| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online December 23, 2004; 10.1104/pp.104.051938 Plant Physiology 137:308-316 (2005) © 2005 American Society of Plant Biologists 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 DevelopmentPlant Cell Biology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia
Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67Cdc25 protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr.
Cell cycle control is essential for the initiation and maintenance of meristems and for the regulation of organogenesis. Auxin and cytokinin hormones are implicated in cell cycle control since they strongly influence the division of cells that are in culture and are means by which Agrobacterium infection causes cell proliferation (e.g. for review, see Srivastava, 2002  ). Cytokinin influences cell division activity in embryos and mature plants through altering the size and activity of meristems as seen when cytokinin levels are altered, by transgenes such as IPT or CKX (Medford et al., 1989; Werner et al., 2001), and by mutations such as amp1 (Chaudhury et al., 1993). Furthermore, defects in cytokinin signal transduction that have been caused by mutation of the receptor gene AHK4 (wol mutant allele) or knockouts in the AHK gene family can result in insufficient division of vascular precursors in the root (Mähönen et al., 2000) or general suboptimal cell division in meristems (Nishimura et al., 2004). It has therefore been suggested that morphogenic effects of cytokinin may primarily occur through influence on cell cycle regulation (e.g. Werner et al., 2001).
There is evidence that cytokinin regulates the cell cycle at both the G1/S phase and G2/M phase progressions. Entry into S phase requires hormone dependent accumulation of D-cyclins in cultured cells from both plant and animal kingdoms. D-cyclin accumulation increases activity of cyclin-dependent protein kinase (CDK) enzymes that release transcription factors for genes of DNA replication (for review, see Gutierrez et al., 2002
A contribution of cytokinins to regulating the G2/M phase progression is indicated by several lines of evidence, including the requirement for cytokinin and the synthesis of cytokinin at mitotic initiation. In the cell line of Nicotiana plumbaginifolia studied here, when cytokinin is limiting, arrest occurs in G2 phase indicated by a 2C nuclear-DNA content (John et al., 1993
An eventual impact of cytokinin on CDK enzymes can be anticipated from the central role of these enzymes in division, which has been indicated by slower division when CDK is mutated (Hemerly et al., 1995
We now report that the key effect of cytokinin on G2/M progression in tobacco cells is exerted on CDK through posttranslational modification of phosphorylation. In yeast (Saccharomyces cerevisiae) and animal cells phosphorylation of CDK at Tyr-15, and to a lesser extent at Thr-14, restrains entry into nuclear division. Mitosis is initiated when inhibiting phosphate is removed by the activator phosphatase Cdc25 (Millar et al., 1991
We now report evidence that posttranslational activation of CDK is a primary mechanism of cytokinin action at mitosis and that the activation is not a secondary consequence of the many actions of the hormone in other aspects of growth. Extensive actions of cytokinin have been detected in developmental, nutritional, and organelle metabolism and are underlined by numerous effects on transcript levels (for reviews, see Schmülling et al., 1997
Cell Lines Inducibly Expressing cdc25
To test whether an essential action of cytokinin in the cell cycle is the activation of protein Tyr phosphatase activity directed to CDK, we inducibly expressed the fission yeast CDK regulator Cdc25 phosphatase by joining the cdc25 gene to a modified plant promoter described by Schena et al. (1991) Three independent cell lines were analyzed biochemically and had similar properties. All contained yeast cdc25 DNA (detected in Southern blots; data not shown) and in 0.01 to 10 µM dexamethasone all accumulated 2 kb cdc25 mRNA and 67 kD Cd25 protein in an inducer dependent manner (Fig. 1, A and B). Effects of inducer on division were tested in cells that had been arrested at the G2 phase hormonal control point by depletion of auxin and cytokinin followed by provision of auxin only. Effect on division was tested by induction with 0.01 to 1.0 µM dexamethasone (Fig. 1C), and a sharp optimum concentration of 0.1 µM inducer was observed in independent clones, consistent with a requirement for a critical optimum Cdc25 activity. No cell division was observed without inducer or in untransformed cells treated with dexamethasone (Fig. 1C). Results from one representative transgenic cell line are shown.
