Peter C. L. John

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34cdc2-like protein, recoverable in the p13suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.

PLANT MITOSIS PROMOTING FACTOR DISASSEMBLES THE MICROTUBULE PREPROPHASE BAND AND ACCELERATES PROPHASE PROGRESSION IN TRADESCANTIA

The regulation of mitosis in higher plant cells has been investigated by microinjecting protein kinase from the metaphase‐arresting (met1) mutant ofChlamydomonas. Biochemical characterization of this enzyme complex confirms the presence of a p34cdc2/cyclin B‐like kinase. The enzyme was injected into living stamen hair cells ofTradescantia virginianain which microtubules (MTs) were visualized using fluorescent analogue cytochemistry and confocal laser scanning microscopy. Microinjection of this p34cdc2/cyclin B‐like kinase caused rapid disassembly of the preprophase band of MTs but not of interphase‐cortical, spindle or phragmoplast MTs. Effects of the enzyme on the cytomorphology of live prophase cells were also monitored using video microscopy. We found that injection of this enzyme accelerated chromatin condensation and nuclear envelope breakdown. This indicates the presence and function in plants of an enzyme that can initiate nuclear division similar to the maturation or mitosis promoting factor (MPF) of animal cells. These studies provide the first direct evidence that the mitotically‐active form of plant MPF can drive disassembly of preprophase band MTs, chromosome condensation and initiation of mitosis in plant cells.

Cyclin/cdk complexes: their involvement in cell cycle progression and mitotic division

SummaryDNA replication and mitosis are dependent on the activity of cyclin-dependent protein kinase(CDK) enzymes, which are heterodimers of a catalytic subunit with a cyclin subunit. Cyclinbinding to specific individual proteins is thought to provide potential substrates to Cdk. Protein binding by cyclins is assessed in terms of its mechanism and biological significance, using evidence from diverse organisms including, substrate specificity in animal Cdk enzymes containing D-,A-, and B-type cyclins and extensive cyclin gene manipulations in yeasts. Assembly of protein complexes with cyclin/Cdk is noted and the capacity of the cyclin-dependent kinase subunit Cks, in such complex, to extend the range of Cdk substrates is documented and discussed in terms of cell cycle regulation. Cell cycle progression involves changing abundance of individual cyclins, due to changing rates of their transcription or proteolysis, with consequent changes in the substrates of CDK through the cell cycle. Some overlap of the functions of individual cyclins in vivo has been identified byn cyclin deletions and is suggested to follow a pattern in which cyclins well enough to preserve viability as groups of cyclins are removed by proteolysis. Cyclin accumulation is particularly important in terminating the G1 phase , when it raises CDK activity and starts events leading to DNA replication. It is suggested that plants share this mechanism. The distribution of cyclins and Cdk in maize root tip cells during mitosis and cytokinesis indicates the presence of Cdk1 (Cdc2a) and cyclin CycBlzm;2 at the mature and disassembling preprophase band and the presence of CycBlzm;2 at condensing and condensed chromosomes. Both observations correlate with the earlier-reported capacity of injected metaphase cyclin/CDK to accelerate preprophase band disassembly and chromosomes condensation and with observations correlate with the earlier-reported capacity of injected metaphase cyclin/CDK to accelerate preprophase band disassembly and chromosome condensation and with observation of the location of Cdk and cyclins in other laboratories. Additionally CycBlzm;2 is seen at the nuclear envelope during its breakdown, which correlates with an acceleration of the process by injected metaphase cyclin B/CDK. A phenomenon possibly unique to the plant kingdom is the persistence of mitotic cyclins after anaphase. Participation of cyclins in cytokinesis is indicated by the concentration of the mitotic cyclin CycA1;zm;1 at the phragmoplast. It is suggested that cyclins have a genral function of spatially focusing Cdk activity and that in the plant cell the concentrations cyclins are important mediators of CDK activity at the cytoskeleton, chromosomes, spindle, nuclear envelope, and phragmoplast.

Cell division and endoreduplication: doubtful engines of vegetative growth.

