Summary of PhD. thesis

Summary of PhD. thesis Cell division represents one of fundamental attributes of all living creatures. Basic molecular mechanisms operating during cell proliferation seem to be evolutionary conserved among eukaryotes. The cell cycle is divided in four subsequent phases; the most regulatory events are concentrated in G 1 /S and G 2 /M checkpoints. The key regulatory proteins, cyclin-dependent kinases (CDKs), govern the progress through the whole cell cycle. Their function is strictly dependent on catalytic cyclin subunits. The corresponding cyclin partner binding CDK determines the time window of the specific CDK/cyclin complex action in individual cell cycle phase. To become fully active, the complex requires further posttranslational modification including activatory phosphorylation and dephosphorylation of the CDK on specific amino acid residues. Plant cell cycle, besides well-conserved mechanisms common to all eukaryotes, exhibits other specific mechanisms resulting from plant survival strategy. The G 1 /S transition is strongly affected by external and internal signals, mainly phytohormones and metabolites, reflecting the elementary conditions suitable for accomplishment of the whole cell cycle. The central molecule responding to these signals at G 1 /S is D cyclin, whose expression is regulated by cytokinin and sucrose (Riou-Khamlichi et al., 1999; Riou-Khamlichi et al., 2000). The control of plant G 2 /M transition, however, still remains a bit obscure. The cell at G 2 /M is primarily monitoring whether the DNA replication has been finished, and in other eukaryotes, it consequently activates CDK phosphatase Cdc25 that is serving as allor-nothing positive signal for the entry into mitosis (e.g. Moreno et al., 1990; O'Farrell, 2001). Although there has been great effort to identify a homologue to this enzyme in plants, unfortunately the regulatory protein responsible for dephoshorylation of higher plant CDK at G 2 /M is so far unknown. Nevertheless, it was documented for several times that this regulation is operating in plants and moreover it was shown to be under the positive control of cytokinins (Zhang et al., 1996; Zhang et al., 2005). Recently, the screening of Arabidopsis and rice genomes revealed a small gene coding for Cdc25 CDK phosphatase catalytic domain (Landrieu et al., 2004). This gene is, however, unable to complement yeast CDC25 mutant strains and moreover its overexpression in plants does not exhibit phenotypic changes (Landrieu et al., 2004; Sorrell et al., 2005). Based on these findings, Boudolf et al. (2006) suggested recently that plants could lack Cdc25 phosphatase and that its role might have been evolutionarily replaced by a B-type CDK-dominated 1

pathway. However, this hypothesis neglects the fact that CDK undergoes before mitosis activatory dephosphorylation and inhibition of this action (e.g. by application of lovastatin - inhibitor of isoprenoid-type cytokinin synthesis) causes arrest of the cells in G 2 phase (Laureys et al., 1998). Moreover, as described by Sorrell et al. (2002) and Orchard et al. (2005) the G 2 /M specific CDKB is most likely phosphoregulated at the same evolutionary conserved amino acid residues as CDKA is. All these arguments together with the fact, that the counteracting CDK kinase Wee1 at G 2 /M transition has been repeatedly identified in higher plants (Sun et al., 1999; Sorrell et al., 2002; Gonzalez et al., 2004) strongly support the important role of CDK activatory dephosphorylation in plants. As the search for plant homologue fails until now it is useful to employ the plants carrying foreign cdc25 gene and thus to study the effect of this regulatory step on plant cell cycle dependent processes. The results concerning plants with introduced fission yeast (model organism