Synchronous Induction of Cdc25 Phosphatase, CDK Activation, and Mitosis
In subsequent experiments, cells were brought to arrest at the cytokinin control point in late G2 phase through incubation without added hormone, followed by incubation with auxin (2,4-D) alone, to bring cells to arrest with 2C DNA content (Zhang et al., 1996 To investigate how well cdc25 supported progression to mitosis, division was followed in a culture that was divided and treated with either the transgene inducer dexamethasone or the cytokinin kinetin, or remained untreated. Mitosis was dependent upon stimulation but was completed as rapidly when initiated by cdc25 expression as by hormone (Fig. 2). The daughter cells resulting from Cdc25 induction were fully viable, indicating that mitosis initiated by mitotic phosphatase is functionally normal and suggesting that CDK activation by phosphatase could be a natural part of cytokinin action. The daughter cells could proliferate indefinitely with dexamethasone replacing exogenous cytokinin, and they yielded a 9-fold increase in cell number in 7 d as did control cultures with cytokinin.
To test whether induced mitosis involved appearance of Cdc25 enzyme activity, we developed an assay for Cdc25 Tyr phosphatase activity that was based upon the in vitro activation of CDK1 (Cdc2) protein kinase enzyme of fission yeast. Yeast CDK1 was prepared for use as an assay reagent in a form that is responsive to activation by Cdc25 by extraction from yeast cells containing a temperature conditional mutation in Cdc25 phosphatase. At the restrictive temperature of 35°C, this mutation caused accumulation of phosphate on Tyr-15 of CDK1, which inhibited its H1 histone kinase activity (Millar et al., 1991 ). This Tyr-phosphorylated CDK1 was used as substrate in the first phase of a Cdc25 phosphatase activity assay that involved in vitro removal of the inhibitory phosphate from the CDK1 by Cdc25. Then, in a second phase of the Cdc25 activity assay, antibody-bound phosphatase enzyme was removed and the extent of its action was measured by the resulting CDK1 H1 histone kinase activity. Induced Cdc25 enzyme activity was detected after 6-h induction with dexamethasone (Fig. 3, left) in enzyme purified by anti-GST-Cdc25 antibody. Specificity of Cdc25 enzyme recovery by the antibody was indicated by low activity recovered by preimmune serum and serum precompeted with freeze/thaw-inactivated GST-Cdc25.
To test the hypothesis that induced Cdc25 activity in Nicotiana stimulated mitosis through activation of cell cycle kinase, CDKA kinase was recovered with antibody specific for its carboxyterminal peptide, and its H1 histone kinase activity was assayed. CDKA was selected for study as the class of plant CDK that has most extensive involvement in the cell cycle, being required for both DNA replication and mitosis (Mironov et al., 1999 ). Members of this class of CDK are recognized by their close sequence similarity to the CDK1 of other eukaryotes (Joubès et al., 2000) and by their capacity to complement mutated CDK1 function in yeast, as has been shown for Nicotiana CDKA (Setiady et al., 1996). CDKA activity was found to increase markedly, after induction of Cdc25 with dexamethasone, in G2 phase-arrested cells (Fig. 3, right). Specific recovery of the CDKA kinase was indicated by elimination of recovery after precompetition of the antibody with free CDKA carboxyterminal peptide. These indications that Cdc25 acted by affecting CDKA were further tested by direct measurement of Tyr phosphate in the enzyme, which revealed a progressive decline in phosphate through the first 12 h after induction with dexamethasone (Fig. 4). To more rigorously test whether the decline in phospho-Tyr in CDK followed induction of Cdc25 activity and correlated with the rise in CDKA protein kinase activity, the timing of the increase in these enzyme activities was measured. Following induction with dexamethasone, Cdc25 activity rose rapidly (Fig. 5) and remained elevated until 12 h, which correlated with the period during which phosphotyrosine in CDKA declined to a basal level (Fig. 4) and CDKA activity increased to its peak (Fig. 5), consistent with the hypothesis that the decline in phosphotyrosine in CDKA was caused by Cdc25 activity and resulted in rising CDKA activity.
