The role and regulation of D-type cyclins in the plant cell cycle

Transcription

1 Plant Molecular Biology 43: , Dirk Inzé (Ed.), The Plant Cell Cycle Kluwer Academic Publishers. Printed in the Netherlands. 621 The role and regulation of D-type cyclins in the plant cell cycle Marcel Meijer and James A. H. Murray Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK ( author for correspondence; j.murray@biotech.cam.ac.uk) Key words: D-type cyclins, differentiation, G 1 /S control, plant cell cycle, proliferation, retinoblastoma protein Abstract The G 1 phase of the cell cycle represents a period of commitment to cell division, both for cells stimulated to resume division from a resting or quiescent state, and for cells involved in repeated cell cycles. During this period, various signals that affect the cells ability to divide must be assessed and integrated. G 1 culminates in the entry of cells into S phase, when DNA replication occurs. In addition, it is likely that several types of differentiation decision may be taken by cells in the G 1 phase. In both animals and plants, it appears that D-type cyclins play an important role in the cell cycle responses to external signals, by forming the regulatory subunit of cyclin-dependent kinase complexes. The phosphorylation targets of D-cyclin kinases in mammalian cells are the retinoblastoma (Rb) protein and close relatives. Unphosphorylated Rb can associate with E2F transcription factors, preventing transcription of genes under E2F control until the G 1 /S boundary is reached. The conservation of Rb and E2F proteins in plants suggests that this pathway is therefore conserved in all higher eukaryotes, although it is absent in fungi and yeasts. Here we review the current understanding of the roles and regulations of D-type (CycD) cyclins in plants. Introduction The co-ordination of cell division with cell growth and differentiation is necessary to create complex multicellular organisms, and is achieved within the framework of a specific developmental plan that defines the characteristics of the particular organism (White-Cooper and Glover, 1995; Meyerowitz, 1997). Plants also need to modulate this primary pattern of growth and development in order to respond flexibly to changes in their environment, since they are unable to physically move to optimal locations (De Veylder, 1998; Francis, 1998). The control of cell division in plants must therefore sense and interact with external signals. Almost half a century ago, Howard and Pelc (1953) introduced the terminology of the eukaryotic cell division cycle, recognising the fundamental importance of the separation of DNA replication (S phase) from cell division (M phase) by the two gap phases (G 1 and G 2 ) in the sequence G 1 -S-G 2 -M. This arrangement allows for the precise control of DNA replication and mitosis, and it is not surprising that an ordered series of molecular and cellular processes define the order and control of the cycle. Cells that temporarily or permanently lose the capacity to divide normally stop in the G 1 phase with 2C DNA content, although in plants there appears to be much more flexibility, with both G 1 and G 2 being important cell cycle exit points. It should be noted however that the frequent occurrence of endoreduplication (chromosome replication without subsequent mitosis) can create G 1 cells with 4C or higher DNA content (see below). Non-cycling or quiescent cells are often said to be in G 0 to distinguish them from actively cycling G 1 cells, although there is no molecular definition of this state in plants (see Loeffler and Potten, 1996). Progression through the eukaryotic cell cycle is largely regulated at two principal control points, one late in G 1 phase and the other at the G 2 /M boundary. A further important control exists at the metaphaseanaphase transition, and no doubt further subsidiary controls also exist. Transit through these control [77]

2 622 points requires activated kinase complexes, consisting of a cyclin-dependent serine/threonine protein kinase (CDK) bound to a cyclin (for review, see Pines, 1995b). CDK activity is dependent on cyclin binding, which also determines substrate specificity and subcellular localisation of the CDK complex. The cell cycle is driven forward by the sequential activation and destruction of CDK activities, and this indicates that CDKs and cyclins play central roles in the regulation of cell cycle commitment and progression (for review, see Pines, 1995b). In this paper, we focus on the molecules that regulate transit through G 1 and entry into S phase in plant cells. This period not only includes the point of commitment to cell division, but may also represent the time during which differentiation decisions are made. Although there are parallels between yeast and animal controls during this stage in the cell cycle, not all processes and molecules involved are equivalent between the two systems. We discuss recent work suggesting that the proteins involved in G 1 /S controls in plants are more closely related to mammals than to fungi, and that decisions on proliferation and differentiation events may therefore be made in analogous ways in mammals and plants. Cell cycle control points The term cell cycle tends to imply an ever-rolling cycle of events, which is true for exponentially growing cell suspensions, but might not be very relevant in vivo. In all multicellular organisms, cells are part of organs, and have defined spatial relationships with their neighbouring cells in creating higher-order structures. Cells must sense when they are required to divide, and when division must be ceased or modified, to allow differentiation into