IN THIS ISSUE

To Shape a Plant—The Cytoskeleton in Plant Morphogenesis

How different tissue and organ morphologies are reproducibly achieved is among the oldest questions in the study of developmental biology. Because plant cells are constrained by their cell walls and cannot migrate, plant morphology is generated by coordinately regulating the directions in which cells divide and expand. In most cells, expansion is maximal in the direction perpendicular to the previous division plane. For example, cells in the root are arranged in longitudinal files, and divisions perpendicular to the long axis of the root (anticlinal divisions) add more cells to a file. Expansion of the resulting daughter cells is greatest along the root axis, causing the root to extend in length.

The cytoskeleton plays an important role in governing the orientation of both cell division and cell expansion. Cell expansion during interphase is controlled in large part by parallel microtubules (MTs) in the cell cortex that are cross-bridged to each other and to the plasma membrane (Kropf et al. 1998 ). Most commonly, these MTs are oriented transverse to the long axis of the cell; they regulate cell expansion by guiding the deposition of cellulose microfibrils in the cell wall. Microfibrils are deposited in the same orientation as the underlying MTs, usually wrapping around and girdling the cell. Because microfibrils cannot be stretched lengthwise, this pattern of deposition causes anisotropic growth, in which expansion in the direction transverse to the orientation of cellulose microfibrils (and therefore transverse to MT orientation) predominates (Baskin et al. 1994 ). MTs, therefore, indirectly regulate growth anisotropy.

Both MTs and F-actin play important roles in orienting division planes. Early in the G2 phase of the cell cycle, the cortical array of interphase MTs gives way to a cortical MT band, termed the preprophase band (PPB), which is localized to the future division site. The PPB is transient and disappears as the mitotic spindle forms. Studies in several plant cell types have shown that spindle position is not strictly constrained by that of the PPB; indeed spindles are often observed in skewed orientations (Palevitz 1993 ; Goddard et al. 1994 ). As mitosis proceeds, the phragmoplast, a ring of antiparallel, interdigitating MTs and F-actin, forms in the midzone of the disassembling spindle. The phragmoplast MTs and F-actin are believed to participate in the transport of vesicles containing new membrane and wall materials to the growing cell plate. The phragmoplast and forming cell plate are initially perpendicular to the spindle, but as they expand toward the cortex of the cell, they realign so that fusion occurs at the site previously marked by the PPB. Therefore, in contrast to animals, fungi, and protists, in which spindle alignment is the major determinant of division plane orientation (Allen and Kropf 1992 ; Kropf 1997 ), establishment of division planes in angiosperm cells depends on 1) positioning the PPB and 2) guiding the phragmoplast to this predetermined cor-tical site.

Very little is known about PPB positioning or phragmoplast guidance. On pages 1875–1888 of this issue, Cleary and Smith analyze the cytoskeleton of the tangled1 (tan1) mutant of maize and present data indicating that Tan1 may be involved in both processes. In wild-type maize leaves, PPBs and cell divisions are either transverse or longitudinal with respect to the long axis of the cell. However, in tan1 mutants, leaf cell divisions are nearly randomly oriented (Smith et al. 1996 ). In the present study, Cleary and Smith show that PPBs in tan1 mutants are not randomly oriented, but are mainly positioned transverse to the cell axis. Longitudinal PPBs are almost never observed, leading the authors to propose that PPBs are positioned transversely by default. Tan1, then, may be involved in overriding the default pathway to specify the formation of longitudinal PPBs.

The fact that PPBs do not predict division planes in the cells of tan1 mutants suggests an additional defect in phragmoplast guidance. In tan1 mutants, phragmoplast position correlates with spindle orientation but not with PPB position, indicating that the phragmoplast forms in the correct location in the center of the spindle but fails to realign and fuse at the PPB site. This phenotype is reminiscent of the effects of disrupting F-actin by treating cells with cytochalasin. Following cytochalasin treatment, the phragmoplast is no longer guided to the PPB site; instead, fusion occurs wherever the cell plate happens to meet the cortex. This causes division plane orientation to be more random than it is in untreated control cells (Palevitz 1993 ).

The similarity between the tan1 phenotype and cytochalasin treatment stimulated Cleary and Smith to analyze F-actin arrays. During PPB formation in both wild-type and tan1 leaves, an area devoid of cortical F-actin, called the actin-depleted zone (ADZ), forms in the area that surrounds the narrowing MT band. In many plant cells, the ADZ persists throughout cytokinesis, and the phragmoplast and cell plate fuse with the parental wall at the site marked by the ADZ (Staehelin and Hepler 1996 ). It is therefore possible that the ADZ in tan1 mutant cells is not properly maintained during cytokinesis, permitting phragmoplast fusion to occur anywhere on the inner surface of the cells. Because it was not possible to consistently observe the ADZ during cell plate formation, however, the role of the ADZ in defining the site of phragmoplast fusion remains unclear.

The results reported by Cleary and Smith are intriguing, and further analyses of the Tan1 gene and the function of its product promise to provide insights into the mechanism by which division planes are determined in plant cells. It will be possible to address a number of important questions after we know what kind of protein the Tan1 gene encodes. For example, how might Tan1 function in both PPB specification and phragmoplast guidance? Is it a component of the phragmoplast itself, or does it localize to the ADZ at the cortical division site? Could Tan1 be a regulatory protein, such as a kinase? Indeed, kinases are associated with the PPB where they could activate many different molecules, some of which may be involved in phragmoplast guidance (Mineyuki et al. 1991 ; Colasanti et al. 1993 ).