A Normal Mechanism in Plant Mitosis
To explore the possibility that Cdc25 induced division because Tyr phosphatase activity directed to CDKA is a normal part of plant mitosis, a control portion of culture was examined after treatment with cytokinin instead of dexamethasone (Fig. 6). The induction of mitosis by hormone involved a very similar increase in Cdc25-like phosphatase activity recoverable by anti-GST-Cdc25 that showed a slightly slower initial increase than was observed when the transgene was induced (Fig. 5) but again occurred in the period up to 12 h during which CDKA activity increased. The absence of a lower total Cdc25-like catalytic activity than when yeast enzyme was expressed (Fig. 5) is consistent with the evidence from other kingdoms that different amounts of the mitotic inducer phosphatase are accommodated and activity is stabilized by homeostatic controls that act on Cdc25 enzyme (Kovelman and Russell, 1996
The hypothesis was further investigated by testing the potential for activation by Cdc25. Through the prophase period of 3 to 12 h extracted CDK activity increased, but total activity after in vitro activation was relatively constant (Fig. 7), therefore showing that in vivo-activated enzyme could not be additionally activated by phosphatase and indicating that the previous in vivo activation had been by phosphatase. Since CDKA amount is essentially constant through the division cycle of cultured Nicotiana cells (Setiady et al., 1996 ; Zhang et al., 1996) this indicates that the rising CDK activity in plant prophase is due to progressive removal of inhibitory Tyr phosphate from CDK. The persistence of some capacity for activation by Cdc25 up to 12 h correlates with persistence of a basal level of phosphotyrosine in the enzyme (Fig. 4). Anaphase was shown to involve a different activity control since the sharp decline in CDK activity after 12 h could not be reversed by incubation with GST-Cd25 (Fig. 7), consistent with activity in anaphase being limited by another cause such as the proteolytic elimination of B-cyclins (King et al., 1995), which has been observed in maize and Nicotiana (Mews et al., 1997; Genschik et al., 1998). The same relative timing of phosphatase and CDK activities and mitosis was consistently observed in different experiments and in all three transgenic lines that were investigated. Furthermore, when phosphatase and CDK activity peaks were a few minutes earlier or later in different cultures, mitosis was proportionately earlier or later (data not shown), consistent with phosphatase activation of CDK again being the determinant of mitotic timing.
A further indication that cytokinin is required as inducer of Cdc25 phosphatase activity was the consistently low level of Cdc25-like activity in cells limited by cytokinin, as at 0 h in Figures 3, 5, 6, and 8 and the rise in Cdc25-like activity when stimulated by cytokinin. To test more rigorously whether such rise is normally involved at mitosis, nontransgenic cells were compared with transgenic cells as a source of CDK-activating phosphatase recoverable by anti-GST-Cdc25, and plant CDK was used as the substrate enzyme to test activation capacity. For this purpose, plant CDK enzyme that was phosphorylated on Tyr (as shown in Fig. 4) was taken from cells arrested at the G2 control point (Fig. 8, lanes 1–3) or after 3-h stimulation with cytokinin (Fig. 8, lanes 4–6) when activation of CDK was in progress (as shown in Fig. 6). A CDK Tyr phosphatase activity, unambiguously of plant origin because from nontransgenic cells (lane 5), was found to be slightly more active than Cdc25 enzyme recoverable from transgenic cells that had also been induced for 6 h (lane 6), indicating that a plant Cdc25-like Tyr phosphatase participates in plant mitosis.