of eukaryotic cell cycle regulation) cdc25 gene (Spcdc25) confirmed that the yeast protein is functional in plants fulfilling the same role as in donor organism (Bell et al., 1993; Zhang et al., 1996; Zhang et al., 2005). Given the Spcdc25 phosphatase overexpression in plants affects the duration of G 2 phase and promotes the entry into mitosis we could expect that the processes dependent on cell division regulation would be influenced. Bell et al. (1993) transformed tobacco with fission yeast Spcdc25 cdna and showed that there is a dramatic impact of Spcdc25 overexpression on plant habitus and ontogenesis without any detail analysis. These findings stimulated us to study the effect of Spcdc25 expression on different plant processes closely related to cell division regulation and supply further pieces to the mosaic of morphological, developmental and biochemical changes induced by overexpression of the mitotic activator thus to propose a model of the interaction with plant growth and development. In this thesis the results analysing the impact of Spcdc25 expression on processes differing primarily in organisation levels under in vitro conditions, i.e. on de novo organ formation (paper 1), on the flowering onset (paper 3) and on cell suspensions characteristics (paper 4,5), were included. A new methodical approach used as a part of the Spcdc25 detection experiments was also incorporated in the thesis (paper 2). 2

The main conclusions on the effect of Spcdc25 expression are as follows: The changes in de novo organ formation mimic the cytokinin effect as there is promotion of shoot formation and restriction of rhizogenesis in transgenics. Spcdc25 and sucrose act synergistically on flower induction thus confirming the key role of cell division and saccharide signalling in flowering onset. The acceleration of the entry into mitosis substantially influenced the orientation of cell division and cells expressing Spcdc25 become cytokinin-independent at G 2 /M transition. Besides changes in rate and orientation of cell division, a shift in carbohydrate status was found, indicating the interaction of cell cycle regulation with plant metabolism being a complex one. In conclusion, the results further confirm the existence and importance of CDK activatory dephosphorylation at plant G 2 /M transition and support the model of cytokinin stimulation of this regulatory step. The results also indicate the possibility, that the decision about mitosis onset generates a signal emanated towards plant metabolism. 3

Shrnutí disertační práce Buněčné dělení je jednou z elementárních vlastností všech živých organismů. Základní mechanismy regulující proces buněčné proliferace na molekulární úrovni jsou evolučně konzervovány napříč eukaryotickou říší. Buněčný cyklus se standardně dělí na čtyři následné fáze, přičemž hlavní regulační kroky se soustřeďují do dvou kontrolních bodů, na přechodu G 1 /S a G 2 /M fáze. Klíčové regulační proteiny, cyklin-dependentní kinázy (CDK), řídí postup celým buněčným cyklem. Jejich funkce je striktně závislá na katalytické cyklinové podjednotce. Odpovídající cyklinový partner interagující s CDK totiž určuje dobu aktivity konkrétního CDK/cyklinového komplexu v jednotlivých fázích cyklu. Pro získání úplné aktivity vyžaduje komplex další posttranslační modifikace, mezi které patří i aktivační fosforylace a defosforylace CDK na specifických aminokyselinových reziduích. Rostlinný buněčný cyklus disponuje vedle evolučně konzervovaných mechanismů i dalšími regulačními procesy, které souvisejí s odlišnou životní strategií rostlin. Průchod G 1 /S kontrolním bodem je silně závislý na signálech z vnějšího a vnitřního prostředí, především fytohormonech a metabolitech, buňka tak monitoruje příznivost podmínek pro dokončení celého buněčného cyklu. Limitujícím faktorem