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Effect of BAP and IAA on the expression of G1 and G2 control points and G1-S and G2-M transitions in root meristem cells of Vicia faba
Cell Biology International
Cell Biology International 27 (2003) 559–566
www.elsevier.com/locate/cellbi
Effect of BAP and IAA on the expression of G1 and G2 control points and G1-S and G2-M transitions in root meristem cells of Vicia faba Justyna T. Polit, Janusz Maszewski*, Andrzej Kaz´mierczak Department of Cytophysiology, University of Ło´dz´, 90-231 Ło´dz´, ul. Pilarskiego 14, Poland Received 26 April 2001; accepted 31 October 2001
Abstract Excised, carbohydrate-starved root meristems of Vicia faba subsp. minor have been used to investigate the impact of the auxin indole-3-acetic acid (IAA) and the cytokinin benzyl-6-aminopurine (BAP) on (1) the expression of Principal Control Points (PCPs) during the G1- and G2-phases of the cell cycle, and (2) the dynamics of sucrose-mediated resumption of DNA replication and mitosis (G1-to-S and G2-to-M transitions). Compared with the excised root tips starved in mineral medium without hormones, stationary phase meristems induced during continuous treatment with BAP, IAA, or a mixture of BAP+IAA, increased the number of G2 cells, producing characteristic profiles of nuclear DNA content. In medium containing 2% sucrose, BAP accelerated PCP1/S and PCP2/M, whereas continuous treatment with IAA resulted in marked prolongation of both transitions. Since the PCPs regulate progression through the key events of interphase and mitosis by interacting with cyclin dependent kinases (CDKs), these results seem to correspond with current data indicating functional connections between phytohormones, nutritional signals, gene expression and the cell division cycles in plants. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cell cycle checkpoints; Phytohormones; DNA replication; Mitosis; Protein kinases; Root meristems; Vicia faba
1. Introduction Despite the diversity of developmental processes in various organisms, the number of key surveillance mechanisms by which biological control of proliferation is exerted and types of underlying molecular phenomena are surprisingly limited. A series of elaborate signalling pathways, known as checkpoints, have evolved to control the cell division cycles in eukaryotes (Elledge, 1996; Tyson et al., 1995). Distinct regulatory events which govern progression through the cell cycle seem to converge upon a common point in late G1, called START (in yeasts) or Restriction Point (in mammals), at which cells become committed to enter S phase. Here, feedback signals conveying information about the downstream processes can delay the G1/S transition, so as to prevent the cell from triggering DNA replication until certain * Corresponding author E-mail address: [email protected] (J. Maszewski).
monitored events are completed. Before entering mitosis, at the G2 checkpoint, the cell must ensure that DNA replication is complete or damage is repaired, and that all systems are ready for chromatin condensation. Before separation of sister chomatids to daughter cells at the third control point during M phase, each cell must make sure that all chromosomes are properly aligned in one plane halfway between the spindle poles (Rieder and Khodjakov, 1997). The existence of checkpoint control systems in plant cells was first recognized about 30 years ago in Pisum sativum, Vicia faba and Helianthus annuus, in response to carbohydrate starvation of roots (Van’t Hof and Kovacs, 1972). When their apical parts are excised and incubated under conditions restrictive to growth (in mineral medium without sucrose), DNA synthesis and cell division cease and stationary phase meristems are produced with cells accumulated in G1 and G2. The discrete phases at which root meristem cells arrest in G1 and G2 are considered to be metabolic
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00081-7
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blocks that prevent the initiation of DNA replication and mitosis and which are relieved when exogenous carbohydrate is replenished (Van’t Hof and Kovacs, 1972). The reproducibility of this phenomenon in plant cells that cease dividing in vivo due to dormancy, or in vitro due to starvation, was the basis for the PCP hypothesis (reviewed by Van’t Hof, 1985). The ratio of cells with either G1 or G2 DNA content (i.e. the proportion of cells blocked at PCP1 and PCP2) in nutritionally starved root meristems was found to have a genetic basis (species-specific) and was similar to that seen in mature root tissues of unstressed seedlings (Evans and Van’t Hof, 1974). Although these findings imply that the phase in which plant cells accumulate is consistent and independent, regardless of the cause of the cell cycle arrest (Van’t Hof, 1985), a number of phenomena have been described suggesting that the nature of the factors inducing the block plays a major role in determining expression of respective PCPs. For example, the general priority of the G1 checkpoint is strongly reflected in cultured plant cells exposed to nitrogen starvation (Gould et al., 1981). On the other hand, PCP2 has been shown to be promoted by trigonelline (5-methylnicotinic acid), a natural substance transported from cotyledons to differentiating root and shoot tissues of legumes, where its activity results in preferential arrest in the G2 phase of the cell cycle (Evans et al., 1987). Progression through S and M phases is triggered by two basic components of the cell cycle machinery, CDKs and their regulatory subunits, the cyclins (Renaudin et al., 1998), which are characterized by brief periods of synthesis and ubiquitin-mediated proteolysis (Chun et al., 1996; Plesse et al., 1998). The activity of CDK may be regulated in a variety of ways in response to extracellular signals and relies on both positive and negative elements including transcription, the amount of cyclin, phosphorylation by kinases, dephosphorylation by phosphatases, and the abundance of CDK inhibitors (CKIs; Peter, 1997). From among a multitude of specific factors affecting cell proliferation in higher plants, phytohormones seem to play the most prominent role in creating functional links between the reception and transduction of internal and external signals, developmental cues, and the expression of cell cycle genes (e.g. Soni et al., 1995; Tre´hin et al., 1998; Zhang et al., 1996). Consistent with cytological and physiological observations, auxin and cytokinin have long been recognized as essential regulators controlling the dynamics of meristem cell populations in vivo and the key inducers required for the in vitro culture of plant cells. Despite recent discoveries regarding the relationships between auxins, cytokinins and the expression levels of various CDK genes, we are still a long way from understanding the molecular aspects of the cell cycle machinery in plants. In particular, very little information is available
concerning the influence of plant growth regulators on the expression of PCPs in plants. Thus, the aim of the work described here was to obtain an insight into how IAA and BAP influence the expression of G1- and G2-phase checkpoints of the cell cycle in root meristems of V. faba. Furthermore, using the block-and-release method, we tested the effects of IAA, BAP, and a mixture of both, on the resumption of DNA replication and mitosis (G1-to-S and G2-to-M transitions). 2. Materials and methods 2.1. Plant material Seeds of V. faba subsp. minor were germinated on wet filter paper in Petri dishes at 23 (C in the dark. After 4 days of germination, seedlings with equal-sized primary roots (3 cm) were selected for further experiments. 2.2. Treatment of excised and nutrient-starved roots Excised apical parts of roots (1 cm long; containing meristems, some differentiating and elongating cells) were surface sterilized with 0.1% acetone–chloroform for 30 min, washed several times with distilled water and transferred to 100 ml Erlenmeyer flasks (10 root tips/ flask) filled with either 20 ml sterile White’s (1943) mineral medium alone (controls), or the same medium containing 0.56 µM 6-benzylaminopurine (BAP; Sigma), 5.4 µM idole-3-acetic acid (IAA; Sigma), or 0.56 µM BAP+5.4 µM IAA. The concentrations of IAA and BAP were selected according to earlier in vitro studies (Zhang et al., 1996). Roots were cultivated at 23 (C in a water-bath shaker (100 rpm) for 3 days, fixed and stained as described below. Nuclear DNA content was evaluated by means of cytophotometry using a Jenamed2 microscope (Carl Zeiss, Jena, Germany) with the computer-aided IMAL512 system (IMAL Co, Ło´dz´, Poland) for image analysis. Absorbance of Feulgen-stained samples was measured at 565 nm (Maszewski and Kaz´mierczak 1998). 2.3. Medium-transfer experiments After 3 days of starvation, roots were transferred to 50 ml Erlenmeyer flasks (five root tips/flask) filled with 15 ml sterile White’s (1943) medium containing 2% sucrose, or to the same medium plus 0.56 µM BAP, 5.4 µM IAA, or 0.56 µM BAP+5.4 µM IAA, and cultivated at 23 (C in a water-bath shaker (100 rpm) for 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 h. Before each sampling period, root tips were incubated for 1 h with 30 µM 5#-bromo-2#-deoxyuridine (BrdUrd; Sigma). 2.4. Immunocytochemistry Excised root segments were washed in distilled water for 15 min at 0 (C, meristems were cut off and fixed for
