BACKGROUND: During the last three decades, the cell cycle and its control by cyclin-dependent kinases (CDKs) have been extensively studied in eukaryotes. This endeavour has produced an overall picture that basic mechanisms seem to be largely conserved among all eukaryotes. The intricate regulation of CDK activities includes, among others, CDK activation by CDC25 phosphatase at G₂/M. In plants, however, studies of this regulation have lagged behind as a plant Cdc25 homologue or other unrelated phosphatase active at G₂/M have not yet been identified. SCOPE: Failure to identify a plant mitotic CDK activatory phosphatase led to characterization of the effects of alien cdc25 gene expression in plants. Tobacco, expressing the Schizosaccharomyces pombe mitotic activator gene, Spcdc25, exhibited morphological, developmental and biochemical changes when compared with wild type (WT) and, importantly, increased CDK dephosphorylation at G₂/M. Besides changes in leaf shape, internode length and root development, in day-neutral tobacco there was dramatically earlier onset of flowering with a disturbed acropetal floral capacity gradient typical of WT. In vitro, de novo organ formation revealed substantially earlier and more abundant formation of shoot primordia on Spcdc25 tobacco stem segments grown on shoot-inducing media when compared with WT. Moreover, in contrast to WT, stem segments from transgenic plants formed shoots even without application of exogenous growth regulator. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size due to a shortening of the G₂ phase together with high activity of cyclin-dependent kinase, NtCDKB1, in early S-phase, S/G₂ and early M-phase. Spcdc25-expressing tobacco ('Samsun') cell suspension cultures showed a clustered, more circular, cell phenotype compared with chains of elongated WT cells, and increased content of starch and soluble sugars. Taken together, Spcdc25 expression had cytokinin-like effects on the characteristics studied, although determination of endogenous cytokinin levels revealed a dramatic decrease in Spcdc25 transgenics. CONCLUSIONS: The data gained using the plants expressing yeast mitotic activator, Spcdc25, clearly argue for the existence and importance of activatory dephosphorylation at G₂/M transition and its interaction with cytokinin signalling in plants. The observed cytokinin-like effects of Spcdc25 expression are consistent with the concept of interaction between cell cycle regulators and phytohormones during plant development. The G₂/M control of the plant cell cycle, however, remains an elusive issue as doubts persist about the mode of activatory dephosphorylation, which in other eukaryotes is provided by Cdc25 phosphatase serving as a final all-or-nothing mitosis regulator.

• MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• cytokininy metabolismus   MeSH
• eukaryotické buňky cytologie   MeSH
• fosfatasy cdc25 metabolismus   MeSH
• fosforylace MeSH
• G2 fáze * MeSH
• geneticky modifikované rostliny MeSH
• mitóza * MeSH
• morfogeneze MeSH
• Schizosaccharomyces enzymologie   MeSH
• tabák cytologie   embryologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklin-dependentní kinasy MeSH
• cytokininy MeSH
• fosfatasy cdc25 MeSH

Here, the tobacco (Nicotiana tabacum) 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 flowering and the number of leaves formed before flowering. Three sucrose treatments (3, 5 or 7% (weight/volume)) were used and measurements of leaf endogenous soluble carbohydrates were performed. In the 3% treatment, cdc25 plants flowered but WT plants did not. 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 fewer leaves compared with wild type. 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. Our data demonstrate that Spcdc25 expression and sucrose act synergistically to induce precocious flowering.

• MeSH
• časové faktory MeSH
• fungální proteiny genetika   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• kořeny rostlin růst a vývoj   MeSH
• květy účinky léků   metabolismus   MeSH
• metabolismus sacharidů MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• ras-GRF1 genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• sacharosa farmakologie   MeSH
• Schizosaccharomyces * genetika   MeSH
• tabák účinky léků   genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fungální proteiny MeSH
• proteiny buněčného cyklu MeSH
• ras-GRF1 MeSH
• sacharosa MeSH

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 culture 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.