pro průchod G 1 /S fází je D cyklin, jehož exprese je pod kontrolou cytokininů a sacharidů (Riou-Khamlichi et al., 1999; Riou-Khamlichi et al., 2000). Regulace G 2 /M přechodu rostlinného buněčného cyklu však zůstává nadále poněkud nejasná. Buňka v tomto kontrolním bodě primárně monitoruje, zda byla zcela dokončena replikace DNA. U ostatních eukaryot následně dochází k aktivaci CDK fosfatázy Cdc25, jež slouží jako definitivní signál stimulující zahájení mitózy (např. Moreno et al., 1990; O'Farrell, 2001). Přes velkou snahu identifikovat rostlinný homolog Cdc25 fosfatázy, bylo pátrání po proteinu zodpovědném za aktivační defosforylaci rostlinné CDK v G 2 /M fázi zatím neúspěšné. Přesto však bylo opakovaně prokázáno, že tato regulace u rostlin probíhá a je pod pozitivní kontrolou cytokininů (Zhang et al., 1996; Zhang et al., 2005). V nedávné době se při prohledávání genomových sekvencí Arabidopsis a rýže podařilo objevit malý gen kódující katalytickou podjednotku Cdc25 fosfatázy, jež však není schopna komplementovat kvasinkové CDC25 mutantní kmeny (Landrieu et al., 2004). Nadexprese tohoto genu v rostlinách navíc nevyvolává fenotypické změny (Sorrell et al., 2005). Na základě těchto výsledků prezentovali Boudolf a kol. (2006) novou originální představu o mechanismu regulace 4

G 2 /M fáze rostlinného buněčného cyklu, kde nastínili možnost, že rostliny postrádají Cdc25 fosfatázu a že její role by mohla být evolučně nahrazena regulační drahou řízenou CDKB kinázou. Tato představa však opomíjí důležitý fakt, že pro průchod G 2 /M fází je nutná defosforylace CDK a že inhibice tohoto kroku (např. aplikací lovastatinu - inhibitoru syntézy izoprenoidních cytokininů) vede k zastavení buněk v G 2 fázi (Laureys et al., 1998). Je také nutno připomenout, že G 2 /M specifická CDKB velmi pravděpodobně podstupuje (de)fosforylaci na evolučně konzervovaných aminokyselinových reziduích podobně jako CDKA (Sorrell et al., 2002; Orchard et al., 2005). Všechny tyto skutečnosti spolu s faktem, že u vyšších rostlin byla opakovaně prokázána existence inaktivační Wee1 kinázy fosforylující stejná aminokyselinová místa na molekule CDK (Sun et al., 1999; Sorrell et al., 2002; Gonzalez et al., 2004), podporují rozhodující úlohu aktivační defosforylace CDK u rostlin při vstupu do mitózy. Jelikož se dosud nepodařilo identifikovat rostlinný homolog tohoto proteinu, je studium rostlin s introdukovaným cizorodým cdc25 genem stále opodstatněné a aktuální a skýtá možnost studovat vliv tohoto regulačního kroku na růst a vývoj rostlin. Výsledky získané pomocí rostlin s vneseným cdc25 genem z poltivé kvasinky (modelový organismus pro studium regulace buněčného cyklu) (Spcdc25) potvrdily, že kvasinková fosfatáza je funkční v rostlinných buňkách a plní stejnou regulační roli jako v donorovém organismu (Bell et al., 1993; Zhang et al., 1996; Zhang et al., 2005). Vycházíme-li z poznatků, že nadexprese Spcdc25 fosfatázy u rostlin ovlivňuje délku G 2 fáze a urychluje vstup do mitózy, lze předpokládat, že budou ovlivněny jakékoli procesy závislé na regulaci buněčného dělení. Bell et al. (1993) transformovali tabák Spcdc25 cdna z poltivé kvasinky a v orientačních experimentech zaznamenali významné změny rostlinného habitu a vývojové posuny vyvolané nadexpresí Spcdc25. Tato zjištění vedla naši pracovní skupinu k myšlence testovat ovlivnění dalších vývojových procesů spojených se změnou intenzity buněčného dělení, doplnit tak mozaiku vývojových, morfologických a biochemických změn vyvolaných expresí mitotického aktivátoru a na základě těchto dat vytvořit návrh možných interakcí s rostlinnými regulačními drahami. Experimenty uvedené v předložené práci byly zaměřené na zhodnocení vlivu exprese Spcdc25 na vybrané růstové a vývojové procesy v podmínkách in vitro, a to na odlišných úrovních organizovanosti kultury, tj. zakládání orgánů de novo (publikace 1), regulaci nástupu kvetení (publikace 3) a charakteristiky buněčných kultur (publikace 4, 5). Do uvedené práce byl zařazen i článek týkající se nového metodického přístupu využitého při detekci Spcdc25 transkriptu (publikace 2). 5

