Endogenous cytokinin oscillations control cell cycle progression of tobacco BY-2 cells

The significance of cytokinins for the progression of the cell cycle is well known. Cytokinins contribute to the control of the expression of D-cyclins and other cell cycle genes, but knowledge as to how they affect the progression of the cell cycle is still limited. Highly synchronized tobacco BY-2 cells with clearly defined cell cycle stages were employed to determine cytokinin patterns in detail throughout the entire cycle. Concentrations of trans-zeatin, and of some other cytokinins, oscillated during the course of the cell cycle, increasing substantially at all four phase transitions and decreasing again to a minimum value during the course of each subsequent phase. Addition of exogenous cytokinins or inhibition of cytokinin biosynthesis promoted the progression of the cell cycle when the effects of these manipulations intensified the endogenous fluctuations, whereas the progression of the cycle was retarded when the amplitude of the fluctuations was decreased. The results show that the attainment of low concentrations of cytokinins is as important as the transient increases in concentration for a controlled progression from one phase of the cell cycle to the next. Cytokinin oxidase/dehydrogenase activity also showed fluctuations during the course of the cell cycle, the timing of which could at least partly explain oscillations of cytokinin levels. The activities of the enzyme were sufficient to account for the rates of cytokinin disappearance observed subsequent to a phase transition.     

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle

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 premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.     

A low content in zeatin type cytokinins is not restrictive for the occurrence of G1/S transition in tobacco BY-2 cells

Theories on the importance of cytokinins in G1/S transition control are manifold and contradictory. By establishing a double A(phi-PZ block, maximal synchronization of a BY-2 suspension culture was obtained to investigate the effect of cytokinin depletion on G1/S transition. Lovastatin was used as a specific inhibitor of cytokinin biosynthesis. Flow cytometry showed that the G1/S transition occurred regardless of the cytokinin drop. This observation indicates an extremely low dose requiry for that stage of the cell cycle. It is very likely that precisely the downregulation of zeatin type cytokinins matters for the G1/S transition to occur, since cytokinin addition at early G1 blocked the cycle at G1/S.     

The role of cytokinins in rooting of stem slices cut from apple microcuttings

ABSTRACT

We examined the role of cytokinins in rooting of 1-mm stem slices cut from microcuttings of the apple rootstock ‘Jork 9?s. Various types of cytokinins inhibited the rooting of apple stem slices to different extents. Highest inhibition was obtained with thidiazuron and benzylaminopurine. Remarkably, isopentenyladenine and isopentenyladenosine enhanced rooting at low concentration (at the optimal concentration of 0.1 μM by 53 and 19%, respectively). We also examined the effect of lovastatin and simvastatin. These drugs are putative cytokinin-synthesis inhibitors. Both inhibited rooting and inhibition was partially reversed by simultaneous addition of zeatin. Moreover, in the presence of lovastatin a higher concentration of zeatin had to be applied to achieve inhibition of rooting than in the absence of the drug. This data indicates that these compounds indeed inhibited cytokinin synthesis. One-day pulses with lovastatin strongly blocked rooting when given just after cutting the slices but had no effect after that. Adding zeatin simultaneously reversed inhibition completely. In conclusion, our data confirm that cytokinins may strongly inhibit rooting but they also show that at low concentration, certain cytokinins enhance rooting. Moreover, synthesis of cytokinin is essential during root formation. We hypothesise that cell division initiated by a relatively high endogenous level of cytokinins just after cutting the slices is a necessary, initial step in adventitious root formation.     

Dose-dependent effects of lovastatin on cell cycle progression. Distinct requirement of cholesterol and non-sterol mevalonate derivatives

The mevalonate pathway is tightly linked to cell proliferation. The aim of the present study is to determine the relationship between the inhibition of this pathway by lovastatin and the cell cycle. HL-60 and MOLT-4 human cell lines were cultured in a cholesterol-free medium and treated with increasing concentrations of lovastatin, and their effects on cell proliferation and the cell cycle were analyzed. Lovastatin was much more efficient in inhibiting cholesterol biosynthesis than protein prenylation. As a result of this, lovastatin blocked cell proliferation at any concentration used, but its effects on cell cycle distribution varied. At relatively low lovastatin concentrations (less than 10 microM), cells accumulated preferentially in G(2) phase, an effect which was both prevented and reversed by low-density lipoprotein cholesterol. At higher concentrations (50 microM), the cell cycle was also arrested at G(1) phase. In cells treated with lovastatin, those arrested at G(1) progressed through S upon mevalonate provision, whereas cholesterol supply allowed cells arrested at G(2) to traverse M phase. These results demonstrate the distinct roles of mevalonate, or its non-sterol derivatives, and cholesterol in cell cycle progression, both being required for normal cell cycling.     