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20 min (at 0 (C) in freshly prepared 4% formaldehyde in 10 mM Tris–HCl buffer (pH 7.2), with 10 mM EDTA2Na, 100 mM NaCl and 0.1% Triton X-100. After fixation, roots were washed thoroughly with several changes of distilled water. To prevent non-specific binding of antibodies to cytoplasmic structures, cell nuclei were isolated by crushing individual root tips between two slides (Van’t Hof, 1975; Marciniak and Maszewski, 1989). A suspension of nuclei collected from drops which formed at the corners of both slides was spread on to Polysine microslides and air-dried at 4 (C. Because the anti-BrdUrd antibodies do not recognize BrdUrd embedded within double-stranded DNA, immunofluorescent staining requires partial DNA denaturation (Dolbeare et al., 1983; Levi et al., 1987). Cell nuclei were hydrolysed in 1.5 M HCl for 3.5 h at 25 (C. After washing with Tris–HCl buffer with 0.5% Triton X-100, slides were air dried as before. The procedure for immunofluorescent staining was performed according to Levi et al. (1987), with some modifications. Mouse monoclonal anti-BrdUrd antibodies (NOVOCASTRA) were diluted 1:10 in Tris–HCl buffer with 0.5% Triton X-100, dropped on to slides and incubated overnight in a humidified chamber at 4 (C in the dark. Slides were then washed three times (10 min each) in the same buffer and incubated for 45 min with FITC-conjugated goat anti-mouse monoclonal antibodies (SIGMA) diluted 1:10 in Tris–HCl buffer with 0.5% Triton X-100 (at 25 (C in a dark humidified chamber). After rinsing in buffer for 10 min, the slides were washed twice (10 min each) in Tris–HCl buffer without Triton, air-dried and embedded in a PBS/ glycerol mixture (9:1) with 2.3% DABCO (Sigma). Negative controls, to examine non-specific labelling, consisted of slides incubated with the FITC-labelled antibody alone. The slides were analysed using an Optiphot-2 fluorescence microscope (Nikon) equipped with a B-2A filter (=450–490 nm). For each experimental series, the BrdUrd-labelling indices were estimated based on measurements taken from 2000 cell nuclei per sample. 3. Results 3.1. Resolution of PCP1 and PCP2 in carbohydrate-starved root meristems The DNA distribution pattern for populations of proliferating cells (intact seedlings; Fig. 1A) reveals a good separation of nuclei around the 2C DNA value, a considerable fraction of nuclei with 4C DNA content, and a less distinct class of cells having intermediate amounts of nuclear DNA (2.5–3.5C). Taking into account the BrdUrd labelling index (42.0%) and the relative number of mitotic cells (14.0%), the approximate share of cells with 2C and 4C nuclear DNA values can be estimated at a ratio of 2:1 (Table 1).
Fig. 1. Frequency distributions (%) for nuclear DNA content (a.u.; arbitrary units) in root meristem cells of Vicia faba subsp. minor: control (A), carbohydrate-starvation without hormones (B), with medium containing 0.56 µM BAP (C), 5.4 µM IAA (D), mixture of 0.56 µM BAP and 5.4 µM IAA (E).
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Table 1 Percentage of G1 and G2 cells in root meristems of Vicia faba subsp. minor and the ratio of G1:G2 cell nuclei in intact seedlings (Control), after excision and 3-day starvation in White’s mineral medium without phytohormones (S), or in medium containing BAP (0.56 µM), IAA (5.4 µM), or BAP+IAA Experimental series
G1
G2
G1:G2
Control* S BAP IAA BAP+IAA
28 60 39 22 27
16 40 61 78 73
1:0.6 1:0.7 1:1.6 1:3.5 1:2.7
* Estimation based on cytophotometric and BrdUrd immunocytochemical measurements.
After excision and prolonged starvation of root tips in carbohydrate-free mineral medium, the frequency distribution pattern of meristem cells with varying nuclear DNA content changes considerably (Fig. 1B). Deprivation of essential nutrients results in complete cessation of both DNA replication and mitotic divisions, and the histogram shows segregation of nuclear C values typical of the biphasic G1–G2 distribution. Compared with the control plants, the proportion of cell nuclei with 2C and 4C DNA values calculated for the stationary phase merstems is slightly shifted in favour of the G2 cells, decreasing the ratio of G1- to G2-arrested cells to about 1.5:1 (Table 1). 3.2. Influence of BAP, IAA, and BAP+IAA on the resolution of PCP1 and PCP2 in carbohydrate-starved root meristems Root meristems starved for 72 h in medium containing 0.56 µM BAP, 5.4 µM IAA, or a mixture of both growth regulators, are comprised of two populations of cells having either 2C or 4C DNA content. Depending on the experimental series, however, there are important differences between the proportions of cells accumulated in the G1 and G2 phases of the cell cycle (Fig. 1C–E, Table 1). Despite an overall similarity with the bimodal separation of meristem cell populations, the frequency distribution of nuclear DNA–Feulgen values evaluated for excised root tips cultivated in the presence of BAP reveals an increased percentage of G2-arrested cells (Fig. 1C). The area covering the right part of the histogram, indicating the prevailing fraction of G2 cells, is roughly twice as large as that delineating the population of G1 cells with unreplicated DNA. In medium containing IAA, root meristem cells arrested almost entirely in the G2 phase (Fig. 1D). This process was accompanied by a significant condensation of chromatin, which resulted in poor resolution of nuclear fractions accumulated around the 2C and 4C values. A considerable decrease in the proportion of G1 to G2 cells was also noted after the
Fig. 2. Recovery of DNA replication (BrdUrd labelling index, %) and cell division (mitotic index, %) in excised, carbohydrate-starved root meristem cells of Vicia faba subsp. minor cultivated in White’s mineral medium containing 2% sucrose (3 h intervals).
prolonged culture of excised root tips in mineral medium containing both BAP and IAA (Fig. 1E). Again, however, as was the case in root meristems starved for 72 h in carbohydrate-free medium or grown in medium supplemented with BAP, the histogram drawn for nuclear DNA C-values is split into two separate populations, with the proportion of G1 to G2 cells evaluated to be about 1:2.5 (Table 1). 3.3. Sucrose-mediated resumption of DNA replication and mitosis in excised root meristems When excised and starved root tips of V. faba subsp. minor are supplied with exogenous carbohydrate (2% sucrose), the G1-arrested cells initiate DNA replication, while the cells blocked in G2 start mitotic divisions. In order to evaluate the time needed to traverse from PCP1 to S and from PCP2 to M, successive BrdUrd pulse labellings followed by immunocytochemistry were used and mitotic activities were analysed within 30 h after the transfer of root tips into medium supplemented with sucrose (Fig. 2). Although the appearance of S phase cells could already be seen after 12 h of recovery, ‘the delay of DNA replication’, defined as the time when the BrdUrd labelling index attains 50% of its maximum level, was estimated to be about 16 h (Table 2). After the first wave of cells entering S phase, and a slight decrease in the profile of the labelling index at the next two sampling periods, the second flow of cells incorporating BrdUrd was observed between 27–30 h of stimulation (Fig. 2). Based on the counts of mitotic cells in excised root meristems transferred to sucrose-rich medium, the delay in the shift from G2 to M phase was found to be about 3 h shorter than that seen in G1 cells traversing to S phase (Fig. 2). Adopting the same time-course of analyses, two peaks of mitotic index were seen at 15 and 21 h of recovery, with a drop in between, succeeded by a period of relatively low and constant frequency of cells entering mitotic division.
