* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Leaf initiation: the integration of growth and cell division
Survey
Document related concepts
Endomembrane system wikipedia , lookup
Cell encapsulation wikipedia , lookup
Signal transduction wikipedia , lookup
Tissue engineering wikipedia , lookup
Extracellular matrix wikipedia , lookup
Programmed cell death wikipedia , lookup
Cell culture wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cell growth wikipedia , lookup
Cellular differentiation wikipedia , lookup
Transcript
Ó Springer 2006 Plant Molecular Biology (2006) 60:905–914 DOI 10.1007/s11103-005-7703-9 Leaf initiation: the integration of growth and cell division Andrew J. Fleming Department of Animal and Plant Sciences University of Sheffield, Western Bank, S10 2TN, Sheffield, UK (author for correspondence; e-mail a.fleming@sheffield.ac.uk) Received 26 January 2005; accepted in revised form 21 May 2005 Key words: cell division, cell growth, development, leaf, morphogenesis Abstract The shoot apical meristem of higher plants is characterized by a conserved pattern of cell division, the functional significance of which is unclear. Although a causal role for cell division frequency and orientation in morphogenesis has been suggested, supporting data are limited. An alternative interpretation laying stress on the control of growth vector and its integration with networks of transcription factors and hormonal signals is discussed in this review. Introduction The shoot apical meristem (SAM) is distinguished by its position within the plant, its ontogeny and its cellular organization. Histological observations in a number of plants show that the SAM is characterized by the following cellular pattern: an outer layer (or layers) of cells in which cell division is predominantly anticlinal (termed the tunica) and an inner body of tissue (corpus) in which cell division orientation is more random (i.e., not restricted to anticlinal) (reviewed in Steeves and Sussex, 1989). An example of this organization is shown for a tobacco SAM in Figure 1A, B. This commonality of form raises the questions: how does such an organization arise and what is its functional significance? In this review, I will discuss both these issues and attempt to integrate knowledge obtained from recent approaches manipulating parameters of cell division and growth into an understanding of how the SAM functions as a centre of morphogenesis in the plant. In particular, I will take the viewpoint that aspects of biophysics can be used to interpret many of the observations that have been made. The acquisition of the tunica/corpus organization in the SAM The SAM first becomes identifiable at the late globular stage of embryogenesis via expression of the STM marker gene (encoding a homeodomain protein) in a region between the developing cotyledons (Long et al., 1996). As development proceeds, the small dome-shaped structure characteristic of the SAM becomes apparent and this occurs concomitantly with the cellular patterning diagnostic for the tunica/corpus organization. During this process, various gene expression patterns are established within the SAM which are indicative of and required for normal meristem function (reviewed in Veit, 2004). How or whether these SAM-specific patterns of gene expression influence the cellular patterning characteristic of the SAM is unclear, although (as will be discussed later) some workers have made interesting correlations between the expression of specific homeodomain proteins and the flexibility (or otherwise) of cell division patterns. One alternative possibility is that the specific cellular patterning observed in the SAM is not a direct outcome of the specific 906 Figure 1. The shoot apical meristem shows a pattern of cell division. (A) Longitudinal section through a tobacco SAM. (B) The cellular outlines of the SAM in (A) have been couloured to indicate the tunica layer (red) in which cell division is anticlinal and the corpus (blue) in which cell division is not restricted to an anticlinal orientation. (C) The same cellular outline has been coloured to indicate the clonal nature of the different cell layers in the SAM. The pattern of anticlinal cell divisions in the outer layers means that tissue derived from the LI layer (red) the LII layer (blue) and the LIII layer (white) are clonally distinct. (D) Longitudinal section through a tobacco SAM in which cell division orientation has been locally disrupted by overexpression of a gene encoding phragmoplastin. (E) Cellular outline of the SAM shown in (D). (F) As in (E) but the outer cell layers have been coloured to highlight the abnormal pattern of cell division, in particular the difficulty of assigning the mauve coloured cells to a particular layer (compare with (C)). (Data adapted from Wyrzykowska and Fleming, 2003). patterns of gene expression but rather a consequence of the specific growth form instigated by these gene products. The classical SAM is a dome structure composed of cells which are undergoing consecutive rounds of growth and cell division. For a given increase in meristem mass, cells located at the surface are liable to undergo