Building a Root: The Control of Patterning and Morphogenesis during Root Development

Transcription

1 The Plant Cell, Vol. 9, , July 1997 O 1997 American Society of Plant Physiologists Building a Root: The Control of Patterning and Morphogenesis during Root Development John W. Schiefelbein, James D. Masucci, and Haiyang Wang Department of Biology, University of Michigan, Ann Arbor, Michigan, INTRODUCTION Roots are plant organs adapted to acquire water and nutrients from the environment. The rapid exploratory growth habit of roots makes them well suited for experimental studies of organogenesis in plants. In this regard, one particularly advantageous feature of root development is that tissues and cell types arise from an apical meristem that is relatively simple in structure. Moreover, root development is not complicated by the formation of lateral appendages at the apex (Esau, 1965). The resulting regular arrangement of tissue and cell types simplifies the analysis of pattern formation and enables us to predict accurately the developmental history and fate of each of the tissues and cells from their location. Furthermore, cellular studies of root development are aided by the transparent nature of roots, and genetic analyses profit from the fact that roots of numerous seedlings growing on media of defined composition can be observed. In this review, we describe studies that are beginning to uncover the fundamental mechanisms controlling root development, with particular emphasis on the analysis of tissue and cell-type patterning. These studies point toward the general view that patterning in the root is largely guided by positional cues that are established during embryogenesis and maintained during postembryonic root development. Moreover, recent genetic and physical manipulations of developing root cells have led to new insights into the plasticity of root development. CELLULAR ORGANIZATION AND STRUCTURE OF ROOTS Roots develop in a largely continuous fashion from controlled cell proliferation and morphogenesis at the root apex. In most roots, the cell lineages are easily identified as orga- To whom correspondence should be addressed. schiefelo umich.edu; fax nized columns (or files) of cells along the length of the root that result from repeated transverse divisions in the meristematic region. Thus, the cell files resemble a cellular assembly line, with each cell in a file more developmentally advanced than the one beneath it (Figure 1 B). A basic feature of roots is their radial pattern, which is made up of concentric rings (or layers) of tissues. The three fundamental types of tissue are the protoderm (e.g., the epidermis), the ground tissue (e.g., the cortex), and the vascular tissue. The Arabidopsis root possesses these tissues in a radial array that is striking in its simplicity and regularity (Figures 1A and 1C). In the Arabidopsis primary root, there are single cell-thick rings of epidermis, cortex, endodermis, and pericycle tissues surrounding the central stele, with a constant number of eight cells per ring for the cortex and endodermis layers (Dolan et al., 1993). In many plant species, including Arabidopsis, the files of root cells can be traced back to specific sets of cells in the meristematic region, termed initials (von Guttenberg et al., 1955; Dolan et al., 1993; Scheres et al., 1994). Based on anatomical and labeling studies, each set of initials is thought to generate a specific tissue(s) by regulated cell divisions (Figures 16 and 1 D; Dolan et al., 1993; Baum and Rost, 1996). Together, the entire cluster of initials and the special cells at the center of the cluster (termed the quiescent center) make up the promeristem, which is believed to constitute the minimal group of root meristem cells (Clowes, 1961). To trace back further the lineage of the cells that make up the Arabidopsis root meristem, Scheres et al. (1994) used anatomical and clonal analyses to develop a fate map that defines the embryonic origin of the root meristem. This work showed that the promeristem is composed of cells derived from each of the two cells (apical and basal) that are generated by the first zygotic division of the embryo. The basal cell gives rise to the quiescent center and columella root cap regions, whereas cells derived from the apical cell give rise to the remainder of the promeristem. This study also showed that the cells generating the three basic tissue types are separated by distinct clonal boundaries during embryogenesis and that the architecture of the root promeristem is formed by the heart stage of embryogenesis (Scheres et al., 1994; see Laux and Jürgens, 1997, in this issue).