Hlavní závěry vyplývající z disertační práce Změny v de novo organogenezi vyvolané expresí Spcdc25 simulují vliv cytokininů; exprese Spcdc25 podporuje zakládání prýtů a potlačuje rhizogenezi. Spcdc25 a sacharóza působí synergicky na květní indukci, a potvrzují tak rozhodující úlohu buněčného dělení a sacharidů v přepnutí vývojového programu. Urychlení nástupu mitózy významně ovlivňuje orientaci buněčného dělení a buňky s expresí Spcdc25 se stávají při průchodu G 2 /M fází nezávislé na cytokininu. Exprese Spcdc25 vedle změn v rychlosti a orientaci buněčného dělení vyvolatelných cytokininem mění i sacharidový status buňky, což nasvědčuje komplexním interakcím buněčného cyklu s rostlinným metabolismem. V souhrnu výsledky přinášejí další důkaz pro existenci a významnost aktivační defosforylace CDK v G 2 /M fázi rostlinného buněčného cyklu a podporují i model regulace tohoto kroku cytokininem. Zároveň naznačují i možnost, že rozhodnutí o vstupu do mitózy generuje signál vysílaný směrem do metabolismu buňky. 6

References: Bell MH, Halford NG, Ormrod JC, Francis D. 1993. Tobacco plants transformed with cdc25, a mitotic inducer gene from fission yeast. Plant Mol Biol 23(3): 445-451. Boudolf V, Inze D, De Veylder L. 2006. What if higher plants lack a CDC25 phosphatase? Trends Plant Sci 11(10): 474-479. Gonzalez N, Hernould M, Delmas F, Gevaudant F, Duffe P, Causse M, Mouras A, Chevalier C. 2004. Molecular characterization of a WEE1 gene homologue in tomato (Lycopersicon esculentum Mill.). Plant Mol Biol 56(6): 849-861. Landrieu I, da Costa M, De Veylder L, Dewitte F, Vandepoele K, Hassan S, Wieruszeski JM, Corellou F, Faure JD, Van Montagu M, Inze D, Lippens G. 2004. A small CDC25 dual-specificity tyrosine-phosphatase isoform in Arabidopsis thaliana. Proc Natl Acad Sci U S A 101(36): 13380-13385. Laureys F, Dewitte W, Witters E, Van Montagu M, Inze D, Van Onckelen H. 1998. Zeatin is indispensable for the G2-M transition in tobacco BY-2 cells. FEBS Lett 426(1): 29-32. Moreno S, Nurse P, Russell P. 1990. Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast. Nature 344(6266): 549-552. O'Farrell PH. 2001. Triggering the all-or-nothing switch into mitosis. Trends in Cell Biology 11(12): 512-519. Orchard CB, Siciliano I, Sorrell DA, Marchbank A, Rogers HJ, Francis D, Herbert RJ, Suchomelova P, Lipavska H, Azmi A, Van Onckelen H. 2005. Tobacco BY- 2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle. Plant Journal 44(2): 290-299. Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JA. 1999. Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science 283(5407): 1541-1544. Riou-Khamlichi C, Menges M, Healy JM, Murray JA. 2000. Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Mol Cell Biol 20(13): 4513-4521. Sorrell DA, Chrimes D, Dickinson JR, Rogers HJ, Francis D. 2005. The Arabidopsis CDC25 induces a short cell length when overexpressed in fission yeast: evidence for cell cycle function. New Phytologist 165(2): 425-428. Sorrell DA, Marchbank A, McMahon K, Dickinson JR, Rogers HJ, Francis D. 2002. A WEE1 homologue from Arabidopsis thaliana. Planta 215(3): 518-522. Sun Y, Dilkes BP, Zhang C, Dante RA, Carneiro NP, Lowe KS, Jung R, Gordon- Kamm WJ, Larkins BA. 1999. Characterization of maize (Zea mays L.) Wee1 and its activity in developing endosperm. Proc Natl Acad Sci U S A 96(7): 4180-4185. Zhang K, Letham DS, John PC. 1996. Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase. Planta 200(1): 2-12. Zhang KR, Diederich L, John PCL. 2005. The cytokinin requirement for cell division in cultured Nicotiana plumbaginifolia cells can be satisfied by yeast cdc25 protein tyrosine phosphatase. Implications for mechanisms of cytokinin response and plant development. Plant Physiology 137(1): 308-316. 7