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Table 2 Duration of G1-to-S (G1/S) and G2-to-M (G2/M) transitions (hours) in carbohydrate-starved root meristem cells of Vicia faba subsp. minor in White’s mineral medium supplied with 2% sucrose (Sucrose), the same medium containing sucrose and 0.56 µM BAP (Sucrose+BAP), 2% sucrose and 5.4 µM IAA (Sucrose+IAA), or 2% sucrose with a mixture of 0.56 µM BAP and 5.4 µM IAA (Sucrose+BAP+IAA). Compared with the control (sucrose only), the shortened () or extended (+) transit times are given in brackets Experimental series
G1/S
G2/M
Sucrose Sucrose+BAP Sucrose+IAA Sucrose+BAP+IAA
16.0 7.5(8.5) 16.5(+0.5) 10.5(5.5)
13.0 10.5(2.5) 16.0(+3.0) 10.5(2.5)
Fig. 3. Recovery of DNA replication (BrdUrd labelling index, %) in excised, carbohydrate-starved root meristem cells of Vicia faba subsp. minor cultivated in White’s mineral medium containing 2% sucrose with 0.56 µM BAP, 5.4 µM IAA and a mixture of 0.56 µM BAP and 5.4 µM IAA (3 h intervals).
3.4. Influence of BAP, IAA, and BAP/IAA mixture on the sucrose-mediated resumption of DNA replication and mitosis in excised root meristems To examine the influence of auxin and cytokinin on the delay of DNA replication and mitosis, the carbohydrate-starved root meristems of V. faba subsp. minor were returned to complete recovery medium (with 2% sucrose) containing either BAP, IAA, or a mixture of both growth regulators. Depending on the experimental series performed using media supplied with phytohormones (Figs. 3 and 4), the upper levels of BrdUrd-labelling indices and the maximum frequencies of mitotic cells are lower than those estimated in the control series. The waves of cells entering S and M phases are significantly longer, demonstrating an increased heterogeneity of G1 and G2 cell populations. These data provide strong evidence that BAP and IAA have a profound influence on the dynamics of both the G1-to-S (Fig. 3) and G2-to-M (Fig. 4) transitions. However, the effect of cytokinin on cells with nuclear 2C or 4C DNA content appears to be opposite to that of auxin.
Fig. 4. Recovery of cell division (mitotic index, %) in excised, carbohydrate-starved root meristem cells of Vicia faba subsp. minor cultivated in White’s mineral medium containing 2% sucrose with addition of 0.56 µM BAP, 5.4 µM IAA and a mixture of 0.56 µM BAP and 5.4 µM IAA (3 h intervals).
The delay of DNA replication in roots transferred to complete mineral medium supplemented with BAP was reduced to about half the time needed to cover the flow of cells from G1 to S in root meristems provided with sucrose alone. In contrast, the recovery of carbohydratestarved root meristems in medium containing IAA resulted in about 3 h excess delay in the entrance to S phase. In the experimental series with the mixture of BAP and IAA, a compromise develops between the two phytohormones, bringing about a moderate acceleration in the transition of G1 cells into the onset of DNA replication (Fig. 3; Table 2). The general kinetic aspects of the response of G2 cells to the various block-and-release exposures are remarkably similar to those seen previously in the G1 cell population. Compared with the control series of carbohydrate-starved roots transferred to medium supplied with 2% sucrose, the continuous refeeding of excised root meristems either in medium containing BAP, or BAP plus IAA, decreases the delay of G2-to-M transition by about 3 h (Fig. 4; Table 2). Incubation in medium with IAA results in the opposite effect, and the cells advance to the G2/M boundary after an additional delay of about 7 h. 4. Discussion Phytohormones have long been recognized as signalling factors playing a pivotal role in nearly every aspect of plant development. Over the last few years it has become increasingly clear that some of them, mostly auxins and cytokinins, are part of an interacting biochemical network involved in the transcriptional control of the cell cycle genes (Carle et al., 1998; Kapros et al., 1992; Soni et al., 1995). Despite accumulating evidence of numerous links between growth regulators and the molecular mechanisms that underlie cell cycle progression, the extent to which they affect the system of G1
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and G2 control points in plants is currently unknown. Adopting an original model of excised root meristems, which contributed to the Principal Control Point hypothesis (Van’t Hof and Kovacs, 1972; Van’t Hof 1985), our present studies were undertaken in the hope of getting answers to the two following questions: 1. How do cytokinins (BAP) and auxins (IAA) influence the expression of PCP1 and PCP2? 2. How do these plant growth regulators change the sucrose-mediated release of G1- and G2-arrested cells into the S and M phase of the cell cycle? Meristem cell populations in excised roots of V. faba subsp. minor become blocked in either G1 (PCP1) or G2 (PCP2). Lack of both mitotic and BrdUrd incorporating cells suggests that deprivation of essential nutrients brings about complete cessation of cellular proliferation, resulting in the production of stationary phase meristems. Although both checkpoints operate in root meristem cells starved for 72 h in mineral medium containing BAP, IAA or a mixture of both, the percentage of cells blocked with either 2C or 4C nuclear DNA content varies significantly depending on the experimental series. Incubation in medium containing BAP allows a considerable additional number of cells to proceed past the G1 checkpoint and arrest after having reached 4C nuclear DNA content. A significantly higher rise in the frequency of cells blocked at the G2 control point is observed in root meristems maintained in the presence of IAA. Thus, the behavioural response of root meristem cell populations is clearly not fixed and the expression of G1 and G2 control points depends largely on the ‘regulatory context’ established by the relationship between the innate (genetic) instructions and extracellular (phytohormonal) signals. The quantitative distribution patterns of G1- and G2-arrested root meristem cells of V. faba subsp. minor starved in media supplied with either BAP or IAA appear to correlate with the results obtained by Zhang et al. (1996) in experiments performed using suspensioncultured cells of Nicotiana plumbaginifolia. Following an 8 day period of starvation without cytokinin and auxin and subsequent 2 day incubation with kinetin, the cells were found to be arrested in both G1 and G2 phases of the cell cycle. In contrast, cells that lacked cytokinin, being incubated with 2,4-D (auxin) only, arrested almost entirely in the G2 phase. Although some caution must be used in drawing parallels between results obtained by means of different experimental assays, it seems likely that a common connection exists between the changes in frequency of G1 and G2 cell populations seen in experiments with excised root tips of V. faba and those obtained using suspension cell cultures of N. plumbaginifolia. Neither DNA replication nor mitosis occurs in the absence of the appropriate CDK-cyclin complex, and
the timing of each event can be advanced by premature CDK activation (Jacobs, 1997). The control points regulate progression through the key transitions of the cell cycle by interacting with the subsequent period of CDK activity, inhibiting it until the cell “senses” an abundance of nutrients and sufficient metabolic capabilities needed for DNA replication or progression into mitosis. Data concerning the cells in higher plants, including (1) their size-dependent control over the onset of DNA replication (John, 1996), (2) the occurrence of p34cdc2-like proteins recognized by antibody against the internal PSTAIRE peptide (e.g. John et al., 1993; Polit, 1998), and (3) the presence of distinct classes of cyclins and CDKs, which drive the progression through the major events of the cell cycle (Sundaresan and Colasanti, 1998), indicate a strong analogy between PCP1, the START control in yeasts, and the restriction point in mammals. Functional conservation of cell cycle control mechanisms among the eukaryotes becomes even more evident at the stages preceding the G2-to-M transition. In both plants and animals, the amounts of A and B cyclins are regulated transcriptionally and show similar expression profiles, with peaks in late G2 and M. Consequently, the activated p34cdc2 kinase phosphorylates a set of specific substrates and promotes chromatin condensation, nuclear envelope breakdown, disassembly of the nucleolus and spatial organization of cytoskeletal polymers (John, 1996). The reduction of G1-to-S transitions in carbohydratestarved root tips restored to recovery medium supplemented with BAP, and the opposite effect observed in meristems incubated in medium containing IAA, correlates well with the phytohormone-regulated expression of cyclin D homologs from Arabidopsis thaliana, referred to as cyclins (Soni et al., 1995). Cyclins 2 and 3, known to play a role during the G1/S transition, have been found to be directly involved in linking plant growth regulators, nutritional status, and cell cycle progression in a manner similar to that of mammalian cyclin D in response to growth factors. In suspension cell cultures of A. thaliana, the cytokinin-dependent induction of mRNA synthesis for 3 cyclin is apparently antagonized by the concomitant addition of auxin (2,4D), so that in the presence of sucrose a smaller induction of transcription is detected using RNA gel blots. In sucrose-free medium, no accumulation over basal 3 cyclin mRNA levels is observed. Concurrently, the levels of 2 cyclin are considerably increased in response to the availability of sucrose. Clearly, the results obtained in our present work correspond well with the biochemical data recorded in suspension-cultured plant cells, demonstrating the stringent requirement of late G1 cells for cytokinin in promoting their re-entry into S phase (Soni et al., 1995; see also Carle et al., 1998; Riou-Khamlichi et al., 2000).