Currently, there is little information to indicate whether plant cell division and development is the collective effect of individual cell programming (cell-based) or is determined by organ-wide growth (organismal). Modulation of cell division does not confirm cell autonomous programming of cell expansion; instead, final cell size seems to be determined by the balance between cells formed and subsequent tissue growth. Control of growth in regions of the plant therefore has great importance in determining cell, organ and plant development. Here, we question the view that formation of new cells and their programmed expansion is the driving force of growth. We believe there is evidence that division does not drive, but requires, cell growth and a similar requirement for growth is detected in the modified cycle termed endoreduplication.

Association of the plant p34cdc2-like protein with p13suc1: implications for control of cell division cycles in plants

SummaryThecdc2-like p34 protein of wheat is closely similar to the p34cdc2 enzyme of yeasts and animals since both have binding affinity for p13suc1 protein encoded by the fission yeastsuc1 gene. In yeast p13suc1 participates in modulation of p34cdc2 activity and is necessary for completion of mitosis. Wheat p34 that was affinity purified on p13suc1 contained the PSTAIR amino acid sequence that is present in all known p34cdc2 homologues and it also had H1 histone protein kinase activity that was independent of calcium and cyclic AMP, which is characteristic of p34cdc2 in vitro. Functional equivalence with the known cell cycle control protein supports our earlier proposition that the high level of this homologue in wheat leaf meristem is important in determining where cell division can occur and the capacity for resumption of cell division. The affinity of plant p34 for fission yeast p13suc1 suggested that a similar association occurs in plants. We have detected a plant homologue of pl3suc1 using affinity-purified antibody raised against the fission yeast protein, in wheat, pea andChlamydomonas. We conclude that the distribution of p13suc1 homologue in plants is likely to be ubiquitous. Thesuc1 gene function inSchizosaccharomyces pombe is essential for mitosis, in which it is required for inactivation of p34cdc2. We propose that association of plant p34 with p13suc1 is similarly involved in plant mitosis.

The plant cell cycle: conserved and unique features in mitotic control

Somatic plant cells can use a hormone checkpoint in late G2 phase. Here cytokinin stimulates removal of phosphotyrosine from p34cdc2 kinase and concurrently capacity for activation of the kinase by Cdc25 phosphatase declines while activity of the kinase increases and cells enter mitosis. Processes unique to plant mitosis are driven by the mitotically active kinase since the enzyme taken from plant cells in metaphase, when injected, can disassemble the preprophase band microtubules that form in G2 phase at the site of the future cross wall. This action is specific, since microtubules are not depolymerised when in interphase cytoplasmic array, or spindle, or phragmoplast. Plant metaphase kinase acts as MPF by accelerating chromosome condensation and nuclear envelope breakdown.

Levels of p34cdc2-like protein in dividing, differentiating and dedifferentiating cells of carrot

P34cdc2 is a key cell-cycle protein in fission yeast that is necessary for progress in the cell cycle from the G1 to the S phase and from G2 through mitosis. Homologues of p34cdc2 have been found in all eukaryotes that have been investigated. Levels of p34cdc2-like protein were studied by quantitative Western blotting in developing cotyledons of Daucus carota L. (carrot) seedlings, in expiants from the same seedlings transferred to tissueculture media with and without 2,4-dichlorophenoxyacetic acid (2,4-D), and in nutrient-starved suspension cultures derived from carrot callus. During the cessation of cell division, which accompanies development of the cotyledon to maturity, there was a 16-fold decline in the level of the p34cdc2-like protein. Auxin-stimulated dedifferentiation in excised tissue from mature cotyledons was accompanied by restoration of the level of p34cdc2-like protein, and the responding cells formed a callus. These data support our earlier proposition, based upon evidence from wheat leaf, that changes in the level of p34cdc2-like protein act in the switch between cycling and differentiation. Persisting high levels of p34cdc2-like protein in suspension cultures, when division was stopped by nutrient limitation, indicated that decline of the protein was not an inevitable consequence of the cessation of division. Decline of p34cdc2 in differentiation may therefore be a regulated process that determines exit from the cell cycle and the converse increase in p34cdc2 may be a regulated process controlling dedifferentiation and resumption of cell division.