The Arabidopsis AtIPT8/PGA22 gene encodes an isopentenyl transferase that is involved in de novo cytokinin biosynthesis

Cytokinin plays a critical role in plant growth and development by stimulating cell division and cell differentiation. Despite many years' research efforts, our current understanding of this hormone is still limited regarding both its biosynthesis and signaling. To genetically dissect the cytokinin pathway, we have used a functional screen to identify Arabidopsis gain-of-function mutations that enable shoot formation in the absence of exogenous cytokinins. By using a chemical-inducible activation tagging system, we have identified over 40 putative mutants, designated as pga (plant growth activators), which presumably were affected in key components of cytokinin biosynthesis and signaling pathway. Here, we report a detailed characterization of pga22, a representative mutant from this collection. A gain-of-function mutation in the PGA22 locus resulted in typical cytokinin responses. Molecular and genetic analyses indicated that PGA22 encodes an isopentenyl transferase (IPT) previously identified as AtIPT8. Plants of the pga22 mutant accumulated at remarkably higher levels of isopentenyladenosine-5'-monophosphate and isopentenyladenosine when analyzed by mass spectrometry, suggesting that AtIPT8/PGA22 is a functional IPT that may direct the biosynthesis of cytokinins in planta via an isopentenyladenosine-5'-monophosphate-dependent pathway.     

Effect of inhibition of phenylalanine ammonia-lyase activity on growth of alfalfa cell suspension culture: Alterations in mitotic index, ethylene production, and contents of phenolics, cytokinins and polyamines

The effect of inhibition of phenylpropanoid biosynthesis on the growth of Medicago sativa L. suspension culture was studied. 2-Aminoindan-2-phosphonic acid (AIP), a potent inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), caused a marked reduction in the amount of hydroxycinnamic acid derivatives in a few hours after cell inoculation into AIP medium. The treatment of alfalfa suspension culture with this inhibitor increased the extractable PAL activity and elevated ethylene production during the growth cycle. The addition of AIP (10 μ M ) stimulated cell division activity during the growth cycle, although the onset of cell division was slightly delayed. The maxima of cytokinin content as well as of the mitotic index were postponed in AIP-treated cells, however, the unchanged content of cytokinins did not correlate with increased mitotic activity of treated cells. The decreased level of hydroxycinnamic acid derivatives, which represent the phenolic conjugation partners of free polyamines (PAs), influenced the rate of PA conjugation. Consequently, the balance between free and conjugated PAs was shifted in favor of the free PA form. A potential role of the reduction of the pool of phenolic acids in the enhancement of cell division of alfalfa cell suspension culture is discussed.     

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).     

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels

To investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8- to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quantification of endogenous cytokinins demonstrated that FPS1S overexpression specifically reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The finding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.     

Cytokinin N-glucosylation inhibitors suppress deactivation of exogenous cytokinins in radish, but their effect on active endogenous cytokinins is counteracted by other regulatory mechanisms

The prolongation of the effect of exogenous cytokinins by inhibition of their inactivation was studied in Raphanus sativus L. cv. Rampouch. As in radish the main way of cytokinin inactivation is their N- glucosylation, inhibitors of this pathway, papaverine, theophylline and olomoucine, as well as two olomoucine analogues, bohemine and roscovitine, were tested. The latter ones, which function as potent inhibitors of cyclin-dependent kinases, have also been found to effectively inhibit cytokinin N- glucosylation. Incubation (24 h) of de-rooted radish seedlings with inhibitors resulted in c . 50% decrease of the conversion of model cytokinins, [³H]dihydrozeatin and [³H]N⁶-benzyladenine, to the corresponding 7 N- glucosides. Simultaneously the level of the non-metabolized cytokinin bases was elevated. The activity of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was suppressed in a dose dependent manner. The concentration of physiologically active endogenous cytokinins was not increased significantly by inhibitor application. The inhibition of N- glucosylation was in the case of olomoucine, bohemine and roscovitine accompanied by the accumulation of physiologically nonactive cis -zeatin derivatives. The impact of inhibitors on the endogenous cytokinin pool seems to be balanced by the mechanisms involved in the maintenance of cytokinin homeostasis.     

Competency of Nicotiana tabacum L. stem tissues to dedifferentiate is associated with differential levels of cell cycle gene expression and endogenous cytokinins

The expression of the mitotic cyclin Arath; CYCB1;1 and of the cyclin-dependent kinase Arath; CDC2a was located by beta-glucuronidase histochemical detection and in situ hybridization, and was quantified by 4-methylumbelliferyl beta- D-glucuronide assay in tobacco stem tissues during both in vivo differentiation and in vitro dedifferentiation. The changes in localization of endogenous cytokinins were also determined during both processes using quantitative analysis and in situ immunocytochemistry. The CDC2a promoter was expressed continuously during stem development, with particularly high expression in the shoot apical meristem and in the internal and external primary phloem. CYCB1 expression was not restricted to the dividing cells; its expression in the shoot apical meristem was particularly high in the leaf-forming peripheral cells but the gene was also expressed throughout development in the internal and external phloem in which the rate of cell division was reduced or zero. Following in vitro culture, the internal phloem cells appeared to be particularly competent to re-enter the cell cycle within a short lag phase while the pith tissue reactivated later. In culture, cells that resumed division were found to accumulate cytokinins. The high competency of primary phloem to dedifferentiate was associated with its capacity to express CDC2a and CYCB genes and the presence of high cytokinin levels, providing some insights into the determinants of competency for resuming cell division.     