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The vast amount of information accumulated by the recent physiological studies on plant cells indicate that the hormonal control point operating in late G2 phase is responsible for triggering the transition into mitosis by rapid activitation of p34cdc2 kinase (John, 1996). Most cellular systems used in the analysis of cell cycle gene expression seem to require exogenous auxin alone, probably because endogenous levels of cytokinin synthesis are sufficient to fulfil the needs for continued growth (Zhang et al., 1996). Moreover, the hormone withdrawal studies performed using Nicotiana tabacum mesophyll protoplasts have shown that a delay in auxin addition has a greater effect on cell division than a delay in cytokinin addition (Carle et al., 1998). Experiments with pith parenchyma cells excised from N. tabacum stem segments, as designed by Zhang et al. (1996), indicate that auxin (NAA) and cytokinin (BAP) induce a greater than 40-fold increase in a p34cdc2-like protein, but without cytokinin the H1 histone kinase is inactive. Cytological and biochemical data consistent with this hypothesis suggest that the presence of adequate cytokinin can stimulate the catalytic capabilities of MPF by a highly specific cdc25 phosphatase which decreases the abundance of p34cdc2 phosphotyrosine and allows the cells to proceed towards prophase. This has been confirmed by pith cells without cytokinin having been found arrested in late G2 phase with a p34cdc2-like protein of high Y–PO4, which could be removed by cytokinin or the cdc25 enzyme (Zhang et al., 1996). A model of this kind seems to provide an easier and more reasonable explanation for the shortened PCP2-M intervals in root meristems of V. faba restored to recovery medium supplemented with BAP. As was the case with the G1 counterparts relieved from the block imposed by PCP1, a diametrically opposed effect could be observed in roots resupplied with sucrose and IAA. In all these cases, however, it is important to bear in mind that the observed effects are not only due to the exogenous hormones, but are rather caused by the interaction of the applied plant growth regulators with internal factors produced by the excised parts of the root (De Veylder et al., 1998). It may be that the prolonged delay in cell progression from G2-to-M results from a disturbance in the balance between the two regulators, which need to be in precise and highly specific proportions to one another in order to activate MPF kinase. The induction of stationary phase meristems and our block-and-release experiments clearly show that exogenous plant hormones exert a profound influence on the expression of G1- and G2-phase checkpoints in root meristems of V. faba. It seems reasonable to believe that the complex biochemical mechanisms involved in PCPs are based on an interplay between genetic (innate) and extragenetic information. Presumably, regulatory systems triggering DNA replication and mitosis integrate both nutritional and hormonal information, and thus
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delay or block the G1-to-S and G2-to-M transitions if the requirements for CDK-dependent activity are not complete. Acknowledgement This work was supported by the National Committee of Scientific Research, grant 6PO4C 02617. References Carle SA, Bates GW, Shannon TA. Hormonal control of gene expression during reactivation of the cell cycle in tobacco mesophyll protoplasts. Plant Growth Regul 1998;17:221–30. Chun KT, Mathias N, Goebl MG. Ubiquitin-dependent proteolysis and cell cycle control in yeast. In: Meijer L, Guidet S, Vogel L, editors. Progress in cell cycle research 2. New York (USA): Plenum Press, 1996;115–27. De Veylder L, Van Montagu M, Inze´ D. Cell cycle control in Arabidopsis. In: Francis D, Dudits D, Inze´ D, editors. Plant cell division. London (UK)/Miami (USA): Portland Press, 1998;1–19. Dolbeare F, Gratzner HG, Pallavicini MG, Gray JW. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc Natl Acad Sci U S A 1983;80:5573–7. Elledge SJ. Cell cycle checkpoints: preventing an identity crisis. Science 1996;274:1664–72. Evans LS, Tramontano WA, Gill R. A natural substance that regulates the cell cycle in complex plant tissues. Phytochemistry 1987; 26:2891–3. Evans LS, Van’t Hof J. Is the nuclear DNA content of mature root cells prescribed in the root meristem? Am J Bot 1974;61:1104–11. Gould AR, Everett NP, Wang TL, Street HE. Studies on the control of the cell cycle in cultured plant cells. I. Effect of nutrient limitation and nutrient starvation. Protoplasma 1981;106:1–13. Jacobs T. Why do plant cells divide? Plant Cell 1997;9:1021–9. John PCL. The plant cell cycle: conserved and unique features in mitotic control. In: Meijer L, Guidet S, Vogel L, editors. Progress in cell cycle research 2. New York (USA): Plenum Press, 1996;59–72. John PCL, Zhang K, Dong C, Didierich L, Wightman F. 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 1993;20:503–26. Kapros T, Bo¨gre L, Ne´meth K, Bako´ L, Gyo¨rgyey J, Wu SC, Dudits D. Differential expression of histone H3 gene variants during cell cycle and somatic embryogenesis in alfalfa. Plant Physiol 1992; 98:621–5. Levi M, Sparvoli E, Sgorbati S, Chiatante D. Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography. Physiol Plant 1987;71:68–72. Marciniak K, Maszewski J. Replicon size and the mean rate of DNA synthesis during root cell differentiation in Vicia faba subsp. minor and major. Biol Zbl 1989;108:241–8. Maszewski J, Kaz´mierczak A. Repression of genetic activity in root meristem cells by peptidic factor derived from male sex organs of Chara. Biol Plant 1998;41:357–68. Peter M. The regulation of cyclin-dependent kinase inhibitors (CKIs). In: Meijer L, Guidet S, Philippe M, editors. Progress in cell cycle research 3. New York: Plenum Press, 1997;99–108. Plesse B, Fleck J, Genschik P. The ubiquitin-dependent proteolytic pathway and cell cycle control. In: Francis D, Dudits D, Inze´ D, editors. Plant cell division. London, Miami: Portland Press, 1998;145–63.
