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Structure, Development, and Morphogenesis |
Department of Biology, Bellarmine University, Louisville, Kentucky 40205 USA
Received for publication June 5, 2001. Accepted for publication December 7, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: chimeras • founder cells • gymnosperms • Juniperus chinensis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), whereas "stem cells" (Barlow, 1978
; Poethig, 1984
) are those from which both differentiated and meristematic cells of an organ are derived. Stem cells can be held in reserve, as are those that belong to the quiescent center in roots (Barlow, 1978
), those in a proximal slow-growing region of the monocot leaf basipetal to the intercalary meristem (Korn, 1994
), and two cells in the maize leaf primordium fated to become part vascular bundle and part mesophyll (Langdale et al., 1989
). Stem cells are usually equivalent to initial cells such as the apical cells in mosses and ferns as well as members of any persistent multicellular meristem. "Founder cells" (Poethig, 1984
) are a transient group of cells of one organ that become enlisted to form a new organ. For example, about 25 pericyclic cells in radish and Arabidopsis roots give rise to a branch root (Sussex et al., 1995
). The size of these groups, either of an embryo, meristem, or the founder group, is the apparent cell number, which is the reciprocal of the sectorial fraction of the affected organ or individual (Coe and Neuffer, 1978
). The shoot apical meristem of Arabidopsis, for example, is composed of about 110 cells arranged in three layers (Irish and Sussex, 1992
).
Chimeras have been particularly useful in studies of the number of cell layers of founder cells. Satina and Blakeslee (1940)
used polyploid cytochimeras in their pioneering studies and found that three layers of the shoot apical meristem contribute to the developing leaf. Many investigations followed using chimeras, especially with the more discernable variegated, or chlorophyll-deficient, chimeras (Derman, 1947
). These variegated chimeras also revealed unequivocally that three layers of one organ give rise to another organ, although some variability occurs as to which layers of the parental organ give rise to which tissues in the derived organ (Derman, 1960
; Irish and Sussex, 1992
).
Conifer development has been examined by sectorial analysis but not to the same extent as in flowering plants. Hejnowicz (1959)
and Derman (1960)
recognized that leaf variegation in Juniperus Sabina nana Laws arose as periclinal chimeras produced by cell replacement at the stem apex. Derman (1960)
, studying J. Sabina nana Laws, and Ruth, Klekowsky, and Stein (1985)
, studying J. davurica Expansa Variegata Hornibr., found sector sizes that suggest the shoot apex has one to three apical initials. Recently, I reevaluated these sector patterns in six species of the Cupressaceae and concluded that there is only one stem apical initial (Korn, 2001
). Although I used sector patterns in the six conifer species to evaluate the ongoing status of the shoot apex, these sectors in leaves and axillary branches also offered an opportunity to analyze the number of founder cells that give rise to derived organs. This study explores what sector types reveal about the quantitative aspects of organogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Stem apices in gymnosperms are thought to be composed of loosely organized cells rather than distinct cell layers (Johnson, 1951
); however Hejnowicz (1959)
noted two somewhat stable cell layers that form true periclinal chimeras in J. sabina nana Laws, an observation supported by Podheim (1971) for ten other species of the Cupressaceae. On the basis of these findings the terminology for the histological organization adopted here is "tunica" for the L1 layer and "corpus" for the L2 layer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The partial sector (ps) is a white region in a mostly green leaf or a green region in a white leaf that runs along the leaf edge from the base but not to the tip (Figs. 2, 3). It is found in about 20% of all sectorial leaves and ranges in size from 1/2 to 1/24 of leaf width. The width of 100 small partial sectors were measured in terms of number of cell files (Fig. 7A). The data fall into five discrete groups with averages of approximately 5.6, 11.2, 16.8, 22.4, and 28.0. A typical leaf, as based on ten counts, is surrounded by about 144 cell files. The average for the smallest peak (5.6) divided by the number of cell files around a leaf (144) gives a sector value of 5.6/144, or 1/25.7 of the complete leaf. Sometimes the size of partial sectors changes abruptly from many cell files to just a few (Fig. 4). A parallel calculation was made by measuring the width of ten of these smallest sectors as well as the circumference of their leaves in millimeters and when the average of the former was divided by that of the latter, the average fraction was 1/25.1, a result close to that calculated from file number.
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The core sector (cs) is light green to dark yellow and does not run along the leaf edge. Transections reveal an embedded dark green sector that appears light green from the outside because it is partially masked by white mesophyll (Figs. 3, 6E). A core sector is usually somewhat round, variable in size and often in multiples. The shape of ten core sectors was taken as the fraction of the lengths of the radial and tangential axes (Fig. 6J, a/b), or the aspect ratio. The mean (±1 SD) fraction is 1.06 (±0.4), indicating that the typical core sector is somewhat circular, although there is a significant amount of variation. This circularity of core sectors indicates that leaf expansion is isodiametric, and no lateral growth gradient is present as may occur in dicot leaves. The aspect ratio value for leaf shape by the a/b fractional calculation, based on five measurements, is 0.48; this value is to be expected because the leaf is clearly wider than it is thick (Fig. 6I). When viewed externally, some core sectors appear to be segmented in that two are separated along the same longitudinal axis of the leaf (Fig. 6E), but when leaves with such sectors were dissected, it was found that the sectors are actually connected. This finding suggests that more core sectors occur than can be recognized by external appearances alone. Core sectors are found in about 8% of sectorial leaves.
