JXB Advance Access published online on July 3, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm148
RESEARCH PAPER |
Okadaic acid (1 µM) accelerates S phase and mitosis but inhibits heterochromatin replication and metaphase–anaphase transition in Vicia faba meristem cells
mierczak
Department of Cytophysiology, University of 
ód, 90-231 ód, ul. Pilarskiego 14, Poland
* To whom correspondence should be addressed. E-mail: justpoli{at}poczta.onet.pl
Received 30 March 2007; Revised 31 May 2007 Accepted 1 June 2007
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Abstract |
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Protein kinases and phosphatases are the foremost agents which take part in cell cycle regulation in both plants and other eukaryotes. Protein kinases are a very well examined group of proteins with respect to chemical structure and function. Nowadays protein phosphatases, including PP1 and PP2A belonging to the PSP family, are the focus of interest. Okadaic acid (OA) which is a specific inhibitor of protein phosphatase activity is widely used to study them. In the present research, the involvement of OA-sensitive phosphatases in the regulation of progression of the plant cell cycle was analysed (in planta) using Vicia faba root meristems synchronized with hydroxyurea and divided into five series. Each series was treated with 1 µM OA for 3 h for different time periods corresponding to the consecutive cell cycle phases. The results showed that in the OA-treated cells DNA replication and mitosis began earlier than in the control cells, since G1 and G2 phases were significantly shorter and the H1 histone kinases activity was higher. Moreover, autoradiography and morphological analyses of mitotic figures revealed that the OA-treated cells entered mitosis before the end of heterochromatin replication. An immunocytochemical search showed that earlier initiation of S phase in the OA-treated cells correlated with more abundant phosphorylation of Rb-like protein in comparison with the control cells. OA also induced significant condensation of metaphase chromosomes and blocked metaphase–anaphase transition.
Key words: Okadaic acid, plant cell cycle, protein phosphatases, Vicia faba
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Introduction |
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An increase in the number of plant cells takes place only in meristems during a cell cycle, which is divided into four distinct phases: G1, S, G2, and M. Genomes of plants, like those of other eukaryotic organisms, encode countless proteins which, in the presence of other molecules, regulate progression of the cell cycle (Rubin et al., 2000; Vandepoele et al., 2002; Robbens et al., 2005). Amonst these are cyclin-dependent kinases (CDKs; Joubès et al., 2000; Menges and Murray, 2002), cyclins (Renaudin et al., 1996), tyrosine kinases, e.g. wee1 (Robbens et al., 2005), CDK inhibitors (CKIs; De Veylder et al., 2001; Zhou et al., 2002), distinct classes of protein phosphatases (PSPs and PTPs; Q Lin et al., 1998; Luan, 1998; Suh et al., 1998; Fordham-Skelton et al., 1999; Bollen and Beullens, 2002; Khadaroo et al., 2004; Landrieu et al., 2004), mitogen-activated protein kinases (MAPKs; Luan, 1998; Heberle-Bors, 2001; Soyano et al., 2003), as well as proteins from the retinoblastoma pathway, Rb–E2F–DP (Dyson, 1998; Shen, 2002). All of them play an important role during each phase of the cell cycle as well as at the control points, which ensure successful completion of each phase before the next one is initiated. Complexes of particular CDKs with particular cyclins form the SPF (S-phase-promoting factor) and MPF (M-phase-promoting factor) which are the positive regulators of the G1–S and G2–M transitions, respectively (Doree et al., 1989; Jacobs, 1992; Dutta and Stillmann, 1992; Zhang et al., 1996; Borgne et al., 1999). SPF and MPF, by sequentially phosphorylating substrate proteins (e.g. pRb/E2F before replication and lamins or histones before mitosis), activate entry into and progression through the cell cycle. The activity of SPF and MPF is self-regulated by a feedback reaction or by other kinases and phosphatases, as well as interaction with specific inhibitors (Q Lin et al., 1998; X-H Lin et al., 1998). Protein phosphorylation and dephosphorylation are, among others, the key events by which yeast, animals, and plants regulate the cell cycle, and are catalysed by protein kinases and phosphatases, respectively. These enzymes are highly specific, and their expression and activity are tightly controlled by extracellular and intracellular signals (Hunter, 1995; Berndt, 1999; Heberle-Bors, 2001; Stals and Inzé, 2001; Bollen and Beullens, 2002; Vandepoele et al., 2002; Robbens et al., 2005). The phosphorylation status can affect the subunit composition, enzymatic activity, subcellular localization, and turnover of proteins (transcription factors, histones, lamins), leading to intracellular rearrangements. As a consequence, the cells are directed to consecutive stages of the cycle or stopped at the control points (Berndt, 1999; Stals and Inzé, 2001; Bollen and Beullens, 2002).