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Polit J. Indirect semiquantitative determination of p34cdc2 levels in G1 and G2 cells of the carbohydrate-starved root meristems in Vicia faba var. minor. Acta Soc Bot Polon 1998;68:255–9. Renaudin JP, Savoure´ A, Philippe H, Van Montague M, Inze´ D, Rouze´ P. Characterization and classification of plant cyclin sequences related to A- and B-type cyclins. In: Francis D, Dudits D, Inze´ D, editors. Plant cell division. London, Miami: Portland Press, 1998;67–98. Rieder CL, Khodjakov A. Mitosis and checkpoints that control progression through mitosis in vertebrate somatic cells. In: Meijer L, Guidet S, Philippe M, editors. Progress in cell cycle research 3. New York: Plenum Press, 1997;301–12. Riou-Khamlichi C, Mendes M, Healy JMS, Murray JAH. Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Mol Cell Biol 2000;20:4513–21. Soni R, Carmichael JP, Shah ZH, Murray JAH. A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. Plant Cell 1995;7:85–103. Sundaresan V, Colasanti J. Cyclin-dependent kinases in higher plants: spatial and temporal control of cell division. In: Francis D, Dudits D, Inze´ D, editors. Plant cell division. London, Miami: Portland Press, 1998;47–65.
Tre´hin C, Planchais S, Glab N, Perennes C, Tregear J, Bergounioux C. Cell cycle regulation by plant growth regulators: involvement of auxin and cytokinin in the re-entry of Petunia protoplasts into the cell cycle. Planta 1998;206:215–24. Tyson JJ, Novak B, Chen K, Val J. Checkpoints in the cell cycle from a medeler’s perspective. In: Meijer L, Guidet S, Tung HYL, editors. Progress in cell cycle research 1. New York: Plenum Press, 1995;1–8. Van’t Hof J. DNA fiber replication in chromosomes of a higher plant (Pisum sativum). Exp Cell Res 1975;93:95–104. Van’t Hof J. Control points within the cell cycle. In: Bryant JA, Francis D, editors. The cell division cycle in plants. Cambridge, London, New York, New Rochelle, Melbourne, Sydney: Cambridge University Press, 1985;1–13. Van’t Hof J, Kovacs CJ. Mitotic cycle regulation in the meristem of cultured roots: The principal control point hypothesis. In: Miller MN, Kuehnert CC, editors. The dynamics of meristem cell. New York, London: Plenum Press, 1972;15–30. White PR. A handbook of plant tissue culture. Lancaster (PA): Catell, 1943. Zhang K, Letham DS, John PCL. Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase. Planta 1996;200:2–12.
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Effect of BAP and IAA on the expression of G1 and G2 control points and G1-S and G2-M transitions in root meristem cells of Vicia faba Justyna T. Polit, Janusz Maszewski*, Andrzej Kaz´mierczak Department of Cytophysiology, University of Ło´dz´, 90-231 Ło´dz´, ul. Pilarskiego 14, Poland Received 26 April 2001; accepted 31 October 2001
Abstract Excised, carbohydrate-starved root meristems of Vicia faba subsp. minor have been used to investigate the impact of the auxin indole-3-acetic acid (IAA) and the cytokinin benzyl-6-aminopurine (BAP) on (1) the expression of Principal Control Points (PCPs) during the G1- and G2-phases of the cell cycle, and (2) the dynamics of sucrose-mediated resumption of DNA replication and mitosis (G1-to-S and G2-to-M transitions). Compared with the excised root tips starved in mineral medium without hormones, stationary phase meristems induced during continuous treatment with BAP, IAA, or a mixture of BAP+IAA, increased the number of G2 cells, producing characteristic profiles of nuclear DNA content. In medium containing 2% sucrose, BAP accelerated PCP1/S and PCP2/M, whereas continuous treatment with IAA resulted in marked prolongation of both transitions. Since the PCPs regulate progression through the key events of interphase and mitosis by interacting with cyclin dependent kinases (CDKs), these results seem to correspond with current data indicating functional connections between phytohormones, nutritional signals, gene expression and the cell division cycles in plants. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Cell cycle checkpoints; Phytohormones; DNA replication; Mitosis; Protein kinases; Root meristems; Vicia faba
1. Introduction Despite the diversity of developmental processes in various organisms, the number of key surveillance mechanisms by which biological control of proliferation is exerted and types of underlying molecular phenomena are surprisingly limited. A series of elaborate signalling pathways, known as checkpoints, have evolved to control the cell division cycles in eukaryotes (Elledge, 1996; Tyson et al., 1995). Distinct regulatory events which govern progression through the cell cycle seem to converge upon a common point in late G1, called START (in yeasts) or Restriction Point (in mammals), at which cells become committed to enter S phase. Here, feedback signals conveying information about the downstream processes can delay the G1/S transition, so as to prevent the cell from triggering DNA replication until certain * Corresponding author E-mail address: [email protected] (J. Maszewski).
