Triggering the cell cycle in plants

The development of plants, compared with that of animals, is influenced much more by the environment in which they grow, suggesting that plants have evolved mechanisms that relay environmental signals to control cell division and ultimately plant growth. Most of the divisional activity in plants is localized in small groups of cells, called meristems (see Box 1), that are already present in the embryo and are active during most of the life cycle of the plant. How environmental cues trigger changes in cell-division activity in meristems that is, how the plant connects environmental changes with molecular changes in the machinery controlling the cell cycle is largely unknown. The cell cycle consists of the alternating phases of DNA replication (S phase) and chromosome separation (mitosis, or M phase) interrupted by gaps known as (interval between M and S phases) and G2 (interval between S and M phases). Important controls operate at the transition points as cells move from into S phase, and from G2 into M phase, primarily through the regulated kinase activity of cyclin-dependent kinases (CDKs). Both the S and G2 M phase transitions can be controlled in plant cells in response to changing conditions. For example, during the early stages of root nodule initiation in pea and alfalfa, the root cortical cells susceptible to Rhizobium infection are in G0/ (Ref. 1). By contrast, before germination of seeds, the cells of the embryo are arrested in, or partly in and partly in G2, depending on the species 2. An example of G2 control is found in radish lateral root primordia, which are derived from pericycle cells arrested in G2. These cells move into M phase upon auxin stimulation 3 and then continue to proliferate, producing a lateral root primordium that eventually emerges from the side of the primary root. In addition to these specific developmental controls, the cell cycle in plants plays an important role in growth responses to the environment. If the grass Dactylis is exposed to increased levels of CO 2, cells in the shoot meristem increase their rate of proliferation, resulting in faster growth. This occurs partly through an increase in the number of cells actively involved in division, and partly through a shortening of the cell cycle, particularly the phase 4. Interestingly, both the shoot and root apical meristems are a mosaic of fast and slow cycling cells, and the main difference between these two populations is in the length of (Refs 5 and 6), suggesting that this phase is the most responsive to signals that change cell-cycle length. The significance of controls in commitment to the cell cycle has been shown in yeast, flies and mammals 7, and, as excellent reviews covering the complete plant cell cycle have appeared recently 8 10, we will limit ourselves here to a discussion of the S transition in plants and how this is controlled. At the end of the review, we turn to the intriguing question of how cell division patterns are coordinated within the meristem. The cyclin D retinoblastoma E2F pathway Eukaryotic cells in phase have several options. The most obvious is that, in the presence of sufficient Triggering the cell cycle in plants Bart G. W. den Boer and James A. H. Murray In essence, the mitotic cell cycle in eukaryotes involves the duplication and separation of chromosomes, coupled to the process of dividing one cell into two. Cytokinesis is therefore the culmination of a series of events that were triggered during phase, and brings the daughter cells back to the starting position in for another possible round of division. In all eukaryotes, progression through the cell cycle is controlled by cyclindependent kinases that bind to positive regulators called cyclins. This review explores some of the pathways that trigger the plant cell cycle, with emphasis on the phase. Examples include signalling pathways involving glutathione and cellular redox potential, the possible existence of a DNA-damage checkpoint, and the plant hormones auxin and cytokinin. rogress in understanding the link between cell proliferation, cell differentiation and the cell-cycle machinery in a developmental context is discussed. stimuli, they commit to further cell division and progress into S phase with the initiation of DNA synthesis. However, there are several other cell fates, including differentiation, programmed cell death and the adoption of a quiescent state (G0) (Fig. 1). We will first summarize the molecular events associated with the first option, the S