specialised organs to occur (Francis, 1998). Cells, therefore, must be able to integrate information on nutrient availability and environmental conditions, the positional and developmental context of the cell, and intracellular information, such as DNA damage, in order to determine whether to continue to divide or not. This integration of information operates through controls at defined points in the cell division cycle to ensure completion of one phase before the next one is initiated (Hartwell and Weinert, 1989). The two main control points in the cell cycle are at the G 1 /S transition and at the G 2 /M transition. Progression through these control points is mediated by the activation of cyclin-cdk complexes (for review, see Pines, 1995b). The first CDK to be described was encoded by the cdc2 gene of Schizosaccharomyces pombe (Simanis and Nurse, 1986) and genetic analysis revealed that the cdc2 gene product, p34 cdc2, is required for both control points. Subsequently, cdc2 homologues have been isolated for many organisms, including human and several plant CDK genes (reviewed by Jacobs, 1995). In contrast to yeast, in higher eukaryotes, multiple CDKs regulate different stages of the cell cycle (reviewed by Pines, 1995a). In most plant cell types, the primary control point probably operates during G 1 phase, as for mammalian cells and yeast. Indeed, classical studies on cell suspension cultures showed that plant cells arrest in G 1 or G 2 when starved of nutrients or hormones, with the G 1 arrest being the more stringent (reviewed by Bayliss, 1985). In intact plants, many differentiated plant cells also have a G 1 (2C) DNA content, indicating exit from the cell cycle in G 1. However, significant proportions of cells stop dividing with a 4C DNA content and can therefore be interpreted as undergoing G 2 arrest. The status of these cells is complicated by the frequency of endoreduplication in differentiated plant cells (Bayliss, 1985; reviewed by Traas et al., 1998). Such 4C cells may, therefore, represent cells in G 1 that have undergone an endoreduplication event rather than G 2 arrest (Bayliss, 1985). Nevertheless, it is likely that in most cells the most important decision to divide or differentiate operates in late G 1 phase. The second main control point operates in G 2 and determines the entry into mitosis. In both yeast and mammals, this transition is largely controlled by kinase activity of the cdc2 gene product. In association with mitotic cyclins (A- and B-type cyclins), the p34 cdc2 phosphorylates a set of substrates at the G 2 /M, driving cells into mitosis (reviewed by Norbury and Nurse, 1992). Both A-like and B-like cyclins have been isolated in plants, and it is likely that the B-like cyclins are regulators of G 2 /M (reviewed by Renaudin et al., 1998). The ability of a B-like cyclin to accelerate growth in Arabidopsis suggests that this G 2 control point may be a limiting factor in root cell division (Doerner et al., 1996). The control of G 1 /S transition in mammalian cells In this section, we briefly outline the current view on G 1 /S transition controls in mammalian cells. These controls in mammalian cells and budding yeast (Sac- [78]

3 623 charomyces cerevisiae) are discussed in more detail in a number of recent reviews (Morgan, 1997; Johnson and Walker, 1999; Pavletich, 1999). External signals impinge on the cell cycle at a principal point in G 1, called the restriction point (R) in mammalian cells, and depending on the nature of the signals, cells either commit to another round of cell division or exit the cell division cycle and adopt alternative differentiation pathways (Pardee, 1989). Progression through R is mediated by D-type cyclins, whose transcription is absolutely dependent on serum growth factors (Matsushime et al., 1991; Ajchenbaum et al., 1993; Ando et al., 1993; reviewed by Sherr, 1993, 1994). Transcript levels decline rapidly upon growth factor removal and reappear again upon addition. Cyclin-D-dependent CDKs direct phosphorylation of the retinoblastoma (Rb) protein in mid-to-late G 1 phase, thereby driving cells through R and allowing activation of E2F controlled genes, which are required for S phase. E-type cyclins accumulate transiently in late G 1 phase, forming kinase complexes that accelerate the phosphorylation of Rb, thereby irreversibly driving cells across the G 1 /S boundary (for review, see Sherr, 1996). Recent evidence suggests functional differences in the phosphorylation of Rb protein by cyclin D-CDK4/CDK6 and cyclin E-CDK2 complexes (Mittnacht, 1998), since in vitro different sites on Rb are phosphorylated (Kitagawa et al., 1996; Zarkowska et al., 1997). A defining characteristic of D-type cyclins is the presence of a specific motif consisting of the sequence LxCxE (single-letter code, x being any amino acid) that is responsible for the binding of D-type cyclins to Rb and related proteins. Interestingly, the same motif is shared by the transforming proteins of several DNA tumour viruses, which inactivate Rb as part of their infection and replication mechanism. Isolation of plant D-type cyclins G 1 /S control in mammalian cells is of primary importance for understanding both cellular proliferation and differentiation. These issues are also important for plants, where the majority of post-embryonic division activity is concentrated in the meristematic regions. Moreover, many plant cells can dedifferentiate in response to wounding, pathogen attack or exogenous application of plant hormones, suggesting more plasticity in controlling cell cycle entry and exit than that normally found in mammalian cells. It was therefore interesting to investigate