There is much more information concerning kinases and their accessory proteins, e.g. cyclins, than about phosphatases; thus recently the latter have received more investigative attention and now it is clear that phosphatases function not only by counterbalancing the protein kinases but also by taking a leading role in many signalling events (Q Lin et al., 1998; X-H Lin et al., 1998; Luan, 1998; Rubin et al., 1998; Schönthal, 1998; Suh et al., 1998; Berndt, 1999; Fordham-Skelton, 1999; Janssens and Goris, 2001; Bollen and Beullens, 2002; Khadaroo et al., 2004; Landrieu et al., 2004).
The large group of protein phosphatases has been divided into families on the basis of their substrate specificity, structure, and sensitivity towards inhibitors (Hunter, 1995; Cohen, 1997; Bollen and Beullens, 2002). The first family includes protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSP). PTPs together with MAPKs share in signal transduction pathways (Xu et al., 1998; Rodríguez-Zapata and Hernandez-Sotomayor, 1998; Fordham-Skelton et al., 1999). However, a very important phosphatase, cdc25, required for the human G1–S transition and regulation of MPF during G2–M transition, belongs to the DSP family (Hunter, 1995; Khadaroo et al., 2004; Landrieu et al., 2004). The presence and possible role of this kind of proteins in plant cells are still unclear, though cdc25-like proteins have been discovered (Khadaroo et al., 2004; Landrieu et al., 2004; Robbens et al., 2005). The next largest family (PSPs) contains serine/threonine-specific protein phosphatases, classified into the PPP and PPM subfamilies. The PPP subfamily includes the phosphatases PP1, PP2A, PP2B, and PP3–PP7, whereas the PPM family includes PP2C (Hunter, 1995; Cohen, 1997; Bollen and Beullens, 2002; Kerk et al., 2002). The enzymes from the PSP family share high sequence similarity (except for PP2C; Schweighofer et al., 2004). Almost all types of protein phosphatases known in yeasts and mammals have also been identified in various higher plants as products of multigene families (Suh et al., 1998; Q Lin et al., 1998; Andreeva and Kutuzov, 2001; Kerk et al., 2002). Most of them are composed of more than one catalytic or variable regulatory subunit, hence there is the possibility of forming many holoenzymes. For example, although in PP2A there are only two catalytic subunit isoforms, the number of potential combinations with 22 different regulatory subunits is very large, and a total of about 75 different dimeric and trimeric holoenzymes can be generated (Janssens and Goris, 2001).
The marine sponge toxin okadaic acid (OA), a specific PSP inhibitor, has greatly facilitated the study of these enzymes in vitro because each enzyme from this family shows different sensitivity to this drug. For instance, the IC50 for OA is around 20–100 nM with respect to PP1 activity and 0.1–1.0 nM with respect to PP2A. PP2B is inhibited by doses over 5000 nM, and PP2C is not sensitive to OA (Schönthal, 1998). Experiments with OA and many other phosphatase inhibitors have demonstrated that plant PSPs play a role in the control of metabolism and development, auxin transport, the signal transduction pathway, as well as cell cycle regulation (Paulson et al., 1996; Favre et al., 1997; Schönthal, 1998; Berndt, 1999; Ayaydin et al., 2000; Janssens and Goris, 2001; Garriga et al., 2004; Polit and Maszewski, 2005). During plant cell division, both PP1 and PP2A control proper chromosome condensation and mitotic progression, whereas PP2A contributes to the control of mitotic kinase activities and microtubule arrangement (Ayaydin et al., 2000).