transition, and emphasize the important parallels found between mammals and plants. In mammalian cells, the retinoblastoma protein (Rb) and its relatives, p107/p130, are important in preventing cells from progressing into S phase, by binding members of the E2F family of transcription factors that are present on promoters driving S-phase specific genes. Recently, it has been recognized that the active recruitment of histone deacetylase by prb is important in keeping E2Fresponsive genes switched off during, by creating an inactive chromatin structure 11. It is only when Rb itself gets inactivated by phosphorylation in late that genes under E2F control are relieved from repression and their subsequent expression can provide the activities needed for S-phase entry 12. hosphorylation of Rb in is a two-step process involving the sequential action of cyclin D CDK Bart den Boer is at Aventis CropScience N.V., Jozef lateaustraat 22, B-9000, Gent, Belgium; and James Murray is at the Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT. E-mails: bart.denboer@ aventis.com; j.murray@ biotech.cam.ac.uk trends in CELL BIOLOGY (Vol. 10) June 2000 0962-8924/00/$ see front matter 2000 Elsevier Science Ltd. All rights reserved. 245 II: S0962-8924(00)01765-7

G0 rogrammed cell death CDK4 and CDK6 (the CDK partners of D-type cyclins), whereas members of the CI/KI family act more broadly by inhibiting the kinase activity associated with cyclin D-, E- and A-dependent kinases 15. stem cell Differentiate De-differentiate FIGURE 1 Options for cells in plants. Newborn cells can start another round of division ( stem cell ) or exit the cycle (non-cycling cells). These cells die (programmed cell death), return into the cell cycle or differentiate. In contrast to animals, differentiated plant cells can more readily de-differentiate and re-enter the cell cycle, given the appropriate signals. ABA Cytokinin Sucrose and cyclin E CDK complexes 13. It is initiated following stimulation by mitogens, which induce and maintain D-type cyclin expression. This is followed by a wave of cyclin E expression in late (Ref. 14). The activities of these cyclin CDK complexes is in turn constrained by CDK inhibitor proteins (CKIs). In mammals, these proteins fall into two families based on their structure and CDK targets. The INK4 family specifically inhibits the catalytic subunits of ICK1 cycd3 cycd2 CDK-a cycd CDK-a Auxin? CAK ICK1 cycd CDK-a START E2F Rb E2F Rb E2F inactive E2F active /S transition S lants In plants, homologues of most of the key players in the Rb pathway (Fig. 2) have been identified, and most show structural and functional similarities to their animal counterparts. As yeasts do not contain direct homologues of proteins involved in the Rb pathway, the backbone of this pathway appears to be conserved between animals and plants, but not fungi 16,17. There are three main classes of plant D-type cyclin (CycD) genes 18, but these are not related to the individual groups of mammalian D-type cyclins, indicating that the increase in gene number in the cyclin-d family might have occurred independently in animals and plants. As in animal cells, the transcription of some types of CycD is inducible during cell-cycle entry by mitogens, but generally stays relatively constant once cells are involved in continuous proliferation 19. Like animals, plants contain several types of CDKlike genes, including direct homologues of fission yeast cdc2 +, which contains a consensus amino acid sequence STAIRE in its cyclin-binding region. Direct homologues of cdc2 are called CDK1 or cdc2 in mammals, and cdc2a or CDK-a in plants. lants have no direct equivalents of other mammalian CDKs, but have plant-specific CDK variants with the consensus sequence TALRE (CDK-b1) or TTLRE (CDK-b2). These are unique among CDKs in showing cell-cycle regulation of expression, being transcribed only from S until M phase 9. Differences are seen in the expression timing of the TALRE and TTLRE subgroups 10. In mammalian cells, the archetypal S-phase genes S-phase genes FIGURE 2 Model for S transition in plants. Cytokinin- and sucrose-induced D-type cyclins bind to cyclindependent kinase-a (CDK-a) to form inactive heterodimers. Regulation of kinase activity after binding the cyclin might occur either by an inhibitor (ICK1) or by phosphorylation by an activating kinase (CAK). hosphorylation of the retinoblastoma protein Rb by CDK-a complexes releases the transcription factor E2F, which is the active molecule required to enter S phase. The phosphorylation of plant CDK-a