whether plant controls operating in G 1 phase were more closely related to those of yeast or mammals. Plant D-type cyclin (CycD cyclin) cdnas were first isolated from Arabidopsis (Soni et al., 1995) and alfalfa (Dahl et al., 1995) by their ability to functionally complement yeast strains that were defective in two of their three G 1 (CLN) cyclins, with the third cyclin under control of a galactose promoter (Xiong et al., 1991). They were defined as D-type cyclins on the basis of low sequence homology to mammalian D-type cyclins, and the presence of the conserved LxCxE motif. Plant CycD proteins as a class have higher homology to animal D-type cyclins than any other class of cyclins, although residues are identical between plant and animal D-type cyclins at only about 20 25% of positions (Renaudin et al., 1996). Subsequently, CycD cyclin cdnas have been isolated from Antirrhinum (Gaudin et al., 2000), Helianthus (Freeman and Murray, unpublished results), tobacco (Sorrell et al., 1999; Nakagami et al., 1999; M. Sekine, personal communication), Pisum sativum (Shimizu and Mori, 1998), tomato (A. Kvarnheden, personal communication; C. Chevalier, unpublished) and Chenopodium (Renz et al., 1997; Fountain et al., 1999) by screening cdna libraries with CycD cyclin cdna probes. These CycD cyclins form three distinct groups designated CycD1, CycD2 and CycD3 (Figure 1; Renaudin et al., 1996; Murray et al., 1998). Using a two-hybrid screen in yeast with Cdc2aAt as a bait, De Veylder et al. (1999) recently isolated a fourth D-type cyclin in Arabidopsis. At this stage it is unclear whether this CycD cyclin belongs to a separate CycD group or is part of the CycD2 group (Figure 1). It is important to note that all the groups of plant CycD cyclins are more closely related to each other than to any of the animal D-type cyclins, and that the numbering of the CycD groups in plants is not related to the numbering of different cyclin D s in animals (Renaudin et al., 1996). When multiple genes have been identified in one group (e.g. CycD3) the suffix number indicates only the order of isolation in a given species. Thus, CycD3;1 from tobacco is not most closely related to CycD3;1 from Antirrhinum (see Figure 1). In fact, the relationship between the different CycD3 cyclins suggests the probable existence of three subgroups, which we propose to designate CycD3a, CycD3b, and CycD3c (Figure 1), and for three species (An- [79]

4 624 Figure 1. CycD cyclin relationships. The relationship between plant CycD cyclins was determined by generating a multiple alignment of the sequences using the CLUSTALX program. This alignment was then optimised by manual editing, and displayed using the PILEUP program in the GCG package. Cyclin nomenclature is according to Renaudin et al. (1996). Antma, Antirrhinum majus (Gaudin et al., 2000); Arath, Arabidopsis thaliana (Soni et al., 1995; De Veylder et al., 1999; T. Jack, personal communication); Cheru, Chenopodium rubrum (Renz et al., 1997; Fountain et al., 1999) Heltu, Helianthus tuberosus (D. Freeman and J. A. H. Murray, unpublished data); Lyces, Lycopersicon esculentum (A. Kvarnheden, personal communication; C. Chevalier, unpublished data); Medsa, Medicago sativa (Dahl et al., 1995); Nicta, Nicotiana tabacum (Sorrell et al., 1999; Nakagami et al., 1999; M. Sekine, unpublished data); Pissa, Pisum sativum (Shimizu and Mori, 1998). The three distinct groups of CycD cyclins, CycD1, CycD2/4 and CycD3, are indicated on the right. The relationship between the different CycD3 cyclins suggests the probable existence of at least three subgroups. These have been designated CycD3a, CycD3b and CycD3c. tirrhinum, tomato and tobacco) CycD3s have been isolated from more than one subgroup. We suggest the use of the subgroup designation CycD3a, CycD3b, CycD3c, as foreseen by Renaudin et al. (1996), is useful to indicate sequence relationships within the CycD3 group. The relationship of a second Arabidopsis CycD3 gene (CycD3;2, T. Jack, personal communication) to the subgroups is currently unclear. The presence of multiple CycD3 genes raises the question of functional redundancy of these genes, and the extent to which they may have distinct or overlapping roles, but the greater conservation of subgroup members between species than similarity with other subgroups in the same species indicates a possible conserved function. Interestingly, distinct CycD3s that have been isolated from both Antirrhinum and tobacco are differentially expressed in Antirrhinum meristems (Gaudin et al., 2000) and tobacco cell suspension cultures (Sorrell et al., 1999). CycD3a (Antma;CycD3;1) in Antirrhinum is expressed in organ primordia only, whereas CycD3b (Antma;CycD3;2) is probably expressed in all dividing cells (Doonan, 1998; Gaudin et al., 2000). Moreover, differences exist in the regulation of subgroup members by external signals (Gaudin et al., 2000). Furthermore, unlike the Arabidopsis CycD3;1 gene, which is highly cytokinin-inducible (see below; Riou-Khamlichi et al., 1999), cytokinin did not induce the alfalfa CycD3 gene and its overexpression had no obvious effect (reported in Inzé et al., 1999). Taken together, these results suggest that cyclins in different CycD groups and subgroups may have distinct functions, although the extent to which these are non-overlapping will have to await the identification of insertion mutants. Characteristics of plant D-type cyclins As mentioned above, a defining characteristic of all cyclin D proteins identified to date is the presence of the Rb interaction motif LxCxE or a very closely related sequence near their N-terminus. In mammalian D cyclins, this motif is within a few amino acids