The aim of the research was to determine the requirements for protein phosphatases (sensitive to lower OA concentration) in the consecutive phases of the plant cell cycle and during transition from one phase to another (in planta). Vicia faba root meristems synchronized by double hydroxyurea (HU) block and divided into five series were treated with 1 µM OA for 3 h for different time periods corresponding to the consecutive cell cycle phases.
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Materials and methods |
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Seed germination
Seeds of V. faba var. minor cv. Nadwi
la
ski (Center for Seed Production in Sobiejuchy, Poland) were washed, sown on wet filter paper in Petri dishes, and germinated in the dark at room temperature for 4 d. For experiments, the seedlings with equal-sized primary roots (2 cm) were selected.
Cell cycle synchronization
The seedlings were placed in Petri dishes (ø14 cm) filled with 15 ml of Hoagland's Nutrient Solution (HNS) containing 1.25 mM HU, prepared according to the protocol of Dole
el et al. (1999), and cultivated for 18 h. Afterwards, the seedlings were thoroughly washed, incubated in HNS for 6 h, and then transferred again to the HNS with 1.25 mM HU for 12 h (Polit, 2007; Table 1).
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Incubation with OA
Seedlings with the cell cycle synchronized in S phase (after the second HU block) were washed with distilled water, placed in Petri dishes, and grown in HNS without HU for 1–18 h (control series) or in HNS supplemented with 1 µM OA for 3 h periods corresponding to the consecutive cell cycle phases (G1, S, G2, M; see Table 1), i.e. first series—in S phase before initiation of heterochromatin replication, second to fifth hour of incubation; second series—at the stage of heterochromatin replication, fourth to seventh hour of incubation; third series—immediately before and in G2 phase just before mitosis, fifth to eighth hour of incubation; fourth series—at the final stages of mitosis just before and during G1 phase, 10th to 13th hour of incubation; fifth series—at the beginning of the next S phase, 12th to 15th hour of incubation.
The samples of roots (five at every experimental point) were collected after every hour.
Autoradiography
One hour before each sampling period, the roots of seedlings were pulsed for 1 h in 25 µCi cm–3 of [6-3H]thymidine solution (1 ml per five roots; specific activity 40 Mbq mmol–1; Amersham) in HNS with or without HU or OA (according to the protocol, see Table 1). The seedlings were then washed in distilled water for 15 min, and 1 cm root segments were fixed for 1 h in cold Carnoy's mixture of absolute ethanol and glacial acetic acid (3:1; v/v). After fixation, the roots were rinsed with absolute ethanol, rehydrated, hydrolysed in 4 M HCl for 2 h, and stained with Schiff's reagent (pararosaniline; Sigma-Aldrich) according to standard methods (Maszewski and Kamierczak, 1998). Meristems were cut off and squashed in a drop of 45% acetic acid on poly-L-lysine-coated microslides (PolysineTM; Menzel-Gläser, Braunsweig, Germany). Slides were covered with EM-1 emulsion (Amersham-Elkabe, Warszawa, Poland) and exposed for 30 d, developed, and fixed according to the standard methods.
Analyses of indices
Analyses were carried out using a Jenamed-2 microscope (Carl Zeiss, Jena Germany). Labelling and mitotic indices, in the root meristems of each experimental series, were estimated based on the measurements of 5000 cells per sample.
Immunocytochemistry
Immunocytochemical detection of phosphothreonine was performed using mouse monoclonal anti-phosphothreonine fluorescein isothiocyanate (FITC)-conjugated antibodies (
TPab-FITC), according to Polit and Maszewski (2004). The specificity of the antibody was confirmed by the use of phosphothreonine antibody inhibitor (Sigma-Aldrich) as the negative control.