growth preferentially perpendicular to the radius of the dome to provide the increased surface area required to maintain the meristem geometry. Maintenance of cell division in an anticlinal orientation would allow this to occur, providing a supply of new cells to undergo expansion to generate area. This concept invokes the ability of plant tissue to respond to specific patterns of growth-associated physical stress by adjusting the preferred orientation of cell division. There are indeed data from in vitro cultured and intact tissue that cell division orientation can be modulated by the vector of impinging forces (e.g., Linthilac and Vescky, 1984; Wyner et al., 1996). However, the most convincing data showing that biophysical stress pattern can initiate biologically relevant responses in term of both cellular organization and gene expression come from the animal field. Thus, using microfabricated matrices which allow the controlled, measured application of force to cultured cells and, simultaneously, the measurement of cellular response in terms of cytoskeletal organization and gene expression, it has been shown that applied force can elicit specific, reproducible outcomes (Tan et al., 2003; Ingber, 2003). Whether plant cells/tissues show similar responses remains to be demonstrated, but the application of such approaches in plant biology is overdue. 907 In biophysical terms, in the example given above the growing meristem surface would take up a minimal surface energy conformation (Green, 1992). Although there are few experimental data to support the idea that such minimal energy conformations are causally involved in plant tissue architecture, it is interesting to note that recent research in the field of animal biology indicates that, at least in some circumstances, the formation of cellular arrangement does indeed seem to be driven by such processes. For example, the drosophila compound eye is composed of many repeating units, termed ommatidia. At the centre of each ommatidium lies a group of four cells arranged in a characteristic pattern. Modelling of minimal surface energy conformations shows that the endogenous pattern reflects the optimum solution to reducing tensile stress between the four cells. Mutations exist which result in fewer or more cells in this region and this leads to new cellular pattern. The patterns produced varies depending on the number of cells involved, but any specific pattern formed is found to be that predicted to result in minimal surface energy within the group of cells (Hayashi and Carthew, 2004). Such data suggest that groups of cells can respond to and alter their relative arrangement to form particular patterns of physical stress. Again, investigation of the situation in plant tissues has been limited. The functional significance of the tunica/corpus organization Irrespective of the mechanism by which the tunica/ corpus organization is achieved, it has a significant outcome on the destination of cells derived from the SAM. The layered structure leads to a constraint on the final position that daughter cells can take up in the plant body. Thus, cells in the plant epidermis are derived from the outer layer of the SAM (LI), cells in the sub-epidermal layers from the inner tunica layer of the SAM (LII) and the innermost tissue of the plant from the innermost tissue of the SAM (LIII) (Figure 1C). Does this distinct clonal origin of different cell layers have a significance for the functioning of the plant? The evidence is mixed. Early work using genetic chimeras created by grafting techniques indicated that the genotype of particular layers of the plant could influence the phenotype of whole organs. For example, in flowers of Camellia the presence of stamens and pistils seemed to be dictated by the genotype of the epidermal LI layer (reviewed in Szymkowiak and Sussex, 1996). Such experiments supported biophysical views of the importance of the epidermis in controlling plant growth. As will be explained later in this article, theoretical considerations of the physical stress patterns generated in growing plant tissue suggest that the outer cell layers of any young organ are likely to be under physical tension. Regulation of tissue response to this tension would afford a mechanism for the control of growth and would place special emphasis on the biophysical/molecular parameters of the outer cell layers of the plant. However, experiments looking at other characteristics in chimeras (such as floral organ number) indicated that often the inner layer genotypes (LII, LIII) played a major role in determining phenotype (Szymkowiak and Sussex, 1992). The interpretation of some of these analyses is complicated by the fact that it is not always clear to what extent each layer contributed to the organs under analysis, but it is clear that it is not always the LI layer genotype that controls plant phenotype. The possible importance of the LI layer in morphogenesis will be returned to later in this review. More recent molecular and cell biological approaches have revealed a potential mechanism to explain some of these classical observations on chimeras. The key to these new