Journal of Experimental Botany, Vol. 57, No. 1, pp. 25–32, 2006 doi:10.1093/jxb/eri268 Advance Access publication 29 November, 2005

FOCUS PAPER

The co-ordination of cell division, differentiation and morphogenesis in the shoot apical meristem: a perspective Andrew J. Fleming* Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK Received 13 May 2005; Accepted 14 July 2005

Abstract Whether morphogenesis is cell division-driven or organismal-based has been a long-running debate in plant biology. This article is a summary of a series of experiments aimed at distinguishing these alternate views by local manipulation of parameters of cell division frequency, orientation, and growth within the shoot apical meristem. These data, put in the context of other investigations in this area, support an organismal view of plant morphogenesis and support the idea that the cell wall plays a key role in the mechanism by which this is achieved. At the same time, the data indicate that the intimate but variable relationship between cell growth and division within the organism means that cell proliferation can indirectly influence this process, leading to a context-dependent influence on morphogenesis. Finally, cell growth and proliferation are intimately related with the process of differentiation as cells exit the meristem. In the final part of the article the molecular mechanism by which these basic cellular parameters are intertwined is discussed. Key words: Cell division, cell wall, leaf, meristem, microinduction, morphogenesis.

Introduction The first description of the cellular nature of biological material was made over 300 years ago by Robert Hooke. Although the concept that plant material consisted of discrete compartments became rapidly accepted, it was not until the middle of the 19th century that it became clear that animals too were multicellular in nature. Since then the cellular basis of life has become a central dogma which underpins our understanding of biology. It is, therefore, perhaps surprising that there are still some basic questions

about cells which remain open to debate. In particular, it remains very unclear how cell size and number are integrated into the whole organism and what precise role cells play in driving growth and morphogenesis (Day and Lawrence, 2000; Nijhout, 2003; Rudra and Warner, 2004). In plants, new cells can be generated essentially throughout the organism but specific organs (the meristems) are sites of high rates of cell proliferation. More importantly, the cells of the meristem are pluripotent so that their progeny can become committed to a spectrum of developmental fates. With respect to the shoot apical meristem (which will be the focus of this article) there is the added complication that cells derived from the meristem do not simply undergo particular patterns of differentiation, they may also become incorporated into new structures, the leaves. Thus, the process of differentiation must be coordinated with the process of morphogenesis if a fully functional organism is to be produced. The relationship between cell proliferation and differentiation, and the connection between cell proliferation and morphogenesis, will be discussed in the remainder of this article.

Cell proliferation and morphogenesis: is there a causal link? From simply looking at a section through an apical meristem, the mechanism involved in generating a new leaf primordium seems obvious (Fig. 1A). Surely cell proliferation simply increases at the presumptive site of leaf formation and this leads to the formation of a new organ? From this simple viewpoint cell division is driving morphogenesis. However, although cell proliferation is certainly associated with leaf formation (Laufs et al., 1998), a number of data indicate that things are not that simple. For example, transgenic plants in which cell division rate has been altered generally do not display massive changes in

* Fax: +44 (0)114 222 0002. E-mail: a.fleming@sheffield.ac.uk ª The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected]