For immunocytochemical detection of phospho-Rb(S807/811), root meristems fixed for 30 min in 4% formaldehyde buffered with phosphate-buffered saline (PBS) were rinsed three times in PBS for 5 min each and treated with the enzyme mixture (1% cellulose Onozuka R-10, 1% pectolyase Y-23', and 1% pectinase) in PBS for 40 min. After washing with PBS (three times for 5 min each), the cells were attached to Super Frost Plus glass slides, and incubated in 5% bovine serum albumin (BSA; w/v) with 0.5% Triton X-100 buffered with PBS for 60 min. After washing with PBS/0.5% Triton X-100 (PBT), the samples were processed for immunostaining. The antibodies against phospho-Rb(S807/811) (Cell Signaling), which do not recognize Rb phosphorylated at other sites, as indicated by the manufacturer, were overlaid onto the cells. After incubation (overnight at 4 °C) the slides were washed three times (5 min each) with PBT and incubated in the secondary anti-rabbit FITC-conjugated antibody (Cell Signaling) diluted 1:200 in PBT for 2 h at room temperature in the dark. The samples were then washed twice (5 min each) with PBT and then for 5 min in PBS, and embedded in PBS/glycerol mixture (9:1) with 2.3% DABCO (Sigma-Aldrich). Observations were made using an Optiphot-2 fluorescence microscope (Nikon, Warszawa, Poland) equipped with a B-2A filter (
=450–490 nm) for FITC. Negative control sections incubated with non-immune serum in place of primary antibody or without primary antibody were consistently free from immunostaining (Polit and Kamierczak, 2007). Images were recorded by a DXM 1200 CCD (Nikon) camera.
Determination of kinase activity
To determine kinase activity, the plant tissue samples were prepared by cutting 1 mm long apical parts with a razor blade from four V. faba roots, which were quickly placed in an Eppendorf tube, weighed, and frozen on solidified carbon dioxide. Then the samples were actively ground in an Eppendorff tube, using a glass pestle, with the extraction buffer (EB) containing 40 mM Tris (pH 7.5), 20 mM MgCl2, and protease inhibitor cocktail (Sigma) at a ratio of 50 µl of EB per mg FW. After centrifugation for 10 min at 15 000 g (4 °C), the pellet was re-extracted with 50 µl of EB and then centrifuged for 10 min (4°C). The supernatants were then combined, mixed, and used immediately to measure the kinase activity.
Protein kinase activity was assayed according to the manufacturer's instructions (Promega) with a Kinase-GloTM Luminescent Kinase Assay Kit (Promega) which consists of the Kinase-GloTM Buffer and the lyophilized Kinase-GloTM Substrate (thermostable UltraGlowTM Recombinant Luciferase). Both components were equilibrated to room temperature and the entire volume of Kinase-GloTM Buffer was transferred into the bottle containing Kinase-GloTM Substrate to reconstitute the lyophilized enzyme. The contents were mixed to obtain a homogeneous solution forming the Kinase-GloTM Reagent. Kinase-GloTM Reagent was immediately dispensed into single-use aliquots and stored at –20 °C. Before use, the appropriate amounts of Kinase-GloTM Reagent were thawed and equilibrated to room temperature.
The reaction mixture was prepared in an Ependorff tube with sequentially added 20 µl of kinase extract, 5 µl of ATP (16 µM), 5 µl of histone H1 (20 µM) as a substrate for kinase, and 25 µl of Kinase-GloTM Reagent. After 10 min to stabilize the luminescent signals, they were recorded using a GLOMAXTM 20/20 luminometer (TBS-20/20n). The kinase activity was calculated as the difference between the luminescence of the control sample (without kinase extracts) containing 16 µM ATP and the luminescence of the rest of the ATP not used for histone phosphorylation by kinase. A unit of activity is expressed as the amount of ATP used by kinase extracted from 1 g FW during 1 min of reaction (U=µg ATP g–1 FW min–1).
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Results |
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Effect of OA on the initiation of heterochromatin replication
In the first experimental series, Hoagland's medium was supplemented with OA between the second and fifth hour post-incubation (Table 1; Fig. 1A, B), when about 90% of the control meristem cells replicated and among them about 60% (third hour) to 10% (fifth hour) poorly incorporated [3H]thymidine. In the other cells, the intensity of this process was substantial or high. Moreover, about 50% of the control meristem cells (fifth hour) replicated heterochromatin (Fig. 2A, B) which was visualized by clusters of autoradiographic grains over replicating nuclei (Fig. 3A; Polit, 2007 press). After 1 h OA treatment, the number of replicating cells was not significantly different and the intensity of this process was slightly lower in comparison with the control series (see indices of [3H]thymidine labelling and of intensity Figs 1A, 2A, and 3). During the next 2 h, labelling indices dropped significantly as a result of nearly complete disappearance of the cells replicating heterochromatin (Figs 1A and 2A). At the same time, the first few cells with a chromatin condensation level characteristic of very early prophase were observed (Figs 1B and 2B). Immunocytochemical studies with the use of anti-phosphothreonine antibodies in OA-treated cells revealed a high level of phosphorylation of threonine residues in the area of the nuclear envelope in comparison with the control (Fig. 4).
