The mechanism of leaf morphogenesis

Transcription

1 Planta (2002) 216: DOI /s REVIEW Andrew J. Fleming The mechanism of leaf morphogenesis Received: 25 April 2002 / Accepted: 15 July 2002 Ó Springer-Verlag 2002 Abstract Whether cell division is a driving force in plant morphogenesis has long been debated. In this review, the evidence for the existence of cell division-dependent and cell division-independent mechanisms of plant morphogenesis is discussed. The potential mechanisms themselves are then analysed, as is our understanding of the regulation of these mechanisms and how they are integrated into development, with particular emphasis on data arising from the investigation of leaf morphogenesis. The analysis indicates the existence of both cell division-dependent and cell division-independent mechanisms in leaf morphogenesis and highlights the importance of future investigations to unravel the co-ordination of these mechanisms. Keywords Development Æ Cell cycle Æ Cell wall Æ Morphogenesis Abbreviation CDK: cyclin-dependent kinase frequency to be deterministic in plant morphogenesis. This expectation is not met by reality. Not only is it clear that the vast majority of differentiation depends on cell position rather than clonal parentage, it has also become apparent that in many circumstances normal plant size and shape can be achieved despite major alteration in factors driving cell division. In this review, I will first survey the evidence for the existence of cell divisiondependent and cell division-independent mechanisms of plant morphogenesis before going on to examine the actual mechanisms themselves. Finally, I will consider the control and integration of these two mechanisms into plant development, with special emphasis on leaf formation. Since morphogenesis in plants is intimately linked with growth, an understanding of the control of plant form also requires an understanding of the control of organ size. Details on this aspect of morphogenesis will also be discussed. Introduction Plants are made of cells. It is an intuitive expectation, therefore, that the number of cells produced in a plant, the size of those cells, and where and when those cells are generated should play a major role in controlling plant size and shape. Indeed, since processes of cell migration and cell death are much more limited in plants during most phases of development compared to their animal counterparts, one might expect cell division pattern and Dedicated to Nikolaus Amrhein, Zu rich, on the occasion of his 60th birthday. A.J. Fleming (&) Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), Universita tsstrasse 2, 8092 Zu rich, Switzerland andrew.fleming@ipw.biol.ethz.ch Fax: Evidence that a cell division-independent mechanism for morphogenesis exists in plants At the single-cell level it is clear that change of form (linked with cell growth) can occur without division (Folkers et al. 1997). However, what is the situation in a multicellular organ, such as a leaf? Significant advances have been made over the last decade in our understanding of the proteins involved in regulating the plant cell cycle (Potuschak and Doerner 2001; Stals and Inze 2001). These data have shown that plant cells contain a machinery which, although it has some peculiarities, in general terms is similar to that described in other eukaryotes. One consequence of this work has been the generation of transgenic plants in which specific modulators of the cell cycle have been over- or under-expressed and, at least in some cases, this has led to successful modulation of the cell cycle (i.e., increased and decreased cell division rate; Hemerley et al. 1995; Doerner et al. 1996; McKibbin et al. 1998;

2 18 Cockcroft et al. 2000). The most surprising observation from these experiments has been that direct modulation of the cell cycle (and, thus, of cell division) generally leads to only minimal alteration in plant morphogenesis [although note the work of Wang et al. (2000) and de Veylder et al. (2001) described in the next section]. Effects on plant growth rate are certainly observed (Cockcroft et al. 2000), but these seem only to influence the time taken until normal size and shape are achieved. One criticism of these data has been that morphogenesis might depend on local variations in cell division rate and that the experiments performed did not alter these local gradients (Meyerowitz 1996). Experiments from our own laboratory approached this question by locally inducing cell division within the apical meristem, the site of leaf organogenesis (Wyrzykowska et al. 2002). As shown in Fig. 1A, these manipulations led to an accumulation of smaller cells on one flank of the apical meristem. Despite these changes, meristem function and leaf formation proceeded normally (Fig. 1B). These experiments demonstrate that local alteration in cell division rate within the meristem does not disturb organogenesis. Plant cell division is characterised not only by the rate at which this process occurs but also by the fact that the new cell wall (generated within the dividing cell) can take up a number of orientations. The orientation of new wall insertion is expected to have a major influence on both the differentiation of the daughter cells generated and the future growth characteristics of these cells (Steeves and