A Key Early Event in the Stimulation of Mitosis
Cytokinin is necessary in cultured cells for both the initiation of mitosis and the dephosphorylation of Tyr in CDK (Zhang et al., 1996
We suggest that cytokinin stimulation of Tyr dephosphorylation in CDK is a normal part of prophase progression in plants. Consistent with this, cells that were synchronously progressing through prophase without induction of a transgene contained CDKA that declined in capacity to be activated by Cdc25 as the enzyme became progressively activated in vivo (Fig. 7) and as its phosphotyrosine content declined (Fig. 4). Furthermore, a plant Tyr phosphatase activity rose following cytokinin stimulation coincident with mitotic activation of CDKA, and the phosphatase was able to activate yeast and plant CDK (Figs. 3, 6, and 8). Identity of the plant mitotic-Tyr phosphatase remains uncertain although it was recovered in immunoprecipitates with antibody against the catalytic region of yeast Cdc25. Close structural similarity of the plant Tyr phosphatase with Cdc25 cannot be assumed since genome sequencing has not yet shown a plant protein phosphatase with extensive similarity to Cdc25. Furthermore, it is not certain that the plant mitotic inducer must be very similar since Cdc25 is not the only mitosis inducing phosphatase in yeast but rather works in tandem with the protein phosphatase Pyp3 (Millar et al., 1992
Our findings indicate that cytokinin, in addition to stimulating G1/S progression through induction of D-cyclins (for review, see Murray et al., 2001
In plant tissues there are also indications from transgene effects that cytokinin regulation of meristem activity involves modulation of CDK phosphorylation. Because cdc25 expression, while substituting for cytokinin, increased the proportion of CDK that was dephosphorylated and active, we hypothesize that high cytokinin is signaled by Tyr-dephosphorylated CDK. Several transgenic and hormone treatment studies are consistent with this. Increased cell division and initiation of growth in lateral shoots follows both raised cytokinin level (e.g. Pillay and Railton, 1983
In the root also, in spite of high exogenous cytokinin being inhibitory to formation of lateral organs (for review, see Srivastava, 2002
Cell Culture
Suspension cultures of Nicotiana plumbaginifolia were grown in CS V medium with 9 µM synthetic auxin (2,4-D) and 0.23 µM kinetin and were brought to arrest at the cytokinin control point by omission of kinetin, and mitotic activity and cell numbers were monitored after fixing protoplasting and DNA staining, as described by Zhang et al. (1992
Polyclonal antibodies were raised in rabbits. The carboxyterminal peptide KRITARNALEHEYFKDIGYVP of the tobacco (Nicotiana tabacum) CDKA, which has been shown by complementation to be a functional homolog of Cdc2 (Setiady et al., 1996
CDK and Cdc25 enzymes were extracted from cells that were frozen and ground in liquid nitrogen. CDK was extracted at 0°C in NDE buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 15 mM dithiothreitol (DTT), 3 µg mL–1 leupeptin, 20 mM EGTA, pH 7.4, 80 mM
Cdc25 was extracted from cell grindate in PDE buffer, containing 25 mM MOPS, pH 7.2, 100 mM NaCl, 10 mM DTT, 5 mM EDTA, 1 mM EGTA, 1% NP40, 50 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride, leupeptin 3 µg mL–1, aprotinin 20 µg mL–1. Immunoprecipitates of Cdc25 were obtained with 10 µL of antibody added to soluble protein from 5 mL of culture (5–10 mg protein) for 3 h at 4°C, sedimented with 35 µL protein A beads, then washed for 10-min intervals three times with HDW buffer supplemented with 2 µM spermidine and then washed once with HBK buffer supplemented with 2 µM spermidine. Cdc25 assays were conducted in two stages; first, immunoprecipitates of Cdc25-like protein from 500 µg plant protein were incubated for 30 min at 30°C in Cdc25 assay buffer (Millar et al., 1991
Phosphotyrosine was detected in CDKA enzyme that was recovered by antibody, essentially as for CDK activity assay, from 10 mg soluble plant protein with NDE buffer modified by increase of sodium vanadate to 2.5 mM and addition of 1 mM phosphotyrosine, and the immune complex was washed twice with HDW buffer supplemented to 1 mM with sodium vanadate. After separation by SDS PAGE on 12% gel a western blot was probed with anti-phosphotyrosine mouse monoclonal (PY99, Santa Cruz Biotechnology, Santa Cruz, CA) and [125I]-second antibody then detected by PhosphorImager, as described (Zhang et al., 1996
RNA was extracted from cells ground in liquid nitrogen into 2 volumes of 10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% SDS, and 2 volumes of phenol:chloroform:iso-amylalcohol (25:24:1, v/v) at 4°C and fractionated. Northern blots were probed with the 650-bp BglII-XbaI fragment of cdc25 made radioactive by nick translation (Sambrook et al., 1989 Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to requisite permission from any third party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.
The authors acknowledge the assistance of Phillip Larkin with electroporation and the kind provision of plasmid pGEX25-BD by Dr. Paul Russell. Received August 17, 2004; returned for revision November 3, 2004; accepted November 10, 2004.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.051938. * Corresponding author; e-mail john{at}rsbs.anu.edu.au; fax 61–2–61254331.
