1 Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208104, New Haven CT 06520, USA
2 Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093, USA
Present address: Tufts School of Veterinary Medicine, North Grafton, MA, USA
*Author for correspondence (e-mail: timothy.nelson{at}yale.edu)
Accepted April 16, 2001
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SUMMARY |
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Key words: Maize, tangled1, Bundle sheath cell, Cell fate
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INTRODUCTION |
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Several studies suggest that positional information for the differentiation of M cells is provided by adjacent provascular or procambial sites. In light-grown plants, M cells adjacent to veins accumulate appropriate C4 enzymes. In contrast, M cells more than two cells from the BS ring, as found in the leaf sheath and husk leaves, accumulate photosynthetic enzymes in a distinct pattern supporting the C3 pathway (Langdale et al., 1988b). Mutations in the C4 grass Panicum maximum that increase leaf interveinal spacing (and hence the maximal distance that an M cell can be from a vein) produce a corresponding increase in C3 photosynthetic characteristics in the leaf (Fladung, 1994). In C3-C4 intermediate species of Flaveria, C4 photosynthesis is limited to the M cells immediately adjacent to veins (Edwards and Ku, 1987; Cheng et al., 1988; Moore et al., 1988). Surveys of C3, C4, and C3-C4 intermediate grass species revealed that interveinal distances range predictably from two M cells in C4 species to many M cells in C3 species, consistent with the requirement for vein-adjacency for C4-type M cells (Hattersley and Watson, 1975; Hattersley, 1987). These observations suggest that veins or their procambial precursors provide a spatial signal for the C4 differentiation of M cells that acts over a limited distance. According to this model, M cells in the maize leaf develop in a C3 pattern by default and in a C4 pattern only through the influence of closely neighboring veins (Langdale and Nelson, 1991).
The basis for BS cell determination is less clear. BS cells occur in a single layer around a vein, making it difficult to distinguish positional effects from lineage effects. Histological studies of vein ontogeny in NADP-ME type C4 grasses have demonstrated that BS cells surrounding a vein are predominantly, perhaps entirely, derivatives of procambial cells (Dengler et al., 1985; Nelson and Dengler, 1992; Bosabalidis et al., 1994; Dengler et al., 1996; Sud and Dengler, 2000). Analyses of genetic clonal sectors in maize and in the C4 grass Stenotaphrum secundatum confirmed that the BS lineage is distinct from that which produces M cells, presumably from the time procambial strands are distinct from ground cells (Langdale et al., 1989; Sud and Dengler, 2000). In the maize study, however, rare clonal sectors were found in which a subset of the BS cells surrounding a vein was included in a sector with a neighboring M cell. One interpretation of such events is that an M cell was formed from the procambial lineage. Alternatively, a procambial cell (BS precursor) and a ground cell (M precursor) may have been derived from a division at the site of the sectoring event. A third interpretation, that the marked BS cells were generated from a non-procambial lineage, is unlikely because the sectors included, in cross section, several of the BS cells surrounding the vein, but only a single M cell.
Are the BS cells found in C4 plants influenced by their vein-adjacent position to become specialized for the C4 pathway, as is the case for M cells, or are they programmed entirely by their procambial lineage? The clonal studies cited above support the first possibility, suggesting that cells from the procambial lineage can assume an M cell fate if cell division pattern places them distant enough from the vein. However, this idea is challenged by the observation, investigated here, that in the maize tangled1 (tan1) mutant, clusters of BS-like cells extend various distances from veins. This aspect of the tan1 phenotype provides a novel opportunity to investigate the roles of cell position and cell lineage in specification of BS cell fate.
tan1 was originally described in reference to the altered cell division orientations and frequencies that were observed in the leaves (Smith et al., 1996). These are associated with changes in the spatial orientation of cytoskeletal arrays in dividing cells (Cleary and Smith, 1998). tan1 mutants exhibit abnormally oriented divisions and a reduction in normally oriented divisions in both the transverse and longitudinal directions. The resulting plants are reduced in stature and have leaves with a crepe paper-like surface, although with normal overall shape (Smith et al., 1996). Here we show that in tan1 leaves, abnormally late divisions within the procambial lineage give rise to BS-like cells in aberrant locations. Despite their non-vein-adjacent positions, these cells differentiate as C4-type BS cells. This is a rare example of lineage-dependent cellular differentiation in plants, where cell fates are generally dictated by positional information.