• MeSH
• cytokininy farmakologie   MeSH
• fungální proteiny biosyntéza   fyziologie   MeSH
• genetická transkripce MeSH
• kořeny rostlin účinky léků   růst a vývoj   MeSH
• kultivační média MeSH
• kyseliny indoloctové farmakologie   MeSH
• proteiny buněčného cyklu biosyntéza   fyziologie   MeSH
• ras-GRF1 biosyntéza   fyziologie   MeSH
• RNA biosyntéza   MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• tabák účinky léků   genetika   růst a vývoj   MeSH
• výhonky rostlin účinky léků   růst a vývoj   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• cytokininy MeSH
• fungální proteiny MeSH
• kultivační média MeSH
• kyseliny indoloctové MeSH
• proteiny buněčného cyklu MeSH
• ras-GRF1 MeSH
• RNA MeSH

Experimental spinach plants grown in soil with (5, 10 and 20 ppm) arsenic (As) contamination were sampled in 21 days after As(V) contamination. Levels of As in spinach samples (from 0.31 ± 0.06 µg g-1 to 302.69 ± 11.83 µg g-1) were higher in roots and lower in leaves, which indicates a low ability of spinach to translocate As into leaves. Species of arsenic, As(III) and As(V), were represented in favor of the As (III) specie in contaminated variants, suggesting enzymatic arsenate reduction. In relation to predominant As accumulation in roots, changes in malondialdehyde levels were observed mainly in roots, where they decreased significantly with growing As contamination (from 11.97 ± 0.54 µg g-1 in control to 2.35 ± 0.43 µg g-1 in 20 ppm As). Higher values in roots than in leaves were observed in the case of 5-methylcytosine (5-mC). Despite that, a change in 5-mC by As contamination was further deepened in leaves (from 0.20 to 14.10%). In roots of spinach, expression of the CDC25 gene increased by the highest As contamination compared to the control. In the case of total phenolic content, total flavonoid content, total phenolic acids content and total antioxidant capacity were higher levels in leaves in all values, unlike the roots.

• Klíčová slova
• CDC25, arsenic species, epigenetics, membrane damage, secondary metabolites, spinach,
• Publikační typ
• časopisecké články MeSH

Mammalian oocytes are arrested at meiotic prophase I. The dual-specificity phosphatase CDC25B is essential for cyclin-dependent kinase 1 (CDK1) activation that drives resumption of meiosis. CDC25B reverses the inhibitory effect of the protein kinases WEE1 and MYT1 on CDK1 activation. Cdc25b-/- female mice are infertile because oocytes cannot activate CDK1. To identify a role for CDC25B following resumption of meiosis, we restored CDK1 activation in Cdc25b-/- oocytes by inhibiting WEE1 and MYT1, or expressing EGFP-CDC25A or constitutively active EGFP-CDK1 from microinjected complementary RNAs. Forced CDK1 activation in Cdc25b-/- oocytes allowed resumption of meiosis, but oocytes mostly arrested at metaphase I (MI) with intact spindles. Similarly, approximately a third of Cdc25b+/- oocytes with a reduced amount of CDC25B arrested in MI. MI-arrested Cdc25b-/- oocytes also displayed a transient decrease in CDK1 activity similar to Cdc25b+/+ oocytes during the MI-MII transition, whereas Cdc25b+/- oocytes exhibited only a partial anaphase-promoting complex/cyclosome activation and anaphase I entry. Thus, CDC25B is necessary for the resumption of meiosis and the MI-MII transition.

• Klíčová slova
• Anaphase I, CDC25B, Meiotic maturation, Mouse oocytes, Resumption of meiosis,
• MeSH
• anafáze MeSH
• anafázi podporující komplex metabolismus   MeSH
• fosfatasy cdc25 MeSH
• meióza * MeSH
• metafáze MeSH
• myši MeSH
• oocyty * metabolismus   MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• anafázi podporující komplex MeSH
• Cdc25b protein, mouse MeSH Prohlížeč
• fosfatasy cdc25 MeSH

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a -/- mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of oocytes.

