Laurie G. Smith, Division of Biological Sciences, University of California, San Diego

May, 2006

During plant development, cell walls ensure that the relative positions of cells change little, if any. Consequently, the cellular organization of a mature plant tissue closely reflects the pattern of cell division during its development. In some species and tissue types, a virtually invariant sequence of oriented divisions elaborates a characteristic cell pattern. For example, stereotypical division patterns in the root tips of Azolla (a fern) and Arabidopsis (a dicot) establish the very regular arrangement of cells in these tissues (Gunning et al., 1978; Dolan et al., 1994). In other tissues, such as the maize leaf, division pattern is more variable but nevertheless follows certain general rules that preserve a characteristic cellular organization (Langdale et al., 1989; Sylvester et al., 1990). Therefore, it is perhaps not surprising that plant cells appear to carefully control their division planes.

How does a plant cell preparing to divide choose an appropriate division plane? It was first recognized over 100 years ago that the division planes of most plant cells could be predicted simply from their shapes. In 1863, Hofmeister noted that new cell walls are usually formed in a plane perpendicular to the main axis of cell expansion—that is, perpendicular to the long axis of the mother cell. In 1888, Errera formulated the rule that the plane of division for most cells corresponds to the shortest path that will halve the volume of the mother cell, corresponding to a plane perpendicular to, and bisecting, the long axis of the mother cell. The notion that the plane of division can be dictated simply by cell geometry is further supported by experimental evidence. Spherical cultured cells suspended in semi-solid medium divide in random orientations. However, if these cells are squeezed into slightly oval shapes through application of a compressive force, the majority divide in a plane perpendicular to the oval's long axis (Lynch and Lintilhac, 1997). Although it is not fully known how a plant cell could read its shape and divide accordingly, Lloyd and colleagues proposed a model based on simple mechanical principles that could largely explain cells' ability to follow Hofmeister's and Errera's rules (Lloyd, 1991).

While simple geometrical rules predict the division planes of most cells, many do not follow these rules, and may be responding to local cues of some kind that override the influence of cell shape on the division plane. The asymmetric divisions involved in the formation of many specialized cell types such as stomatal complexes provide a good example. In grasses, stomata form through an invariant sequence of asymmetric divisions illustrated in Figure 1 (Stebbins and Shah, 1960). The first asymmetric division leads to the formation of a small guard mother cell, which will divide later to form a pair of guard cells. Before it divides again, asymmetric division of the guard mother cell's lateral neighbors (subsidiary mother cells) results in the formation of subsidiary cells, which will flank the guard cells. Prior to each of these asymmetric divisions, the mother cell becomes polarized, involving migration of the nucleus to one side of the cell as well as redistribution of the cytoplasm and other cell components. In these cells, the division plane is related to the cell's polarity rather than its overall shape. Polarization of subsidiary mother cells in particular is thought to be controlled by a signal of some kind from the guard mother cell. Other cells that divide in a manner not predicted by their shapes may similarly be responding to extracellular cues, but this remains to be determined.

Figure 1   Formation of stomatal complexes in grasses. Following an asymmetric division giving rise to a guard mother cell, the flanking subsidiary mother cells become polarized and divide asymmetrically to form stomatal subsidiary cells.

The process of cytokinesis itself—the partitioning of daughter nuclei and other cell components after mitosis into separate daughter cells—is quite different in plants compared to other eukaryotes. Whereas animal cells divide by forming a membrane constriction between daughter nuclei and eventually pinching in two, plant cells divide by building a new cell wall between daughter nuclei (called a cell plate while under construction). Cell plate formation is directed by a cytoskeletal structure unique to plant cells called a phragmoplast. As illustrated in Figure 2, a phragmoplast is composed of short microtubules and actin filaments organized into two parallel, interdigitating discs. In most dividing cells, where cytokinesis immediately follows mitosis, the phragmoplast arises from the mitotic spindle between daughter nuclei immediately after mitosis, and then expands radially to complete the formation of the cell plate (Figure 2). Cell plate formation depends most critically on the microtubule component of the phragmoplast (Gunning, 1982). Phragmoplast microtubules appear to direct the kinesin-driven transport of Golgi-derived vesicles containing cell wall components to the phragmoplast equator (Otegui et al., 2001), where they fuse with each other, eventually coalescing to form a new cell wall sandwiched between new plasma membranes (Samuels et al., 1995). A variety of proteins have been implicated in the membrane fusion events involved in cell plate formation, including a dynamin-like GTPase and several components of a SNARE complex (Assaad, 2001; Verma, 2001). In addition, cell plate formation has recently been shown to be regulated by a MAP kinase cascade (Nishihama and Machida, 2001).

Figure 2   Cytoskeletal organization in dividing plant cells. Drawings for prophase and interphase cells represent projections of a three-dimensional view showing both the cell surface and internal features. Drawings for cells in mitosis and cytokinesis represent mid-plane, cross-sectional views showing only the outlines of the cell cortex. During prophase, a cortical preprophase band (PPB) of microtubules (green) and actin filaments (red) lies in the future plane of cell division. Disassembly of the PPB creates an actin depleted zone in the cell cortex that persists and marks the division site throughout mitosis and cytokinesis. After completion of mitosis, a phragmoplast of microtubules and actin filaments arises between daughter nuclei, and guides the movement of Golgi-derived vesicles containing cell wall materials to the cell plate. As cytokinesis proceeds, the phragmoplast expands laterally until it fuses with the parental plasma membrane and cell wall at the cortical division site previously occupied by the PPB. (Smith, L. G. 2001, reprinted by permission from Nature Reviews Molecular Cell Biology.)