Sussex 1989). Nevertheless, mutants in which cell division orientation is virtually randomised still generate all essential organs (Traas et al. 1995) and mutants in which cell division orientation is disrupted to a lesser (but still significant) extent generate plants of approximately normal morphology (Smith et al. 1996). Again, it is possible to argue that it is local patterns of cell division that are important and that in the mutants described above these local patterns were still maintained, thus allowing cell division pattern to drive morphogenesis. To address this point, we have locally overexpressed the dynamin-like protein phragmoplastin within the meristem. Phragmoplastin is involved in cell c Fig. 1A E. Cell division-dependent and cell division-independent mechanisms of morphogenesis. A Longitudinal section through a tobacco (Nicotiana tabacum) meristem in which extra cell divisions have been induced on one flank (arrow; Wyrzykowska et al. 2002). Bar = 30 lm. B Scanning electron micrograph of a tobacco meristem treated as in A. Leaf initiation is normal (Wyrzykowska et al. 2002). Bar = 80 lm. C Tobacco leaf resulting from a primordium in which part of one flank (arrow) has been induced to transiently undergo extra cell divisions. An indentation of the lamina has occurred (Wyrzykowska et al. 2002). Bar = 7 mm. D Tobacco leaf resulting from a primordium in which part of one flank (arrow) has been induced to overexpress expansin protein. An increased lamina expansion has occurred (Pien et al. 2001). Bar = 5 mm. E Scanning electron micrograph of a meristem after local application of expansin protein. Morphogenesis has occurred (Fleming et al. 1999). Bar = 20 lm

3 19 plate formation and overexpression of the protein leads to abnormal planes of cell division (Gu and Verma 1997). Local overexpression of phragmoplastin in the meristem led to a disruption of the normally highly regular pattern of cell division but with no overt alteration in leaf organogenesis (J. Wyrzykowska and A.J. Fleming, unpublished data). Taken together, these studies indicate that plant morphogenesis is dependent neither on the rate nor orientation of cell division. A cell division-independent mechanism of morphogenesis must exist. Evidence that a cell division-dependent mechanism for morphogenesis exists in plants Cytokinesis involves the separation of the mother cell cytoplasm into two daughter cells (Sylvester 2000). In plants, this occurs by the formation of an internal cell wall at a site dictated by the accretion of vesicles containing new membrane and cell wall material. Mutations in genes encoding proteins involved in cell plate formation (such as KNOLLE and KEULE) lead to abnormal or incomplete cell wall formation. As a consequence, although cell division and cytokinesis can occur, the seedlings formed cease growth (Lukowitz et al. 1996; Assad et al. 2000). Combinations of these mutant genes are phenotypically additive and lead to an even earlier cessation of development (Waizenegger et al. 2000). These data indicate that morphogenesis requires a certain minimum level of competence in cell division. More positive data supporting a role for cell division in morphogenesis have come from the manipulation of expression of proteins involved in inhibiting cyclin-dependent kinase (CDK) activity. CDK inhibitors (described as ICKs and KRPs in the plant literature) inhibit the activity of CDK/cyclin complexes. Genes encoding these proteins have been identified and constitutively overexpressed in plants (Wang et al. 2000; de Veylder et al. 2001). This led to the production of smaller plants and to a change in leaf morphology, in particular an increase in leaf serration. These data show that not only can cell division have an influence on plant size, but also that constitutive expression of a cell cycle regulator can result in a non-uniform response in terms of organ morphology. Experiments from our own group also indicate that manipulation of cell division can influence leaf morphogenesis. Thus, local induction of genes encoding proteins with a role in the cell cycle on the flank of young leaf primordia led to a local increase in cell division rate. Subsequently, the induced regions underwent decreased growth relative to the surrounding tissue, thus leading to a change in leaf shape, as shown in Fig. 1C and Wyrzykowska et al. (2002). Taken together, these data indicate that cell division can influence morphogenesis, i.e, that a cell divisiondependent mechanism of morphogenesis exists. Synthesis Some level of cell division is required for normal morphogenesis in multicellular plants. Once this minimal level is achieved, a degree of normal morphogenesis can occur. In some regions of the plant (e.g., shoot apical meristem) morphogenesis can occur despite large changes in the parameters of cell division rate and orientation. In these regions morphogenesis is not driven by cell division and occurs primarily via a cell division-independent mechanism. In other regions of the plant both cell divisiondependent and cell division-independent mechanisms may function. How do these mechanisms work? The mechanism of cell division-independent morphogenesis: the cell wall and growth rate Plant cell growth is a biophysical process involving the balance of a hydrostatic force acting outwards from the