• MeSH
• AMP cyklický metabolismus   MeSH
• exprese genu MeSH
• fosfatasy cdc25 analýza   metabolismus   MeSH
• meióza * MeSH
• myši MeSH
• oocyty chemie   cytologie   enzymologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• AMP cyklický MeSH
• Cdc25a protein, mouse MeSH Prohlížeč
• Cdc25b protein, mouse MeSH Prohlížeč
• fosfatasy cdc25 MeSH

Embryonic stem cells progress very rapidly through the cell cycle, allowing limited time for cell cycle regulatory circuits that typically function in somatic cells. Mechanisms that inhibit cell cycle progression upon DNA damage are of particular importance, as their malfunction may contribute to the genetic instability observed in human embryonic stem cells (hESCs). In this study, we exposed undifferentiated hESCs to DNA-damaging ultraviolet radiation-C range (UVC) light and examined their progression through the G1/S transition. We show that hESCs irradiated in G1 phase undergo cell cycle arrest before DNA synthesis and exhibit decreased cyclin-dependent kinase two (CDK2) activity. We also show that the phosphatase Cdc25A, which directly activates CDK2, is downregulated in irradiated hESCs through the action of the checkpoint kinases Chk1 and/or Chk2. Importantly, the classical effector of the p53-mediated pathway, protein p21, is not a regulator of G1/S progression in hESCs. Taken together, our data demonstrate that cultured undifferentiated hESCs are capable of preventing entry into S-phase by activating the G1/S checkpoint upon damage to their genetic complement.

• MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• checkpoint kinasa 1 MeSH
• checkpoint kinasa 2 MeSH
• cyklin-dependentní kinasa 2 metabolismus   MeSH
• fosfatasy cdc25 metabolismus   MeSH
• G1 fáze * účinky záření   MeSH
• kmenové buňky cytologie   metabolismus   účinky záření   MeSH
• lidé MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinkinasy metabolismus   MeSH
• S fáze * účinky záření   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDC25A protein, human MeSH Prohlížeč
• CDK2 protein, human MeSH Prohlížeč
• checkpoint kinasa 1 MeSH
• checkpoint kinasa 2 MeSH
• CHEK1 protein, human MeSH Prohlížeč
• CHEK2 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH
• fosfatasy cdc25 MeSH
• protein-serin-threoninkinasy MeSH
• proteinkinasy MeSH

Cyclin-dependent kinase two (Cdk2) is the major regulator of the G1/S transition and the target of an activated G1 checkpoint in somatic cells. In the presence of DNA damage, Cdk2 kinase activity is abrogated by a deficiency of Cdc25A phosphatase, which is marked by Chk1/Chk2 for proteasomal degradation. Embryonic stem cells (ESCs) lack a G1 checkpoint response. In this study, we analyzed the G1 checkpoint pathways in mouse ESCs (mESCs) in the presence of DNA double-strand breaks evoked by ionizing radiation (IR). We show that checkpoint pathways, which operate during G1 phase in somatic cells, are activated in mESCs after IR; however, Cdk2 activity is not abolished. We demonstrate that Cdc25A is degraded in mESCs, but this degradation is not regulated by Chk1 and Chk2 kinases because they are sequestered to the centrosome. Instead, Cdc25A degradation is governed by glycogen synthase kinase-3beta kinase. We hypothesize that Cdc25A degradation does not inhibit Cdk2 activity because a considerable proportion of Cdk2 molecules localize to the cytoplasm and centrosomes in mESCs, where they may be sheltered from regulation by nuclear Cdc25A. Finally, we show that a high Cdk2 activity, which is irresponsive to DNA damage, is the driving force of the rapid escape of mESCs from G1 phase after DNA damage.

• MeSH
• aktivace enzymů genetika   MeSH
• buněčné linie MeSH
• buněčný cyklus genetika   MeSH
• CDC geny fyziologie   MeSH
• centrozom enzymologie   MeSH
• checkpoint kinasa 1 MeSH
• checkpoint kinasa 2 MeSH
• cyklin-dependentní kinasa 2 genetika   MeSH
• cytoplazma enzymologie   MeSH
• DNA genetika   účinky záření   MeSH
• embryonální kmenové buňky cytologie   enzymologie   MeSH
• fosfatasy cdc25 genetika   MeSH
• G1 fáze genetika   MeSH
• ionizující záření MeSH
• kinasa 3 glykogensynthasy metabolismus   MeSH
• kinasa glykogensynthasy 3beta MeSH
• myši MeSH
• oprava DNA MeSH
• poškození DNA genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• proteinkinasy genetika   MeSH
• signální transdukce genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Cdc25a protein, mouse MeSH Prohlížeč
• Cdk2 protein, mouse MeSH Prohlížeč
• checkpoint kinasa 1 MeSH
• checkpoint kinasa 2 MeSH
• Chek1 protein, mouse MeSH Prohlížeč
• Chek2 protein, mouse MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH
• DNA MeSH
• fosfatasy cdc25 MeSH
• Gsk3b protein, mouse MeSH Prohlížeč
• kinasa 3 glykogensynthasy MeSH
• kinasa glykogensynthasy 3beta MeSH
• protein-serin-threoninkinasy MeSH
• proteinkinasy MeSH