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Fig. 1. (A) Longitudinal section through the meristem of a tobacco plant. The meristem consists of an orderly array of cells. (B) Hand section through the shoot apical meristem of a Tet::GUS plant which has been micro-induced. This leads to local induction of gene expression, as visualized by blue signal. (C) Section (7 lm) through an apex micro-induced as in (B). Blue signal is restricted to one flank of the meristem and encompasses several cell layers. (D) Diagram of the shoot apex to demonstrate the micro-induction experimental strategy. The meristem (m) generates leaf primordia (P2, P1) in a sequence so that the position and timing of the next incipient primordium (I1) can be predicted. Micro-inductions (red dots) were performed at the opposite side of the meristem (I2) where leaf formation/morphogenesis is not expected until after that at I1. (E) Section (7 lm) through the meristem of a Tet::CycA3;2 plant in which Nicta;CycA3;2 gene expression has been induced on one flank of the meristem (red dot). An accumulation of smaller cells is observed on this flank, but no morphogenesis. (F) Scanning electron micrograph of the apex of a Tet::Expansin plant in which expansin gene expression has been induced at the I2 position (red dot) between primordia P2 and P1. Morphogenesis has occurred to form a bulge at this position. (G) Section (7 lm) through the apex of a wild-type tobacco plant which has been hybridized with an anti-sense probe for NTH15, encoding a KNOX1like protein. Signal (purple) is seen throughout the meristem (including the I2 position) but is excluded from the I1 position. (H) Longitudinal section through the apex of a Tet::phragmoplastin tobacco plant which has hybridized with an anti-sense probe for NTH15. Micro-induction was previously performed at the I2 position of the intact meristem. Signal (purple) is absent from the meristem at both the I2 and I1 positions. (I) Leaf of a Tet::CyclinA3;2 plant in which the primordium was micro-induced on one flank (red dot). Growth of the lamina is inhibited at this position in the leaf. (J) Model to interpret the phenotype observed in (I). Cells on the flank of the leaf primordium (left-hand schematic) normally undergo a switch from meristematic (red cells) to expansion growth (blue cells), as shown in the middle schematic. Micro-induction of cyclin gene expression (red dots in righthand schematic) leads to some cells retaining meristematic growth, while adjacent cells undergo the appropriate switch to expansion growth. Differential growth is produced in the tissue, leading to morphogenesis. (K) Hand-section through the apex of a tomato plant transgenic for an RBCS Pr::GUS construct. GUS activity (yellow) has been visualized with a fluorescent substrate. Signal is absent from the meristem dome (m) but appears as the leaf primordium is formed. (L) Section through the apex of a tobacco plant transgenic for an RBCS Pr::GUS construct. Gus signal has been developed using a histogenic substrate and visualized using dark-field optics so that signal appears red. Signal is absent from the meristem (m) but is visible in the adjacent stem and leaf tissue. Parts (A), (G), and (H) are adapted from Wyrzykowska and Fleming (2003); parts (B), (C), and (F) from Pien et al. (2001); parts (E) and (I) from Wyrzykowska et al. (2002); part (K) from Fleming et al. (1996) (copyright 1996 Blackwell Publishing Ltd and the Society for Experimental Biology); part (L) is Fig. 5B from Mandel T, Fleming AJ, Krahenbunl R, Kuhlemeier C. 1995. Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model. Plant Molecular Biology 29, 995–1004, and is reprinted with kind permission of Springer Science and Business Media.

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plant form, i.e. the basic elements of plant structure are elaborated (Hemerly et al., 1995; Cockcroft et al., 2000; Wang et al., 2000; De Veylder et al., 2001), although in some cases the total size of the plant may be reduced (De Veylder et al., 2002). Similarly, mutants in which the cell division pattern is disrupted still form functional plants (Traas et al., 1995; Smith et al., 1996). Such observations can be interpreted as supporting an organismal theory of plant development in which some endogenous (unspecified) mechanism sets the size and shape of an organ or organism independent of cell division processes (Kaplan, 2001). In this interpretation, the observed cell divisions that accompany morphogenesis are simply a means by which the increased volume accompanying morphogenesis is chopped up into usable units (cells) which allow compartmentation and specialization of function within the plant body. As to the nature of this unspecified size- and shapesetting mechanism, although the molecular details are obscure, many workers in this field would incorporate an emphasis on the cell wall. Plant growth and morphogenesis (which is essentially differential growth) are dependent on physical characteristics of the cell wall, so any size- or shape-setting mechanism which is cell division independent is likely to include control of cell-wall parameters (Green, 1999; Cosgrove, 2000; Fleming, 2002). Efforts to reconcile these different views on the mechanism of plant morphogenesis have been discussed in a number of articles (Meyerowitz, 1996; Doonan, 2000; Fleming, 2002; Tsukaya, 2003). To investigate these two distinct interpretations of the role of cell division in morphogenesis, a series of experiments was designed to alter parameters of cell proliferation and growth (as controlled by cell-wall parameters) in local regions of the shoot apical meristem. The question posed was: Is there a difference in the efficacy of these parameters in influencing plant morphogenesis? Although the use of specific promoter elements to direct gene expression to specific regions of the meristem is a viable approach to this problem (Deveaux et al., 2003), at the time the experiments were started the availability of such promoter elements was very limited. Even now, the regions of the meristem that can be precisely targeted by such methods remain restricted. Therefore a novel technique was developed to manipulate gene expression in the apical meristem. The approach (which was termed micro-induction) was inspired by work from the Tickle laboratory working on chick limb development (Cohn et al., 1995). This group had shown by the insertion of FGF (fibroblast growth factor)-loaded beads into the flanks of developing embryos that a localized source of this growth factor was sufficient to induce limb formation. At the time, a number of chemically inducible promoters had been described in plants and it was reasoned that if one could manipulate a local source of inducer on to the surface of a meristem then a local gradient of inducer (and, thus, of transgene expression) would follow.