2 1090 The Plant Cell Jjjjg^ endodermis lateral root cap Figure

3 Patterning in Roots 1091 PAlTERNING OF TISSUE TYPES WlTHlN THE ROOT A fundamental question in root development centers on the nature of the radial patterning mechanism that defines the type of tissue that forms within each concentric ring of cells. The fact that each tissue alises from a specific set of initials led to the formulation of the histogen concept, which posits that each set of meristem initials is programmed to generate cells that will form only one tissue type (Hanstein, 1870). An alternative view is that positional information is of major importance in defining the characteristics of the initials and the fate of their daughters, a model supported by studies of shoot development (Poethig, 1987, 1997, in this issue). Recently, an elegant set of laser ablation studies designed to address this issue was conducted on cells in the Arabidopsis root promeristem (van den Berg et al., 1995). After ablation, the developmental fates of root cells were analyzed by tissue-specific marker gene expression. In one experiment, the ablation of quiescent center cells led them to collapse, and the available space became occupied by cells originating from the vascular initials. These displaced cells took on characteristics typical of quiescent center cells, as assessed by their subsequent expression of a columelld quiescent center marker gene. Similarly, the ablation of cortical cell initials enabled pericycle cells to expand into the cortical cell spaces, where they initiated the expression of a cortex rnarker gene. The position-dependent switch in the fate of cells that cross radial tissue boundaries implies that positional cues rather than lineage-based determinants direct the tissue-type characteristics of the root meristem initials and their descendants. Additional evidence that the radial pattern of root tissue types is not strictly controlled by lineage-dependent factors has been provided by the analysis of the fass and tonneau (ton) mutants of Arabidopsis (Torres-Ruiz and Jürgens, 1994; Scheres et al., 1995; Traas et al., 1995). The normally regular pattern of cell division within the root meristem is disrupted in these mutants, and unorganized cell divisions lead to an excessive number of cells and cell layers. If lineage information controlled tissue-type patterning, then the fass and ton roots would be expected to exhibit a disorganized collection of tissue types in inappropriate cell layers. However, despite the disruption in cell division patterns, fass and ton roots possess an organized radial pattern of tissue types (Torres-Ruiz and Jürgens, 1994; Scheres et al., 1995; Traas et al., 1995). Laser ablation studies were also able to provide insight into the directionality of the positional signals that define the root tissue types (Van den Berg et al., 1995). When cells located above (proximal to) a cortical initial were ablated such that contacts with more mature cortical cells were disrupted, the cortical initial failed to behave it did not undergo characteristic divisions). These observations imply that cortical initials require information from differentiating cortical cells located above them to guide their fate (van den Berg et al., 1995). Extrapolating further, it is possible that the entire radial pattern of root tissues is controlled by a mode of topdown transfer of information from more mature tissues that continuously guides the development of the immature cells and maintains the previously established pattern. What is the molecular basis of the positional cues and how are they conveyed to guide radial tissue patterning? One promising approach that is likely to shed light on this question is the identification and analysis of mutants exhibiting abnormal root tissue patterning; such mutants may define genes with a role in the production or propagation of positional signals. Severa1 radial patterning mutants have been identified in Arabidopsis by screening for seedlings with retarded root growth (Benfey et al., 1993; Scheres et al., 1995). These include the recessive mutants shortroot (shr), which lacks the endoderrnis tissue; scarecrow (scr), which lacks one of the two ground tissue layers; gollum (glm), which alters the organization of vascular tissue and pericycle; and wooden leg (wol), which alters the vascular tissue. The comparative analysis of the shr and scr mutants has been particularly revealing because it suggests that different processes related to tissue patterning are affected in each of these mutants. The shr and scr mutants are similar in that each has a single ground tissue layer, whereas wild-type roots possess two layers (cortex and endodermis) that are derived from a common set of initials via an asymmetric division (Figure 1D). Because this single tissue layer in the shr mutant has cortex characteristics and because endodermal cells are not generated in the multicellular fass shr double mutant, the SHR gene may be required to specify endodermis identity (Scheres et al., 1995). By contrast, the single ground tissue layer in the scr mutant has characteristics of Figure 1. (continued). (A) Transverse section of an Arabidopsis root from the late meristematic region. A single layer of lateral root cap cells surrounds the epidermis. At this stage of development, the two differentiating epidermal cell types can be distinguished cytologically; the eight trichoblasts possess densely staining cytoplasm compared with the neighboring atrichoblasts. Bar = 10 pn. (6) Median longitudinal section of an Arabidopsis root apex. The cell files converge to the promeristem region (outlined in red). (C) Colorized drawing of the transverse section shown in (A). (D) Colorized drawing of the promeristem region outlined in (6). The red lines indicate the planes of division that occur in the initials; white lines indicate the secondary divisions that occur in the corticavendoderma1 and the lateral root cap/epidermal initials.