of the initiation methionine. All plant CycD cyclins, with the exception of tobacco CycD3;4, contain the same Lx- CxE motif near the N-terminus. In contrast, CycD3;4 contains a LxCxD motif, in which aspartate replaces the glutamate residue. In common with animal D-type cyclins, all CycD cyclins isolated to date, with the exception of Arabidopsis CycD2;2 (CycD4), contain at least one acidic residue (D or E) at positions 1or 2 relative to the LxCxE motif (Renaudin et al., 1996; De Veylder et al., 1999). All cyclins contain a defining homologous region of ca. 100 amino acids known as the cyclin box, which is involved in the interactions with the CDK partner (Lees and Harlow, 1993; Jeffrey et al., 1995; Renaudin et al., 1998). The corresponding region in CycD cyclins has relatively low homology to mitotic cyclins, but nine residues are invariant between the cyclin box of all cyclins of the A, B, and D classes in animals and plants, including five residues that have been shown experimentally to be essential for catalytic activity. [80]

5 625 The majority of cyclins are destroyed rapidly at certain points in the cell cycle and this ability to activate turn-over of the cyclins is central to cell cycle control (Murray et al., 1998; Renaudin et al., 1998). In the case of mitotic cyclins, a specific N-terminal sequence called the destruction box targets cyclins for specific ubiquitin-mediated destruction during mitosis (Glotzer et al., 1991; Pines, 1995b; Genschick et al., 1998). The types of destruction motifs in plant mitotic cyclins have recently been reviewed by Renaudin et al. (1998), and analysed by Genschick et al. (1998). G 1 cyclins in both yeast and mammals have short half-lives and their rapid turn-over depends on the presence of so-called PEST sequences, regions rich in these four amino acids, which are characteristic of many proteins with a rapid turn-over (Rechsteiner and Rogers, 1996; Rogers et al., 1996). Examination of all CycD cyclins using the program PESTFIND (Rogers et al., 1996) identified PEST sequences in all CycD cyclins, apart from tobacco CycD2;1 (Sorrell et al., 1999). This suggests that PEST sequences are a general feature of G 1 cyclins and that most plant D cyclins, like their animal homologues, may be shortlived proteins. However, in no case has the role of PEST sequences in determining the half-life of plant CycD protein been experimentally verified. Regulation of D-type cyclins in cell cultures In mammalian cell cultures, the transcription of D- type cyclins is highly growth-factor-dependent (Matsushime et al., 1991; Baldin et al., 1993; Sewing et al., 1993). Growth-factor-starved cells or quiescent cells show low levels of Cyclin D mrna, and, upon addition of growth factor, show rapidly increasing expression levels that reach a maximum after 10 h of serum stimulation and 8 h before DNA synthesis begins (Sewing et al., 1993). However, the abundance of cyclin D1 transcript does not change significantly in cycling cells, suggesting that cyclin D1-kinase activity is post-transcriptionally regulated. Indeed, cyclin D protein levels are high in G 1 cells and decline significantly by late S phase (Matsushime et al., 1991; Sewing et al., 1993). In human cells, D-type cyclins act as growth factor sensors with their expression depending more on extracellular stimuli than on the position of the cell cycle (Sherr, 1993, 1996). Therefore, D-type cyclins are proposed to provide the link between stimuli from the environment and the cell cycle. Support for this hypothesis comes from observations that over-expression of D-type cyclins does not only reduce the length of the G 1 phase, but also partially overrides the need of dividing cells for mitogens (Kato and Sherr, 1993; Zwijssen et al., 1996). Analysis of D-type cyclin expression in partially synchronised Arabidopsis cell suspension cultures showed that CycD3 accumulated rapidly upon release of the G 1 /S block before accumulation of histone H4 expression and the onset of S phase (Soni et al., 1995; Fuerst et al., 1996). CycD1 is expressed at very low levels in liquid cultured cells and CycD2 mrna levels were unaffected by a G 1 /S block and release using low concentrations of cycloheximide. Levels of CycD3 remained relatively constant after the initial accumulation at the G 1 /S boundary (Fuerst et al., 1996), which mirrors the behaviour of CycD1 mrna in mammalian cells (Matsushime et al., 1991; Sewing et al., 1993). In tobacco BY-2 cell suspension cultures, which are highly synchronisable (Nagata et al., 1992), CycD3;2 is induced in G 1 upon re-entering the cell cycle after synchronisation and remains at a constant level throughout the cell cycle (Sorrell et al., 1999). Surprisingly, CycD2;1 and CycD3;1 both showed their greatest abundance in mitotic cells (Sorrell et al., 1999). It could be that these cyclins are required for entry into or progression through mitosis. In proliferating mammalian cells, retinoblastoma proteins are further phosphorylated in G 2 /M before being dephosphorylated in the later stages of mitosis (De- Caprio et al., 1992; Ludlow et al., 1993; Taya, 1997). However, it is also possible that mitotic accumulation is a BY-2 cell-specific phenomenon and not a normal feature of the plant cell cycle. In the human HeLa tumour cell line an increase in human cyclin D1 is also observed in G 2 /M phase, but not in other cell lines or in primary cultures (Motokura et al., 1992). It has been suggested that selection may have occurred in this cell line for altered or deregulated expression of cyclin D1 as a consequence of extended proliferation (Sewing et al., 1993). Similarly, tobacco BY-2 cells have been growing in culture for over 30 years and show an exceptional ability for rapid cell growth (Nagata et al., 1992). Similar to the response of mammalian CycD cyclins to serum growth factors, plant CycD cyclins are regulated in response to exogenous signals known to affect growth of plant cells. Auxin and cytokinin are important plant growth regulators (PGRs or hormones) that are required for most plant cell cultures. In addition to these plant hormones, sucrose is of central [81]

