auxins. Until recently, synthetic auxins and anti-auxins were discovered through biological studies of synthetic auxin analogues and related compounds. The identification of TIR1 as an auxin receptor enabled rational design of novel auxins and anti-auxins based on their predicted binding to TIR1 by in silico screening. Herein, we present a novel auxin analogue which was predicted to be a strong TIR1 antagonist by in silico docking experiments. Its anti-auxin activity was confirmed in vitro by SPR analyses. In vivo, it antagonizes classical auxin responses, such as primary root growth inhibition, expression of GUS and GFP signals in the roots of auxin-responsive reporter lines DR5::GUS and DR5::GFP, respectively, and degradation of DII-VENUS signal in the roots of p35S::DII-VENUS reporter line. In the hypocotyls of MBD::GFP plants, it antagonizes auxin effects such as reorientation of the cytoskeleton and decrease in microtubule density, as well as reduction in cell elongation. However, unlike auxinole, when applied alone it does not induce significant changes to the aforementioned parameters. Our study shows that this new compound acts as a strong anti-auxin in the roots and that this effect is not translated into activity in the shoots, differentiating it from auxinole and making it a promising candidate for potential applications in basic plant research. Supported by the Internal Grant Agency of Palacký University (IGA_PrF_2019_020).