by CAK and the presence of Rb E2F complexes on the promoters of S-phase genes have not been shown to occur in plants but are based on the mammalian S model. STAIRE-containing CDK1 is involved mainly in the G2 M transition and alternative CDKs control the phase (CDK4, CDK6) and the S transition (CDK2). However, in plants, only CDK-a protein (the CDK1 equivalent) has been detected during the phase. CDK-a, however, is presumably not specific for the S transition as its levels are relatively constant during the cell cycle, and CDK-a-associated activity peaks at both the S and the G2 M boundaries. Its activity is supplemented by CDK-b kinase activity during G2 M. CycD cyclins are regulatory partners of plant CDK-a during the S phase transitions. What is the substrate for the cyclin D CDK activity? In mammals, Rb is the preferred substrate of kinases, and Rb-like homologues have been reported in maize 17 and tobacco 20. In tobacco, complexes of CDK-a with CycD3 can be detected in vivo, and, when expressed in insect cells, these can phosphorylate the tobacco Rb-related protein in vitro 20. 246 trends in CELL BIOLOGY (Vol. 10) June 2000

Having established that the upstream components of the cyclin D Rb pathway are conserved in plants and animals, the next obvious question is whether conservation continues downstream: in other words, are there E2F transcription factors in plants? Until recently, the only evidence was indirect, as it was shown that maize Rb1 can bind to human and Drosophila E2F and inhibits the transcriptional activation ability of human E2F 16. This changed with the identification and analysis of wheat and tobacco clones 21,22. Wheat and tobacco E2F were shown to interact respectively with maize Rb1 and tobacco Rb in yeast two-hybrid assays. For wheat E2F, the interaction was also confirmed in vitro and the Rb-binding motif was mapped to the C-terminal end. CDK activity requires activating phosphorylation by CDK-activating kinase (CAK) 23, but there is little evidence that this regulates cell-cycle progression. However, inhibitory phosphorylation by wee1-like kinases is likely to play a significant regulatory role 24. In addition, four CDK inhibitor (CKI) protein genes are reported in plants, but only one, ICK1, has been characterized biochemically 25,26. ICK1 interacts with both CDK-a (cdc2a) and cyclin CycD3 in vitro, is an inhibitor of plant cdc2-like kinases and its C-terminal consensus sequence resembles part of the CDK2-binding domain of the mammalian CKI p27 Kip1 (Ref. 25). Its activity throughout the cell cycle has not been investigated, nor is it clear whether overexpression in plant cells can inhibit progression. Interestingly, ICK1 expression is induced by the stress hormone abscisic acid, although it is not known whether this is a direct effect 25. Thus, although several players involved in the S transition in plants are structurally and functionally similar to their animal counterparts, differences are apparent. For example, a cyclin E homologue has not been identified in plants. This might not come as a surprise as cyclin E and the D-type cyclins bind to different CDKs (CDK2 and CDK4/6 respectively) in animals, and only one CDK activity has been reported at the S transition in plants 9. erhaps plant Rb could be phosphorylated by sequential cyclin D kinase activity as there are three main groups of plant CycD proteins with differential regulation and timing of expression (CycD1 3) 18. athways triggering the cell cycle in plants The rest of this review focuses on four examples of signalling pathways that feed into the plant cell cycle (Fig. 3). The DNA damage and glutathione pathways are not specific to plants, whereas the study of pathways relating to auxin cytokinin and meristem function are plant-specific solutions to the problems of integrating cell division with differentiation and development. DNA damage pathway DNA damage occurs in all living things and our understanding of the control of cell cycle by DNA damage has made enormous progress in yeasts and mammals 27. Mammalian cells are known to arrest in upon DNA damage. This block is induced by the DNA damage FIGURE 3 AR X Y rml1/cad2? GSH Auxin ABA ICK1 tumour suppressor protein p53 (Ref. 28) that activates transcription of p21 CI1, resulting in elevated levels of this CKI. This inhibits the activity of CDKs, leading to a arrest 29. The p53 protein can associate with poly(ad-ribose) polymerase (AR), another protein that senses DNA damage, and inhibition of AR activity leads to loss of p21 upregulation