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MATERIALS AND METHODS |
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Histology
Leaf sections were cleared by dehydrating 5x5 mm sections of fresh leaf tissue through an ethanol series to 100% ethanol. The sections were maintained in 100% ethanol until clear, then stained for 15 minutes with a 0.1% aqueous Toluidine Blue solution. Material for plastic and paraffin wax embedding was prepared by cutting 1-2 mm wide sections of fresh tissue and fixing in 4% paraformaldehyde in Sorensons buffer under vacuum for 1 hour (Sylvester and Ruzin, 1994). The fixative was replaced and the tissue was fixed overnight at 4°C, then dehydrated through an ethanol series to 100% ethanol. Sections for embedding in paraffin wax were put through a Hemo-De (Fisher Scientific):ethanol series and then embedded in Paraplast Plus (Fisher Scientific) for 4 days at 60°C, with 4 changes of Paraplast. 8 µm sections of the embedded material were made using a rotary microtome. Sections were de-waxed in Hemo-De (Fisher Scientific), rehydrated, stained in aqueous 0.1% Toluidine Blue for 10 minutes, dehydrated and mounted. Sections for plastic-embedding were rinsed 3 times in 100% polypropylene oxide after ethanol dehydration and infiltrated overnight at room temperature in a 1:1 mixture of polypropylene oxide:Spurrs resin (Electron Microscope Sciences). The tissue sections were then embedded in Spurrs resin at 65°C overnight. 2 µm sections of the plastic-embedded material were made on a Sorvall MT-2 ultramicrotome using a glass knife. Sections were dried on slides at 60°C, then stained with 1% Toluidine Blue, 1% borax at 60°C for 3 minutes. Microscope observations were made and photographs taken using a Zeiss Axiophot light microscope. Sites and planes of recent cell division were identified by locating pairs of cells with relatively thin separating walls, in transverse sections.
Immunolocalizations
Tissue from the fourth leaf of 2-week-old seedlings was fixed and embedded in Paraplast Plus as described above. Immunolocalizations were performed as previously described (Smith et al., 1992), with the following modifications: the proteinase step was omitted, a dilution of 1:50 was used for all primary antibodies, alkaline phosphatase-conjugated goat anti-rabbit-IgG (Boehringer Mannheim) diluted 1:600 was used as a secondary antibody and the signal was visualized by staining for 2 hours in the substrate 5-bromo-4-chloro-3-indolyphosphate, p-toluidine salt/nitroblue tetrazolium chloride. ME antibodies were as described previously (Rothermel and Nelson, 1989) and PEPCase antibodies were a gift from Dr James Berry (SUNY; Buffalo).
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RESULTS |
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DISCUSSION |
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The formation of veins and their associated bundle sheaths from non-clonal groups of ground cell precursors that can also give rise to M cells suggests that positional information initially specifies the fates of BS cells and other procambial derivatives. BS fate may be conferred on the cells immediately surrounding the vein cells by a positional signal, either from less peripheral procambial cells or from more peripheral non-procambial cells. Alternatively, positional information may act only to set aside the procambial lineage from the mesophyll, whereas the fates of BS cells and other procambial derivatives are subsequently determined by lineage. In this case, the regular pattern of procambial divisions might determine that certain derivatives become BS cells, and might assure that they are in a peripheral position relative to vascular tissue. However, the observation that tan1 veins are surrounded by a continuous bundle sheath, despite irregular patterns of division of the procambial strands, is more consistent with the former possibility that positional information confers BS cell fate on the cells immediately surrounding the vein.
Regardless of the mechanism by which BS fate is initially specified within the procambial lineage, this fate appears to be inherited by the products of abnormally late divisions that occur in tan1 mutant leaves. In mutant leaves, BS cells are formed as many as 6 cells distal to the vascular sheath, but are clonally related to sheath cells. It is possible that the lineage-based mechanism by which these cells differentiate as BS might also depend on positional information. That is, these cells might become BS cells through the action of a positional cue for which only procambial derivatives are primed, signaling either an adjacency to M cells or a distance from the vascular tissue. However, the simplest interpretation is that, once specified as BS, cells remain committed to the BS fate while they continue to divide, and therefore differentiate as BS cells regardless of their final positions. Cells of the bundle sheath are formed 3-4 plastochron intervals from the vein-initiating procambial cell divisions in maize (Nelson and Dengler, 1992). Our observations on tan1 mutants suggest that BS cells and any subsequent daughter cells are committed to BS fate at this time.