Cytokinin-Regulated Mesophyll Cell Division and Expansion during Development of Cucurbita pepo Leaves

The role of cytokinins in the development of mesophyll structure was studied in developing pumpkin Cucurbita pepo L. leaves. Leaves were treated with cytokinins at different stages of growth: when they reached 25 or 50% of their final size (S _max), immediately after leaf growth ceased, and during senescence. At the early stages of leaf development, treatment with exogenous benzyladenine accelerated division of mesophyll cells. At the later stages of development, BA treatment activated expansion of growing cells and those, which have just accomplished their growth. The exogenous cytokinin did not affect the senescent leaf cells. The content of endogenous cytokinins changed during mesophyll development. The juvenile leaves (25% of S _max) were characterized by low level of these phytohormones. In the expanding leaves (50% of S _max), the content of phytohormones increased and decreased when leaf growth ceased. In the senescent leaves, the cytokinin content decreased markedly. It was concluded that the response of mesophyll cells to cytokinin depended on the cell growth phase at the moment of hormone action. Furthermore, in the young leaves, lower cytokinin concentrations were required for division of mesophyll cells in vivo than for cell expansion at the final stage of leaf development.     

Inhibition of growth of cultured tobacco cells at low concentrations of lovastatin is reversed by cytokinin

De novo synthesis of mevalonic acid, which is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase, is the first committed step in the formation of isoprenoid compounds. Various studies have shown that mevalonic acid-derived compounds are required for growth of plant and animal cells, a conclusion supported by the observation that cells treated with lovastatin (a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase) cease growth. We show that Nicotiana tabacum BY-2 cells, which require exogenous auxin for growth in culture but do not require exogenous cytokinin, are growth inhibited by 1 μm lovastatin. However, these cells are capable of growing in the presence of 1 μm lovastatin if 8 μm zeatin is supplied in the medium. Furthermore, benzyladenine, kinetin, and thidiazuron effectively reverse the inhibition of growth of these cells at 1 μm lovastatin, whereas adenine and 6-methyladenine have no effect. These results demonstrate that restoration of growth to lovastatin-treated cells is cytokinin specific and is not caused by metabolism of cytokinin into other isoprenoid compounds. Cytokinin does not effectively reverse the effects of higher concentrations of lovastatin, but mevalonic acid does, consistent with the hypothesis that cytokinin biosynthesis is more sensitive to lovastatin than the biosynthesis of other essential isoprenoid compounds in tobacco cells. This observation suggests that lovastatin can be used to induce cytokinin dependence in cytokinin-autonomous tobacco cell cultures.     

Moss biology and phytohormones--cytokinins in Physcomitrella

Mosses present several advantages for the analysis of phytohormone physiology. Their enormous regeneration capacity, the possibility of controlling their whole life cycle under in vitro culture conditions, as well as the small number of cell types facilitate studies of hormone homeostasis. This review focuses on the metabolism and biosynthesis of cytokinins, mostly summarising data obtained using the moss Physcomitrella patens (Hedw.) B.S.G. which has served as a model system for cytokinin research for many years. A comparison of metabolic differences with respect to seed plants is presented, pointing out an important role of adenosine kinase for the formation of nucleotides during cytokinin interconversion in Physcomitrella. Results on cytokinin biosynthesis in Physcomitrella are summarised with respect to the OVE mutants, which can be considered unique in the plant kingdom due to their strong overproduction of cytokinins. The OVE phenotype is correlated with both increased activity in early stages of cytokinin biosynthesis as well as increased conversion of cytokinin riboside to the base. Cytokinin interconverting reactions can contribute to the increased levels of cytokinins in OVE mutants. Further studies on hormone physiology in moss will help to complete our understanding of hormonal homeostasis by elucidating the situation in an evolutionary early embryophyte.     

Tobacco cells transformed with the fission yeast Spcdc25 mitotic inducer display growth and morphological characteristics as well as starch and sugar status evocable by cytokinin application.

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.     

Effects of synthetic cytokinins on levels of endogenous cytokinins and respiration patterns ofBeta vulgaris cells in suspension

Respiration patterns and growth of cytokinin-dependent cell suspensions ofBeta vulgaris L., precultured in media with or without three different synthetic cytokinins [benzyladenine (BA), kinetin (KIN), and thidiazuron (TDA)], were compared. The content of endogenous cytokinins, especially zeatin and isopentenyladenine, as well as the dry mass yield, were dependent on the kind of synthetic cytokinin present in the culture medium and decreased in the following order: thidiazuron, kinetin, benzyladenine, no cytokinin. The apparent capacity of the alternative pathway, as measured after blocking of the cytochrome pathway by cyanide, was inversely proportional to the content of endogenous cytokinins. Some synthetic cytokinins (e.g., benzyladenine), when exogenously applied, are known to inhibit selectively the alternative pathway. However, this does not necessarily imply that the mechanism of action of endogenous cytokinins on the respiration pattern is limited to a single effect on the alternative pathway. Multiple effects on oxidative processes cannot be excluded.