6 626 importance to plant metabolism as the major transport product of photosynthesis, and a possible signalling molecule in the regulation of a large number of genes (Koch, 1996; Jyung and Sheen, 1997). Sucrose is a favourable candidate for modulating cell division rates because its availability to dividing cells in shoot and root meristems will be a reflection on the overall photosynthetic capacity of the plant and therefore on the environmental conditions to which the plant is exposed (Koch, 1996). Using Arabidopsis suspension cell cultures, it was shown that sucrose can induce both CycD2 and CycD4 in starved suspension cells (Soni et al., 1995; De Veylder et al., 1999). A more comprehensive analysis of all possible combinations of sugars and PGRs in cell cultures that were deprived of cytokinin, auxin and sucrose for 48 h showed that CycD3 was specifically induced by cytokinin and CycD2 by sucrose. Recently, more detailed analysis has shown that cytokinin induction of CycD3 is dependent on the presence of sucrose, and sucrose alone can also induce CycD3 (Riou- Khamlichi et al., submitted). When cycloheximide was used at a concentration that inhibits both protein synthesis and cell cycle progression, CycD2 and CycD3 levels were still stimulated by sucrose. From these experiments it can be concluded that neither cell cycle progression nor de novo protein synthesis is required for increases in CycD2 and CycD3 mrna levels and this is consistent with their proposed roles as sensors of nutrient status in cell cycle control (Riou- Khamlichi et al., submitted). The response of CycD2 and CycD3 to different stimuli suggests that each of them is involved in a separate signal transduction pathway, a conclusion supported by the different responses of the two genes to the presence of inhibitors of protein phosphatases (Riou-Khamlichi et al., submitted). Regulation of D-type cyclins in vivo Data on CycD cyclin expression levels in intact tissues are more limited. In situ hybridisation studies on the two CycD3 homologues in Antirrhinum shoot apical and floral meristems confirmed that CycD3 mrna does not accumulate in a cell-cycle-dependent manner, unlike cyclin B genes that have been studied (Doonan, 1998; Gaudin et al., 2000). In Antirrhinum, CycD1 is expressed throughout the meristem and at low levels in other tissues. Both CycD3 genes of Antirrhinum are expressed within regions that exhibit cell division activity, but CycD3a is only expressed in the peripheral region of the meristem, in particular in organ primordia (Gaudin et al., 2000). CycD3b appears to be generally expressed in dividing cells. In floral meristems, CycD3b expression is modulated in cells surrounding the base of organ primordia and, later in development, in the ventral petals and is repressed in the dorsal stamen. These results add weight to the concept of differential function within apical meristems (Doonan, 1998), and it suggests that changes in cell cycle control could be early events in the switch to a differentiated state (discussed later). In Arabidopsis, CycD3;1 is shown by RNA gel blot analysis to be highly expressed in mature roots and to show somewhat lower expression levels in aboveground and callus tissue (Soni et al., 1995; reviewed by Murray et al., 1998). In situ hybridisation studies on the recently isolated CycD4 in Arabidopsis showed that CycD4 is expressed during vascular tissue development, embryogenesis and formation of lateral root primordia (De Veylder et al., 1999). The three transcripts of CycD1 show differential abundance in different tissues with the longest transcript particularly prevalent in flowers, and to a lesser extent in roots and callus material, whereas in leaves the intermediate and shorter transcripts prevail (Soni et al., 1995). Fusion of the CycD3;1 promoter to the β- glucuronidase (GUS) marker gene in transgenic Arabidopsis resulted in GUS activity in the shoot apical meristem, and in stele cells associated with lateral root primordia. In older roots, more general expression was observed in the stele, but GUS activity was never observed in the root apex (C. Riou-Khamlichi and Murray, unpublished observations). This expression pattern distinguishes CycD3;1 from general cell cycle genes that are expressed in all dividing cells, since tissue specificity is observed. The role of cytokinin in regulating the CycD3;1 cyclin in Arabidopsis has been reported recently (Riou- Khamlichi et al., 1999). In both cell cultures and in whole plants, application of cytokinin induces CycD3. Moreover, leaf tissue from transgenic plants that constitutively express CycD3 were able to generate callus in the absence of exogenous cytokinin, which could be maintained on auxin alone. It has been suggested that cytokinin activates Arabidopsis cell division through induction of CycD3 at the G 1 /S phase transition in whole plants, as well as in tissue culture (Riou-Khamlichi et al., 1999). The only other in planta study on CycD cyclin gene expression levels is on CycD3;Ms in alfalfa (Dahl et al., 1995), which appears to be induced [82]