approaches has been the realization that macromolecules (most notably transcription factors) have the potential to move from cell to cell via plasmodesmata in a regulated fashion (Gillespie and Oparka, 2005). The first indications that such a mechanism of intercellular communication is possible came from work on the homeodomain protein KNOTTED-1. Observation of RNA and protein localization in the SAM revealed a discrepancy which suggested that the protein might move between cells. Later microinjections studies and experiments in which transgene expression was directed to different cell layers indicated that indeed the KN1 protein can move between layers in the SAM (Kim et al., 2002, 2003; Lucas et al., 1995). These data provide a neat explanation for earlier work on this gene which demonstrated that although the phenotype involved altered cellular patterning in the maize 908 leaf epidermis, the phenotype depended on the genotype of sub-epidermal cells (Hake and Freeling, 1986). Following on from these initial studies, work on other transcription factors has revealed that inter-cellular movement is not specific to the KN1 protein. For example, analysis of chimeras of Antirrhinum in which the MADS box gene DEFICIENS (DEF) is expressed only in the LII and LIII layers led to DEF protein accumulation in all three layers of the floral meristem, implying protein movement. Interestingly, reciprocal chimeras in which the DEF gene was only expressed in the LI layer did not lead to DEF protein accumulation in the internal layers, suggesting a restriction in polarity of DEF protein movement (Perbal et al., 1996). On the other hand, a careful analysis of the LEAFY (LFY) transcription factor revealed that the protein could move from the LI to the inner cells of the SAM, indicating that there is no general restriction on polarity of movement of proteins within the SAM (Sessions et al., 2000; Wu et al., 2003). Indeed, the work on the LFY protein suggested that movement of this transcription factor occurred passively throughout the SAM with no particular domain involved in, for example, targeting of the protein. This contrasts with investigation of the KN1 protein which indicated that specific regions of the protein were involved in intercellular movement. Taken together, the overall picture of intercellular protein movement in the SAM is still somewhat confused. Generally, the data support the concept that plasmodesmatal (PD) connections can act as an intercellular pathway for information flow and, moreover, that this flow can be regulated (Foster et al., 2002). However, the mechanism involved and whether different proteins might move via different specific pathways remains to be elucidated. Ignoring this issue of mechanism, one main question relating to the observed potential of intercellular communication between cells and layers within the SAM is its biological relevance. Does it provide the plant with a precise way of channeling information? Alternatively, is it a backup system which ensures that groups of cells contain the same information, thus ensuring that groups of cells take on identities delineated by the regional expression of specific combinations of transcription factors? Or are the observations that have been made in some way artefacts of the experimental approaches taken, i.e., they indicate a potential which is only of significance in the experimental context inflicted on the plant? At present, the jury is still out. However, if the concept of a PD-mediated information ‘‘superhighway’’ within the SAM is true, then the observed conserved cellular patterning in the SAM would have immediate impact. Primary PDs are only formed in the newly formed cell plate. Therefore, in a layer of cells dividing anticlinally, all cells in the layer are connected by primary PDs whereas symplastic connection with an underlying layer requires the formation of secondary PD. Since primary and secondary PD are probably not functionally equivalent (Oparka et al., 1999), the different ontogeny of the PD connections between cells in the SAM could provide a mechanism by which different groups of cells form different potential networks of communication. Two final points in this discussion (which may not clarify but rather add to the debate) are the observations that auxin flux within the SAM (involved in patterning of leaf initiation) occurs predominantly in the outer cell layers (although most probably not via PDs) (Reinhardt et al., 2003a) and that physical ablation of the LI layer blocks leaf initiation (Reinhardt et al., 2003b). These data again suggest that the layered cellular structure of the SAM has functional significance. Manipulation of cell division pattern in the SAM Although the SAM displays a characteristic pattern of cell division, the pattern is not constant. In particular, it has long been noted that at (or just prior) to leaf initiation, a change in pattern is observed at the site of presumptive leaf formation (reviewed in Lyndon, 1990). Careful analysis indicated that this altered pattern reflected not so much an increased frequency of cell division, rather a relaxation of the restriction of anticlinal cell