Publications 1. Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator Petra Suchomelová, DenisaVelgová, Tomáš Mašek, Dennis Francis, Hilary J. Rogers, Angela M. Marchbank, Helena Lipavská (Plant Physiology and Biochemistry, 2004, 42, 49 55) During the last decade, the cell cycle and its control by cyclin-dependent kinases (CDKs) has been extensively studied in eukaryotes. The regulation of CDK activity includes, among others, its activation by Cdc25 phosphatase at G2/M. However, within the plant kingdom studies of this regulation have lagged behind and a plant cdc25 homologue has not been identified yet. Here, we report on the effects of transformation of tobacco (Nicotiana tabacum L., cv. Samsun) with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on de novo plant organ formation, a process dependent on rate and orientation of cell division. On shoot-inducing medium (low 1-naphthylacetic acid (NAA), high 6-benzylaminopurine (BAP)) the number of shoots formed on internode segments cultured from transgenic plants was substantially higher than in the non-transformed controls. Anatomical observations indicated that the shoot formation process was accelerated but with no changes in the quality and sequence of shoot development. Surprisingly, and in contrast to the controls, when on root-inducing medium (high NAA, low BAP) cultured segments from transgenic plants failed to initiate hardly any roots. Instead, they continued to form shoots at low frequencies. Moreover, in marked contrast to the controls, stem segments from transgenic plants were able to form shoots even without the addition of exogenous growth regulators to the medium. The results indicate that Spcdc25 expression in cultured tobacco stem segments mimicked the developmental effects caused by an exogenous hormone balance shifted towards cytokinins. The observed cytokinin-like effects of Spcdc25 transformation are consistent with the concept of an interaction between cell cycle regulators and phytohormones during plant development. 2. Denaturing RNA electrophoresis in TAE agarose gels Tomas Masek, Vaclav Vopalensky, Petra Suchomelova, Martin Pospisek (Analytical Biochemistry, 2005, 336, 46 50) Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris acetate ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and 8

cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals. 3. The fission yeast mitotic activator, cdc25, and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun Martina Teichmanová, Petra Mašková, Petra Vojvodová, Jan Krekule, Dennis Francis, and Helena Lipavská (submitted for publication) The tobacco (Nicotiana tabacum L.) day-neutral (DN) cv. Samsun transformed with the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, was used to study the onset of flowering. Wild type (WT) and cdc25 plants were grown from seeds in vitro until they were 20 cm high. Apical and basal nodes were then subcultured repeatedly and the regenerated plants were used to document time-to-flower and leaf number until flowering. Three sucrose treatments (3, 5 or 7% (w/v)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, WT plants did not flower but cdc25 plants did. Also, the higher sucrose treatments enabled WT flowering; two thirds of the plants flowered at 5%, while all plants flowered at 7% sucrose. However, in all treatments, cdc25 plants exhibited significantly earlier flowering and yet further reductions in leaf number. Remarkably, a typical acropetal flowering gradient in WT plants did not occur in cdc25 plants. In cdc25 leaves, there were significantly higher amounts of endogenous sugars with a higher proportion of sucrose compared with WT. The data are consistent with a synergistic effect of exogenous sucrose and Spcdc25 expression resulting in fewer leaves, and shortened flowering times. 4. Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle Craig B. Orchard, Ilario Siciliano, David A. Sorrell, Angela Marchbank, Hilary J. Rogers, Dennis Francis, Robert J. Herbert, Petra Suchomelova, Helena Lipavska, Abdelkrim Azmi and Harry Van Onckelen (Plant Journal, 2005, 44, 290 299) The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25- expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25- expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited 9

premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable. 5. Tobacco cells transformed with fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application Petra Suchomelová-Mašková, Ondřej Novák, Helena Lipavská (submitted for publication) In plants, the G2/M control of cell cycle remain elusive issue as doubts persist about activatory dephosphorylation - in other eucaryots provided by CDC25 phosphatase serving as a final all-or-nothing mitosis regulator. We report on the effects of tobacco (Nicotiana tabacum L., cv. Samsun) transformation with yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on cell characteristics. Transformed cell suspension cultures showed higher dry mass accumulation during exponential phase and clustered more circular cell phenotype compared to chains of elongated WT cells. Similar cell parameters, as in the transformants, can be induced in WT by cytokinins. Spcdc25 cells after cytokinin treatment showed giant cell clusters and growth inhibition. In addition, Spcdc25 expression leads to altered carbohydrate status: increased starch and soluble sugars with higher sucrose: hexoses ratio, inducible in WT by cytokinin treatment. Taken together, the Spcdc25 transformation had cytokinin-like effect on studied characteristics. However, endogenous cytokinin determination revealed markedly lower cytokinin levels in Spcdc25 transformants. It indicates that the cells sense Spcdc25 expression as an increased cytokinin availability, manifested by changed cell morphology, and in consequence decrease endogenous cytokinin levels. Clearly, the results on cell growth and morphology are consistent with the model of G2/M control including cytokinin-regulated activatory dephosphorylation. Nevertheless, no clear link is obvious between Spcdc25 transformation and carbohydrate status and thus the observed cytokinin-like effect on carbohydrate levels pose a problem. Hence, we propose that Spcdc25-induced higher CDK(s) activity at G2/M generates a signal modifying carbohydrate metabolism to meet high energy and C demands of forthcoming cell division. 10

Curriculum vitae Petra Mašková born on 29 th of April 1975 in Prague Education: since 1999: Dept. of Plant Physiology, Faculty of Science, Charles University in Prague PhD student in the Laboratory of Plant Tissue Culture 2000-2003: Faculty of Science, Charles University, Prague supplementary study of teaching in biology (state examination) 1993-1999: Faculty of Science, Charles University, Prague master degree specialization plant physiology and anatomy diploma thesis: Physiological characteristics of transgenic tobacco plants under in vitro conditions, 1999 Employment: Dept. of Plant Physiology, Faculty of Science, Charles University in Prague 2000-2004: researcher (since 2002 full-time position) since 2004: assistant (since November 2005 maternity leave) Teaching: 1999-2005: Practical course on plant physiology for pregraduate students (exercise in plant tissue cultures) 2003-2005: Advanced practical course on plant tissue cultures for pregraduate and postgraduate students (winter semester) 2004-2005: Practical lesson