4 1092 The Plant Cell both cortex and endodermis, and both tissue types are generated in the scr fass double mutant. These data imply that SCR activity is required for the generation of two distinct ground tissue layers (i.e., it affects the asymmetric division of the cortical/endodermal initial) but that it is not involved in specifying tissue type (Scheres et al., 1995). Molecular cloning and sequencing of the SCR gene suggest that it may encode a transcription factor and, as predicted from the genetic analyses, that it is expressed in the developing ground tissue of the embryo and seedling (Di Laurenzio et al., 1996). As more genes controlling radial patterning are analyzed at the molecular level, it will be interesting to investigate the interplay between pathways that control particular cell divisions and those that control the specification of tissue type. An interesting finding from the study of the radial patterning mutants is that the defect in each mutant is manifested in the tissues of the hypocotyl and the embryonic root as well as the seedling root (Scheres et al., 1995). This implies that the radial pattern is established during embryogenesis and that the mutations cause cell division and/or tissue specification defects that alter radial patterning throughout the entire embryonic axis. Other evidence for overlap in the control of hypocotyl and root development comes from the embryonic pattern mutant monopteros, which displays defects in both of these regions (Berleth and Jürgens, 1993; see also Laux and Jürgens, 1997, in this issue). The findings discussed thus far point to a general explanation of root tissue patterning as being guided by tissuespecific signaling of developing cells by more mature cells, with the initial architecture established by embryonic patterning genes. The patterning of some of the root tissues, however, cannot be explained by this sort of scheme. For example, the quiescent center and the columella root cap tissue do not make up a part of the radial pattern; rather, they represent portions of the apical-basal pattern of seedling tissues. To define the mechanisms responsible for programming these dista1 root tissues and for organizing the root meristem as a whole, mutants have been identified that lack a functional root meristem (Scheres et al., 1996). This group of mutants, which includes hobbit and bombadil, possesses a seedling- or adult-lethal phenotype and exhibits altered development of the hypophyseal cell and/or its derivatives, emphasizing the importance of this set of embryonic cells in generating a functional root meristem (Scheres et al., 1996). Further analysis of these loci is expected to provide new insights into the embryonic signaling events that are responsible for establishing and organizing the root meristem. PAlTERNlNG OF CELL TYPES WlTHlN ROOT TISSUE A central goal in developmental biology is to understand how diverse cell types are generated. More specifically, in tissues that contain cells with more than one developmental fate, how do individual cells acquire their distinct identities? This question is addressed in severa1 of the reviews in this issue (see, e.g., Fukuda, 1997; Larkin et al., 1997; Nelson and Dengler, 1997; Poethig, 1997), but the formation of the root epidermis during root development provides an exceptional model for studying this problem in plants. In most species, there are only two cell types in the root epidermis: cells that produce long hairlike extensions (i.e., root hair cells, which are derived from trichoblasts) and cells that do not &e., hairless cells, which are derived from atrichoblasts; Cormack, 1949, 1962; Cutter, 1978). The arrangement of these two cell types within the epidermis varies in different plant species. In some plants, there is no apparent pattern of root hair and hairless cells, and each newly formed epidermal cell appears to be capable of differentiating into either cell type (Leavitt, 1904). In other species, including many monocots, epidermal cell fate is linked to an asymmetric cell division, with the smaller daughter cell differentiating into a root hair cell and the larger daughter cell generating one or more mature hairless cells (Sinnott and Bloch, 1939; Avers, 1963; Cutter and Feldman, 1970). In a third group of plants, which includes Arabidopsis and other members of the Brassicaceae, a distinct position-dependent pattern of epidermal cell types is generated (Cormack, 1935; Bunning, 1951; Dolan et al., 1994; Galway et al., 1994). In this case, trichoblasts form in the crevice between underlying cortical cells (outside an anticlinal cortical cell wall), whereas atrichoblasts develop over a cortical cell (outside a periclinal cortical cell wall; Figures 2A and 26). The simple correlation between cell position and cell-type differentiation in the epidermis of Brassica species implies that lateral signaling events between cortical and epidermal cells help to define cell identity. At what point during epidermis development does this signaling take place? Trichoblasts and atrichoblasts exhibit cytological differences within the meristematic region of the root, which indicates that cell-type specification begins at an early stage of development (Figure 1A Cormack, 1962; Cutter, 1978; Dolan et al., 1994; Galway et al., 1994). However, it appears that developmental plasticity is also a feature of root epidermis cell differentiation. Indeed, three different experimental approaches show that epidermal cells that have embarked on one differentiation pathway can switch to the alternate pathway at a relatively late stage. These analyses include surgical experiments in which atrichoblasts can be induced to form root hairs when they are physically separated from the underlying cortical cells (Bunning, 1951), anatomical studies in which trichoblast daughter cells from rare longitudinal divisions develop as hairless cells (F. Berger, G. Hung, L. Dolan, and J.W. Schiefelbein, unpublished results), and pharmacological experiments in which hormone treatments induce root hair production on atrichoblasts (Tanimoto et al., 1995; Masucci and Schiefelbein, 1996). Arabidopsis mutations that disrupt the normal pattern of epidermal cell types have also been identified. Two of these