division in the inner tunica layer(s). These observations raise the question of whether the altered cell division pattern associated with leaf initiation plays a causal role in the process. A number of data suggest that this is not the case. For example, mutants in which cell division orientation throughout the plant is altered can lead to a relatively mild phenotype (Smith et al., 909 1996). Even when growth is severely disrupted by mutations which alter cell division orientation, the plant can still generate a structure in which morphogenesis is recognizably occurring (Traas et al., 1995). These data suggest that if cell division orientation is normally involved in leaf initiation, the plant contains some mechanism for coping with its disruption. Experiments in which cell proliferation has either been promoted or repressed throughout the plant also indicate that leaf initiation, and morphogenesis in general, is not dependent on a particular set pattern of cell division. For example, promotion of cell proliferation by overexpression of a cyclinD2 led to an increased growth rate but plant form was normal (Cockcoft et al., 2000). Repression of cell proliferation can lead to the generation of smaller plants (e.g., by overexpression of E2F/DP factors or CDK inhibitors (De Veylder et al, 2002) or the formation of plants of normal size (e.g., by overexpression of a dominant negative form of CDK (Hemerley et al., 1995)), however in all cases plant morphology is hardly affected. The average size of the constituent cells of these plants can be influenced by these manipulations, but the overall form of the organs produced is not drastically changed, and the initiation of the organs appears to be totally normal. Most of these manipulations have involved changing cell division rates and patterns throughout the plant at all stages of development, raising the question of whether gradients of cell proliferation were maintained against an altered background level of cell division. Experiments performed in my own group addressed this issue by locally and transiently promoting cell proliferation in the SAM (by overexpression of a cyclinA and a cdc25 protein) (Wyrzykowska et al., 2002). Leaf initiation was neither promoted nor inhibited by this manipulation. Similarly, disruption of the anticlinal pattern of cell division in the tunica layer via overexpression of a gene encoding phragmoplastin (which disrupts phragmoplast formation) did not alter the ability of the SAM to form normal leaves (Figure 1D–F) (Wyrzykowska and Fleming, 2003). It should be noted, however, that some data show that altered expression of cell cycle genes does alter plant morphogenesis. For example, when a cyclinD3 gene was over expressed throughout the plant abnormal leaves were generated (Riou-Khamlichi et al., 1999). Since cyclinD proteins lie at the interface of various inputs to the cell cycle, it is possible that manipulation of expression of some of these proteins might feed back to these inputs. Indeed, the manipulation of cyclinD3 expression described above was shown to influence plant response to the growth regulator cytokinin. Such effects might lead to numerous outcomes on SAM function in addition to those directly related to cell proliferation. For example, recent data have implicated cytokinin in the control of meristem size and function (Giulini et al., 2004), thus any manipulation altering cytokinin perception is likely to have a profound influence on meristem function. Taken together, the simplest interpretation of the data available is that cell division frequency and orientation in the SAM are not causally involved in leaf formation. Does this mean that cell division is simply a means by which the plant volume is split into compartments to allow for biophysical integrity and for cellular differentiation (Kaplan, 2001)? A number of data suggest that this is not the case. Instead, it seems that cell division pattern during the early stages of leaf initiation may be closely related to the patterns of transcription factor gene expression that occur at the onset of organogenesis and that the downstream targets of these transcription factors could act to set or promote particular patterns of growth and cellular patterning required for appropriate differentiation within the leaf. One of the most conspicuous patterns of gene expression observed in the SAM is the absence of transcripts encoding members of the KNOX (homeodomain-encoding) gene family at the presumptive site of leaf formation (Jackson et al., 1994). This coincides with the region of altered cell division pattern describe above. Based on a series of observations of cellular patterning in leaves following ectopic expression of KNOX genes, Matsouka and colleagues suggested that such homeodomain proteins might play a regulatory role in maintaining the flexibility of cell division within organs (Sato et al., 1996; Tamaoki et al., 1997). Moreover, further work demonstrated that certain KNOX proteins can directly interact with the promoter region (and repress expression) of a gene encoding a GA20-oxidase, which catalyses a key step in biosynthesis of gibberellic acid (GA) (Sakamoto