cell (turgor) with a tensile force within the surrounding cell wall (Cosgrove 2000). Growth requires a controlled imbalance between these forces to allow inward flux of water and the stretching of the cell wall until a new physical equilibrium is established. The body of evidence available indicates that while the internal turgor pressure remains generally constant during growth processes, the cell wall undergoes regulated changes in extensibility (relaxation), which allows regulated increase in cell size (Cosgrove 2000). Theoretically, this regulation in cell wall extensibility could direct where and when growth in plant tissue occurs (Green 1999). Evidence in support of such a mechanism has come from the analysis and manipulation of a particular family of cell wall proteins, termed expansins. Expansins were first identified in cucumber seedlings (McQueen-Mason et al. 1992). Since then, expansinrelated proteins have been identified in the majority of plant groupings, as well as some non-plant organisms (Li et al. 2002). Expansin proteins are characterised by their in vitro ability to loosen cell wall material (hence, their name). This activity, coupled with the general correlation of high expansin gene expression and tissue extension led to the suggestion that they played an endogenous role in the cell wall loosening process required for growth. Causal evidence from the study of living tissue has appeared over the last few years. Thus, directed over- and under-expression of an expansin gene in leaf vascular tissue using a specific promoter element led to a change in leaf size and morphology (Cho and Cosgrove 2000) and local induction of expansin activity on the flanks of young leaves led to a local increase in tissue growth and lamina expansion, as shown in Fig. 1D and by Pien et al. (2001). Moreover, localised expansin activity within the meristem was sufficient to induce morphogenesis (Fig. 1E), leading to the formation of leaves (Fleming et al. 1997; Pien et al. 2001). Although the mechanism of expansin action is still

4 20 obscure, the experiments described above support the contention that modulation of cell wall loosening controls cell growth and that, when these manipulations are performed at a local tissue level, the discontinuities in cell wall extensibility induced are sufficient to induce relative differences in growth rate which become visible as altered morphogenesis. If cell wall relaxation controls growth rate, an important question is how the vector of this growth is controlled. Recent analysis of the ANGUSTIFOLIA mutant (in which leaves are narrower and thicker than normal) has implicated elements associated with regulation of the microtubule cytoskeleton and the cell wall in this process (Folkers et al. 2002; Kim et al. 2002). The mechanism of cell division-dependent morphogenesis: cell division and growth rate Morphogenesis requires a change in relative growth rate and, as described above, requires changes in biophysical parameters of the tissue. The precise mechanism by which cell division can influence these parameters is unclear, but presumably factors associated with the cell cycle (such as CDK/cyclin complexes) impact on target proteins which regulate these growth processes. This relatively indirect pathway allows a considerable degree of flexibility in the relationship between cell division and growth rate. For example, in monocot leaves the highest rates of tissue expansion are measured in the region of the leaf where cell division has ceased or is undergoing cessation (Tardieu and Granier 2000), whereas in dicot leaves cell division rate and tissue expansion tend to be highly correlated (Granier et al. 2000). A further example of the potentially complex interaction of cell division and growth is provided by the observed nonuniformity of response of tissue to uniform expression of regulators of the cell cycle, implying either a differential sensitivity of tissue in terms of cell division to the same input of regulator or a differential output of growth rate in response to the same input of altered cell division (Wang et al. 2000; de Veylder et al. 2001). Finally, cell division can influence growth indirectly via its linkage with differentiation. For example, in the context of leaf development a key early event is the establishment of a functional vascular tissue. If altered cell division pattern were to disrupt vascular differentiation then transport processes required to supply materials for growth would be impaired, leading to retardation of growth and altered morphogenesis. To summarise, the data indicate that cell division has the potential to influence morphogenesis, but that the outcome is dependent on the anatomical and developmental context in which the daughter cells are generated. The link of cell division to growth rate may be very indirect (via altered differentiation) but can involve a mechanistic relationship with growth. The nature of this relationship is discussed further in the next section. The integration and control of division-dependent and division-independent mechanisms of morphogenesis The above discussion indicates that both divisiondependent and division-independent mechanisms of morphogenesis exist. This raises the questions: How are these mechanisms co-ordinated? Is one mechanism superior to the other? At