5 Patterning in Roots 1093 patterning mutants, transparent testa glabra (ttg) and possess root hairs on essentially every root epidermal cell. This implies that the role of the TTG and GL2 genes is either to promote hairless cell differentiation or to repress root hair cell differentiation (Galway et al., 1994; DiCristina et al., 1996; Masucci et al., 1996). Although the TTG gene has not been cloned, ttg mutations can be functionally complemented by expression of the maize R cdna under the control of the strong cauliflower mosaic virus 35s promoter (Lloyd et al., 1992; Galway et al., 1994). These data indicate that TTG is or activates a homolog of R, which encodes a protein related to basic helix-loop-helix transcriptional activators (Ludwig et al., 1989; see also Larkin et al., 1997, in this issue). The GL2 gene encodes a homeodomain protein (Rerie et al., 1994) and is preferentially expressed in the differentiating hairless epidermal cells within the meristematic and elongation regions of the root (Figures 2C and 2D; Masucci et al., 1996). Thus, the emerging picture is that TTG and GL2 may encode transcription factors that are expressed in a cell position-dependent manner to specify the hairless cell type in the root epidermis. Furthermore, because GL2 affects only a subset of the hairless cell characteristics that are affected by TTG (Galway et al., 1994; Masucci et al., 1996), TTG may act at an early stage to regulate the expression of GL2 and other as yet unidentified genes (Figure 3). The TTG and GL2 genes are known also to affect trichorne (shoot epidermal hair) formation in Arabidopsis, which provides evidence for overlap in the genetic control of epidermis development in the shoot and root (Galway et al., 1994; Marks, 1994; Masucci et al., 1996; see Larkin et al., 1997, in this issue). This outcome was unexpected because the cell-type specification mechanisms operating in these two tissues appear to be different. In the root epidermis, cell-type specification appears to rely on the positional relationship between epidermal cells and underlying cortical cells, whereas in the shoot epidermis, cell-type specification appears to be based on the sensing of trichome density. A further interesting aspect of TTG and GL2 is that they affect epidermal hair formation in opposite ways in root and shoot organs; they are required for the formation of hairless cells in the root and for hair-bearing (trichome) cells in the shoot. Although the situation is unclear at present, it is possible that TTG and GL2 encode general epidermis transcription factors that have been recruited to play distinct roles in cell-type specification in shoots and roots during angiosperm evolution. The analysis of another group of Arabidopsis mutants has been instrumental in linking root hair formation to the action of plant hormones, particularly ethylene and auxin. Mutations affecting the CONSTITUTIVE TRIPLE RESPONSEl (CTR7) locus, which encodes a Raf-like protein kinase proposed to negatively regulate the ethylene signal transduction pathway (Kieber et al., 1993), cause root hairs to form on epidermal cells that normally are hairless (Dolan et al., 1994). The hairless root phenotype of the dwad (dwf; auxin-resistant) and auxin resistant2 (a ; auxin-, ethylene-, and abscisic acid-resistant) mutants implicate auxin as another possible regulator of root hair formation (Mizra et al., 1984; Wilson et al., 1990). In addition, the hairless phenotype of the root hair defective6 (rhd6) mutant can be suppressed by the inclusion of 1 -arnino-cyclopropane-1-carboxylic acid (ACC; an ethylene precursor) or indole-3-acetic acid (IAA; an auxin) in the growth media, further implicating ethylene and auxin action in root hair initiation (Figure 3; Masucci and Schiefelbein, 1994). Finally, pharmacological studies with wild-type Arabidopsis seedlings show that root hair formation is enhanced by treatment with ACC (Tanimoto et al., 1995) and reduced by treatment with aminoethoxyvinylglycine (AVG), which is an ethylene biosynthesis inhibitor (Masucci and Schiefelbein, 1994; Tanimoto et al., 1995), or with Ag+, which is an inhibitor of ethylene perception (Tanimoto et al., 1995). Given the involvement of hormones in epidermal cell differentiation, one attractive possibility is that ethylene and/or auxin may act as a diffusible signal responsible for epidermal cell-type patterning. According to this notion, TTG and GL2 may represent downstream transcription factor genes that are regulated by the hormones in a cell position-dependent manner. To test this possibility, the relationship between the TTGIGL2 pathway (negative regulators of root hair cell differentiation) and the ethylene/auxin-related pathway (positive regulators of root hair cell differentiation) has been analyzed (Masucci and Schiefelbein, 1996). By contrast with the above model, the results from epistasis tests and GL2 promoterreporter gene analyses indicate that the ethylene/auxin pathway does not regulate the TTGIGL2 pathway. In addition, studies of the developmental timing of the hormone effects indicate that ethylene and auxin pathways