monitored events are completed. Before entering mitosis, at the G2 checkpoint, the cell must ensure that DNA replication is complete or damage is repaired, and that all systems are ready for chromatin condensation. Before separation of sister chomatids to daughter cells at the third control point during M phase, each cell must make sure that all chromosomes are properly aligned in one plane halfway between the spindle poles (Rieder and Khodjakov, 1997). The existence of checkpoint control systems in plant cells was first recognized about 30 years ago in Pisum sativum, Vicia faba and Helianthus annuus, in response to carbohydrate starvation of roots (Van’t Hof and Kovacs, 1972). When their apical parts are excised and incubated under conditions restrictive to growth (in mineral medium without sucrose), DNA synthesis and cell division cease and stationary phase meristems are produced with cells accumulated in G1 and G2. The discrete phases at which root meristem cells arrest in G1 and G2 are considered to be metabolic
1065-6995/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1065-6995(03)00081-7
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blocks that prevent the initiation of DNA replication and mitosis and which are relieved when exogenous carbohydrate is replenished (Van’t Hof and Kovacs, 1972). The reproducibility of this phenomenon in plant cells that cease dividing in vivo due to dormancy, or in vitro due to starvation, was the basis for the PCP hypothesis (reviewed by Van’t Hof, 1985). The ratio of cells with either G1 or G2 DNA content (i.e. the proportion of cells blocked at PCP1 and PCP2) in nutritionally starved root meristems was found to have a genetic basis (species-specific) and was similar to that seen in mature root tissues of unstressed seedlings (Evans and Van’t Hof, 1974). Although these findings imply that the phase in which plant cells accumulate is consistent and independent, regardless of the cause of the cell cycle arrest (Van’t Hof, 1985), a number of phenomena have been described suggesting that the nature of the factors inducing the block plays a major role in determining expression of respective PCPs. For example, the general priority of the G1 checkpoint is strongly reflected in cultured plant cells exposed to nitrogen starvation (Gould et al., 1981). On the other hand, PCP2 has been shown to be promoted by trigonelline (5-methylnicotinic acid), a natural substance transported from cotyledons to differentiating root and shoot tissues of legumes, where its activity results in preferential arrest in the G2 phase of the cell cycle (Evans et al., 1987). Progression through S and M phases is triggered by two basic components of the cell cycle machinery, CDKs and their regulatory subunits, the cyclins (Renaudin et al., 1998), which are characterized by brief periods of synthesis and ubiquitin-mediated proteolysis (Chun et al., 1996; Plesse et al., 1998). The activity of CDK may be regulated in a variety of ways in response to extracellular signals and relies on both positive and negative elements including transcription, the amount of cyclin, phosphorylation by kinases, dephosphorylation by phosphatases, and the abundance of CDK inhibitors (CKIs; Peter, 1997). From among a multitude of specific factors affecting cell proliferation in higher plants, phytohormones seem to play the most prominent role in creating functional links between the reception and transduction of internal and external signals, developmental cues, and the expression of cell cycle genes (e.g. Soni et al., 1995; Tre´hin et al., 1998; Zhang et al., 1996). Consistent with cytological and physiological observations, auxin and cytokinin have long been recognized as essential regulators controlling the dynamics of meristem cell populations in vivo and the key inducers required for the in vitro culture of plant cells. Despite recent discoveries regarding the relationships between auxins, cytokinins and the expression levels of various CDK genes, we are still a long way from understanding the molecular aspects of the cell cycle machinery in plants. In particular, very little information is available
concerning the influence of plant growth regulators on the expression of PCPs in plants. Thus, the aim of the work described here was to obtain an insight into how IAA and BAP influence the expression of G1- and G2-phase checkpoints of the cell cycle in root meristems of V. faba. Furthermore, using the block-and-release method, we tested the effects of IAA, BAP, and a mixture of both, on the resumption of DNA replication and mitosis (G1-to-S and G2-to-M transitions). 2. Materials and methods 2.1. Plant material Seeds of V. faba subsp. minor were germinated on wet filter paper in Petri dishes at 23 (C in the dark. After 4 days of germination, seedlings with equal-sized primary roots (3 cm) were selected for further experiments. 2.2. Treatment of excised and nutrient-starved roots Excised apical parts of roots (1 cm long; containing meristems, some differentiating and elongating cells) were surface sterilized with 0.1% acetone–chloroform for 30 min, washed several times with distilled water and transferred to 100 ml Erlenmeyer flasks (10 root tips/ flask) filled with either 20 ml sterile White’s (1943) mineral medium alone (controls), or the same medium containing 0.56 µM 6-benzylaminopurine (BAP; Sigma), 5.4 µM idole-3-acetic acid (IAA; Sigma), or 0.56 µM BAP+5.4 µM IAA. The concentrations of IAA and BAP were selected according to earlier in vitro studies (Zhang et al., 1996). Roots were cultivated at 23 (C in a water-bath shaker (100 rpm) for 3 days, fixed and stained as described below. Nuclear DNA content was evaluated by means of cytophotometry using a Jenamed2 microscope (Carl Zeiss, Jena, Germany) with the computer-aided IMAL512 system (IMAL Co, Ło´dz´, Poland) for image analysis. Absorbance of Feulgen-stained samples was measured at 565 nm (Maszewski and Kaz´mierczak 1998). 2.3. Medium-transfer experiments After 3 days of starvation, roots were transferred to 50 ml Erlenmeyer flasks (five root tips/flask) filled with 15 ml sterile White’s (1943) medium containing 2% sucrose, or to the same medium plus 0.56 µM BAP, 5.4 µM IAA, or 0.56 µM BAP+5.4 µM IAA, and cultivated at 23 (C in a water-bath shaker (100 rpm) for 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 h. Before each sampling period, root tips were incubated for 1 h with 30 µM 5#-bromo-2#-deoxyuridine (BrdUrd; Sigma). 2.4. Immunocytochemistry Excised root segments were washed in distilled water for 15 min at 0 (C, meristems were cut off and fixed for
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20 min (at 0 (C) in freshly prepared 4% formaldehyde in 10 mM Tris–HCl buffer (pH 7.2), with 10 mM EDTA2Na, 100 mM NaCl and 0.1% Triton X-100. After fixation, roots were washed thoroughly with several changes of distilled water. To prevent non-specific binding of antibodies to cytoplasmic structures, cell nuclei were isolated by crushing individual root tips between two slides (Van’t Hof, 1975; Marciniak and Maszewski, 1989). A suspension of nuclei collected from drops which formed at the corners of both slides was spread on to Polysine microslides and air-dried at 4 (C. Because the anti-BrdUrd antibodies do not recognize BrdUrd embedded within double-stranded DNA, immunofluorescent staining requires partial DNA denaturation (Dolbeare et al., 1983; Levi et al., 1987). Cell nuclei were hydrolysed in 1.5 M HCl for 3.5 h at 25 (C. After washing with Tris–HCl buffer with 0.5% Triton X-100, slides were air dried as before. The procedure for immunofluorescent staining was performed according to Levi et al. (1987), with some modifications. Mouse monoclonal anti-BrdUrd antibodies (NOVOCASTRA) were diluted 1:10 in Tris–HCl buffer with 0.5% Triton X-100, dropped on to slides and incubated overnight in a humidified chamber at 4 (C in the dark. Slides were then washed three times (10 min each) in the same buffer and incubated for 45 min with FITC-conjugated goat anti-mouse monoclonal antibodies (SIGMA) diluted 1:10 in Tris–HCl buffer with 0.5% Triton X-100 (at 25 (C in a dark humidified chamber). After rinsing in buffer for 10 min, the slides were washed twice (10 min each) in Tris–HCl buffer without Triton, air-dried and embedded in a PBS/ glycerol mixture (9:1) with 2.3% DABCO (Sigma). Negative controls, to examine non-specific labelling, consisted of slides incubated with the FITC-labelled antibody alone. The slides were analysed using an Optiphot-2 fluorescence microscope (Nikon) equipped with a B-2A filter (=450–490 nm). For each experimental series, the BrdUrd-labelling indices were estimated based on measurements taken from 2000 cell nuclei per sample. 3. Results 3.1. Resolution of PCP1 and PCP2 in carbohydrate-starved root meristems The DNA distribution pattern for populations of proliferating cells (intact seedlings; Fig. 1A) reveals a good separation of nuclei around the 2C DNA value, a considerable fraction of nuclei with 4C DNA content, and a less distinct class of cells having intermediate amounts of nuclear DNA (2.5–3.5C). Taking into account the BrdUrd labelling index (42.0%) and the relative number of mitotic cells (14.0%), the approximate share of cells with 2C and 4C nuclear DNA values can be estimated at a ratio of 2:1 (Table 1).
Fig. 1. Frequency distributions (%) for nuclear DNA content (a.u.; arbitrary units) in root meristem cells of Vicia faba subsp. minor: control (A), carbohydrate-starvation without hormones (B), with medium containing 0.56 µM BAP (C), 5.4 µM IAA (D), mixture of 0.56 µM BAP and 5.4 µM IAA (E).
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Table 1 Percentage of G1 and G2 cells in root meristems of Vicia faba subsp. minor and the ratio of G1:G2 cell nuclei in intact seedlings (Control), after excision and 3-day starvation in White’s mineral medium without phytohormones (S), or in medium containing BAP (0.56 µM), IAA (5.4 µM), or BAP+IAA Experimental series
G1
G2
G1:G2
Control* S BAP IAA BAP+IAA
28 60 39 22 27
16 40 61 78 73
1:0.6 1:0.7 1:1.6 1:3.5 1:2.7
* Estimation based on cytophotometric and BrdUrd immunocytochemical measurements.