7 627 before the onset of DNA synthesis. CycD3;Ms expression levels are high in the root and is limited to the pericycle, endodermis and outer cortex (Dahl et al., 1995). In summary, there is still a lot to learn about the expression of CycD cyclins. So far, there is clear evidence for tissue- and meristem-specific expression, and cell cycle behaviour that may be similar to that in mammalian cells. It has been established that CycD3 in Arabidopsis is stimulated by cytokinin, but additional signals, such as sucrose, nitrate, cell cycle progress and cellular position in specific tissues also play a role. Nevertheless, all results clearly implicate plant CycD cyclins as important integrators of proliferating signals in G 1 phase. Interactions of D-type cyclins with CDKs and Rb proteins Progression through the eukaryotic cell cycle is mediated by the phosphorylation of key substrates by cyclin-dependent kinases (CDKs). Potential substrates of CDKs include cytoskeletal proteins (e.g. lamins), chromatin-associated proteins (e.g. histone H1), and regulatory proteins, such as Rb (reviewed by Nigg, 1993). The substrate specificity of different CDKs is thought to be regulated by the targeting of CDKs to distinct cellular compartments by binding of CDKs to different types of cyclins (Pines, 1995b). The interaction of CycD cyclins in plants with CDKs has been shown with a yeast two-hybrid approach. In Arabidopsis, a CDK (Cdc2aAt; De Veylder et al., 1997) was used as a bait to identify putative substrates of CDKs. Using this approach, cyclin D1;1 was identified as a partner of Cdc2aAt and Cdc2bAt (De Veylder et al., 1997). Likewise, recent results using immunoprecipitation with specific antisera show that in Arabidopsis both CycD2 and CycD3 associate with Cdc2aAt. Kinase activity could be assayed using histone H1 as a substrate with both CycD2 and CycD3 immunoprecipitates (S. Healey and J.A.H. Murray, manuscript in preparation). As mentioned earlier, all D-type cyclins contain N- terminal LxCxE motifs, which are capable of binding the pocket domain of Rb (Dowdy et al., 1993; Kato et al., 1993). The Rb family of proteins play an important role in mammalian cell cycle by controlling transit through G 1 and inhibition of inappropriate cell proliferation (Weinberg, 1995). Rb exerts its negative regulatory control by inactivating the E2F family of transcription factors (Sherr, 1996), through recruitment of histone deacetylases (HDAC) to the promoters of E2F-regulated genes (reviewed by Brehm and Kouzarides, 1999). The Msi1 protein identified in tomato interacts with Rb, and is a homologue of a human component of an HDAC complex (Ach et al., 1997b; Figure 2). E2F activity is normally essential for S phase, and Rb inhibits its activation by binding E2F via a region of Rb containing two conserved sequence blocks, which form the so-called A/B pocket domain (reviewed by Dyson, 1998). This pocket domain is also required for the growth restraining ability of Rb, and cyclin D interaction (Kato et al., 1993). Binding and inactivation of E2F by Rb is regulated by Rb phosphorylation. During most of G 1, Rb is hypo-phosphorylated, but phosphorylation of Rb by the cyclin-dependent kinase complexes cyclin D/CDK4, cyclin E/CDK2 and cyclin A/CDK2 inactivates Rb (Sherr, 1996). This activates E2F and leads to S-phase onset. Mutational analysis has shown the requirement for the intact LxCxE motif in D-type cyclins as well as integrity of the pocket domain of Rb (Dowdy et al., 1993; Ewen et al., 1993). The recent isolation of E2F cdna clones from wheat, tobacco and Arabidopsis reinforces the idea that regulation of G 1 /S transition is more closely related to that found in animals than that in yeast (Ramirez-Parra et al., 1999; Sekine et al., 1999; de Jager et al., manuscript in preparation). The importance of cyclin D-Rb interactions was shown by several lines of evidence. First, the cyclin D component of the cyclin D/CDK4 complex links Rb to environmental clues, since cyclin D is an unstable protein whose transcription is totally dependent on serum growth factors in the growth media (Sewing et al., 1993; Sherr, 1993). Second, at least one residue on Rb that is required for Rb inactivation by phosphorylation can only be phosphorylated by cyclin D/CDK4 (Kitagawa et al., 1996), so S-phase entry is ultimately dependent on cyclin D/CDK activity. Finally, cyclin D-kinases are unnecessary for progression through the restriction point in cells that lack Rb, suggesting that Rb may be the only essential substrate of cyclin D/CDK4 (Sherr, 1996). The recent isolation of Rb homologues in plants (Grafi et al., 1996; Xie et al., 1996; Ach et al., 1997a) and the interaction of these homologues with plant CycD cyclins through the conserved LxCxE motif (Ach et al., 1997a; Huntley et al., 1998) indicates that the pathway of G 1 /S control involving cyclin D- mediated Rb phosphorylation and E2F activation is [83]

8 628 Figure 2. Model for control of G 1 /S transition in plants, indicating the stimulatory and inhibitory signals that are incorporated into control of a START-like point in plant cells, and modulate the activity of CDKs. CycD2 and CycD3 respond to sucrose, whereas only CycD3 is induced by cytokinin. CDK inhibitors (CKI) prevent kinase activity of cyclin-cdk complexes. ICK1, a CDK inhibitor from Arabidopsis, was isolated and it was shown that ICK1 interacts with both CycD3 and Cdc2a (Wang et al., 1998) and is induced by abscisic acid (ABA). This suggests that ABA might provide an inhibitory mechanism for the cell cycle, operating through CycD activity. Plants also contain homologues of human Rb-binding proteins (RbAp48; Ach et al., 1997b), itself a homologue of yeast MSI1. RbAp48 is a component of a human histone deacetylase complex, and its plant homologue MSI1 