Beeckman T, Burssens S, Inzé D (2001) The peri-cell-cycle in Arabidopsis. J Exp Bot 52: 403–411 Biddington NL, Dearman AS (1982) The involvement of the root apex and cytokinins in the control of lateral root emergence in lettuce seedlings. Plant Growth Regul 1: 183–193 Bulavin DV, Higashimoto Y, Demidenko ZN, Meek S, Graves P, Phillips C, Zhao H, Moody SA, Appella E, Piwnica-Worms H, et al (2003) Dual phosphorylation controls Cdc25 phosphatases and mitotic entry. Nat Cell Biol 5: 545–551[CrossRef][Web of Science][Medline] Chaudhury AM, Letham DS, Craig S, Dennis ES (1993) amp1: a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering. Plant J 4: 907–916[CrossRef][Web of Science] Cleary AL, Fowke LC, Wang H, John PCL (2002) The effect of ICK1, a plant cyclin-dependent kinase inhibitor, on mitosis in living plant cells. Plant Cell Rep 20: 814–820[CrossRef]
Genschik P, Criqui MC, Parmentier Y, Derevier A, Fleck J (1998) Cell cycle-dependent proteolysis in plants: identification of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132. Plant Cell 10: 2063–2075 Gorst JR, Sek FJ, John PCL (1991) Level of p34cdc2-like protein in dividing, differentiating and dedifferentiating cells of carrot. Planta 185: 304–310 Gutierrez C, Ramirez-Parra E, Castellano M, Carlos del Pozo JC (2002) G1 to S transition: more than a cell cycle engine switch. Curr Opin Plant Biol 5: 480–486[CrossRef][Web of Science][Medline]
Haberer G, Kieber JJ (2002) Cytokinins: new insights into a classic phytohormone. Plant Physiol 128: 354–362 Hata S, Kouchi H, Suzuka I, Isii T (1991) Isolation and characterisation of cDNA clones for plant cyclins. EMBO J 10: 2681–2688[Web of Science][Medline] Hemerly A, de Almeida Engler J, Bergounioux C, Van Montagu M, Engler G, Inzé D, Ferriera P (1995) Dominant negative mutants of Cdc2 kinase uncouple cell division from iterative plant development. EMBO J 14: 3925–3936[Web of Science][Medline] Hemerly AS, Ferriera PCG, de Almeida Engler J, Van Montagu M, Engler G, Inzé D (1993) cdc2a expression in Arabidopsis is linked with competence for cell division. Plant Cell 5: 1711–1723[Abstract] Hoth S, Ikeda Y, Morgante M, Wang X, Zuo J, Hanafey MK, Gaasterland T, Tingey SV, Chua NH (2003) Monitoring genome-wide changes in gene expression in response to endogenous cytokinin reveals targets in Arabidopsis thaliana. FEBS Lett 554: 373–380[CrossRef][Web of Science][Medline] Hush JM, Wu L, John PCL, Hepler LH, Hepler PK (1996) Plant mitosis promoting factor disassembles the microtubule preprophase band and accelerates prophase progression in Tradescantia. Cell Biol Int 20: 275–287[CrossRef][Web of Science][Medline] John PCL, Zhang K (2001) Cytokinin control of cell proliferation in plant development. In D Francis, ed, The Plant Cell Cycle and Its Interfaces, CRC Press, Boca Raton, FL, pp 190–211 John PCL, Zhang K, Dong C, Diederich L, Wightman F (1993) p34cdc2 related proteins in control of cell cycle progression, the switch between division and differentiation in tissue development and stimulation of division by auxin and cytokinin. Aust J Plant Physiol 20: 503–526 Joubès J, Chevalier C, Dudits D, Heberle-Bors E, Inzé D, Umeda M, Renaudin JP (2000) CDK-related protein kinases in plants. Plant Mol Biol 43: 607–620[CrossRef][Web of Science][Medline] Kakimoto T (2003) Perception and signal transduction of cytokinins. Annu Rev Plant Biol 54: 605–627[CrossRef][Medline] King RW, Peters JM, Tugendreich S, Rolfe M, Hieter P, Kirschner MW (1995) A 20S complex containing CDC27 and CDC16 catalyses the mitosis-specific conjugation of ubiquitin to cyclin B. Cell 81: 279–288[CrossRef][Web of Science][Medline] Klee H, Romano C (1994) The roles of phytohormones in development as studied in transgenic plants. CRC Crit Rev Plant Sci 13: 311–324