The role of hydrogen sulfide (H2S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H2S metabolism, cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H2S. On another hand, a dose-dependent inhibition was obtained using the H2S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H2S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H2S signaling in the timing of amphibian oocytes meiosis resumption.

• Klíčová slova
• Xenopus laevis, cell cycle, hydrogen sulfide, meiosis, oocyte,
• MeSH
• apoptóza účinky léků   MeSH
• cyklin B metabolismus   MeSH
• cystathionin-beta-synthasa antagonisté a inhibitory   metabolismus   MeSH
• cystathionin-gama-lyasa antagonisté a inhibitory   metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• faktor podporující zrání metabolismus   MeSH
• fosfatasy cdc25 metabolismus   MeSH
• katalasa metabolismus   MeSH
• meióza účinky léků   MeSH
• metafáze účinky léků   MeSH
• oocyty chemie   enzymologie   metabolismus   MeSH
• profáze meiózy I účinky léků   MeSH
• proteinkinasy metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny Xenopus metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• sulfan metabolismus   MeSH
• sulfidy metabolismus   farmakologie   MeSH
• sulfurtransferasy antagonisté a inhibitory   metabolismus   MeSH
• superoxiddismutasa metabolismus   MeSH
• viabilita buněk účinky léků   MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3-mercaptopyruvate sulphurtransferase MeSH Prohlížeč
• CDC25C protein, human MeSH Prohlížeč
• CDK1 protein, Xenopus MeSH Prohlížeč
• cyklin B MeSH
• cystathionin-beta-synthasa MeSH
• cystathionin-gama-lyasa MeSH
• faktor podporující zrání MeSH
• fosfatasy cdc25 MeSH
• katalasa MeSH
• proteinkinasy MeSH
• proteiny buněčného cyklu MeSH
• proteiny Xenopus MeSH
• reaktivní formy kyslíku MeSH
• sodium bisulfide MeSH Prohlížeč
• sulfan MeSH
• sulfidy MeSH
• sulfurtransferasy MeSH
• superoxiddismutasa MeSH

In plants, the G2/M control of cell cycle remains an elusive issue as doubts persist about activatory dephosphorylation--in other eukaryotes provided by CDC25 phosphatase and serving as a final all-or-nothing mitosis regulator. We report on the effects of tobacco (Nicotiana tabacum L., cv. Samsun) transformation with fission yeast (Schizosaccharomyces pombe) cdc25 (Spcdc25) on cell characteristics. Transformed cell suspension cultures showed higher dry mass accumulation during the exponential phase and clustered more circular cell phenotypes 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 led 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 a cytokinin-like effect on studied characteristics. However, endogenous cytokinin determination revealed markedly lower cytokinin levels in Spcdc25 transformants. This 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 poses 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.

• MeSH
• buněčný cyklus účinky léků   genetika   fyziologie   MeSH
• cytokininy metabolismus   farmakologie   MeSH
• fungální proteiny genetika   fyziologie   MeSH
• geneticky modifikované rostliny účinky léků   genetika   metabolismus   MeSH
• kultivované buňky MeSH
• proteiny buněčného cyklu genetika   fyziologie   MeSH
• ras-GRF1 genetika   fyziologie   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• sacharidy analýza   MeSH
• Schizosaccharomyces genetika   MeSH
• škrob analýza   MeSH
• tabák cytologie   genetika   růst a vývoj   MeSH
• transformace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• fungální proteiny MeSH
• proteiny buněčného cyklu MeSH
• ras-GRF1 MeSH
• sacharidy MeSH
• škrob MeSH