in response to DNA damage 30. AR is catalytically activated by DNA strand breaks, and is responsible for the poly(ad-ribosylation) of various nuclear proteins using NAD as substrate 31. Although there is evidence that major DNA repair mechanisms found in other species also occur in plants 32, it is not known whether a checkpoint control exists in plants. However, it is interesting to note that one of the Arabidopsis AR genes is transcriptionally activated by the genome instability resulting from a mutation in the DNA ligase I gene 33, suggesting that at least part of the DNA damage signalling pathway might be conserved. However, with over 85% of the Arabidopsis genome sequence already completed, no proteins homologous to p53 have been found. As p53 is also intimately involved in the control of apoptosis in animals 34,35, its possible absence in plants raises the possibility of different links between the cell cycle, DNA damage responses and programmed cell death 36. GSH-dependent control of the S transition Glutathione (GSH) is an abundant and ubiquitous thiol with proposed functions in the adaptation of plants to extreme temperatures, tolerance to xenobiotics and to biotic and abiotic environmental stresses 37. Until recently, a direct link between GSH Cytokinin cycd3/cdk-a S otential signalling pathways feeding into the S transition in plants. Genome instability transcriptionally activates poly(ad-ribose) polymerase (AR). In mammalian systems X = p53 and Y = p21, but their homologues have not been identified in plants. The rml1/cad2 gene encodes the first enzyme of glutathione (GSH) biosynthesis. When the intracellular GSH concentration falls below a threshold level, the S transition is blocked in dividing root cells. Depletion of auxin arrests cells in, and abscisic acid (ABA) induces the inhibitor ICK1 transcriptionally. ICK1 can interact with both cycd3 and CDK-a (cdc2a). Cytokinin activates cycd3 transcription, and constitutive cycd3 expression can rescue the cytokinin requirement of callus. trends in CELL BIOLOGY (Vol. 10) June 2000 247

(a) 1 2 0 levels and the cell-division cycle was lacking, but this changed when Vernoux and colleagues uncovered a role for intracellular GSH in the S transition in plants 38 during the cloning of the ROOTMERISTEM- LESS (RML) gene. RML encodes the first enzyme of GSH biosynthesis ( -glutamylcysteine synthetase). Moreover, depletion of intracellular GSH by addition of an inhibitor of GSH biosynthesis to Arabidopsis or tobacco seedlings or tobacco BY-2 cells abolishes cell division. Interestingly, the cell-division block in seedlings affects only the root meristem, and the shoot apical meristem is unaffected. Is there a physiological significance to the block of cell division caused when intracellular GSH concentration is reduced artificially? In other words, does the plant modulate its endogenous GSH levels during normal development to change the cell-division cycle? An indication that this could be the case is that, in Arabidopsis roots, high levels of GSH (measured by a reporter) are associated with proliferating cells such as epidermal and cortical initials, whereas reduced levels of GSH were found in the slowly cycling cells of the quiescent centre that have an extended (Ref. 39). It would be interesting to see whether an increase in GSH levels could stimulate FIGURE 4 Cell proliferation in the shoot apex. (a) Scanning electron micrograph of a tobacco shoot apical meristem (SAM). Leaf primordia are initiated on the flanks of the apical meristem. 0 is the youngest primordium, 1 the next oldest primordium, and 2 the oldest. Hatched line indicates the plane of section in (b). Bar, 100 m. (b) Cross-section through the SAM and flanking leaf primordia. Note the layered configuration of the dome (L1 and L2). roliferating cells in the peripheral zone are displaced towards the leaf primordia where eventually they will exit the cell cycle and differentiate. Black arrows indicate the direction of cell displacement. Bar, 100 m. BOX 1 GLOSSARY (b) rimordium A group of cells that will develop into a new organ. Meristem group of cells acting as a source of cells for all main plant organs. ericycle layer between the endodermis and the conducting tissue, from which lateral roots arise. Cortex bulk tissue in stem and root that lies between the epidermis and the central tissues. Quiescent centre small group of cells at the centre of the root meristem that rarely divides. L1 L2 Arabidopsis quiescent cells to enter the cell cycle, as was shown in maize for