Kovelman R, Russell P (1996) Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe. Mol Cell Biol 16: 86–93 Laureys F, Dewitte W, Van Montagu M, Inzé D, Van Onckelen H (1998) Zeatin is indispensable for the G2-M transition in tobacco BY-2 cells. FEBS Lett 426: 29–32[CrossRef][Web of Science][Medline]
Mähönen AP, Bonke M, Kauppinen L, Riikonen M, Benfey PN, Helariutta Y (2000) A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes Dev 14: 2938–2943
Mailand N, Falck J, Lukas C, Syljuasen RG, Welcker M, Bartek J, Lucas J (2000) Rapid destruction of human Cdc25A in response to DNA damage. Science 288: 1425–1429 McKibbin RS, Halford NG, Francis D (1998) Expression of fission yeast cdc25 alters the frequency of lateral root formation in transgenic tobacco. Plant Mol Biol 36: 601–612[CrossRef][Web of Science][Medline]
Medford JI, Horgan R, El-Sawi Z, Klee HJ (1989) Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentyl transferase gene. Plant Cell 1: 403–413 Mészáros T, Miskolczi P, Ayaydin F, Pettkó-Szandtner A, Peres A, Magyar Z, Horváth GV, Bakó L, Fehér A, Dudits D (2000) Multiple cyclin-dependent kinase complexes and phosphatases control G(2)/M progression in alfalfa cells. Plant Mol Biol 43: 595–605[CrossRef][Web of Science][Medline] Mews M, Sek FJ, Moore R, Volkmann D, Gunning BES, John PCL (1997) Mitotic cyclin distribution during maize cell division: implications for the sequence diversity and function of cyclins in plants. Protoplasma 200: 128–145[CrossRef] Millar JBA, Lenaers G, Russell P (1992) Pyp3 PTPase acts as a mitotic inducer in fission yeast. EMBO J 11: 4933–4941[Web of Science][Medline] Millar JBA, McGowan CH, Lenaers G, Jones R, Russell P (1991) p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast. EMBO J 10: 4301–4309[Web of Science][Medline]
Mironov VV, De Veylder L, Van Montagu M, Inzé D (1999) Cyclin-dependent kinases and cell division in plants—the nexus. Plant Cell 11: 509–522 Murray JAH, Doonan J, Riou-Khamlichi C, Meijer M, Oakenfull EA (2001) G1 cyclins, cytokinins and the regulation of the G1/S transition. In D Francis, ed, The Plant Cell Cycle and Its Interfaces. CRC Press, Boca Raton, FL, pp 19–41 Nefsky B, Beach D (1996) Pub1 acts as an E6-AP-like protein ubiquitin ligase in the degradation of cdc25. EMBO J 15: 1301–1312[Web of Science][Medline]
Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C (2004) Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. Plant Cell 16: 1365–1377 Norbury C, Nurse P (1992) Animal cell cycles and their control. Annu Rev Biochem 61: 441–470[CrossRef][Web of Science][Medline] Patton WF, Lam L, Su Q, Lui M, Erdjumentbromage H, Tempst P (1994) Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting. Anal Biochem 220: 324–335[Medline]
Pillay I, Railton ID (1983) Release of axillary buds from apical dominance in intact, light grown seedlings of Pisum sativum L. following application of cytokinin. Plant Physiol 71: 972–974
Preuss SB, Britt AB (2003) A DNA-damage-induced cell cycle checkpoint in Arabidopsis. Genetics 164: 323–334 Redig P, Shaul O, Inzé D, Van Montagu M, Van Onckelen H (1996) Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells. FEBS Lett 391: 175–180[CrossRef][Web of Science][Medline] Reichheld J-P, Vernoux T, Lardon F, Van Montagu M, Inzé D (1999) Specific checkpoints regulate plant cell cycle progression in response to oxidative stress. Plant J 17: 647–656[CrossRef][Web of Science]
Rhind N, Russell P (2001) Roles of the mitotic inhibitors Wee1 and Mik1 in the G2 DNA damage and replication checkpoints. Mol Cell Biol 21: 1499–1508
Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JAH (1999) Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science 283: 1541–1544 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Sauter M, Kende H (1992) Gibberellin induced growth and regulation of the cell division cycle in deepwater rice. Planta 188: 362–368[Web of Science]