on plant tissue cultures at 3 rd Age University consultant of diploma theses: Denisa Velgová (2003), Martina Smoloňová (2003), Petra Vojvodová (2005), Lucie Uchytilová (2006) Grants: Changes in de novo organogenesis of tobacco induced by cdc25 gene from S. pombe FRVŠ (2001) participation in grant projects: Use of transgenic plants in teaching of plant physiology - FRVŠ (1997) De novo regeneration on transgenic tobacco segments with introduced foreign gene participating in cell cycle regulation - GAUK (1998-2000) Critical parameters of autonomous flowering regulation in herbs - GAUK (2000-2002) Research centre: Signalling pathways in plants - MŠMT (2000-2004) The role of cell division in plant morphogenesis: the use of transgenic tobacco with inducible yeast mitotic activator - GAUK (2003), GAUK (2005-2006) 11

Staying Abroad: laboratory of Dr. Dennis Francis, University of Cardiff, Great Britain (2003, 1 week) laboratory of Dr. Jean-Francois Morot-Gaudry, INRA,Versailles, France (1997, 1 week) Other Scientific Activities: 2004-2005 Member of the Society of Experimental Plant Biology (SEBR) 2004-2005 Member of the Federation of European Societies of Plant Biology (FESPB) Publications: Suchomelová-Mašková, P., Novák, O., Lipavská, H.: Tobacco cells transformed with fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application (submitted for publication) Teichmanová, M., Mašková, P., Vojvodová, P., Krekule, J., Francis, D., Lipavská, H.: The fission yeast mitotic activator, cdc25, and sucrose induce early flowering synergistically in the day-neutral Nicotiana tabacum cv. Samsun (submitted for publication) Orchard, C.B., Siciliano, I., Sorrell, D.A., Marchbank, A., Rogers, H.J., Francis, D., Herbert, R.J., Suchomelova, P., Lipavska, H., Azmi, A., Van Onckelen, H.: 2005, Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle, Plant Journal, 44, 290 299. Masek, T., Vopalensky, V., Suchomelova, P., Pospisek, M.: 2005, Denaturing RNA electrophoresis in TAE agarose gels, Analytical Biochemistry, 336, 46 50. Suchomelová, P., Velgová, D., Mašek, T., Francis, D., Rogers, H.J., Marchbank, A.M., Lipavská, H.: 2004, Expression of the fission yeast cell cycle regulator cdc25 induces de novo shoot formation in tobacco: evidence of a cytokinin-like effect by this mitotic activator, Plant Physiology and Biochemistry, 42, 49 55. Suchomelová, P., Albrechtová, J., Lipavská, H., Pechan, P.M.: 2001, Anatomical analysis of de novo organogenesis on internode stem segments of tobacco transformed with fission yeast cdc25 gene, Scripta fac. sci. nat. univ. Masaryk. Brun, 27, 39-43. Oral presentations: Mašková-Suchomelová P., Lipavská H.: 2005, Cytokinin effect on cell morphogenesis can be invoked by fission yeast cdc25 expression, 6th Symposium Recent Advances in Plant Biotechnology, From Laboratory to Business, České Budějovice, Book of Abstracts, p. 107. Suchomelová, P., Lipavská, H., Velgová, D., Smoloňová, M.: 2002, Cell cycle regulation in plants: Growth and developmental changes resulting from transformation of tobacco with fission yeast cdc25 gene, 13th Congress of FESPP, Hersonissos, Crete, Greece, Book of Abstracts, invited lecture. Suchomelová P., Lipavská H., Velgová D.,Smoloňová M., Krekule J.: 2001, The effects of fission yeast mitotic activator cdc25 expression in tobacco, IXth Days of Plant Physiology, České Budějovice, Book of Abstracts, awarded lecture. 12