can promote root hair outgrowth after epidermal cell-type characteristics have developed (Masucci and Schiefelbein, 1996). Taken together, the results suggest that the TTGIGL2 pathway acts upstream of or independently from the ethylene/ auxin pathway to define the pattern of cell types in the root epidermis (Figure 3). lnteresting aspects of this model are the implications that all newly formed epidermal cells in the Arabidopsis root may initially have the capacity to differentiate into root hair cells and that the action of the TTGIGL2 pathway is required to induce the hairless cell fate (i.e., a root hair cell may represent the default fate). An important future goal is to identify additional components of the epidermal cell patterning mechanism, particularly regulators of the TTG and GL2 genes. CONTROL OF MORPHOGENESIS IN ROOTS The molecular mechanisms responsible for regulating the structure and shape of plant organs and their component cells are largely unknown. Because roots exhibit a regular and simple structure and their development is experimentally accessible, they have been widely used in studies of organ and cell morphogenesis. The defined complement of

6 1094 The Plant Cell A C CJ Epidermis Cortex Figure

7 Patterning in Roots 1095 cell types and cell shapes and the ease with which genetic screens and molecular cloning experiments can be conducted make the Arabidopsis root a particularly useful system for these studies. One of the interesting issues related to morphogenesis in plants is the relative contribution of cell division to the size and shape of organs (see also Jacobs, 1997; Poethig, 1997, in this issue). lnsight into this issue has been obtained from the analysis of roots from transgenic Arabidopsis that overexpress a mitotic cyclin gene, which normally acts as a key regulator of cell division (Doerner et al., 1996). These transgenic roots exhibit an increased cell division rate, which leads to increases in cell number and organ growth &e., longer roots) but causes no significant changes in meristem organization or root morphology (Doerner et al., 1996). Similarly, the basic morphology of Arabidopsis roots is not altered by the application of redox agents that stimulate or inhibit cell proliferation (Sánchez-Fernández et al., 1997), nor is it altered in transgenic plants expressing modified versions of a cyclin-dependent kinase (Hemerly et al., 1995). These observations imply that the form of a plant organ may be largely insensitive to changes in cell number, which supports the concept of a higher order regulation of organ shape, rather than control at the leve1 of the cell (Kaplan and Hagemann, 1991). Although cell proliferation may not represent a primary regulator of organ morphology, some cell division activity is obviously required for organ formation. This point has been clearly emphasized by the study of the rootmeristemless (rml7 and rm12) mutations of Arabidopsis (Cheng et al., 1995). Embryogenic root development is normal in these mutants, but cell divisions in the postembryonic root are lacking or greatly reduced, which leads to exceedlingly small roots (Cheng et al., 1995). These analyses suggest that the RML genes are required to activate cell division in the apical cells of roots that have reached a critical size. An interesting future goal is to determine whether the RML proteins act only as a "trigger" to instigate root meristem activity or whether they are required continuously to maintain (and perhaps to regulate) cell proliferation in the root meristem. RHD6 TTG 1 GL2 et hylene/auxin-regulated root hair outgrowth Figure 3. Tentative Model for the Control of Cell-Type Differentiation in Arabidopsis Root Epidermis. The TTG and GL2 genes are proposed to act on developing epidermal cells in a specific position (those located over cortical cells) to inhibit the action of the RHD6 gene product and the ethylenelauxin pathway, thereby preventing these cells from adopting the root hair fate and generating the wild-type pattern of root hair and hairless cell types. Evidence for this model is summarized in the text. Arrows indicate positive action; T-bars indicate negative regulation; question marks indicate unclear relationships. The ongoing analysis of severa1 additional root morphology mutants is contributing to our understanding of the genetic and molecular mechanisms that regulate organ morphogenesis in Arabidopsis (Baskin et al., 1992, 1995; Benfey et al., 1993; Holding et al., 1994; Hauser et al., 1995). In many of these mutants, the abnormal root morphology is Figure 2. (continued). (A) Schematic drawing of an Arabidopsis root in surface view in the root hair developmental zone. Files of epidermal cells consist entirely of root hair cells or hairless cells. (B) Schematic drawing of a transverse section from the mature portion of an Arabidopsis seedling root. Root hair cells are located in the clefts between adjacent cortical cells. Only three root hairs are shown in this figure because the length of epidermal cells generally prevents all eight root hair projections from being visible in a single transverse section. (C) Spatial expression pattern of the GL2 promoter-p-glucuronidase reporter gene fusion construct in the root apex of a 4-day-old seedling. The GL2 promoter directs expression preferentially in a cell file-specific manner in the epidermis. Bar = 20 pm. (D) Expression pattern of the GL2 promoter-p-glucuronidase reporter gene fusion construct in a transverse root section from the late meristematic region of a 4-day-old seedling. The GL2 promoter directs expression preferentially in epidermal cells located over the cortical cells (atrichoblasts). Bar = 1 O pm.