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Effect of OA on heterochromatin replication
In the second experimental series, the medium was supplemented with OA between the fourth and seventh hour of culture (Table 1; Fig. 1C, D), when in the control meristems heterochromatin replication was observed in about 10% (fourth hour) and 50% (fifth to sixth hour) of cells, while 65% of cells (seventh hour) were in G2 phase (Figs 2C and 3A; Polit, 2007). In this case, the heterochromatin replication process was not blocked so drastically; at the sixth hour it occurred in >20% of the cells. It seems that the blockade took place only in the cells most retarded in the cycle which, according to the control series, accounted for 20% of the population (Fig. 2C). The correct completion of replication was proved by the rapid appearance of many normal mitotic figures in most of the cells (Fig. 1D). During the sixth hour, when in the control few prophases were observed, in the OA-treated meristems they constituted about 20% (Fig. 1D). In this case, the time of G2 phase was significantly shortened. While in the control 65% of the cells were in G2 phase at the seventh hour, in the OA-treated material at the same time a similar percentage of the cells were already in prophase, and the mitotic index reached its maximum (
90%) during the eighth hour, 2 h earlier than in the control (Figs 1D and 2D). At that time, the activity of H1 histone kinase was also higher in OA-treated meristems than in the control (Fig. 5A). During the ninth and 10th hours, some mitotic cells harboured chromosomes different from the rest (Fig. 6A–H), but they were not numerous (7% of population). At the later stages of division in these chromosomes (especially near centromeres and telomeres which are easily discernible during metaphase and anaphase), intensively Feulgen-stained distinct oval areas (resembling interphase heterochromatin—chromocentres) were observed. They indicated a different condensation level and seemed common for both chromatids (Fig. 6I, J). During telophase, these structures prevented equal migration of chromatids to two poles of a cell, and more chromosomes with elongated chromatid arms were observed at one pole. It seems that this phenomenon reflected the onset of the mitotic condensation of chromosomes in the cells in which activation of heterochromatin replication had been blocked (at the sixth hour); thus the S phase not finished (Fig. 6A–H).
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Effect of OA on G2 phase duration and at the beginning of mitosis
In the third experimental series, the medium was supplemented with OA between the fifth and eighth hour, when the control meristem cells were finishing replication, going through G2 phase, and entering mitosis (Table 1; Fig. 1E, F). In this case, replication of heterochromatin was not blocked and OA drastically shortened the time of G2 phase, and the cells completing [3H]thymidine incorporation started condensation of mitotic chromosomes with hardly any interval. Due to this, at the ninth hour of culture, 50% out of the total of 75% of mitotic cells were already in telophase (Figs 1E, F, and 2F
) while in the control this was only 10% (Fig. 3B). Correctly dividing cells (40% in the 10th hour) started a new cell cycle and DNA synthesis just after telophase without a clear G1 gap (Figs 1E and 2E, F). In the control series, about 66% of G1 cells were observed in the 11th hour of culture (Polit, 2007). At the same time in the OA-treated meristems, about 50% of the cells replicated DNA incorporating [3H]thymidine in euchromatin with weak or medium intensity (Figs 1E, F and 2E, F). During the eighth and ninth hours, a few cells with changed mitotic figures, mainly metaphases, appeared among the mitotic cells (Fig. 7). However, these changes were different from those observed in the previous experimental series. Two kinds of changed metaphase plates were observed: scattered (Fig. 7A–C) and condensed (Fig. 7D–F); chromosomes in them were highly condensed, which was reflected by a significant shortening of their arms (Fig. 7A–F). Changed anaphases and telophases were rare, but chromosome segregation was also incorrect (Fig. 7G–I). The number of such changed mitotic figures did not exceed 8%, but their number increased to 40% and 65% after prolonged incubation with OA from the fifth to the ninth and 10th hour, respectively (data not shown).