Given their distinct mode of cytokinesis, it is not surprising that plant cells also appear to utilize mechanisms different from those used by other eukaryotes to control their division planes. Unlike animal cells, in which the plane of division is determined during mitosis by the position of the mitotic spindle, the division planes of plant cells are determined earlier in the cell cycle, well before mitosis. Throughout prophase, a belt of cortical microtubules and actin filaments called the preprophase band (PPB) precisely predicts where the edges of the cell plate will attach to the parental membrane and wall at the conclusion of cytokinesis (Figure 2; Wick, 1991a). Although the PPB itself is disassembled upon entry into mitosis, some sort of imprint of its position is evidently left behind, because a variety of observations demonstrate that the phragmoplast is actively guided to this site as it expands during cytokinesis. For example, classic experiments have shown that if the spindle or nascent phragmoplast is displaced from the plane defined by the former PPB, the expanding phragmoplast will generally migrate or rotate to attach the cell plate at the former PPB site (Ota, 1961). Therefore it is thought that the PPB plays some role in establishing a division site in the cell cortex or plasma membrane during prophase that guides phragmoplast expansion during cytokinesis, but little is known about the nature of the division site or the role of the PPB in establishing this site.

Given the well established roles of cytoskeletal filaments in directing the intracellular movement of various cell components, an attractive hypothesis for the function of the PPB is that it guides the deposition of one or more molecules at the division site that remain there after the PPB itself is disassembled, and which later serve as landmarks to attract the expanding phragmoplast during cytokinesis (Mineyuki and Gunning, 1990). Observations of vesicle accumulation and cell wall thickening in the vicinity of the PPB in some cell types have suggested that localized secretion directed by the PPB might be involved in establishing the division site. However, a recent study in which dividing cells in culture were treated with brefeldin A (a drug that disrupts the secretory pathway) during prophase and then allowed to complete cytokinesis in the absence of the drug convincingly demonstrated that secretion during prophase is not required for division site establishment (Dixit and Cyr, 2002). In fact, no molecules that readily serve as markers of the division site have been identified.

An interesting feature of the cortical division site that may be related in some way to its function is that it is relatively deficient in F-actin. During prophase, actin filaments are found within the PPB and elsewhere in the cell cortex. Upon entry into mitosis, the F-actin component of the PPB is disassembled along with the microtubule component, but F-actin elsewhere in the cell cortex remains, creating an "actin-depleted zone" (ADZ) corresponding to the position of the former PPB (Cleary et al., 1992). In a wide variety of cell types, the ADZ has been shown to persist and mark the division site throughout mitosis and cytokinesis. It has been proposed that the ADZ could indirectly function to guide the expanding cell plate to the division site if the edges of the phragmoplast are repelled by cortical F-actin elsewhere. Alternatively, the local deficiency of cortical F-actin at the division site might reflect, or even be required to maintain, the presence of other molecules at the division site that function as positive landmarks to guide phragmoplast/cell plate expansion.

Related to the problem of understanding the nature of the division site and the role of the PPB in its establishment is the question of how the phragmoplast interacts with the division site as it expands. While the mechanisms governing this interaction are not known, it seems that cytoplasmic F-actin plays a significant role. It has long been known that treatment of dividing cells with actin-disrupting drugs causes them to divide in aberrant orientations (Wick, 1991b). This is probably due mainly to disruption of F-actin cables connecting the dividing nucleus and the edges of the phragmoplast to the cell cortex, predominantly at the division site (Figure 2). It is likely that these actin cables help to guide the phragmoplast to the division site. Moreover, recent work involving treatment of dividing cells with drugs affecting actin-myosin interactions suggest a role for one or more myosins in generating a force that promotes the lateral expansion of the cell plate and guides it toward the division site (Molchan et al., 2002).

Clearly there are many important, unanswered questions regarding how plant cells select and establish their division planes early in the cell cycle, and how the phragmoplast and associated cell plate are guided to the previously established division site during cytokinesis. How can these problems be tackled? Recent availability of complete genome sequences of Arabidopsis and rice combined with tools for reverse genetic analysis has opened new doors for analysis of the contributions of individual proteins suspected to be involved based on their amino acid sequences. However, one cannot predict all of the proteins involved based on sequence alone. To identify components of the molecular mechanisms involved in the spatial regulation of cytokinesis, we and others have taken a forward genetic approach by isolating mutants in which cells divide in aberrant orientations (Smith, 2001). Proteins encoded by some of the corresponding genes have recently been identified and studied. The maize Tan1 gene is required for properly oriented divisions in developing leaves and other tissues (Smith et al., 1996). Analysis of the cytoskeleton in tan1 mutants showed that the Tan1 gene is required for guidance of phragmoplasts to sites formerly occupied by a PPB (Cleary and Smith, 1998). The Tan1 gene is specifically expressed in young regions of developing leaves where cells are actively dividing. It encodes a highly basic protein that can bind to microtubules in vitro and belongs to a family of proteins preferentially associated with the cytoskeletal structures that are misoriented in tan1 mutants (PPBs, spindles and phragmoplasts; Smith et al., 2001). Thus TAN1 may be directly involved in division plane establishment and/or the interaction of the phragmoplast with the division site. The Arabidopsis TON2 gene is also required for properly oriented cell divisions, but unlike Tan1, TON2 is required for PPB formation (Traas et al., 1995). TON2 was recently shown to encode a protein likely to function as a regulatory subunit for a protein phosphatase 2A, which may directly regulate some aspect of cytoskeletal reorganization critical for formation of the PPB (Camilleri et al., 2002). Fitting all of this information into a cohesive model for division plane control will require identification of additional components involved in this process, analysis of their intracellular localization patterns and biochemical functions, and elucidation of the interactions among these these components.

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