This approach involved the dissection and exposure of the shoot apical meristem, precluding the use of the model plant Arabidopsis on technical grounds. Therefore tobacco was chosen as a compromise system in which dissection of the apical meristem was feasible and in which a reliable tetracycline-inducible promoter system was established (Gatz et al., 1992). Experiments using a GUS reporter gene construct under tetracycline-inducible transcriptional regulation (Tet::GUS) showed that the technique worked and that, depending on the inducer concentration and size of lanolin bead used, it was possible to induce transgene expression within a fraction of an apical meristem (Fig. 1B, C; Pien et al., 2001). The fact that this approach led to the induction of gene expression in a reasonable fraction of the apical meristem was important, since the view was taken that altered gene expression in only a single cell or a few cells would be unlikely to alter the developmental cellular and growth dynamics of the meristem as a whole. Having established the technique, the question remained as to which gene products to manipulate. With respect to cell proliferation there was already a significant body of data (reviewed in Potuschak and Doerner, 2001; Inze, 2005). The plant cell cycle is, in essence, very similar to that described for other eukaryotes, depending as it does on a controlled sequence of phosphorylation and dephosphorylation events to drive the various phases of the cycle. Importantly, it had been shown that overexpression of specific regulators of the cell cycle could (in a given context) promote cells to divide faster or over a longer time period than they normally would. For example, Shen’s group in Strasbourg had shown that misexpression of a cyclin (Nicta;CycA3;2) gene in tobacco led (at the whole plant level) to a phenotype consistent with the encoded protein promoting cell proliferation (Yu et al., 2003). In addition, Francis and colleagues in the UK had created a series of transgenic tobacco plants in which a yeast cdc25 was under inducible transcriptional regulation and had shown that in the root the elevated expression of this transgene promoted cell proliferation (McKibbin et al., 1998). Since these two genes were relatively well characterized, had been cloned into a plant system compatible with the micro-induction approach, and had been shown to display phenotypes consistent with a promotion of cell proliferation, efforts were focused on local induction of these genes within the apical meristem. An important part of this strategy was the attempt to promote cell division in cells that were already primed to enter the cell cycle, i.e. an attempt was being made to persuade cells to do something that they are already predisposed to do. Another important aspect of the experimental strategy was the fact that the apical meristem is a pattern-generating machine in which leaf primordia are produced at regular intervals of time and space. This enabled the inductions to be focused on the region of the meristem (I2) that, normally, would not form a new leaf primordium within the next 48–72 h (Fig. 1D).

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Therefore the question was posed whether promotion of cell proliferation in this region was sufficient to induce premature morphogenesis. The results for both Nicta;CycA3;2 and cdc25 were clear; cell proliferation was locally promoted after the manipulation but meristem growth was normal (Fig. 1E; Wyrzykowska et al., 2002). These results support the hypothesis that cell proliferation on its own is not sufficient to drive morphogenesis. Since cell division did not seem to influence morphogenesis in the meristem, what about parameters affecting tissue growth independent of any direct influence on cell proliferation? To address this question a similar experimental approach to that described above, but using molecular tools to modify cell wall extensibility, was undertaken, focusing in particular on a family of cell-wall proteins termed expansins. Cell wall extensibility and morphogenesis: is there a causal link? Although key details on their mechanism of action are still obscure, expansins have the ability when supplied exogenously to increase cell-wall extensibility (McQueen-Mason et al., 1992). A large number of expansin genes have been characterized and two general aspects have emerged (reviewed in Cosgrove, 2000; Lee et al., 2001). Firstly, in the in vitro assays used to determine expansin activity, different expansin proteins from the same species seem to have similar activity profiles. At the same time, different expansin genes often show very distinct expression patterns. Although not proven, these generalizations suggest that a key to expansin function is when and where the genes are expressed rather than any particular functional differences between the proteins. Thus, targeting ectopic foreign expansin gene expression to the apical meristem seemed a reasonable strategy to influence tissue growth in this organ. A key aspect to this strategy was that cells in the meristem are poised to undergo massive expansion. It was reasoned that these cells might be primed to respond to an increase in expansin gene expression by an increase in growth, i.e. again the cells would be made to do what they were predisposed to do. Having created and characterized a series of transgenic plants in which expansin gene expression was inducible (Tet::Expansin plants), local inductions were performed at the I2 position of the apical meristem. This manipulation was sufficient to induce morphogenesis (Fig. 1F; Pien et al., 2001). Moreover, the bulges induced grew out to form phenotypically normal leaves. These data support the organismal view of morphogenesis, i.e. that local altered growth characteristics are likely to be the key to initiating morphogenesis, with cell division in the meristem being coupled to growth so that the increased volume generated is split into appropriate compartments or cells. The observed cell division is not driving growth. The nature of