Suchomelová, P., Albrechtová,J., Lipavská,H., Pechan,P.: 2000, Anatomical analysis of de novo organogenesis on internode stem segments of tobacco transformed with fission yeast cdc25 gene, Book of Abtracts, Němcův cytologický den, Brno. Abstracts from conferences published in periodicals: Suchomelová P., Uchytilová L., Lipavská H.: 2004, Tobacco cells transformed with fission yeast cdc25 gene display characteristics evocable by cytokinin application, 14th Congress of FESPP, Cracow, Poland, Acta Physiol. Plant., 26, 3 Suppl., 161. Suchomelová,P., Lipavská,H., Albrechtová,J., Pechan,P.M.: 2000, Expression of S.pombe cdc25 in tobacco lead to a shift in the timing, frequency and type of de novo organogenesis, 12th Congress of FESPP, Budapest, Hungary, Plant Physiology and Biochemistry, Vol. 38, Suppl.. Posters: Uchytilová L., Mašková-Suchomelová P., Lipavská H.: 2005, Effect ot time limited S.pombe cdc25 expression on cytokinin-independent de novo shoot formation in tobacco, 6th Symposium Recent Advances in Plant Biotechnology, From Laboratory to Business, České Budějovice, Book of Abstracts, p. 132. Mašková-Suchomelová P., Vojvodová P., Lipavská H.: 2005, Tobacco with yeast mitotic activator a useful model to study the role of cell division in plant morphogenesis and development, Plant Physiology Conference of PhD Students and Young Scientists, Modrá, Slovakia, Book of Abstracts, p. 25. Uchytilová L., Suchomelová P., Mašek T., Lipavská H.: 2004, Cytokininindependent de novo shoot formation in tobacco with controlled expression of fission yeast cdc25, X. Days of Plant Physiology, Bratislava, Slovakia, Book of Abstracts, p. 36. Lipavská H., Hudec L., Suchomelová P., Konrádová H.: 2003, Making use of PEG treatment needs precise search for optimum concentration, COST 843, Quality enhancement of plant production through tissue culture Working Group 2 Advanced propagation techniques, San Remo, Italy, Book of Abstracts. Smoloňová M., Suchomelová P., Krekule J., Lipavská H.: 2003, Flowering in day neutral tobacco as affected by fission yeast mitotic activator cdc25 transformation, 5th Symposium Recent Advances in Plant Biotechnology, Stará Lesná, Tatry, Slovakia, Book of Abstracts, p. 90. Smoloňová, M., Suchomelová, P., Krekule, J., Lipavská, H.: 2002, Kvetení tabáku ovlivněné transformací kvasinkovým genem urychlujícím dělení buněk, Book of Abstracts, Výzkum geneticky modifikovaných organizmů v ČR. Lipavská H., Suchomelová P.: 2001, The growth characteristics of tobacco tissue cultures as influenced by transformation with fission yeast cdc25 gene, IXth Days of Plant Physiology, České Budějovice, Book of Abtracts. Smoloňová M., Suchomelová P., Lipavská H., Krekule J.: 2001, The effect of introduction of cdc25 gene on flowering of Nicotiana tabacum, IXth Days of Plant Physiology, České Budějovice, Book of Abstracts. Suchomelová P., Lipavská H., Velgová D., Smoloňová M., Krekule J.: 2001, Fission yeast cdc25 gene expression in tobacco influenced the plant morphogenesis and cell characteristics of tissue cultures, Plant Physiology Days of Young Scientists, Praha, Book of Abstracts. 13

Suchomelová,P., Lipavská,H., Albrechtová,J., Pechan,P.: 1999, Changes in de novo organogenesis on tobacco internodal stem segments induced by transformation with cdc25 gene from fission yeast, III. International Symposium From Cells to Crops Recent Advances in Plant Biotechnology, Tatry, Slovakia, Book of Abstracts. Suchomelová,P., Albrechtová,J., Lipavská,H., Pechan,P.: 1999, Quantitative analysis of structural changes in de novo organogenesis on tobacco internode stem segments induced by transformation with cdc25 gene from fission yeast, Conference on Stereology, Spatial Statistics and Stochastic Geometry S 4 G, Praha, Book of Abstracts. Suchomelová,P., Lipavská,H., Pechan,P.: 1998, De novo organogenesis of tobacco transformed with cdc25 gene from Schizosaccharomyces pombe, VIIIth Days of Plant Physiology, Olomouc, Book of Abstracts. Suchomelová,P., Lipavská,H., Pechan P.: 1997, Vliv transformace (Nicotiana tabacum L.) genem cdc25 ze Schizosaccharomyces pombe na de novo organogenezi indukovanou změnami v exogenní rovnováze růstových regulátorů, XIV. Biologické dny, Praha, Book of Abstracts. 14