et al., 2001). A significant body of 910 evidence indicates that GA can influence the plant cell cytoskeleton and, thus, the vector of plant cell expansion, although there is some discussion on the mechanism involved (Wasteneys, 2000). Thus, it is highly plausible that KNOX proteins can influence cell division pattern via modulation of GA levels (Hay et al., 2002). In this scenario, the KNOX proteins would not dictate the orientation of cell division directly, rather they (or their absence) would provide the permissive signal to allow GA-mediated cell expansion to occur. The actual outcome of elevated GA on cell division/ expansion is likely to be highly context dependent, i.e., different tissues would respond differently to the same input of altered KNOX/GA switching. In the context of the SAM, two questions arise: Why does the expression of particular KNOX genes decrease at the site of presumptive leaf formation? Why does the permissive condition for GAmediated expansion in the SAM lead to growth in a new direction (i.e., perpendicular to the surface of the SAM)? With respect to the decrease in KNOX gene expression, the likeliest explanation is that these genes are targets for the flux of auxin predicted to occur at the site of presumptive leaf formation, although this has yet to be shown (Reinhardt et al., 2003a). With respect to the new vector of growth, a number of data indicate that the architecture of the cell wall plays a key role, as discussed in the next section. The role of the cell wall in providing the growth vector for leaf initiation Plant growth is essentially a matter of biophysics (Green 1992, 1994, 1999). It depends on the equilibrium of internally generated turgor pressure and tensile forces within the cell wall which restrain the tendency of the cell to expand and (eventually) explode. This is shown diagrammatically in Figure 2. Each cell within the SAM generates a turgor pressure which acts equally in all directions. Cells within the SAM thus both exert and experience force on all sides. Due to symplastic connections between cells, this hydrostatic pressure will tend to equalize itself throughout the body of the SAM. However, cells located at the epidermis (LI layer) lack cellular neighbours towards the outside. To balance the outward acting forces, a tensile stress must develop within the outer cell walls to counteract and balance the internally generated force. It is noticeable that in real SAMs the outer cell wall of the LI layer is thickened, as expected of a structure designed to contain or withstand relatively high tensile stress. In the diagram in Figure 2A the tissue is in equilibrium, i.e., the total outwardly acting forces are balanced by the tensile stress in the outer cell walls. If, however, the architecture of this wall were locally altered so that the walls in this region became weaker (less resistant to the tensile force developed within it), then the tissue would tend to bow outwards as a consequence of the internallygenerated outwardly acting forces (Figure 2B). Provided there was a mechanism by which the architecture of the cell walls could re-establish its previous stress-resistant quality, then (after a certain amount of give in the material) a new biophysical equilibrium would be established but the tissue would have a new shape, i.e., morphogenesis would have occurred. In the context of the SAM, such a mechanism would provide a new vector of growth upon which the release of KNOX-mediated repression of GA biosynthesis could act to fix and amplify outward growth of a new leaf primordium. However, does such a biophysical mechanism exist in plants? A number of data suggest that it does. Although our understanding of the control of plant cell wall architecture is still very much incomplete, a number of investigators have identified components of the cell wall that can modulate its extensibility. The prime candidate as an endogenous regulator of cell wall extensibility is the protein expansin. Expansins are encoded by relatively large gene families in all higher plants so far investigated. Although their mechanism of action remains obscure, they have the ability to loosen the cell wall matrix to increase its extensibility (Cosgrove, 2000) and specific members of the expansin gene family are expressed at the site of presumptive site of leaf formation (Cho and Kende, 1998; Reinhardt et al., 1998), i.e., they are present at the appropriate time and place to act at the very earliest stages of leaf formation. Moreover, local overexpression of an expansin gene is sufficient to induce morphogenesis at the SAM in a manner consistent with the model portrayed in Figure 2 (Pien et al., 2001). Although data showing that lack of expansin gene expression blocks leaf formation are lacking, the results so far 911 (A) LI (B) LI (C) LI Figure 2. Local loosening of the SAM cell wall is sufficient to induce morphogenesis. (A) A theoretical group of cells towards the edge of the SAM dome. Each cell generates an internal, force (turgor) indicated by the crossed-arrows. This acts outwards equally in all directions. Cells located internally both exert and experience a force