Schena M, Lloyd AM, Davies RW (1991) A steroid-inducible gene expression system for plant cells. Proc Natl Acad Sci USA 88: 10421–10425 Schmülling T, Schäffer S, Romanov G (1997) Cytokinins as regulators of gene expression. Physiol Plant 100: 505–519[CrossRef]
Schuppler U, He P-H, John PCL, Munns R (1998) Effect of water stress on cell division and Cdc2-like cell cycle kinase activity in wheat leaves. Plant Physiol 117: 667–678
Setiady YY, Sekine M, Hariguchi N, Kouchi H, Shinmyo A (1996) Molecular cloning and characterisation of a cDNA clone that encodes a Cdc2 homolog from Nicotiana tabacum. Plant Cell Physiol 37: 369–376 Srivastava LM (2002) Plant Growth and Development. Academic Press, San Diego, pp 303–379 Suchomelova P, Velgova D, Masek T, Francis D, Rogers HJ, Marchbank AM, Lipavska 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 Physiol Biochem 42: 49–55[CrossRef][Web of Science][Medline] Taylor BH, Larkin PJ (1988) Analysis of electroporation efficiency in plant protoplasts. Aust J Biotechnol 1: 52–57 Valente P, Tao W, Verbelen J-P (1998) Auxins and cytokinins control DNA replication and deduplication in single cells of tobacco. Plant Sci 134: 207–215[CrossRef] Van't Hof J (1974) Control of the cell cycle in higher plants. In GM Padilla, IL Cameron, A Zimmerman, eds, Cell Cycle Controls. Academic Press, New York, pp 77–85 Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98: 10478–10492 Wightman F, Schneider EA, Thimann KV (1980) Hormonal factors controlling the initiation and development of lateral roots. II. Effects of exogenous growth factors on lateral root formation in pea roots. Physiol Plant 49: 304–314[CrossRef] Wyrzykowska J, Pien S, Shen WH, Fleming AJ (2002) Manipulation of leaf shape by modulation of cell division. Development 129: 957–964
Zetterberg A, Larsson O (1985) Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells. Proc Natl Acad Sci USA 82: 5365–5369
Zhang K, Yasumasa T, John PCL (1992) Mitotic arrest in tobacco caused by the phosphoprotein phosphatase inhibitor okadaic acid. Plant Cell Physiol 33: 677–688 Zhang K, Letham DS, John PCL (1996) Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2 -like H1 histone kinase. Planta 200: 2–12[Web of Science][Medline] This article has been cited by other articles:
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ASPB Publications | PLANT PHYSIOLOGY® | THE PLANT CELL | |
|---|---|---|---|
TOP
ABSTRACT
RESULTS
), and nontransgenic cells treated identically (
). Cells had been arrested in G2 phase, by culture without hormone and then with auxin only, prior to dexamethasone treatment.
) proceeded with similar rapidity. Prior to the sampling period, cells were brought to arrest in G2 phase by culture without hormone and then with auxin only, and at zero time received either cytokinin at normal medium concentration or dexamethasone at 0.1 µM; control received no addition (
) was relatively constant through prophase (3–12 h), indicating that activation in vivo reduced the capacity for further activation by Tyr phosphatase. Low CDK activity after 12 h, while cells progressed through anaphase, could not be increased by phosphatase in vitro and was probably not due to inhibitory phosphorylation.
, as described (Millar et al., 1991
-galactoside for 3 h were loaded at 0.5 µg/lane. 
-32P]ATP, incubated at 30°C for 10 min and the reaction stopped by addition of 10 µL of 1% acetic acid. Phosphate transferred to H1 histone was measured by transfer of 24-µL aliquots to strips of Whatman (Clifton, NJ) p81 paper, then scintillation counting radioactivity in histone after extensive washing in 75 mM phosphoric acid, and was also visualized by neutralizing the reaction mix with NaOH and adjusting to pH 7.8 with 0.5 M Tris-HCl before SDS PAGE on 12% gel and exposure of the fixed, washed, and dried gel in a PhosphorImager. 