the cellular level it is clear that under most circumstances growth determines division, i.e., division occurs only when a cell has reached a particular size (Conlon and Raff 1999). However, we have little idea of how a cell measures its size and transduces this information into a signal to promote (or repress) division. Moreover, it is clear that the size/division relationship is variable. Cells in a meristem are maintained at a particular size, but as cells are removed from the meristem this balance between size and division is shifted. Moreover, this shift occurs in a tissue-specific manner (in fact, in some ways defines tissue identity). The mechanism controlling this shift in size/division relationship is unknown. The best we can say is that this shift is somehow related to differentiation and, indeed, might be actually causally involved in differentiation. This, however, simply moves our level of ignorance to the problem of defining differentiation. Certain cells of the meristem seem to be maintained in an undifferentiated or indeterminate state by a complex cascade of transcription factors (Byrne et al. 2002), but the nature of the molecular changes induced (or repressed) by these factors remains to be elucidated. At the organ level, cell division-dependent and cell division-independent mechanisms of morphogenesis are co-ordinated to generate structures of appropriate size and shape. By balancing these mechanisms the plant can both co-ordinate growth rates with environmental/ nutritional status and can provide a failsafe mechanism by which if one element is removed or not functioning properly, a compensatory mechanism can cut in to ameliorate the situation and maintain at least the basic morphogenic patterns required for plant function (Doonan 2000). The system acts both to restrict growth if cell division is promoted above normal levels, but also to promote growth if cell division is lower than normally required, leading to the formation of approximately normal sized and shaped organs, but containing fewer and larger cells (Hemerley et al. 1995; Jones et al. 1998). The question, of course, is what is the nature of this growth co-ordinator? A similar conundrum in animal biology has led to the proposal of a system for measuring either tissue mass (Potter and Xu 2001) or a morphogen gradient over space so that cells within the gradient can sense discontinuities and respond accordingly by appropriate growth (Day and Lawrence 2000). Although the molecular machinery involved in these proposed sensors remains unknown, some progress has been made in identifying factors that have an input into

5 21 the system. Thus, overexpression of the putative transcription factor AINTEGUMENTA leads to an increase in organ size with little affect on shape (Krizak 1999; Mizukami and Fischer 2000) and overexpression of the ROTUNDIFOLIA3 protein (a cytochrome P450 with a potential function in steroid biosynthesis) leads to increase in leaf length (Kim et al. 1999). This last paper suggests that hormonal factors are likely to influence organ size, as they do in animal systems (Potter and Xu 2001). This expectation seems to be borne out by observations on altered organ size associated with altered hormone transport or perception (Ecker 1995; Zhong and Ye 2001). However, the pleiotropic physiological affects of plant hormones makes direct causal relationships difficult to identify, and its noticeable, for example, that although altered perception to auxin can lead to altered cell size within a leaf, no change in organ size occurs (Jones et al. 1998). Finally, much of the theoretical work on organ size and shape assumes the presence of boundaries within which growth is controlled (Day and Lawrence 2000). In the context of leaf development, the margin appears to be a boundary required for lamina extension to occur. Thus, recent work has identified a number of genes whose products are required for the acquisition of abaxial or adaxial leaf identity (Byrne et al. 2001). Significantly, lamina extension seems to require the juxtaposition of cells possessing abaxial and adaxial identities (Waites and Hudson 1995). The interaction of these domains would thus seem to define a boundary condition required for setting the limits of future lateral growth of the leaf. Bearing in mind the specific cellular processes of division and differentiation that occur in these marginal cells (Poethig and Sussex 1985; Donnelly et al. 1999), and the fact that our own experiments modifying leaf shape involved modulation of these cells (Pien et al. 2001; Wyrzykowska et al. 2002), the future analysis of the mechanism of abaxial/adaxial domain interaction and its downstream targets promises to shed light on a fundamental aspect of leaf morphogenesis as well as exploring the molecular nature of a classically proposed morphogenic patterning mechanism. Acknowledgements I would like to thank members of the group for useful discussions during the preparation of this manuscript. The author is a START Fellow of the Swiss National Science Foundation (SNF). Research in my laboratory is funded both by the SNF and the Swiss Federal Institute of Technology. This research was enabled by the generous provision of space, equipment and materials by Nikolaus Amrhein. 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