in all directions. Cells located in the outer layer (epidermis, LI) lack neighbours on one side. The outward acting force of these cells in this direction is balanced by the development of a tensile force within the outer cell walls. The forces are in equilibrium and no growth occurs. (B) If the architecture of the cell wall is locally altered (red colour) so as to loosen the cell wall, then the restraining tensile force within this region of the wall will be dissipated. (C) As a consequence, providing the cells in this region have sufficient growth potential (e.g., sufficient energy and materials for growth), the tissue will bulge outwards until a new biophysical equilibrium is achieved, i.e. sufficient tensile stress is attained in the outer cell walls to restrict growth. strongly suggest that modulation of cell wall extensibility at the site of leaf formation (presumably downstream of the auxin patterning process) provides the differential growth impulse for leaf initiation. One consequence of viewing the initial steps of leaf morphogenesis from a biophysical perspective is the observation that, since the orientation of cell division in plant cells tends to occur perpendicular to the principal axis of growth, the new vector of growth associated with leaf formation will automatically lead to a new pattern of cell division. As shown schematically in Figure 3, in the outer layers of the SAM growth occurs preferentially parallel to the surface plane of the tissue. As discussed in the first part of this article, this might simply be a consequence of the biophysical stress patterns in the growing dome which necessitate that the surface tissue maintains a high rate of surface area increase to maintain the integrity of the dome structure. This pattern of growth will lead to the orientation of cell division in these surface cells being preferentially anticlinal. If the vector of growth switches to perpendicular to the surface of the SAM, then the cells within the growing bulge will now tend to divide in a periclinal orientation (Figure 3B). Does this switch in cell division orientation have an outcome for the tissue in which it is occurring? As outlined above, a change of cell division pattern is not necessary for, nor does it disrupt, leaf initiation. However, such new cellular patterns are likely to lead to new patterns of PD connections. Although the functional significance of PD connections within the SAM is still being unraveled (as discussed in a previous section), this might present a mechanism which ensures that groups of cells entering a particular developmental pathway share relevant 912 (A) LI (B) LI Figure 3. A new vector of growth can feed back onto the pattern of cell division and the potential pattern of intercellular contacts. (A) A theoretical group of cells towards the edge of the SAM dome. Growth is restricted parallel to the surface plane of the tissue (large arrows). New cell walls are laid perpendicular to the vector of growth, thus transport through primary plasmodesmata will be restricted within the cell layers (double-headed arrows). (B) As a consequence of the new vector of growth which defines leaf formation (large arrow), cell division orientation within the tissue is altered, i.e., periclinal divisions occur. Primary plasmodesmata within these new cell walls allow new potential pathways of information flux. transcriptional information. Thus, the switch in cell division pattern which must accompany leaf initiation could provide a fool-proof mechanism to ensure that the cells within the new organ form a PD network-defined entity separate from the SAM. Actual evidence that altered cell division in the SAM has a consequence on gene expression pattern has come from work in my own group (Wyrzykowska and Fleming, 2003). Disruption of anticlinal cell divisions in the tunica via local overexpression of phragmoplastin led to a local decrease in transcripts encoding a Knotted-like transcription factor (NTH15) and, simultaneously, accumulation of transcripts encoding a Phantastica-like transcription factor implicated in leaf formation. Similar changes were also observed when cell proliferation was locally promoted in the SAM. The mechanism by which such altered patterns of cell division and gene expression are linked remain totally unclear, as is their functional significance (since in both instances meristem growth and leaf formation appeared unimpaired). Nevertheless, these data demonstrate the potential for information flow not only from the gene to cell division but also from cell division pattern to the gene. The above model lays special emphasis on the outer layers of the SAM and recent data based on micro-ablation of cells within the SAM have provided evidence that the physical integrity of the LI layer does indeed play a special role in organogenesis and that the LI layer influences cell division pattern in the internal layers. Thus, physical removal of the LI layer prevents leaf initiation at that site and, at the same time, leads to growth in that region which is accompanied by periclinal cell divisions (Reinhardt et al., 2003b). These data are consistent with the idea that the