8 1096 The Plant Cell dueto excessive cell expansion that is largely restricted to a particular tissue. For example, the major morphological abnormality in the cobra and root epidermal blebbing mutants is excessive swelling of the epidermis, the sabre mutant displays abnormal expansion primarily in the cortex, and the lion s tail mutation principally causes abnormal cell enlargement in the stele (Baskin et al., 1992; Benfey et al., 1993; Hauser et al., 1995). These results indicate that organ morphogenesis is not likely to be governed by a single overriding pathway but probably relies on the independent control of the orientation and extent of cell expansion in different tissues. Studies of Arabidopsis root morphology mutants have also called into question the long-held view that the orientation of microtubules plays a primary role in influencing the orientation of cell expansion (Baskin et al., 1994; Hauser et al., 1995). The cloning of some of the root morphogenesis genes is beginning to shed light on the molecules involved in coordinated organ growth. The SABRE gene product encodes a nove1 protein that appears to be required to counter the effects of ethylene during radial cell expansion (Aeschbacher et al., 1995). The RHD3 gene, which is required for appropriate cell enlargement throughout the Arabidopsis plant, encodes a protein with putative GTP binding motifs, and RHD3-related proteins have been identified in yeast, rice, and the parasite Entamoeba histolytica (Wang et al., 1997). Together, these studies show that despite the complexity of organ and cell morphogenesis, the root is a suitable subject for untangling the interrelated molecular mechanisms that control these processes. CONCLUDING REMARKS A related feature of root development emphasized by these studies is the developmental plasticity exhibited by cells or tissues. There are mw numerous examples in which root cells that begin to differentiate in one manner switch to a different program upon a change in their position or in response to externa1 factors. This plasticity may be important to ensure proper cell and tissue patterning in the event of rare abnormal cell divisions or organ damage. An important future goal in studies of root patterning is to identify the molecular nature of the positional cues. Achieving this objective will probably require more sophisticated genetic screens and the use of tissue- or cell-specific markers to identify pattern mutants rather than structural (e.g., cell division) or morphological (e.g., cell expansion) mutants. For example, it would be particularly informative to identify and analyze mutants that possess roots with a normal number of cell layers but inappropriate tissue types occupying one or more of those layers. With regard to the molecular basis for the positional cues, there are many possibilities. Positional information may be communicated by a cell wall component(s) (Berger et al., 1994; Freshour et al., 1996), by diffusible signals that act symplastically (Duckett et al., 1994), or by some other, as yet unknown, signaling mechanism. ACKNOWLEDGMENTS We thank our colleagues in the root development community, including Philip Benfey, Fred Berger, Liam Dolan, Chen-Yi Hung, Jocelyn Malamy, Mike May, and Ben Scheres, for their helpful discussions and for providing manuscripts before publication. A general theme that has emerged from studies of root patterning is that positional cues play a critical role in controlling the identity of the component tissues and cells. This conclusion may be somewhat unexpected, given the strict correlation between cell lineage and cell/tissue types in the roots of some species, such as Arabidopsis. Nonetheless, the examples of genetic and physical manipulations of root development outlined in this review show that the history of a cell or tissue is less important than is its position in determining its developmental fate. Thus, it may be that the strict correlation between cell lineage and cell-type patterns found in some roots (e.g., Arabidopsis) is simply the result of a regular array of cell divisions overlaid on a defined set of positional cues. Furthermore, the fact that the roots of some species lack such a lineage-correlated pattern of cell and tissue types may reflect a difference in their meristem structure (e.g., different cell division patterns) rather than a difference in the basic patterning mechanism, because the same tissues are arranged in basically the same way in all roots. Thus, a common set of positional cues that transcends taxonomic boundaries may be used to direct the patterns of tissue and cell types during root development. REFERENCES Aeschbacher, R.A., Hauser, M.