Section of Cell and Developmental Biology, U.C. San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0116, USA
*Author for correspondence (e-mail: lsmith{at}biomail.ucsd.edu)
Accepted 9 April 2002
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SUMMARY |
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Key words: Maize, Leaf development, Cell division, tangled
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INTRODUCTION |
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The problem of how plant cells orient their divisions appropriately during development is one of longstanding interest, but remains largely unsolved (Smith, 2001). Over 100 years ago, plant biologists recognized relationships between cell shape and division plane that apply to most dividing cells. Hofmeisters rule states 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 (Hofmeister, 1863
). Erreras rule states that the plane of division for most plant cells corresponds to the shortest path that will halve the volume of the parental cell (Errera, 1888
). Although it is not fully known how a plant cell would be able to read its shape and divide accordingly, Lloyd and colleagues have proposed a model based on simple mechanical principles that could largely explain cells ability to follow Hofmeisters and Erreras rules (Flanders et al., 1990
; Lloyd, 1991
).
However, not all cell division planes can be accurately predicted by these rules, and appear to be influenced by extracellular cues. For example, cells in the prospective leaf-forming region of the shoot apical meristem divide predominantly in different orientations than do those of similar shapes outside this region (Lyndon, 1972; Cunninghame and Lyndon, 1986
). Observations on stomatal complex development in monocots suggest that newly formed guard mother cells signal their nearest neighbors to divide asymmetrically, forming small daughters adjacent to the guard mother cell that will become part of the complex (Stebbins and Shah, 1960
; Stebbins and Jain, 1960
). Another striking example emerges from laser ablation studies on developing Arabidopsis roots. When an individual cortex-endodermis initial cell within the root is ablated, a neighboring pericycle cell divides in an atypical orientation not predicted by its shape or position to produce a daughter that takes the place and assumes the fate of the ablated cell (van den Berg, 1995
). The nature of the extracellular information these cells apparently respond to and how it is transmitted remain largely unknown, and may vary considerably in different situations. In animal cells, both cell shape and cell-cell communication can play important roles in determining planes of cell division (Goldstein, 2000
). Though much remains to be learned about how cell-cell communication can direct the orientation of animal cell divisions, asymmetrically dividing cells in the early C. elegans embryo provide a relatively well understood example. Here, EMS cells signal neighboring P2 cells via the wingless/WNT pathway to re-orient their division planes (Schlesinger et al., 1999
).
In the maize leaf primordium, as pointed out earlier, rectangular epidermal cells divide in both transverse and longitudinal orientations. For an elongated cell, a longitudinal division plane is not predicted by Hofmeisters and Erreras rules. This raises the question of what role cell-cell interactions might play in division plane selection in this tissue. We have explored this question through a mosaic analysis of the tan mutation, in which we closely examined the boundaries of tan mutant sectors in otherwise wild-type leaves. If the Tan gene is involved in sending or controlling a signal that orients proliferative cell divisions, we would expect to find that it acts non cell-autonomously. That is, we would expect wild-type cells to rescue genotypically mutant cells nearby so that they appear wild type. However, we found that the tan mutant phenotype is not rescued or influenced by adjacent wild-type cells, demonstrating that the wild-type Tan gene acts cell-autonomously to promote normal division orientations.
At sector boundaries, the juxtaposition of aberrantly divided mutant cells with wild-type cells gave us the opportunity to further explore the role of cell-cell communication in division plane determination by asking what impact mutant cells have on the divisions of neighboring wild-type cells. Although the Tan gene itself acts cell-autonomously, the proper orientation of proliferative divisions may nevertheless depend on cross-talk between adjacent, dividing cells involving the products of other genes. In this case, alterations in the signals sent by aberrantly dividing mutant cells could change the division planes of adjacent wild-type cells. Indeed, such cell-cell interactions have been invoked to explain how leaves of normal shape might form in tan mutants in spite of the high frequency of aberrantly oriented divisions. Meyerowitz (Meyerowitz, 1996) proposed that through local coordination of division orientations, tan mutant cells may divide so as to compensate for each others mistakes, essentially correcting for each other to achieve an overall division pattern that permits the elaboration of normal leaf shape. In a fully mutant leaf, this idea is not readily testable because of the difficulty in recognizing such corrective divisions. However, corrective divisions in wild-type cells adjacent to mutant cells would be recognizable if they were oriented differently from other wild-type cell divisions. Our results show that proximity to tan mutant cells does not substantially alter the division orientations of wild-type cells. These observations argue against the idea that the division planes of proliferatively dividing maize leaf epidermal cells are governed by short-range communication with their nearest neighbors, and implicate spatial regulation of cell expansion rather than division as the primary determinant of leaf shape in both tan and wild-type leaves.