this coupling is an aspect of the research currently being undertaken that will be discussed later in this article. Although the strategy described above did not assume that expansins represented an endogenous part of the programme of leaf initiation (i.e. interest was mainly in using expansin as a tool), expression analysis has revealed that specific expansin genes are expressed at the appropriate time and place to play a role in leaf initiation (Cho and Kende, 1998; Reinhardt et al., 1998). However, due to the size and complexity of the expansin gene family in model organisms such as Arabidopsis, molecular genetic data showing that expansin gene expression is essential for the normal process of leaf initiation are still lacking. The role of cell proliferation in morphogenesis is context dependent Do the data described above mean that cell division is not required for morphogenesis? The answer is no. There is a tight (but variable) relationship between cell growth and division. If division is blocked then growth will eventually be limited and, since, in plants, growth and morphogenesis are intimately linked, there will be a point at which morphogenesis will not occur. Moreover, in addition to this basic requirement for cell proliferation if multicellularbased morphogenesis is to occur, the tight but variable relationship between cell growth and proliferation can lead to rather complex and, indeed, non-intuitive linkages between cell proliferation and morphogenesis. An example of this was revealed by a series of experiments that were performed using inducible expression of cell cycle-related genes in young leaf primordia. As described, the apical meristem generates new leaf primordia. The question was therefore posed whether this tissue (derived from and closely associated with the apical meristem) showed a similar response to local manipulation of cell proliferation and growth. With respect to induced expansin gene expression the phenotype observed was comparable with that seen in the meristem, i.e. local expansin expression led to local increased growth coordinated with appropriate cell proliferation (although in this context the induced growth had appropriate leaf identity rather than the switching from meristem to leafidentity, as observed in the meristem; Pien et al., 2001). The outcome of local promotion of cell proliferation in leaf primordia was dramatic and counter-intuitive. During the initial phase of induction an accumulation of cells at the site of induction was observed. However, as the leaf grew, the growth rate in the induced region was slower than in the adjoining tissue, leading to an overall lack of lamina growth in the area that had originally been induced to undergo cell proliferation (Fig. 1I; Wyrzykowska et al., 2002). The mechanism involved in this response is open to discussion, but one possibility is that plant cells that are undergoing proliferation have a lower overall rate of growth

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than cells which are exiting the cell cycle to undergo purely expansion growth. These two distinct phases of plant cell development (proliferative- and non-proliferativeassociated growth) have been termed meristematic growth and expansion growth (Chen et al., 2001). Recent observations on suspension culture cells support the hypothesis that these two distinct phases of cell development do indeed display different growth rates (Campanomi and Nick, 2005). Interpreted in the light of these data, it is speculated that the cells on the induced flank of the primordium were maintained in a proliferative growth phase, while adjacent cells were entering a phase of expansion growth (Fig. 1J). This would lead to a differential growth rate and, thus, morphogenesis. Although the induced tissue might later regain an appropriate growth rate as the cells underwent a transition to expansion-type growth, due to the fixation of plant cells relative to one another via the cell wall, the relative positions of the tissues would become more or less fixed in space (Fig. 1J). Irrespective of whether this conjecture is correct, the data demonstrate that the influence of cell proliferation on morphogenesis is highly context dependent. One cannot make simple predictions on what the outcome of altered cell proliferation will be on morphogenesis, a view that the literature in this area supports (Cockcroft et al., 2000; Autran et al., 2002; Dewitte et al., 2003; reviewed in Doonan, 2000; Tsukaya, 2003). Cell division pattern and morphogenesis: is there a causal link? In addition to the role of cell division rate on plant morphogenesis, another long-held view is that the orientation of cell division influences plant growth. Plant cytokinesis involves the formation of the phragmoplast within the dividing cell. This agglomeration of vesicles and cytoskeletal elements dictates the position of the new cell wall which separates the daughter cells following division. According to classical observations, orientation and position of the new cell wall dictates the relative size and shape of the daughter cells, which therefore dictates the future preferred orientation of growth. One can imagine that local control of the orientation of cell division within the apical meristem would control the direction of growth of the daughter cells which, thus, would control leaf primordium outgrowth. Indeed, histological observations reveal that one of the first markers of the presumptive site of leaf initiation is a switch in cell division orientation, although the correlation is not absolute (Lyndon, 1990; Selker et al., 1992). Since experiments simply increasing cell proliferation did not directly address this issue, a series of experiments was performed in which the dynamin-like protein phragmoplastin was used as a tool to locally disrupt the cell division pattern in the meristem. Previous work from the Verma laboratory had shown that overexpression of phrag-