The Tan1 gene encodes a highly basic protein with microtubule-binding activity, and with a localization pattern consistent with its inferred role in the orientation of cytoskeletal structures in dividing cells (Smith et. al., 2001). The observed prolongation of cell division relative to commitment or differentiation in tan1 mutant leaves indicates that Tan1 is required for division arrest in differentiating cells, and suggests that the schedule of cellular differentiation can be controlled independently of the timing of cell division. It is possible that in tan1 leaves, cell division occurs on a normal schedule while cellular differentiation occurs precociously. Alternatively, cellular differentiation may occur on schedule while cell division is prolonged. If this is the case, then Tan1 might play a role in cell cycle regulation in addition to its role in the spatial regulation of cytokinesis (Cleary and Smith, 1998). This possibility might be investigated by examining the expression in tan1 leaves of cell cycle regulators such as cyclin-dependent kinases, which reflect the competency of the cell to divide (Hemerly et al., 1993; Shaul et al., 1996) and mitotic cyclins, which appear to be expressed only in actively dividing cells (Ferriera et al., 1994; Shaul et al., 1996). Alternatively, Tan1 may be indirectly required for the proper timing of cell division because of its role in the orientation of cell division. Following misorientation of subsidiary mother cell divisions in maize by centrifugation (Galatis et al., 1984) or by mutations perturbing these divisions (Gallagher and Smith, 1999; Gallagher and Smith, 2000), aberrant divisions are often corrected by additional, normally oriented divisions. Cell volume and shape may be important factors that influence cell cycle activity (Jacobs, 1997). It may be that when a cell is stimulated to divide but daughter cells of appropriate shape or volume are not produced because the new cell wall is misoriented, one or both daughters can respond again to the same stimulus and re-enter the cell cycle.
Examples in which a cell whose fate is already committed or restricted transmits that state to its progeny are more common in the animal developmental biology literature than in the plant literature. For example, cell lineages leading to germline precursors in C. elegans are progressively restricted through five cell divisions; those to body muscles through more (Sulston and Horvitz, 1977). In contrast, similar studies of plant development have almost universally indicated that cell fates are dictated by their positions within the tissue. For example, clonal analyses of maize leaf development have shown that although patterns of cell division are variable, the final arrangement of various cell types within the leaf is highly predictable (Langdale et al., 1989; Cerioli et al., 1994; Poethig and Szymkowiak, 1995; Hernandez et al., 1999). In some plant organs, patterns of cell division are sufficiently regular that cell fates can be accurately predicted on the basis of lineage, such as in the Arabidopsis root (Dolan et al., 1993; Dolan et al., 1994). Nevertheless, when an individual epidermal or cortex-endodermis initial cell in the Arabidopis root meristem is laser-ablated, a neighboring cell belonging to a different lineage divides so that one of its daughters occupies the position of the ablated cell, and differentiates according to its new position rather than its lineage (van den Berg et al., 1995).
The formation of BS cells at vein-distal positions in the tan1 mutant provides a relatively rare example in plant development of a lineage-committed state that is transmitted to daughter cells. Another such example occurs during stomatal development in dicots. Guard cell pairs are formed from meristemoids, which are produced through asymmetric cell divisions. Before forming a guard cell pair, the meristemoid may undergo additional asymmetric divisions to form non-stomatal cells. However, regardless of how many times it divides, only one guard cell pair is formed by each meristemoid (Kagan et al., 1992; Larkin et al., 1997). This suggests that the meristemoid is determined to form guard cells and passes this state to one of its daughters each time it divides. The transmission of a lineage-committed state from a mother cell to one or both of its daughters may be uncommon in plants because of the nature of the plant body. Unlike animal cells, which can migrate extensively during embryogenesis, plant cells are constrained by their walls to remain at the site where they are initially formed. If cell fate commitments were hard-wired in cells that are still dividing, irregularities in cell division pattern could not be corrected and would therefore perturb the pattern of cellular differentiation. Such mistakes could have deleterious consequences, particularly at early stages of development. For this reason, reliance on positional information for cell fate specification is a strategy that appears to be well suited to plants.
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ACKNOWLEDGMENTS |
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