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MATERIALS AND METHODS |
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Analysis of sector composition
Hand-cut transverse sections of the sectors were made, mounted in water, and observed with a Nikon Eclipse E600 microscope equipped with fluorescence epi-illumination, using a standard rhodamine filter set. Images were acquired with a DAGE MTI CCD72 camera and digitized with a Scion LG3 framegrabber using Scion Image 1.62. Images were collected under both bright-field and epifluorescence conditions to record the distribution of chlorophyll-containing cells. In instances where multiple images needed to be taken to span the entire sector, a composite of adjacent cross sections was created using Adobe Photoshop 4.0.1.
Epidermal peels were also prepared from a portion of each sector (immediately adjacent to the location of the hand cross sections) as described previously (Gallagher and Smith, 1999). Prior to fixation each sector boundary was marked with a Sharpie ink pen so that it could later be aligned with the corresponding hand section; because only guard cells in the epidermis contain chloroplasts, boundaries marked with ink represented lateral boundaries in the mesophyll. For each sector, bright-field and epifluorescence images of epidermal peels were collected as described above and assembled into a composite image of the entire sector in surface view. Guard cells showing chlorophyll autofluorescence were marked on the composite image. Information from cross sections and epidermal peels was compiled to make a complete illustration of each sector showing both mesophyll and epidermal composition as shown in Fig. 2.
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Scanning electron microscopy
Scanning electron micrographs of the surface of wild-type and tan mutant maize leaf primordia were prepared as described previously (Smith et al., 1996).
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RESULTS |
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In the epidermis, only guard cells could be scored as w14 or W14+, because these are the only epidermal cells containing mature chloroplasts. Consequently, lateral sector boundaries in the epidermis could be located to the interval between one row of stomata containing chloroplasts and another without chloroplasts, which could be distinguished by the presence or absence of chlorophyll autofluorescence. Examples of lateral boundaries in the epidermis are seen in Fig. 3A-D where the white asterisks indicate wild-type stomata containing chloroplasts and the black asterisks indicate mutant stomata lacking chloroplasts. In each case, a lateral boundary is located to the interval between the wild-type and mutant stomatal files. As illustrated in Fig. 3A-D, the number of cell files separating wild-type and mutant stomata fluctuates along the length of the sector boundary. Quantitative analysis (Fig. 4) showed that the number of files separating wild-type and mutant stomata was most often 2, 3 or 4 (73% of the time). Thus, most of the time, we could be sure that the true distance from the sector boundary to the nearest marked stomatal file was no more than 4 cells. 29% of the time, the distance was no more than 2 cells. Examples of wild-type and mutant stomata separated by 2 or fewer cells are indicated by numbered black arrowheads in Fig. 3A-D.
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Tan acts cell-autonomously
Inspection of mutant epidermal cells near wild-type epidermal cells or overlying wild-type mesophyll allowed us to determine whether or not tan is cell-autonomous. When lateral sector boundaries in the epidermis and mesophyll coincide, a sharp transition from wild-type to tan-appearing cells is seen between the wild-type and mutant stomatal rows (Fig. 3A,B). Lateral sector boundaries in the epidermis also show the same sharp transition when they overlie wild-type mesophyll (Fig. 3C) or mutant mesophyll (Fig. 3D). Thus, we observed that tan mutant cells always showed the mutant phenotype, even when close to wild-type cells. In fact, many examples of aberrantly divided (presumably mutant) cells were observed in the boundary region immediately adjacent to marked, wild-type stomatal files (black arrows in Fig. 3A-C). Moreover, we found that mutant epidermis overlying wild-type mesophyll (Fig. 3C) appears to have as severe a tan phenotype as mutant epidermis overlying mutant mesophyll (Fig. 3A,B). The fact that mutant epidermal cells are not phenotypically rescued by underlying or adjacent wild-type cells indicates that tan is cell-autonomous in both lateral and transverse dimensions.
Although visual inspection of sectors revealed no effect of nearby wild-type cells on the phenotypes of mutant cells, we considered the possibility that there could be a small effect not apparent from casual observation. To do this, we carried out a quantitative analysis comparing the frequency of aberrantly oriented walls in mutant cells near wild-type cells with that in mutant cells far from wild-type cells. An abnormality index was calculated for selected cell files, which reflected the proportion of cells with oblique or aberrantly located walls, indicative of abnormal planes of cell division (see Materials and Methods for details). As shown in Fig. 5A, abnormality indexes for mutant stomatal files near boundaries with wild-type epidermis or overlying wild-type mesophyll were not significantly different from that for mutant stomatal files nowhere near wild-type cells.