moplastin disrupted the cell division pattern in BY2 cells and led to seedling lethality (Gu and Verma, 1997; GeislerLee et al., 2002). Overexpression of phragmoplastin seems to lead to an overloading of the vesicle transport system to the forming phragmoplast, preventing appropriate orientation. Using the micro-induction system, local overexpression of phragmoplastin disrupted the pattern of cell division in the outer tunica layers of the meristem but no overt outcome on leaf initiation or meristem function was observed (Wyrzykowska and Fleming, 2003). These observations again support an organismal view of the meristem in which altered cell division per se is not sufficient to drive morphogenesis. Gene expression influences cell division and cell division influences gene expression An interesting aspect of these experiments was revealed by an analysis of marker gene expression in the induced meristems. Research from a number of experimental systems has led to the identification of genes whose expression pattern defines particular regions of the meristem and which, moreover, encode proteins which play vital roles in various aspects of meristem function (reviewed in Byrne, 2005). For example, KNOX genes (such as NTH15 in tobacco) play an essential role in the maintenance of meristem indeterminancy and are expressed in a specific pattern so that transcripts within the meristem are excluded from the presumptive site of leaf formation (Fig. 1G; Sakamoto et al., 2001; Wyrzykowska and Fleming, 2003). The expression pattern of KNOX genes is intimately linked with that of ARP transcription factors (such as NtPHAN) whose transcripts accumulate within the developing primordium in a pattern complementary to that shown by KNOX genes. Analysis of the expression patterns of these marker genes revealed that, subsequent to local induction of phragmoplastin, the patterns of NTH15 and NtPHAN were disrupted, although no obvious effect on meristem function was observed (Fig. 1H; Wyrzykowska and Fleming, 2003). One cautionary point here is that there is a significant body of data indicating that the proteins encoded by many genes within the meristem (including KNOX genes) are mobile so that the pattern of transcript does not necessarily indicate the pattern of protein accumulation (Perbal et al., 1996; Sessions et al., 2000; Kim et al., 2002; Wu et al., 2003). Nevertheless, the data suggest that the pattern of cell division within the meristem can influence the pattern of transcript accumulation of at least some genes. The mechanism is unclear, but hypotheses invoking plasmodesmatal-gating of transcript and protein movement within the meristem have been proposed (Haywood et al., 2002), and an altered pattern of cell division would be predicted to influence such symplastic pathways. Thus, in addition to the canonical pathway of gene expression influencing cell division, there is the

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possibility of a feedback mechanism by which an altered cell division pattern can influence gene expression. From meristem to non-meristem: the control of differentiation The experiments described above were focused on unravelling the interaction of cell proliferation, growth, and morphogenesis. As noted at the beginning of this article, these parameters must also be integrated with the process of differentiation if a fully functional organism is to be formed. The micro-induction approach has also been used to investigate this aspect of development, again focusing on the shoot apical meristem. Cells in the meristem are characterized by a number of parameters. Although a relatively high rate of proliferation is one of them, this is in no way exclusive to the meristem. Cells outside the meristem in the young leaf and stem also display relatively high rates of division, although this tends gradually to decrease as the organs mature. By contrast, one of the most dramatic shifts in cellular function between the meristem and non-meristem cell state is the acquistion of photosynthetic machinery. Meristem cells contain proplastids which lack the organized thylakoid structure of functional chloroplasts. Moreover, the cells do not contain chlorophyll or express the array of gene products required for photosynthesis. However, within a very short time of being displaced from the meristem, cells gain the capacity to be photosynthetic, as shown, for example, by the expression of genes encoding RBCS (small subunit, ribulose bisphosphate carboxylase) (Fig. 1K, L; Fleming et al., 1996). Simple exposure of meristems to light is insufficient to trigger this switch, indicating that it is an endogenous programmed event and not simply a result of enclosure of the meristem within the surrounding primordia to create a darkened environment. At the same time as gaining photosynthetic capacity, the cells that are displaced from the meristem enlarge via vacuolation. Simultaneously the rate of cell proliferation decreases. Thus, there is a correlation of decreased proliferation, acquisition of a specific differentiation pathway, and an increase in cell size via vacuolation. The molecular mechanism that controls these decisive events is obscure. For example, modulation of cell cycle genes influences the rate of growth but meristem function is maintained (Hemerly et al., 1995; Cockcroft et al., 2000; Wang et al., 2000; De Veylder et al., 2001, 2002). One potential candidate that might influence this switch is the retinoblastoma family of proteins. Retinoblastoma protein (pRb) was first identified in mammalian cells as a tumour suppressor. Since then it has been characterized as a key player in the cell cycle (Weinberg, 1995; Harbour and Dean, 2000). Specifically, the paradigm has emerged that pRb acts as an intermediary between cyclin/CDK complexes which promote entry into the cell cycle and E2F/DP transcription factors which