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Effect of OA on the final stages of mitosis and G1 phase duration
In the fourth series of experiments, the medium was supplemented with OA between the 10th and 13th hour of culture, when the cells in the control series finished mitosis, moved to G1 phase, and started a new round of replication (Table 1; Fig. 1G, H). No irregularities in the cells leaving mitosis were observed, and chromosome segregation and formation of telophase nuclei were correct. The number of mitotic cells was similar to that in the control. In both the experimental and control series, the populations of cells most retarded in cycle progression, which between the 11th and 13th hour instead of finishing mitosis were merely in the middle of this process, were similar (Figs 1H and 2H). However, some differences appeared in the mitotic figures. The metaphase plates were similar to those observed earlier in the third series of experiments (eighth and ninth hours; Fig. 7A–F). A significant increase in the metaphase index in the 14th, 15th, and 16th hours might indicate that the cell transition from metaphase to anaphase was stopped (Fig. 2H). In the great majority of correctly dividing cells which finished mitosis earlier, the onset of replication was observed. During the 11th hour of culture when 66% of the cells in untreated roots were in G1, the cells in OA-treated roots incorporated [3H]thymidine at a weak or medium level (Figs 1G and 2G). In the replicating cells, no replication inhibition was observed and the labelling index indicated a similar intensity to that in the control (Fig. 2G). In the OA-treated roots, the increase in replication level was accompanied by higher activity of H1 histone kinase than in the control series (Fig. 5B). Immunocytochemical studies of the cells in the OA-treated roots, using anti-phospho-pRb(S807/811) antibodies, revealed a high level of identified phosphorylation in the nuclear area of daughter cells in comparison with the control series (Fig. 8).
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Effect of OA on the beginning of the next S phase
In the last series of experiments the medium was supplemented with OA between the 12th and 15th hour, when most of the control cells replicated (Table 1; Fig. 1I, J). Similarly to the findings in the fourth series, no inhibition of euchromatin replication was observed and >50% of cells incorporated [3H]thymidine similarly to the control (Figs 2I and 3A). During the 18th hour, however, the labelling index decreased by about 20% due to the lack of cells replicating heterochromatin. In the control series, these cells constituted about 20% of the population (Figs 1I and 2I). Between the 15th and 18th hour, the mitotic index increased (Fig. 1J). Constant levels of the phase indices (
18%) between the 14th and 18th hour indicated the presence of the retarded cells finishing the previous mitosis and the appearance of the cells starting a new division (Fig. 2J). The start of OA incubation in the 12th hour (not in the 10th hour as in the fourth series) did not cause incorrect mitotic figures. |
Discussion |
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The influence of OA and thus the role of protein phosphatases at consecutive stages of the cell cycle in asynchronously dividing root meristem cells in planta are difficult to define. The problem consists of the fact that it is impossible to determine the phase of the cell cycle at which a particular cell was influenced by the inhibitor and thus to say whether its division was retarded, inhibited, or accelerated. Synchronization of root meristem cell division seemed to be a good way to deal with this difficulty. In the present report, HU-synchronized cells (accumulated in S phase), with slightly inhibited replication, were used. The analysis of the synchronized cell cycle (after HU removal) in the control V. faba meristems (Polit, 2007) allowed the choice of five periods at specific phases, during which OA was applied. This made it possible to analyse the cell cycle regulation in planta during the transition periods between the consecutive phases. The time of treatment with OA (3 h) and OA concentration (1 µM) were chosen on the basis of both preliminary analyses (data not shown) and the literature (Zernicka-Goetz and Maro, 1993; Buzanska and Wheatley, 1994; Berndt, 1995; Favre et al., 1997; Janssens and Goris, 2001; Polit and Maszewski, 2005).
The results of the present experiments confirm the fact that the regulation of the cell cycle in V. faba root meristems and the control of G2–M and G1–S transitions are governed by protein phosphatases sensitive to 1 µM OA, i.e. PP2A (but the role of PP1 which is less sensitive to OA cannot be excluded).