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Mutant cells do not cause nearby wild-type cells to divide aberrantly
Since we found that Tan acts cell-autonomously, we could also ask how the proximity of mutant cells might affect the divisions of wild-type cells. Do wild-type cells divide in aberrant orientations to somehow compensate for or respond to abnormally dividing mutant cells nearby? As discussed in more detail in the Introduction, this might occur if division planes in normal leaf tissue are determined through some form of cell-cell communication involving the products of genes other than Tan. We found that occasionally, aberrant cell divisions occurred in wild-type epidermal cells adjacent to or overlying mutant cells (e.g., white arrows Fig. 3C,D). These aberrant divisions were rare, however, and did not usually occur near other improper divisions. Even in areas where wild-type and mutant stomata were no more than 2 files apart, the wild-type cells appeared to have divided normally (Fig. 3A-D). In fact, normally divided wild-type cells were often observed directly adjacent to aberrantly divided (presumably mutant) cells in the boundary region (e.g., black arrows Fig. 3A-C). Thus, visual inspection indicated that wild-type cells do not divide aberrantly under the influence of nearby mutant cells.
To determine whether there could be a small effect of mutant cells on wild-type cells, we performed a quantitative analysis of abnormal wall orientations in wild-type cells near mutant cells as described earlier. As shown in Fig. 6A, the abnormality indexes for wild-type stomatal files near boundaries with mutant epidermal cells or overlying mutant mesophyll were not significantly different from the index for wild-type stomatal files far from mutant cells. Moreover, the abnormality indexes for wild type, non-stomatal files near boundaries with mutant epidermal cells (such as those immediately to the left of the files marked with white asterisks in Fig. 3) or overlying mutant mesophyll were also not significantly different from the index for wild type, non-stomatal files far from mutant cells (Fig. 6B). Thus, both visual and quantitative analyses showed that the division planes of wild-type cells are not substantially altered by the proximity of mutant cells.
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DISCUSSION |
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The cell-autonomy of Tan gene function allowed us to address the additional question of how tan mutant cells affect the divisions of neighboring wild-type cells. If division planes in the developing leaf epidermis are governed by short-range cross-talk between neighboring cells involving genes other than Tan, then wild-type cells might respond to the abnormal divisions of adjacent tan mutant cells by dividing differently themselves. Such local interactions allowing cells to compensate for each others aberrant divisions have been proposed to explain how normal leaf shape can be acquired in tan mutant leaves (Meyerowitz, 1996). Therefore, we closely examined wild-type epidermal cells neighboring mutant cells for evidence that aberrant divisions had taken place. We found that there was no significant increase in the frequency of abnormally positioned walls in wild-type epidermal cells overlying mutant mesopyll cells or adjacent to mutant epidermal cells. Owing to the adjustments in wall orientation that could take place during postmitotic cell expansion, we cannot rule out the possibility that minor aberrations in wall orientation were present in wild-type cells immediately following division but were undetectable at maturity. However, our results indicate that the division planes of wild-type cells in close proximity to aberrantly dividing mutant cells were not substantially altered.
This observation lends support to the previously proposed view that the generation of normal leaf shape in both tan and wild-type leaves is achieved primarily through spatial control of cell expansion rather than cell division (Smith et al., 1996; Cleary and Smith, 1998
; Reynolds et al., 1998
). Thus, if mechanisms responsible for orienting cell expansion can operate on abnormally shaped cells to orient their expansion appropriately relative to the leaf as a whole, then a high frequency of abnormally oriented divisions need not alter the overall pattern of leaf growth so long as there is a sufficient number of cells to support growth in the appropriate directions. Consistent with this proposal, a recent study has shown that localized induction of expansin gene expression within tobacco leaf primordia can induce dramatic changes in the pattern of leaf morphogenesis (Pien et al., 2001
). This suggests that the regional variations in wall extensibility within the leaf primordium governed by the pattern of expansin gene expression could play a primary role in determining leaf shape.
Our observation that the division planes of wild-type epidermal cells are unperturbed by aberrantly oriented divisions of adjacent, tan mutant cells argues against the idea that the division planes of proliferatively dividing maize leaf epidermal cells are governed by short-range communication with their nearest neighbors. However, this does not mean that cell-cell interactions play no role in division plane determination in this tissue. While the majority of epidermal cells in the maize leaf primordium may choose transverse division planes simply because of their elongated shapes, some choose longitudinal division planes that are not predicted by shape according to Hofmeisters and Erreras rules. Thus, the high frequency of longitudinally oriented divisions in this tissue remains to be explained, and may involve extracellular influences of some kind. One possibility is that cues stimulating cell expansion in the width dimension can also override the default choice of a transverse division plane to produce longitudinal divisions. Another intriguing possibility is suggested by experiments demonstrating that application of a compressive force to callus cultures as well as to single cells in suspended in semi-solid medium can alter cell division planes (Lintilhac and Vesecky, 1984; Lynch and Lintilhac, 1997
). Thus, cell-cell interactions of a mechanical nature within the developing leaf primordium may cause some cells to divide longitudinally. According to either of these explanations, defects in cell plate-orienting mechanisms in tan mutant cells could account for their high frequency of aberrantly oriented divisions without predicting that these aberrant divisions would interfere with the divisions of adjacent, wild-type cells.
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ACKNOWLEDGMENTS |
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