may play a similar role (Ach et al., 1997b). The presence of an Rb-binding motif in a gemini virus replication protein indicates that viral proteins in plants may drive cells into S phase by directly promoting release of E2F from Rb (Xie et al., 1995; Ach et al., 1997a). conserved among higher eukaryotes. This indicates that G 1 /S control in plants is more closely related to mammalian G 1 /S regulation than to yeast, which does not involve proteins with homology to cyclin D, Rb or E2F (Pines, 1995b; Nasmyth, 1996; Huntley et al., 1998). Recently, Nakagami et al. (1999) have also shown that a tobacco Rb-related protein (NtRb1) can be phosphorylated in vitro by a kinase assembled from tobacco Cdc2a and cyclin Nicta;CycD3;3. An antibody against the cyclin can immunoprecipitate a complex from tobacco BY-2 cells that can also phosphorylate NtRb1, suggesting that NtRb1 phosphorylation is indeed mediated by a CycD kinase in vivo (Nakagami et al., 1999). So far, little is known about the regulation of cyclin-cdk complexes in plants. Studies in yeast and mammalian systems have shown that activation of CDK involves not only binding of a cyclin, but also involves a CDK-activating kinase (CAK) and CDC25 protein phosphatase (Lees, 1995). Another level of regulation of cyclin/cdk complex activity is provided by CDK inhibitors, which stoichiometrically inhibit CDK activity (reviewed by Pines, 1995; Harper and Elledge, 1996). The discovery of a CDK inhibitor in Arabidopsis (ICK1; Wang et al., 1997) indicates that similar regulatory pathways exist in plants. ICK1 was [84]

9 629 subsequently shown to be induced by abscisic acid, and upon ICK1 induction a decrease in histone H1 kinase activity was observed (Wang et al., 1998). ICK1 clones were also identified in yeast two-hybrid screens with CycD3 as bait, and it was subsequently confirmed that ICK1 protein could interact with both Cdc2a and CycD3 by in vitro binding assays (Wang et al., 1998). These results indicate a role for CDK inhibitors in regulation of CycD kinases. Role of D-type cyclins in cell differentiation and development An emerging field is the role of D-type cyclins in cellular differentiation in human tumours. Cyclin D1 was originally identified as a proto-oncogene activated by translocation to a thyroid promoter in parathyroid adenomas (Motokura et al., 1991). Subsequently, it was show that cyclin D1-deficient mice had a reduced body size and a reduced number of cells in their retinas, a tissue that has a very high demand for cyclin D1 (Sicinki et al., 1995). Recent developments in mammalian systems showed that increased expression levels of cyclin D2 is associated with testicular cancer development (Bartkova et al., 1999). In more general terms, it appears that the cyclin D-CDK4 and D-CDK6 complexes interact with the proto-oncogene c-myc. Loss of c-myc causes profound growth defects correlated with a 12-fold reduction in cyclin D1-CDK4 activity (Mateyak et al., 1999). It is therefore imperative that we develop our understanding of the interaction between proliferation and differentiation in plant cells and in particular the reversibility of this switch, its relationship to meristem function, and regulation of cell cycle decision by organ identity genes. Some data already point to the likely importance of D-type cyclins in cellular differentiation in plants. The discovery of the differential expression of two CycD3 genes in Antirrhinum, with one of them only expressed in incipient and developing primordia and the other down-regulated in boundary layers of cells that lie between proliferating zones (Doonan, 1998; Gaudin et al., 2000) hints at the possibility that CycD cyclins are involved in meristem function and the control of proliferation and differentiation. So, although it may seem intuitive that cell cycle regulation should follow on from developmental controls, it is possible that changes in cell cycle regulation could drive downstream differentiation events. This hypothesis finds a parallel in the discovery of the change from controlled cell division to tumourous growth upon disruption of cyclin D2 expression levels in the human testis (Bartkova et al., 1999). This relationship between proliferation and differentiation is likely to be subtle and complex. The structure of the shoot apical meristem (SAM) of dicotyledonous plants illustrates the issues involved. The central zone of the SAM consists of a group of cells whose size and morphology do not change during most of the post-embryonic development of the plant (Laufs et al., 1998; Doerner, 1999; Lenhard and Laux, 1999). This population of cells, the central zone, represents the source of all above-ground tissues. The meristem cells divide anticlinally (division plane perpendicular to the surface), so the progeny cells are pushed into the surrounding peripheral zone. In this zone, leaf or floral primordia are specified and develop in a spiral pattern Cells in the peripheral zone must therefore undergo decisions that will result in the formation of determinate structures, such as leaves and floral organs, which consist of differentiated, non-dividing cells. Thus, the fate of cells in the peripheral zone is different from cells in the central zone, and may be thought of as having undergone a differentiation process. However, few if any morphological differences are found between cells in the two zones, and the rate of cell division is actually faster in cells in the peripheral zones (Lyndon, 1998). Also, these peripheral cells produce progeny cells which will develop into a number of different cell types (Francis, 1998). Nevertheless, all cells in the peripheral zone and their progeny