In plant cells there are two types of CDKs which govern the cell cycle: CDKA (with a PSTAIRE domain) which plays a pivotal role in both the G1–S and G2–M transitions and, unique in plants, CDKB1 or CDKB2 (with a PPTALRE or PPTTLRE domain, respectively) which accumulate at the G2 and M phases and are necessary for the G2–M transition (Porceddu et al., 2001). In plants there are also two distinct classes of CDK-activating kinases (CAKs): CDKD which is functionally related to vertebrate-type CAKs, and the CDKF which is a plant-specific CAK with unique enzymatic specificity (Shimotohno et al., 2004).
These CDKs create complexes with their cell cycle-oscillating partners, cyclins: D-type cyclins essential during G1 phase and for G1FS transition; A-type cyclins, important during control of S–M phases; B-type cyclins, playing a role in the G2–M transition and during M phase; and H-type cyclin, important during the whole cell cycle as part of the CAK (Stals and Inzé, 2001; Shimotohno et al., 2004; Inzé, 2005; Umeda et al., 2005). Similarly to other eukaryotic organisms before mitosis, the CDK–cyclin complexes (MPF) are subject to an inhibitory phosphorylation of a kinase N-terminal residue (tyrosine in yeast, and Thr14 and Tyr15 in mammals) and an activating phosphorylation of Thr161. The inhibitory reaction is catalysed by the WEE1 kinase and is counteracted by the CDC25-like phosphatase, which activates MPF before mitosis (Stals and Inzé, 2001; Zhang et al., 2005). The cells of plant organisms possess the WEE kinase which phosphorylates CDKA (Sun et al., 1999). Furthermore, recently, the unicellular alga Ostreococcus tauri has been found to contain CDC25 (Khadaroo et al., 2004); likewise, a small protein with dual specificity, CDC25-like phosphatase, was identified in Arabidopsis (Landrieu et al., 2004). CDC25 is in turn activated by POLO-like kinases (PLK) and deactivated by PP1 and PP2A. The second activating reaction of MPF is catalysed by CAK, which in turn is negatively regulated, probably by PP2A. Active CDK–cyclin complexes phosphorylate histone H1, lamins, vimentin, and microtubule-associated proteins, initiating mitotic events such as chromosome condensation, nuclear envelope breakdown, and spindle formation (Janssens and Goris, 2001; Stals and Inzé, 2001; Inzé, 2005; Umeda et al., 2005).
In the present studies, OA application at the time when >90% of the cells were at the final stage of replication (second to fifth hour of culture) caused mitotic condensation of chromosomes in some cells, which in the controls was observed at least 2 h later. When added later (fourth to the seventh hour and fifth to the eighth hour), OA drastically shortened the duration G2 phase, increased protein kinase activity, and magnified the effect of a 2 h earlier onset of mitosis. These results are in agreement with the data presented for HU-/OA-treated plant cells (Weingartner et al., 2003) as well as Xenopus oocytes (reviewed in Janssens and Goris, 2001) and with the above-presented model of G2–M transition regulation. These data might indicate that OA, by inhibiting the activity of PP2A, which catalyses CAK dephosphorylation, could block this dephosphorylation, responsible for maintaining this kinase activity. Thus CAK could activate MPF without limitations. A hypothesis might be put forward that in natural conditions PP2A is the main protein which maintains plant cells in G2, allowing time for the correct completion of one important event and the start of the next. This hypothesis corresponds to the observations made by Ayaydin et al. (2000) in experiments performed with endothall (ET). A low concentration of this herbicide in cultured alfalfa cells reduced mostly the activity of PP2A. The studies of Ayaydin et al. (2000) revealed that cdc2MsF kinase was considerably activated by 1 µM ET at the G2–M transition, 4 h earlier than in the untreated cells.