ascorbic acid, another molecule involved in the removal of active oxygen species 40. Thus intracellular redox homeostasis could affect cell-cycle progression by regulating key components of the S transition 37. It is not clear, however, how this would operate at the molecular level. Oxidative stress and GSH-dependent transduction pathways probably also impinge on the animal cell cycle as GSH depletion leads to an arrest of cell-cycle progression in human cells 41. A candidate S cell-cycle factor through which the GSH pathway might have its effect in mammals could be the inhibitor p21, as mammalian cells blocked in due to a low GSH level show high levels of p21 protein 41. Auxin and cytokinin action In higher plants, only two groups of hormones, the auxins and cytokinins, are generally stimulatory to the proliferation of most cell types 42. Many plant tissues, such as leaf, root or stem pieces, can be explanted into culture, where dedifferentiation and proliferation occur to form a callus of largely undifferentiated cells. This process normally occurs if both auxin and cytokinin are present. Conceptually, the link between auxin cytokinin action and cell division has been around for more than 40 years 43, but the progress in understanding the molecular basis of their action in cell proliferation has been very slow until recently. Auxin alone increases the level of a CDK protein in cultured tobacco cells and stem pith explants, but addition of a cytokinin was required for activation of this kinase 44. The -glucuronidase reporter gene under control of the Arabidopsis CDK-a (cdc2a) promoter is inducible by auxins and, to a lesser degree, by cytokinins 45. However, it is often difficult to separate the direct control of cell-cycle genes by hormones from their indirect induction as a consequence of the cell-cycle progress that the hormones provoke. rogress in analysing modes of action has also been hampered by the different types of interaction that both hormones show, depending on the plant species or tissue type 46. At what point of the cell cycle do auxins and cytokinins act? Somewhat confusingly, both hormones have been associated with progression through the S and the G2 M control points. Suspension cells arrest in both and G2 phases after they are transferred to medium containing cytokinin but lacking auxin 44,47. However, most progress has been made in linking cytokinin to the cell cycle. First, cells of certain suspension cultures arrest in G2 when deprived of cytokinin, and these cells have inactive CDK complexes. Addition of cytokinin or tyrosine dephosphorylation restores kinase activity 47, suggesting that cytokinin-depleted cells accumulate CDK cyclin complexes that are 248 trends in CELL BIOLOGY (Vol. 10) June 2000

inactive due to phosphorylation of the threonine 14 and/or tyrosine 15 regulatory residues of the CDK subunit. Second, in tobacco BY-2 cells, the cytokinin biosynthesis inhibitor lovastatin is able to block cells in G2, and this can be reversed by addition of exogenous cytokinin 48. However, proof that cytokinin operates through CDK dephosphorylation in vivo is still lacking. Evidence has emerged that cytokinin also regulates the S transition. Cytokinin application to shoot meristems reduces the size of chromosomal DNA replication units, resulting in the closer spacing of sites of DNA replication initiation. This leads to faster DNA synthesis, thereby providing a mechanism for the shortening of S phase that was observed 49. More recently, CycD3 has been shown to be induced by cytokinin in both cultured cells and intact plants 50, suggesting a role in the cytokinin control of cell division. If CycD3 is a primary target of cytokinin action in S control, constitutive expression of this cyclin should be able to bypass the requirement for cytokinin. In a key experiment, CycD3 was expressed in stable Arabidopsis transformants, and was found to remove the requirement for exogenous cytokinin during callus initiation and growth from leaf pieces 50. The timing of CycD3 induction occurs slightly before the expression of an S-phase marker gene histone H4 in partially synchronized Arabidopsis cells, pointing to its action during the phase 50. It is attractive to speculate that the increase in the number of DNA replication origins caused by cytokinin application might also be due to increased CycD3-associated kinase activity. The different steps in the signalling pathway from cytokinin to CycD3 are unknown but might involve a phosphorelay as used in bacterial twocomponent signalling 