LI layer is somehow intimately involved in the process of leaf morphogenesis, that it normally acts to restrict outward growth, and that increased periclinal cell divisions are linked to and are a consequence of outward growth in the SAM. In the context of the model put forward in Figure 2, removal of the outer cell layer would lead to a loss of the physical restraint on growth provided by this layer, thus outward growth is the predicted (and observed) result. In the context of the model shown in Figure 3, such outward growth should lead to the occurrence of periclinal divisions, again as observed. As to the question of why a leaf primordium does not result from this initial impetus for outward growth by layer ablation, then it seems that the LI layer plays an additional role to that of a biophysical restraining wall, with epidermal-localised auxin flux the prime suspect (Reinhardt et al., 2003a; Fleming, 2005). Summary and future directions Classical views of the SAM have focussed on the role of cell division in the SAM. More recently, molecular genetic studies have identified many of the components involved in regulating meristem function in terms of control of meristem growth and leaf formation. In many of these studies, cell division has been assumed or indirectly implicated as the downstream target of these regulatory systems. In reality, many of the observations can be better interpreted as a consequence of altered growth parameters in the SAM. Although growth and cell proliferation are tightly coupled in the 913 SAM, they are not synonymous terms. Indeed, as outlined in this review, causal data showing that cell division frequency or orientation is directly linked to the process of morphogenesis and leaf formation are very limited. An alternative interpretation laying stress on the control of the vector of growth provides a simpler explanation for the observed data. Unfortunately, our understanding of the molecular control and integration of this basic parameter remains astonishingly limited, both in plants and animals (Rudra and Warner, 2004). Research directed at deciphering the linkage between transcriptional or hormonal regulators of SAM function and non-cell division based target processes may provide the key insights required for future progress. References Cho, H-T. and Kende, H. 1998. Tissue localization of expansins in deep water rice. Plant J. 15: 805–812. Cockcroft, C.E., den Boer, B.G.W., Healy, J.M.S. and Murray, J.A.H. 2000. Cyclin D control of growth rate in plants. Nature 405: 575–578. Cosgrove, D.J. 2000. Loosening of plant cell walls by expansins. Nature 407: 321–326. De Veylder, L., Beeckman, T., Beemster, G.T.S., Engler, J.D., Ormenese, S., Maes, S., Naudts, M., Van der Schueren, E., Jacqmard, A., Engler, G. and Inzé, D. 2002. Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa-DPa transcription factor. EMBO J. 21: 1360–1368. Fleming A.J., 2005. Formation of primordia and phyllotaxy. Curr. Opin. Plant Biol. 8: 53–58. Foster, T.M., Lough, T.J., Emerson, S.J., Lee, R.H., Bowman, J.L., Forster, R.L.S. and Lucas, W.J.A. 2002. Surveillance system regulates selective entry of RNA into the shoot apex. Plant Cell 14: 1497–1508. Gillespie, T. and Oparka, K.J. 2005. Plasmodesmata- gateways for intercellular communication. In: A.J. Fleming (Ed.), Intercellular Communication in Plants, Blackwell Publishing, Oxford, UK, pp. 109–146. Giulini, A., Wang, J. and Jackson, D. 2004. Control of phylloaxy by the cytokinin-inducible response regulator homologue ABPHYL1. Nature 430: 1031–1034. Green, P.B. 1992. Pattern formation in shoots: a likely role for minimal energy configurations of the tunica. Int. J. Plant Sci. 153: S59–S75. Green, P.B. 1994. Connecting gene and hormone action to form, pattern and organogenesis: biophysical transductions. J. Exp. Bot. 45: 1775–1778. Green, P.B. 1999. Expression of pattern in plants: combining molecular and calculus-based biophysical paradigms. Amer. J. Bot 86: 1059–1064. Hake, S. and Freeling, M. 1986. Analysis of genetic mosaics shows that the extra epidermal cell divisions in Knotted mutant maize plants are induced by adjeacent mesophyll cells. Nature 320: 621–623. Hay, A., Kaur, H., Phillips, A., Hedden, P., Hake, S. and Tsiantis, M. 2002. The gibberelin pathway mediates KNOTTED1-type homeobox function in plants with different body plans. Current Biol. 12: 1557–1565. Hayashi, T. and Carthew, R.W. 2004. Surface mechanics mediate pattern formation in the developing retina. Nature 431: 647–652. Hemerley, A., Engler, J.A., Bergounioux, C., Van Montagu, M., Engler, G., Inzé, D. and Ferreira, P. 1995. Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development. EMBO J. 14: 3936–3936. Ingber, D.E. 2003. Mechanosensation through integrins: cells act locally but think globally. Proc. Natl. Acad. Sci. USA 100: 1472–1474. Jackson, D., Veit, B. and Hake, S. 1994. Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 120: 405–413. Kaplan, D.R. 2001. Fundamental concepts of leaf morphology and morphogenesis: a contribution to the interpretation of molecular genetic mutants. Int. J. Plant Sci. 162: 465–474. Kim, J-Y., Yuan, Z., Cilia, M., Khalfan-Jagani, Z. and Jackson, D. 2002. Intercellular trafficking of a KNOTTED1 green fluorescent protein fusion in the leaf and shoot meristem of Arabidopsis. Proc. Natl. Acad. Sci. (USA) 99: 4103–4108. Kim, J-Y., Yuan, Z. and Jackson, D. 2003. Developmental regulation and significance of KNOX protein trafficking in Arabidopsis. Development 130: 4351–436. Linthilac, P.M. and Vesecky, T.B. 1984. Stress-induced alignment of division plane in plant tissues grown in vitro. Nature 307: 363–364. Long, J.A., Moan, E.I., Medford, J.I. and Barton, M.K. 1996. A member of the KNOTTED class of homeodomain proteins encoded by the SHOOTMERISTEMLESS gene of Arabidopsis. Nature 379: 66–69. Lucas, W.J., Bouche-Pillon, S., Jackson, D.P., Nguyen, L., Baker, L., Ding, B. and Hake, S. 1995. Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata. Science 270: 1980–1983. Lyndon, R.F. 1990. Plant Development: the cellular basis. Unwin Hyman, London, UK. Oparka, K.J., Roberts, A.G., Boevink, P., Santa Cruz, S., Roberts, L., Pradel, K.S., Imlau, A., Kotlizky, G., Sauer, N. and Epel, B. 1999. Simple, but not branched, plasmodesmata allow the non-specific trafficking of proteins in developing tobacco leaves. Cell 97: 743–754. Perbal, M.C., Haughn, G., Saedler, H. and Schwarz-Sommer, Z. 1996. Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking. Development 122: 3433–3441. Pien, S., Wyrzykowska, J., McQueen-Mason, S., Smart, C. and Fleming, A. 2001. Local expression of expansin induces the entire process of leaf development and modifies leaf shape. Proc. Natl. Acad. Sci. USA 98: 11812–11817. Reinhardt, D., Wittwer, F., Mandel, T. and Kuhlemeier, C. 1998. Localized upregulation of a new expansin gene predicts the site of leaf formation in the tomato meristem. Plant Cell 10: 1427–1437. Reinhardt, D., Pesce, E-R., Stieger, P., Mandel, T., Baltensperger, K., Bennett, M., Traas, J., Friml, J. and Kuhlemeier, C. 2003a. Regulation of phyllotaxis by polar auxin transport. Nature 426: 255–260. 914 Reinhardt, D., Frenz, M., Mandel, T. and Kuhlemeier, C. 2003b. Microsurgical and laser ablation analysis of interactions between the zones and layers of the tomato shoot apical meristem. Development 130: 4073–4083. Riou-Khamlichi, C., Huntley, R., Jacqmard, A. and Murray, J.A.H. 1999. Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science 283: 1541–1544. Rudra, D. and Warner, J.R. 2004. What better measure than ribosome synthesis?. Genes & Dev. 18: 2431–2436. Sakamoto, T., Kamiya, N., Ueguchi-Tanaka, M., Iwahori, S. and Matsouka, M. 2001. KNOX homeodomain protein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem. Genes & Dev. 15: 581–590. Sato, Y., Tamaoki, M., Murakami, T., Yamamoto, N., KanoMurakami, Y. and Matsouka, M. 1996. Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox gene OSH1 lead to altered morphology of leaves in transgenic tobacco. Mol. Gen. Genet. 251: 13–22. Sessions, A., Yanofsky, M.F. and Weigel, D. 2000. Cell-cell signalling and movement by the floral transcription factors LEAFY and APETALA1. Science 289: 779–781. Smith, L.G., Hake, S. and Sylvester, A.W. 1996. The tangled1 mutation alters cell division orientations throughout maize leaf development without altering leaf shape. Development 122: 481–489. Steeves, T.A. and Sussex, I.M. 1989. Patterns in Plant Development. Cambridge University Press, Cambridge, UK. Szymkowiak, E.J. and Sussex, I.M. 1996. What chimeras can tell us about plant development. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 351–376. Szymkowiak, E.J. and Sussex, I.M. 1992. The internal layer (LIII) determines floral meristem size and carpel number in tomat periclinal chimeras. Plant Cell 4: 1089–1100. Tan, J.L., Tien, J., Pirone, D.M., Gray, D.S., Bhadriraju, K. and Chen, C.S. 2003. Cells lying on a bed of microneedles: An approach to isolate mechanical force. Proc. Natl. Acad. Sci. USA 100: 1484–1489. Tamaoki, M., Kusaba, S., Kano-Murakami, Y. and Matsouka, M. 1997. Ectopic expression of a tobacco homeobox gene, NTH15, dramatically alters leaf morphology and hormone levels in transgenic tobacco. Plant Cell Physiol. 38: 917–927. Traas, J., Bellini, C., Nacry, P., Kronenberger, J., Bouchez, D. and Caboche, M. 1995. Normal differentiation patterns in plants lacking microtubular preprophase bands. Nature 375: 676–677. Veit, B. 2004. Determination of cell fate in apical meristems. Curr. Opin. Plant Biol. 7: 57–64. Wasteneys, G.O. 2000. The cytoskeleton and growth polarity. Curr. Opin. Plant Biol. 6: 503–511. Wu, X., Dinneny, J.R., Crawford, K.M., Rhee, Y., Citovsky, V., Zambryski, P.C. and Weigel, D. 2003. Modes of intercellular transcription factor movement in the Arabidopsis apex. Development 130: 3735–3745. Wymer, C.L., Wymer, S.A., Cosgrove, D.J. and Cyr, R.J. 1996. Plant cell growth responds to external forces and the response requires intact microtubules. Plant Physiol. 110: 425–430. Wyrzykowska, J. and Fleming, A.J. 2003. Cell division pattern influences gene expression in the shoot apical meristem. Proc. Natl. Acad. Sci. USA 100: 5561–5566. Wyrzykowska, J., Pien, S., Shen, W.-H. and Fleming, A.J. 2002. Manipulation of leaf shape by modulation of cell division. Development 129: 957–964.