In addition to its utility in studying the mechanism of division plane alignment, the tan1 mutant may permit analysis of the coordinate regulation of anisotro-pic expansion and division plane orientation during plant morphogenesis. Although the Arabidopsis mutants tonneau and fass are defective in these processes, their study has not shed much light on the issue of coordinate regulation. This is because the orientations of both division and expansion are disrupted in the mutant plants (Torres-Ruiz and Jurgens 1994 ; Traas et al. 1995 ; Fowler and Quatrano 1997 ; McClinton and Sung 1997 ). In tan1 mutants, however, expansion remains anisotropic (based on cell shape) despite the aberrant division planes (Smith et al. 1996 ).

The morphology of tan1 mutant leaves is relatively normal, suggesting that directional cell expansion may compensate for abnormal divisions to achieve correct organ morphology. (Because divisions are randomly oriented in tan1 leaves, the default pattern of cell expansion transverse to the previous division would result in isodiametric leaf expansion, and this clearly does not occur). To begin to assess cell expansion in tan1 mutants, Cleary and Smith analyzed the orientations of interphase cortical MTs that direct cellulose deposition. In wild-type cells, MTs are predominantly transverse with respect to the long axis of the cell, but this correlation between MT orientation and cell shape is less apparent in tan1 mutants. Instead, Cleary and Smith demonstrate that interphase MTs in tan1 leaves coalign across cell boundaries. This observation suggests that cell expansion is controlled on a regional or possibly a whole-leaf basis rather than in individual cells. Further analysis of cell expansion with respect to the long axis of the leaf in wild-type and tan1 mutants is needed to address directly whether directional expansion can compensate for abnormal divisions.

Observations of cytoskeletal arrays in live cells of expanding tissues would aid in understanding many of the issues raised above. For example, analyses of MT dynamics in phragmoplasts of living cells would help clarify how the phragmoplast scans the cortex and finds the appropriate site for fusion. Do phragmoplast MTs grow out and shrink back from incorrect sites? Or do the edges of the phragmoplast contact the cortex and slide to the correct position? Presumably, tan1 phragmoplasts are defective in this searching process.

On pages 1927–1939 of this issue, Marc et al. introduce a new technique for labeling plant MTs in vivo that will make studies of MT dynamics much more feasible. Until now, microinjection of fluorescently labeled tubulin has been the only technique available for observing MT dynamics, and microinjection is formidable in many cell types, including maize leaf epidermal cells. Marc et al. describe an MT reporter gene construct that can be bombarded into many plant cells to generate fluorescently labeled MTs. This construct includes a fusion of the green fluorescent protein (GFP) gene and the sequence encoding the MT binding domain (MBD) of a mammalian MT bundling protein, MT-associated protein 4 (MAP4). The construct generated by Marc et al. includes a "plant optimized" GFP that has improved spectral properties and plant-specific codon usage.

The authors present three lines of evidence to demonstrate that this plant GFP–MBD chimeric protein binds specifically to plant MTs, both in vitro and in vivo. First, a crude preparation of the fusion protein expressed in Escherichia coli cosediments with taxol-stabilized MTs. Second, the fusion protein localizes to MT arrays in fava bean epidermal cells, and third, this localization can be disrupted by MT depolymerizing agents or cold treatment. In addition, labeled MTs remain dynamic; Marc et al.'s time-lapse studies show that labeled MTs assemble, disassemble, and change orientation over the course of several minutes to hours.

The GFP–MBD reporter has several advantages over microinjection of fluorescently tagged tubulin. The method of introduction, particle bombardment, can be used on small cells that are difficult to microinject, works in a variety of plant species, is rapid and efficient, and allows many cells to be labeled at once. Also, the GFP–MBD fusion protein is less likely to affect cytoplasmic tubulin levels (which regulate MT dynamics) than is microinjection of labeled tubulin. Moreover, because the GFP–MBD fusion is not incorporated into MTs, it should label both dynamic and stable MT populations (only dynamic MTs incorporate tubulin following injection). Finally, the optical properties of GFP make the signal quite stable (Ormo et al. 1996 ), allowing repeated imaging without photobleaching for as long as several days. This feature will be especially useful in time-lapse studies.

One potential caveat to this technique is that by binding to MTs, the GFP–MBD may block the binding of endogenous MAPs and thereby increase bundling and change MT dynamics. Indeed, Marc et al. find that after long periods of expression (>10 hours) some cells have fewer and thicker filaments, indicative of increased bundling activity. Given the differences in method of labeling and delivery, it will be interesting to compare MT life spans in fluorescent tubulin–injected cells to GFP–MBD—bombarded cells.

Whether or not there are differences, this new MT reporter offers the potential to gain new insight into mechanisms controlling cell expansion and division by facilitating the investigation of MT dynamics in living cells of tissues and organs undergoing morphogenesis. For example, observing MT dynamics in cells of wild-type and tan1 leaves as they expand would determine the extent to which MT orientation predicts the direction of expansion and whether cell expansion in the tan1 leaves compensates for abnormal division planes.

¹ These authors contributed equally to this article
² kropf@bioscience.utah.edu

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