51 as CycD3 induction was found to be independent of protein synthesis 50. Coordination of cell division in intact plants Understanding plant growth and development requires an understanding of how plant cells grow and divide and how positional information is integrated with cell division. As meristems provide the cells that eventually give rise to the root, leaves, stem and flowers, it is essential to understand the molecular controls underlying cell division in the primary root meristems and shoot apical meristems 52. Here we take the divisional activity of cells present in the shoot apical meristem (SAM) as an example (Fig. 4a). SAMs of higher plants are not homogeneous, as there is a gradient of cell division and growth rate within the apex 53. At the summit (central zone) cells have long cell-cycle times, whereas displaced cells that end up at the flank in emerging primordia divide faster. This reduction in cell-cycle time is due mainly to a shortening of phase, although the G2 phase is reduced in some species 5. This picture of divisional activity is superimposed on the layered structure of the SAM. Cells in the two outermost layers (L1 and L2, Fig. 4b) divide primarily with cell walls normal to the surface of the meristem, so cells in these sheets rarely move from one layer to another 54. It is not known how the pattern of cell division activity, which seems to bear no resemblance to the layered structure of the apex, is coordinated in these different layers, particularly because there are cells with faster and slower cellcycle times in all meristem zones. A little later, when groups of cells develop into organs, individual cells exit the cell cycle and differentiate into specific cell types. During maize leaf development, the exit from occurs from tip to base, and is correlated with Rb expression and loss of celldivision activity 16. Somehow, the fate of cells that are in, and therefore could exit the cell cycle, is influenced by signalling molecules originating from neighbouring cells or cells even further away 55. Little is known about the connections between signalling pathways operating at cell-type level in organs and the celldivision cycle, although several mutations have been identified that disrupt patterned cell division operating in meristems, and some of the affected genes have been cloned. This should allow the identification of molecular pathways describing the connection between known signalling cascades and the cell-cycle machinery, that is, the activity of cyclin CDK complexes. One might have expected to identify cell-cycle components such as cyclins or CDKs by this route, but this was not the case. Most of the mutations are present in genes that encode homeodomain proteins, components of a common signal-transduction pathway or evolutionary conserved members of the piwi family 56,57. Such genes act upstream in cell division control, in the sense that they are probably involved both in the correct patterning of cell division within the meristem and in ensuring the continued proliferation of meristem cells. Recent work on the AINTEGUMENTA (ANT ) gene suggests that it might exert a more direct regulation on the proliferation of meristem cells as overexpression of ANT results in larger organs containing more cells without changes to the morphology of the final organ 58,59. Conversely, a loss-of-function ant mutation decreases floral meristem size and floral organ size by reducing cell number 59. Conclusions and prospects Yeasts, plants and mammals control progression through their cell cycles by using CDKs that bind to cyclins. During evolution, plants and mammals have evolved into multicellular organisms with many different cell types, a change that is marked by the appearance of the cyclin D Rb E2F pathway. erhaps this is important in allowing the differentiation of multiple cell types in complex tissues. lants and animals have elaborated their cell-cycle controls with kingdom-specific changes that allow indeterminate development in plants, characterized by the continuous production of new organs, on the one hand, and the determinate development of animals, on the other. As plants are sessile, environmental changes can have profound effects on their growth, which are likely to be reflected in the signalling pathways that connect to the cell cycle and possibly in the details of the cell-cycle machinery itself. The role of cytokinin is the first example where trends in CELL BIOLOGY (Vol. 10) June 2000 249

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