will eventually differentiate, unlike cells in the central zone. It is likely, therefore, that several types of differentiation decisions are made by plant cells that involve alterations in the control of the cell cycle, suggesting intimate interconnections between proliferation and differentiation. One example would be the loss of stem cell characteristics, corresponding to exit from the central zone of the SAM. Cells undergoing this transition into the peripheral zone differ from cells in the central zone in that they lose the ability to give rise to stem cells, and the progeny of these peripheral zone cells will therefore ultimately differentiate and cease division. The transition from central zone to peripheral zone characteristics may involve aspects of cell cycle control as the proliferation rate increases (Lyndon, 1994). A subset of peripheral zone cells become involved in the initiation of new organ primordia, ei- [85]

10 630 ther leaves or flowers depending on the characteristics of the apical meristem, and the expression of CycD3a (Antma;CycD3;1) in Antirrhinum is a molecular marker for these cells (Doonan, 1998). In addition, in Arabidopsis the expression of the homeobox gene STM is absent from cells participating in the formation of primordia (Long et al., 1996). Further events occur when cells embark on cellspecific differentiation pathways and become morphologically or physiologically distinct. This is likely to correspond to a reduction in cell division rates. One could speculate that Rb-related proteins may be involved in mediating this reduction, or may be associated with differentiation events. In the final stages of determination, a cell ceases all division and exits from the cell cycle either in the G 1 or the G 2 phase. Regardless of cell cycle exit point, such cells might be predicted to lack CycD expression and have high levels of Rb-related proteins. Further types of differentiation events that relate to cell cycle controls are likely in cells that undergo further events such as endoreduplication (Traas et al., 1998), or programmed cell death in processes such as xylogenesis (for review, see Pennel and Lamb, 1997). Conclusions Mammalian D-type cyclins regulate progression of cells through the G 1 phase of the cell division cycle in response to extracellular signals through the interaction with Rb. The plant CycD cyclins that have been analysed so far show conserved regions and expression patterns that have parallels to those found in mammalian systems. This suggests that CycD cyclins may also function as integrators of external signals into the cell cycle via interactions with plant Rbs. The recent discovery that Arabidopsis CycD cyclins can bind both plant and human Rb proteins via the conserved LxCxE motif supports this hypothesis (Ach et al., 1997a; Gutierrez, 1998; Huntley et al., 1998). The requirement for plant growth regulators such as auxins and cytokinins for the growth of cell cultures (Murashige and Skoog, 1962) and for re-entry into the division cycle of quiescent cells has been known for many years (Bayliss, 1985). Studies on tobacco pith explants showed that auxin increased the amount of CDK protein. However, this protein did not have kinase activity unless cytokinin was also present (John et al., 1993). It was proposed that CDKs are induced by auxin (Miao et al., 1993; Murray et al, 1998; reviewed by Mironov et al., 1999), whereas CycD3 represents the cytokinin-limited component required for the G 1 /S transition of the cell cycle (Murray et al., 1998). Other cytokinin targets are likely to be important in other species or cell cycle controls (John et al., 1993). Recent studies on cell suspension cultures of Arabidopsis showed the induction of expression levels of CycD3 by cytokinin (Riou-Khamlichi et al., 1999) and CycD2 and CycD4 by sucrose (De Veylder et al., 1999; Riou-Khamlichi et al., submitted). Here we present a model on the control of G 1 /S transition in plants, incorporating the recent discoveries of inducibility of CycD cyclins by sucrose and cytokinin (Figure 2). Various signals, both stimulatory and inhibitory, are incorporated into control of a START-like point in plant cells (Murray et al., 1994; Murray, 1998) and modulate the activity of CDKs. The strong parallels in controlling G 1 /S transition in plants and mammals suggests that common themes will be found. Future work will be exciting as the involvement of CycD cyclins in plant-specific aspects of both proliferation and differentiation is uncovered. We expect that studies on the cell cycle regulators will incorporate studies on meristematic controls, organ identity and developmental processes. This combined effort will result in the uncovering of the features that are unique to plant growth and development. Acknowledgements We thank Thomas P. Jack, Masami Sekine and Anders Kvarnheden for sharing sequences in advance of publication. References Ach, R.A., Durfee T., Miller, A.B., Taranto, P., Hanley-Bowdoin, L., Zambryski, P.C. and Gruissem. W. 1997a. RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein. Mol. Cell. Biol. 17: Ach, R.A., Taranto, P. and Gruissem, W. 1997b. A conserved family of WD-40 proteins bind to the retinoblastoma protein in both plants and animals. Plant Cell 9: Ajchenbaum, F., Ando, K., DeCaprio, J.A. and Griffin, J.D Independent regulation of human D-type cyclin gene expression during G 1 phase in primary human T-lymphocytes. J. Biol. Chem. 268: Ando, K., Ajchenbaum-Cymbalista, F. and Griffin, J.D Regulation of G 1 /S transition by cyclins D2 and D3 in hematopoietic cells. Proc. Natl. Acad. Sci. USA 90: [86]

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