promote the expression of genes associated with, and required for, entry into the S-phase of the cell cycle. In its hypo-phosphorylated form pRb binds to E2F/DP factors and inhibits transcription of target genes required for entry into the cell cycle. Phosphorylation of pRb by cyclin/CDK complexes alleviates this restriction and allows the E2F/DP transcription factors to initiate entry to the cell cycle. Although the main focus of research has been on the role of pRb in cell proliferation, it has become clear that pRb can also influence differentiation, although to what extent this is an indirect effect of altered cell proliferation and how much a direct influence of pRb has been somewhat obscure. Recent genomic approaches studying the outcome of altered pRb expression have revealed a number of potential transcriptional responses to altered pRb expression which are not overtly linked with the cell cycle, favouring the view that pRb may also have a reasonably direct influence on a spectrum of gene expression events linked with differentiation (Dimova et al., 2003). pRb has, therefore, emerged as a potential pivot in the cellular decision to proliferate or enter a pathway of differentiation. With respect to plants, the situation is unclear. Although plants contain genes encoding retinoblastoma-related proteins (RBRs) their functional analysis has been limited (Xie et al., 1996; Durfee, 2000). RBRs can certainly interact with cyclin/CDKs (Huntley et al., 1998; Nakagami et al., 2002), and experiments in which a truncated RBR cDNA was shot ballistically into tissue culture cells inhibited cell division (Gordon-Kamm et al., 2002). At the level of the whole plant, RBR is an essential protein. Deletion of the single copy RBR gene in Arabidopsis is gametophytic lethal, leading to the formation of multi-nucleate and infertile eggs (Ebel et al., 2004). This lethality precludes simple analysis of the knock-out phenotype in the mature vegetative plants. One approach to tackle this problem has been to use a viralsuppression strategy to down-regulate endogenous RBR levels in developing leaves of tobacco (Park et al., 2005). This strategy revealed that a decreased RBR transcript level led to an inhibition of organ growth and the disruption of epidermal cell characters, consistent with the paradigm of RBR being required to suppress cell proliferation. The function of RBR has also been investigated in the intact plant using the micro-induction technique. This allowed the focus to be on the response of the apical meristem to manipulation of RBR protein levels. After transient induction of RBR gene expression it was observed that meristem cells underwent at least the initial stages of differentiation, including cell expansion and expression of marker genes for photosynthesis (J Wyrzykowska, M Schorderet, S Pien et al., unpublished results). This outcome is very different from that observed when the expression of other cell cycle genes (e.g. Nicta;CycA3;2) is altered in the meristem, suggesting that RBR is affecting other elements than simple cell proliferation and, moreover, that the observed outcome on differentiation is not

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simply related to a decrease in cell proliferation. These data therefore support the idea that RBR can act as a pivot in the cellular decision to divide or differentiate. Conclusion Cells are the basic unit of plants, yet fundamental questions still remain as to how cell proliferation is integrated into the whole organism and how this process is itself related to basic aspects of differentiation. One approach has been to target a very specific and essential organ for plant development (the shoot apical meristem) to observe how the system responds to specific, transient perturbations of parameters of cell division frequency, orientation, and growth. Using gene products as tools, it has been possible to decipher some ground rules of how the meristem (and the leaf primordia derived from it) function as multicellular organs. The challenge is now to identify the endogenous processes/genes controlling the cellular and tissue mechanisms by which morphogenesis occurs, to understand how these end-processes are integrated with the transcriptional networks controlling them, and how these end-processes feed back to these same networks.

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