-T., Feldmann, K.A., and Benfey, P.N. (1995). The SABRE gene is required for normal cell expansion in Arabidopsis. Genes Dev. 9, Avers, C.J. (1963). Fine structure of Phleum root meristem cells. II. Mitotic asymmetry and cellular differentiation. Am. J. Bot. 50, Baskin, T.I., Betzner, A.S., Hoggart, R., Cork, A., and Williamson, R.E. (1992). Root morphology mutants in Arabidopsis thaliana. Aust. J. Plant Physiol. 19, Baskin, T.I., Wilson, J.E., Cork, A,, and Williamson, R.E. (1994). Morphology and microtubule organization in Arabidopsis roots exposed to oryzalin or taxol. Plant Cell Physiol. 35, Baskin, T.I., Cork, A., Williamson, R.E., and Gorst, R. (1995). STUNTED PLANT7, a gene required for expansion in rapidly elongating but not in dividing cells and mediating root growth responses to applied cytokinin. Plant Physiol. 107, Baum, S.F., and Rost, T.L. (1996). Root apical organization in Arabidopsis thaliana. I. Root cap and protoderm. Protoplasma 192,

9 Patterning in Roots 1097 Benfey, P.N., Linstead, P.J., Roberts, K., Schiefelbein, J.W., Hauser, M.-T., and Aeschbacher, R.A. (1993). Root development in Arabidopsis: Four mutants with dramatically altered root morphogenesis. Development 119, Berger, F., Taylor, A., and Brownlee, C. (1994). Cell fate determination by the cell wall in early Fucus development. Science 263, Berleth, T., and Jürgens, G. (1993). The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo. Development 118, Bunning, E. (1951). Uber die Differenzierungsvorgange in der Cruciferenwurzel. Planta 39, Cheng, JA., Seeley, K.A., and Sung, Z.R. (1995). RML7 and RML2, Arabidopsis genes required for cell proliferation at the root tip. Plant Physiol. 107,365376, Clowes, F.A.L. (1 961). Apical Meristems. (Oxford, UK: Blackwell Scientific). Cormack, R.G.H. (1 935). The development of root hairs by flodea canadensis. New Phytol. 34, Cormack, R.G.H. (1949). The development of root hairs in angiosperms. Bot. Rev. 15, Cormack, R.G.H. (1 962). Development of root hairs in angiosperms. II. Bot. Rev. 28, Cutter, E.G. (1978). The epidermis. In Plant Anatomy (London: Clowes and Sons), pp Cutter, E.G., and Feldman, L.J. (1 970). Trichoblasts in Hydrocharis. 1. Origin, differentiation, dimensions and growth. Am. J. Bot. 57, DiCristina, M.D., Sessa, G., Dolan, L., Linstead, P., Baima, S., Ruberti, I., and Morelli, G. (1996). The Arabidopsis Athb-10 (GUBRA2) is an HD-Zip protein required for regulation of root hair development. Plant J. 10, Di Laurenzio, L., Wysocka-Diller, J., Malamy, J.E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M.G., Feldmann, K.A., and Benfey, P.N. (1996). The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell86, Doerner, P., Jorgensen, J.-E., You, R., Steppuhn, J., and Lamb, C. (1996). Control of root growth and development by cyclin expression. Nature 380, Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, R.S., Roberts, K., and Scheres, B. (1993). Cellular organisation of the Arabidopsis thaliana root. Development 119, Dolan, L., Duckett, C., Grierson, C., Linstead, P., Schneider, K., Lawson, E., Dean, C., Poethig, R.S., and Roberts, K. (1994). Clonal relations and patterning in the root epidermis of Arabidopsis. Development 120, Duckett, C.M., Oparka, K.J., Prior, D.A.M., Dolan, L., and Roberts, K. (1994). Dye-coupling in the root epidermis of Arabidopsis is progressively reduced during development. Development 120, Esau, K. (1965). Plant Anatomy. (New York: John Wiley and Sons). Freshour, G., Clay, R.P., Fuller, M.S., Albersheim, P., Darvill, A.G., and Hahn, M.G. (1996). Developmental and tissue-specific structural alterations of the cell-wall polysaccharides of Arabidopsis thaliana roots. Plant Physiol. 110, Fukuda, H. (1997). Tracheary element differentiation. Plant Cell 9, Galway, M.E., Masucci, J.D., Lloyd, A.M., Walbot, V., Davis, R.W., and Schiefelbein, J.W. (1994). The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root. Dev. Biol. 166, Hanstein, J. (1 870). Die Entwicklung des Keimes der Monocotyledon und der Dikotylen. (Bonn, Germany: Botanische Abhandlung). Hauser, M.