PP1 and/or PP2A are known to participate in normal chromosome condensation and mitotic progression: the reactions during metaphase–anaphase transition and the exit from mitosis in plant and animal cells (Paulson et al., 1994, 1996; Ayaydin et al., 2000; Sassa et al., 2003). Ayaydin et al. (2000) revealed that a high concentration of ET which inhibited both phosphatases (PP1 and PP2A) increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. Likewise, treatment of tobacco cells with 12 µM OA resulted in similar chromosome condensation abnormalities, but mitosis was blocked at exit from G2 (Zhang et al., 1992). Vandré and Wills (1992) showed that low OA concentrations resulted in a metaphase-like mitotic block of a pig kidney cell line. Chaudhuri et al. (1997) revealed that most of the HeLa mitotic cells treated for a prolonged period with OA seemed to be arrested at the metaphase–anaphase transition point. In the arrested mitotic cells the chromosomes became highly condensed and remained arranged at the equatorial plate, but with prolonged treatment the chromosomes became either scattered or clumped. The present research revealed similar abnormalities in the fourth series of experiments in some of the most retarded cells which underwent division last and were influenced by OA at the beginning of or during metaphase. In these cells (14th to 16th hour) highly condensed chromosomes formed incorrect mitotic figures and genetic material was randomly distributed to daughter cells. A few similar effects were observed in the third series of experiments, during the 8th and 9th hour of culture. Their number drastically increased when the time of root incubation with OA was prolonged to 4 h or 5 h. A hypothesis may be put forward that 1 µM OA applied just before or at the beginning of mitosis accelerated its onset and did not disturb chromosome condensation. However, during metaphase, OA blocked the proper metaphase–anaphase transition. This means that in plant cells, similarly to the case in other eukaryotic cells, the onset of mitosis requires the inhibition of PP2A activity while the correct process of leaving mitosis needs its reactivation during metaphase–anaphase. This phenomenon may be connected with microtubule organization and phragmoplast maturation, as well as mitotic cyclin degradation and/or MPF deactivation. (Ayaydin et al., 2000; Criqui et al., 2000; Weingartner et al., 2003)
Taking into account the mechanism of MPF activation it is possible to explain nearly complete (second to fifth hour of incubation) or significant (fourth to seventh hour of incubation) blockade of initiation of heterochromatin replication. Activation of MPF leads to phosphorylation of lamins, then their dissociation, and finally breakdown of a nuclear envelope. Immunocytochemical studies with the anti-phosphothreonine antibodies revealed an increase in phosphorylation within a nuclear envelope. The present results indicated that premature MPF activation could bring about this process before DNA replication was finished, thus blocking initiation (but not continuation) of heterochromatin replication at the end of S phase. It would mean that activation of heterochromatin replication in V. faba cells is independent of earlier activated euchromatin replication while it is dependent on the state of phosphorylation/dephosphorylation of proteins, possibly lamins. This idea would partially correlate with the opinion of Steen et al. (2000) who put forward a hypothesis that phosphorylation/dephosphorylation of B-type lamins in interphase may be implicated in the regulation of nuclear envelope–chromatin interactions, which in turn may regulate intranuclear processes such as replication of heterochromatin or redistribution of B-type lamins from the nuclear envelope to foci of DNA replication in HeLa cells.
In the present work, apart from the above-mentioned results, mobilization of the cells to enter replication earlier (similarly to the case with mitosis) was observed. OA applied both at the beginning and at the end of mitosis accelerated the onset of the next DNA replication cycle by about 2 h. This acceleration was not only a simple consequence of an earlier completed mitosis, as the results of the third series of experiments might suggest, but was also a consequence of OA interference with the processes between mitosis and S phase of the next cycle, as the results of the fourth series of experiments confirm. Earlier replication and significant shortening of the duration of the G1 phase might have been connected with an increase in protein kinase activity and the pRb-like pathway which, similarly to the case in animal cells, in plants plays an important role during G1–S transition. During mitosis pRb is dephosphorylated and regains specificity to bind the E2F transcription factor. PP1 and PP2A prevent cells from premature entry into S phase by keeping pRb in a hypophosphorylated state during M–G1 transition and in early G1 phase (Bollen and Beullens, 2002; Polit and Kamierczak, 2007). E2F release occurs only after a series of pRb phosphorylations by CDK–cyclin complexes active during G1 and G1–S transition (Dyson, 1998; De Veylder et al., 2003; de Jager et al., 2005). In the case when OA was present in cells, disturbances in the balance between kinase and phosphatase activities could bring about replication acceleration. Phosphatases blocked by OA could not remain pRb-like in a hypophosphorylated state after the end of mitosis and thus could not make the G1 phase long enough.
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Acknowledgements |
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We thank Symbios Company for lending us the luminometer which allowed us to measure the protein kinase activity, E Damrat-Rogacka for training, and M Fronczak for help in preparing this manuscript in English.
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