After excision and prolonged starvation of root tips in carbohydrate-free mineral medium, the frequency distribution pattern of meristem cells with varying nuclear DNA content changes considerably (Fig. 1B). Deprivation of essential nutrients results in complete cessation of both DNA replication and mitotic divisions, and the histogram shows segregation of nuclear C values typical of the biphasic G1–G2 distribution. Compared with the control plants, the proportion of cell nuclei with 2C and 4C DNA values calculated for the stationary phase merstems is slightly shifted in favour of the G2 cells, decreasing the ratio of G1- to G2-arrested cells to about 1.5:1 (Table 1). 3.2. Influence of BAP, IAA, and BAP+IAA on the resolution of PCP1 and PCP2 in carbohydrate-starved root meristems Root meristems starved for 72 h in medium containing 0.56 µM BAP, 5.4 µM IAA, or a mixture of both growth regulators, are comprised of two populations of cells having either 2C or 4C DNA content. Depending on the experimental series, however, there are important differences between the proportions of cells accumulated in the G1 and G2 phases of the cell cycle (Fig. 1C–E, Table 1). Despite an overall similarity with the bimodal separation of meristem cell populations, the frequency distribution of nuclear DNA–Feulgen values evaluated for excised root tips cultivated in the presence of BAP reveals an increased percentage of G2-arrested cells (Fig. 1C). The area covering the right part of the histogram, indicating the prevailing fraction of G2 cells, is roughly twice as large as that delineating the population of G1 cells with unreplicated DNA. In medium containing IAA, root meristem cells arrested almost entirely in the G2 phase (Fig. 1D). This process was accompanied by a significant condensation of chromatin, which resulted in poor resolution of nuclear fractions accumulated around the 2C and 4C values. A considerable decrease in the proportion of G1 to G2 cells was also noted after the
Fig. 2. Recovery of DNA replication (BrdUrd labelling index, %) and cell division (mitotic index, %) in excised, carbohydrate-starved root meristem cells of Vicia faba subsp. minor cultivated in White’s mineral medium containing 2% sucrose (3 h intervals).
prolonged culture of excised root tips in mineral medium containing both BAP and IAA (Fig. 1E). Again, however, as was the case in root meristems starved for 72 h in carbohydrate-free medium or grown in medium supplemented with BAP, the histogram drawn for nuclear DNA C-values is split into two separate populations, with the proportion of G1 to G2 cells evaluated to be about 1:2.5 (Table 1). 3.3. Sucrose-mediated resumption of DNA replication and mitosis in excised root meristems When excised and starved root tips of V. faba subsp. minor are supplied with exogenous carbohydrate (2% sucrose), the G1-arrested cells initiate DNA replication, while the cells blocked in G2 start mitotic divisions. In order to evaluate the time needed to traverse from PCP1 to S and from PCP2 to M, successive BrdUrd pulse labellings followed by immunocytochemistry were used and mitotic activities were analysed within 30 h after the transfer of root tips into medium supplemented with sucrose (Fig. 2). Although the appearance of S phase cells could already be seen after 12 h of recovery, ‘the delay of DNA replication’, defined as the time when the BrdUrd labelling index attains 50% of its maximum level, was estimated to be about 16 h (Table 2). After the first wave of cells entering S phase, and a slight decrease in the profile of the labelling index at the next two sampling periods, the second flow of cells incorporating BrdUrd was observed between 27–30 h of stimulation (Fig. 2). Based on the counts of mitotic cells in excised root meristems transferred to sucrose-rich medium, the delay in the shift from G2 to M phase was found to be about 3 h shorter than that seen in G1 cells traversing to S phase (Fig. 2). Adopting the same time-course of analyses, two peaks of mitotic index were seen at 15 and 21 h of recovery, with a drop in between, succeeded by a period of relatively low and constant frequency of cells entering mitotic division.
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Table 2 Duration of G1-to-S (G1/S) and G2-to-M (G2/M) transitions (hours) in carbohydrate-starved root meristem cells of Vicia faba subsp. minor in White’s mineral medium supplied with 2% sucrose (Sucrose), the same medium containing sucrose and 0.56 µM BAP (Sucrose+BAP), 2% sucrose and 5.4 µM IAA (Sucrose+IAA), or 2% sucrose with a mixture of 0.56 µM BAP and 5.4 µM IAA (Sucrose+BAP+IAA). Compared with the control (sucrose only), the shortened () or extended (+) transit times are given in brackets Experimental series
G1/S
G2/M
Sucrose Sucrose+BAP Sucrose+IAA Sucrose+BAP+IAA
16.0 7.5(8.5) 16.5(+0.5) 10.5(5.5)
13.0 10.5(2.5) 16.0(+3.0) 10.5(2.5)
Fig. 3. Recovery of DNA replication (BrdUrd labelling index, %) in excised, carbohydrate-starved root meristem cells of Vicia faba subsp. minor cultivated in White’s mineral medium containing 2% sucrose with 0.56 µM BAP, 5.4 µM IAA and a mixture of 0.56 µM BAP and 5.4 µM IAA (3 h intervals).
3.4. Influence of BAP, IAA, and BAP/IAA mixture on the sucrose-mediated resumption of DNA replication and mitosis in excised root meristems To examine the influence of auxin and cytokinin on the delay of DNA replication and mitosis, the carbohydrate-starved root meristems of V. faba subsp. minor were returned to complete recovery medium (with 2% sucrose) containing either BAP, IAA, or a mixture of both growth regulators. Depending on the experimental series performed using media supplied with phytohormones (Figs. 3 and 4), the upper levels of BrdUrd-labelling indices and the maximum frequencies of mitotic cells are lower than those estimated in the control series. The waves of cells entering S and M phases are significantly longer, demonstrating an increased heterogeneity of G1 and G2 cell populations. These data provide strong evidence that BAP and IAA have a profound influence on the dynamics of both the G1-to-S (Fig. 3) and G2-to-M (Fig. 4) transitions. However, the effect of cytokinin on cells with nuclear 2C or 4C DNA content appears to be opposite to that of auxin.
Fig. 4. Recovery of cell division (mitotic index, %) in excised, carbohydrate-starved root meristem cells of Vicia faba subsp. minor cultivated in White’s mineral medium containing 2% sucrose with addition of 0.56 µM BAP, 5.4 µM IAA and a mixture of 0.56 µM BAP and 5.4 µM IAA (3 h intervals).