The effect of ICK1, a plant cyclin-dependent kinase inhibitor, on mitosis in living plant cells

Abstract. The inhibitory activity of Arabidopsisthaliana ICK1, a plant cyclin-dependent kinase inhibitor, has previously been characterised by its effect on plant cyclin-dependent kinase activity in vitro and its effect on growth in transgenic plants. Herein, we examine cyclin-dependent kinase-driven cell-cycle events, probed by testing the sensitivity of living cells to introduced ICK1 protein. The microinjection of ICK1 into individual Tradescantia virginiana stamen hair cells during late prophase and prometaphase resulted in a clear protein-specific increase in the metaphase transit time (time from nuclear envelope breakdown to the onset of anaphase) in a manner dependent on load and injection time. The results indicate a continuing role for cyclin-dependent kinases in mitotic progression and provide in vivo evidence at the cellular level that ICK1 can restrict growth in the plant by inhibiting cell division.

Isolation and partial characterization of conditional cell division cycle mutants inChlamydomonas

SummaryWe have isolated a number of temperature conditional cell division cycle mutants of the unicellular plantChlamydomonas reinhardtii that are defective in single nuclear genes. Cells grow and divide normally at the permissive temperature (21 °C), but arrest in division at the restrictive temperature (33 °C). We have characterized these mutants using DNA probes and immunofluorescence techniques to localize cytoskeletal and microtubule organizing centre proteins. We describe here 3 broad classes of cell cycle mutation which result in cell cycle arrest with: unreplicated DNA (G1 arrest), duplicated DNA (G2 arrest) and multiple nuclei due to defective cytokinesis (cytokinesis arrest). The continuation of nuclear division in mutants blocked in cytokinesis provides support of an earlier hypothesis that stage specific events in theChlamydomonas cell cycle are arranged in separate dependent sequences. The mutants isolated in the present study provide insights into the role of cytoskeletal proteins in the coordination of plant cell division and the means to investigate the molecular mechanisms whereby division by multiple fission is controlled in the unicellular plantChlamydomonas.

Immunodetection of four mitotic cyclins and the Cdc2a protein kinase in the maize root: Their distribution in cell development and dedifferentiation

SummaryCyclin proteins and cyclin-dependent kinases play a key role in the regulation of cell division. We have therefore studied the relationship of the level of four mitotic cyclin proteins and the Cdc2a kinase protein to cell division in maize root tissue with respect to cessation of division as cells leave the primary meristem region, resumption of division in formation of lateral-root primordia, and induced division following wounding. All four mitotic cyclins and Cdc2a were most abundant in dividing cells. The only examined cell cycle protein which was restricted to dividing tissue was cyclin ZmCycB1;2 (previously ZmIb) and may thus be a limiting factor for cell division. All other cyclin proteins, i.e., ZmCycB1;1 (previously ZmIa), ZmCycA1;1 (previously ZmII), and ZmCycB2;1 (previously ZmIII), and the Cdc2a kinase declined shortly after cells had ceased division. The distance from the root tip at which cells ceased division was tissue-specific and reflected the distance at which decrease of cell cycle proteins was detected. Whereas cyclin ZmCycB1;2 rapidly declined to a hardly detectable level in either nucleus or cytoplasm, in the nuclei of nondividing cells there was persistence of Cdc2a and of cyclins ZmCycB1;1, ZmCycCA1;1, and ZmCycB2;1, indicating that there are plant cyclins which are tightly linked to cell division and others that persist, especially in the nuclei, in nondividing cells. The transition from division to differentiation may thus partly be triggered and enforced by the decrease of the cell cycle proteins and especially the decline of cyclins in the cytoplasm. In the resumption of cell division, both in lateral-root formation and in wound response, high nuclear and low cytoplasmic accumulation of cyclin ZmCycB2;1 was the first visible sign of cell dedifferentiation, implying a role for cyclin ZmCycB2;1 in the G0–G1 phase transition. Next, cytoplasmic accumulation of cyclin ZmCycA1;1, followed by a rearrangement of cortical microtubules, was observed and since both the cyclins ZmCycA1;1 and ZmCycB2;1 were found at places of high tubulin concentration, they may function in the microtubule rearrangement for cell division. When the nuclei of dedifferentiating cells had visibly enlarged, all cyclins and Cdc2a accumulated there, possibly contributing to DNA replication and preparation for mitosis. Later, presumably during G2 phase, cytoplasmic accumulation was observed for Cdc2a at low levels, as observed in G2 phase cells of the primary meristem, and for cyclins ZmCycB1;1 and ZmCycB1;2 accumulation was observed above the levels found in undisturbed meristems, suggesting special contributions to late dedifferentiation processes in both wound-induced and lateral meristems.