-T., Morikami, A., and Benfey, P.N. (1995). Conditional root expansion mutants of Arabidopsis. Development 121, Hemerly, A., de Almeida Engler, J., 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, Holding, D.R., McKenzie, R.J., and Coomber, S.A. (1994). Genetic and structural analysis of five Arabidopsis mutants with abnormal root morphology generated by the seed transformation method. Ann. Bot. 74, Jacobs, T. (1997). Why do plant cells divide? Plant Cell9, Kaplan, D.R., and Hagemann, W. (1991). The relationship of cell and organism in vascular plants. BioScience 41, Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, J.R. (1993). CTR7, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell72, Larkin, J.C., Marks, M.D., Nadeau, J., and Sack, F. (1997). Epidermal cell fate and patterning in leaves. Plant Cell9, Laux, T., and Jürgens, G. (1997). Embryogenesis: A new start in life. Plant Cell 9, Leavitt, R.G. (1904). Trichomes of the root in vascular cryptograms and angiosperms. Proc. Boston SOC. Nat. Hist. 31, Lloyd, A.M., Walbot, V., and Davis, R.W. (1992). Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258, Ludwig, S.R., Habera, L.F., Dellaporta, S.L., and Wessler, S.R. (1989). Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc. Natl. Acad. Sci. USA 86, Marks, M.D. (1994). The making of a plant hair. Curr. Biol. 4, Masucci, J.D., and Schiefelbein, J.W. (1994). The rhd6 mutation of Arabidopsis thaliana alters root-hair initiation through an auxinand ethylene-associated process. Plant Physiol. 106, Masucci, J.D., and Schiefelbein, J.W. (1996). Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root. Plant Cell 8, Masucci, J.D., Rerie, W.G., Foreman, D.R., Zhang, M., Galway, M.E., Marks, M.D., and Schiefelbein, J.W. (1996). The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Awbidopsis thaliana. Development 122,

10 1098 The Plant Cell Mizra, J.I., Olsen, G.M., Iversen, T.-H., and Maher, E.P. (1984). The growth and gravitropic responses of wild-type and auxin resistant mutants of Arabidopsis thaliana. Physiol. Plant. 60, Nelson, T., and Dengler, N. (1997). Leaf vascular pattern formation. Plant Cell9, Poethig, R.S. (1987). Clonal analysis of cell lineage patterns in plant development. Am. J. Bot. 74, Poethig, R.S. (1 997). Leaf morphogenesis in flowering plants. Plant Cell9, Rerie, W.G., Feldmann, K.A., and Marks, M.D. (1994). The GLABRAP gene encodes a homeodomain protein required for normal trichome development in Arabidopsis. Genes Dev. 8, Sánchez-Fernández, R., Fricker, M., Corben, L.B., White, N.S., Sheard, N., Leaver, C.J., Van Montagu, M., Inzé, D., and May, M.J. (1997). Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control. Proc. Natl. Acad. Sci. USA 94, Scheres, B., Wolkenfelt, H., Willemsen, V., Terlouw, M., Lawson, E., Dean, C., and Weisbeek, P. (1994). Embryonic origin of the Arabidopsis primary root and root meristem initials. Development 120, Scheres, B., Di Laurenzio, L., Willemsen, V., Hauser, M.-T., Janmaat, K., Weisbeek, P., and Benfey, P.N. (1995). Mutations affecting the radial organization of the Arabidopsis root display specific defects throughout the embryonic axis. Development 121,5342. Scheres, B., McKhann, H., Van den Berg, C., Willemsen, V., Wolkenfelt, H., de Vrieze, G., and Weisbeek, P. (1996). Experi- mental and genetic analysis of root development in Arabidopsis thaliana. Plant Soil 187, Sinnott, E.W., and Bloch, R. (1939). Cell polarity and the differentiation of root hairs. Proc. Natl. Acad. Sci. USA 25, Tanimoto, M., Roberts, K., and Dolan, L. (1995). Ethylene is a positive regulator of root-hair development in Arabidopsis thaliana. Plant J. 8, Torres-Ruiz, R.A., and Jürgens, G. (1994). Mutations in the FASS gene uncouple pattern formation and morphogenesis in Arabidopsis development. Development 120, 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, Van den Berg, C., Willemsen, V., Hage, W., Weisbeek, P., and Scheres, 6. (1995). Cell fate in the Arabidopsis root meristem determined by directional signaling. Nature 378, von Guttenberg, H., Burmeister, J., and Brosell, H.-J. (1955). Studien uber die Entwicklung des Wurzelvegetationspunktes der Dikotyledonen. II. Planta 46, Wang, H.-Y., Lockwood, S., Hoeltzel, M., and Schiefelbein, J.W. (1997). The ROOTHAlR D F CT/V 3 gene encodes an evolutionarily conserved protein with GTP-binding motifs and is required for regulated cell enlargement in Arabidopsis. Genes Dev. 11, Wilson, A., Pickett, F.B., Turner, J.C., and Estelle, M. (1990). A dominant mutation in Arabidopsis confers resistance to auxin, ethylene, and abscisic acid. MOI. Gen. Genet. 222,