The delay of DNA replication in roots transferred to complete mineral medium supplemented with BAP was reduced to about half the time needed to cover the flow of cells from G1 to S in root meristems provided with sucrose alone. In contrast, the recovery of carbohydratestarved root meristems in medium containing IAA resulted in about 3 h excess delay in the entrance to S phase. In the experimental series with the mixture of BAP and IAA, a compromise develops between the two phytohormones, bringing about a moderate acceleration in the transition of G1 cells into the onset of DNA replication (Fig. 3; Table 2). The general kinetic aspects of the response of G2 cells to the various block-and-release exposures are remarkably similar to those seen previously in the G1 cell population. Compared with the control series of carbohydrate-starved roots transferred to medium supplied with 2% sucrose, the continuous refeeding of excised root meristems either in medium containing BAP, or BAP plus IAA, decreases the delay of G2-to-M transition by about 3 h (Fig. 4; Table 2). Incubation in medium with IAA results in the opposite effect, and the cells advance to the G2/M boundary after an additional delay of about 7 h. 4. Discussion Phytohormones have long been recognized as signalling factors playing a pivotal role in nearly every aspect of plant development. Over the last few years it has become increasingly clear that some of them, mostly auxins and cytokinins, are part of an interacting biochemical network involved in the transcriptional control of the cell cycle genes (Carle et al., 1998; Kapros et al., 1992; Soni et al., 1995). Despite accumulating evidence of numerous links between growth regulators and the molecular mechanisms that underlie cell cycle progression, the extent to which they affect the system of G1
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and G2 control points in plants is currently unknown. Adopting an original model of excised root meristems, which contributed to the Principal Control Point hypothesis (Van’t Hof and Kovacs, 1972; Van’t Hof 1985), our present studies were undertaken in the hope of getting answers to the two following questions: 1. How do cytokinins (BAP) and auxins (IAA) influence the expression of PCP1 and PCP2? 2. How do these plant growth regulators change the sucrose-mediated release of G1- and G2-arrested cells into the S and M phase of the cell cycle? Meristem cell populations in excised roots of V. faba subsp. minor become blocked in either G1 (PCP1) or G2 (PCP2). Lack of both mitotic and BrdUrd incorporating cells suggests that deprivation of essential nutrients brings about complete cessation of cellular proliferation, resulting in the production of stationary phase meristems. Although both checkpoints operate in root meristem cells starved for 72 h in mineral medium containing BAP, IAA or a mixture of both, the percentage of cells blocked with either 2C or 4C nuclear DNA content varies significantly depending on the experimental series. Incubation in medium containing BAP allows a considerable additional number of cells to proceed past the G1 checkpoint and arrest after having reached 4C nuclear DNA content. A significantly higher rise in the frequency of cells blocked at the G2 control point is observed in root meristems maintained in the presence of IAA. Thus, the behavioural response of root meristem cell populations is clearly not fixed and the expression of G1 and G2 control points depends largely on the ‘regulatory context’ established by the relationship between the innate (genetic) instructions and extracellular (phytohormonal) signals. The quantitative distribution patterns of G1- and G2-arrested root meristem cells of V. faba subsp. minor starved in media supplied with either BAP or IAA appear to correlate with the results obtained by Zhang et al. (1996) in experiments performed using suspensioncultured cells of Nicotiana plumbaginifolia. Following an 8 day period of starvation without cytokinin and auxin and subsequent 2 day incubation with kinetin, the cells were found to be arrested in both G1 and G2 phases of the cell cycle. In contrast, cells that lacked cytokinin, being incubated with 2,4-D (auxin) only, arrested almost entirely in the G2 phase. Although some caution must be used in drawing parallels between results obtained by means of different experimental assays, it seems likely that a common connection exists between the changes in frequency of G1 and G2 cell populations seen in experiments with excised root tips of V. faba and those obtained using suspension cell cultures of N. plumbaginifolia. Neither DNA replication nor mitosis occurs in the absence of the appropriate CDK-cyclin complex, and
the timing of each event can be advanced by premature CDK activation (Jacobs, 1997). The control points regulate progression through the key transitions of the cell cycle by interacting with the subsequent period of CDK activity, inhibiting it until the cell “senses” an abundance of nutrients and sufficient metabolic capabilities needed for DNA replication or progression into mitosis. Data concerning the cells in higher plants, including (1) their size-dependent control over the onset of DNA replication (John, 1996), (2) the occurrence of p34cdc2-like proteins recognized by antibody against the internal PSTAIRE peptide (e.g. John et al., 1993; Polit, 1998), and (3) the presence of distinct classes of cyclins and CDKs, which drive the progression through the major events of the cell cycle (Sundaresan and Colasanti, 1998), indicate a strong analogy between PCP1, the START control in yeasts, and the restriction point in mammals. Functional conservation of cell cycle control mechanisms among the eukaryotes becomes even more evident at the stages preceding the G2-to-M transition. In both plants and animals, the amounts of A and B cyclins are regulated transcriptionally and show similar expression profiles, with peaks in late G2 and M. Consequently, the activated p34cdc2 kinase phosphorylates a set of specific substrates and promotes chromatin condensation, nuclear envelope breakdown, disassembly of the nucleolus and spatial organization of cytoskeletal polymers (John, 1996). The reduction of G1-to-S transitions in carbohydratestarved root tips restored to recovery medium supplemented with BAP, and the opposite effect observed in meristems incubated in medium containing IAA, correlates well with the phytohormone-regulated expression of cyclin D homologs from Arabidopsis thaliana, referred to as cyclins (Soni et al., 1995). Cyclins 2 and 3, known to play a role during the G1/S transition, have been found to be directly involved in linking plant growth regulators, nutritional status, and cell cycle progression in a manner similar to that of mammalian cyclin D in response to growth factors. In suspension cell cultures of A. thaliana, the cytokinin-dependent induction of mRNA synthesis for 3 cyclin is apparently antagonized by the concomitant addition of auxin (2,4D), so that in the presence of sucrose a smaller induction of transcription is detected using RNA gel blots. In sucrose-free medium, no accumulation over basal 3 cyclin mRNA levels is observed. Concurrently, the levels of 2 cyclin are considerably increased in response to the availability of sucrose. Clearly, the results obtained in our present work correspond well with the biochemical data recorded in suspension-cultured plant cells, demonstrating the stringent requirement of late G1 cells for cytokinin in promoting their re-entry into S phase (Soni et al., 1995; see also Carle et al., 1998; Riou-Khamlichi et al., 2000).
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The vast amount of information accumulated by the recent physiological studies on plant cells indicate that the hormonal control point operating in late G2 phase is responsible for triggering the transition into mitosis by rapid activitation of p34cdc2 kinase (John, 1996). Most cellular systems used in the analysis of cell cycle gene expression seem to require exogenous auxin alone, probably because endogenous levels of cytokinin synthesis are sufficient to fulfil the needs for continued growth (Zhang et al., 1996). Moreover, the hormone withdrawal studies performed using Nicotiana tabacum mesophyll protoplasts have shown that a delay in auxin addition has a greater effect on cell division than a delay in cytokinin addition (Carle et al., 1998). Experiments with pith parenchyma cells excised from N. tabacum stem segments, as designed by Zhang et al. (1996), indicate that auxin (NAA) and cytokinin (BAP) induce a greater than 40-fold increase in a p34cdc2-like protein, but without cytokinin the H1 histone kinase is inactive. Cytological and biochemical data consistent with this hypothesis suggest that the presence of adequate cytokinin can stimulate the catalytic capabilities of MPF by a highly specific cdc25 phosphatase which decreases the abundance of p34cdc2 phosphotyrosine and allows the cells to proceed towards prophase. This has been confirmed by pith cells without cytokinin having been found arrested in late G2 phase with a p34cdc2-like protein of high Y–PO4, which could be removed by cytokinin or the cdc25 enzyme (Zhang et al., 1996). A model of this kind seems to provide an easier and more reasonable explanation for the shortened PCP2-M intervals in root meristems of V. faba restored to recovery medium supplemented with BAP. As was the case with the G1 counterparts relieved from the block imposed by PCP1, a diametrically opposed effect could be observed in roots resupplied with sucrose and IAA. In all these cases, however, it is important to bear in mind that the observed effects are not only due to the exogenous hormones, but are rather caused by the interaction of the applied plant growth regulators with internal factors produced by the excised parts of the root (De Veylder et al., 1998). It may be that the prolonged delay in cell progression from G2-to-M results from a disturbance in the balance between the two regulators, which need to be in precise and highly specific proportions to one another in order to activate MPF kinase. The induction of stationary phase meristems and our block-and-release experiments clearly show that exogenous plant hormones exert a profound influence on the expression of G1- and G2-phase checkpoints in root meristems of V. faba. It seems reasonable to believe that the complex biochemical mechanisms involved in PCPs are based on an interplay between genetic (innate) and extragenetic information. Presumably, regulatory systems triggering DNA replication and mitosis integrate both nutritional and hormonal information, and thus
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