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Stem patterns
The fifth type of sector, noted only in stems, is the reverse kind in which the mother branch (mb) is sectorial and the derivative leaf or axillary branch (ab) is completely green or white (Figs. 5, 6F, gl). The analysis of sectoring for this type is the reverse of that for the other four types of sectors in that a sector in the parental organ is scored instead of in the derived organ. The parental stems of ten completely green axillary stems had green sectors of 3/4, 3/4, 3/4, 3/4, 1/2, 1/2, 1/2, 1/2, 1/4, and 1/4 and ten completely white stems had white sectors of similar white fractions, 3/4, 3/4, 3/4, 1/2, 1/2, 1/2, 1/2, 1/2, 1/2, 1/4, and 1/4. Hence, the smallest fraction of the mother stem circumference that produces a daughter branch is about 1/4.
Data were collected on the minimum circumference of the stem apex that gives rise to a leaf. In a decussate system, a node (n) has two leaves 180° apart; the next (younger) node (n + 1) up the stem also has two leaves 180° apart, each of which is displaced 90° from the two leaves at node n; and the next node (n + 2) has two leaves just above those at node n and in the same orthostichy. Leaves were sought that were completely green except for two white leaves at n + 1 (Fig. 5). The two adjacent leaves at n + 1 were observed for color, and those that were nearly all white were noted and scored but only when the leaves at n + 2 above n were also all green. No branch was found with two all-green n leaves and the two all-white n + 1 leaves. The closest patterns were an all-green n leaf and two n + 1 leaves with 1/8 green sectors each (Table 1, case a) or 1/16 green sectors each (Table 1, case b). Also found were branches with completely white n + 1 leaves and a 7/8 green leaf at node n (Table 1, case b). The fraction of a leaf that shares cell files with an adjacent leaf at the next node is [(5 x 1/8 + 5 x 1/8 + 1 x 1/16)]/11, or 1.9/16, or 0.12; that is, on average, a leaf shares few cell files with leaves in adjacent orthostichies. Since the shoot is decussate, or four-ranked, each leaf is derived from about 1/4 of the circumference of the stem apex.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Sacher (1955)
found that leaves of several species of the Cupressaceae have the typical gymnosperm pattern of leaf development: an initial phase of apical growth, followed by a prolonged second phase in which the apical meristem is replaced with a nearly basal intercalary meristem. The juniper stem establishes files of cells below the apex that extend down the stem, resulting in subtending leaves coming from common cell files and sharing any sectors. Apical growth is necessary for a leaf primordium to emerge (Korn, 2001
) and the summit must grow faster than more basal regions. This apical growth is soon replaced by a permanent, basal meristem. This view is supported by the morphological observation that a leaf primordium is initially composed of isodiametric cells, and as it becomes larger than the stem primordium, tissues are composed of cell files. Partial sectors also support this description; all have one end at the leaf base with over 50% extending acropetally more than half the way along the leaf (Fig. 7B). If there were significant apical growth, the nonsectorial apical region would extend down more; that is, partial sectors would extend only a short way up the leaf. It appears that a group of founder cells gives rise to the leaf primordium, first by apical growth and then by basal cell-file growth. On the basis of known leaf ontogeny, the various types of leaf sectorial patterns can be examined in terms of how they arise. The entire sector comes from a parental stem sector that occupies one side of the founder area or about half of the founder population of cells (Fig. 6A, B). By contrast, the partial sector always runs from the leaf base up, not from the apex down, because the sector in the parental stem occupies only the edge of the founder cell area (Fig. 6C). Multiple leaf sectors are explained by an irregular outline of the sectorial area facing the non-sectorial area within the founder area (Fig. 6D). Core sectors are formed by an internal region of the founder group composed of green cells. These core sectors are most useful in determining the number of layers in the founder group. The tunica layer is colorless and is not involved in sectoring, whereas corpus cells are either green or white and hence determine the sector. Entire and partial sectors are contiguous with the tunica but are located still in the corpus so they are in the first corpus layer, or C1. Core sectors by definition are not next to the tunica, hence they are internal to C1 and so are in a second corpus layer, C2. Multiple core sectors are green and separated by colorless tunica cells (Fig. 6I), which then constitute an even more central region, namely, C3. This central corpus layer could actually be composed of several layers; however, multiple core sectors indicate that there are at least three corpus layers (Fig. 8E).
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The patch of C1 founder cells then telescopes out to form the outer layer of the leaf mesophyll; the outer ring of cells of this patch form cells at the leaf base, with more central C1 cells giving rise to the apical region of the leaf (Fig. 8A–E). The smallest sectors found at the basal region of the leaf average about six cell files wide out of a total of about 144 cells files surrounding the leaf. The value 144/5.6, or approximately 26, is then the approximate number of cells in the outer ring of C1 cells. Cells average six sides in surface view so that one cell is surrounded by six cells, which are, in turn, surrounded by 12 cells, and so on, forming a series of concentric rings (Korn and Spalding, 1973
). In addition to consisting of 26 cells, the outer ring of cells has a shape with a vertical/width ratio of 0.48. To integrate these two features, the C1 field must have a set of concentric rings of 24 outer cells, 18 intercalary cells, and a row of 7 internal cells (Fig. 8F). This set of rings gives a total of 49 C1 cells with a horizontal diameter of 11 cells and a vertical diameter of five cells for a shape fraction of 5/11, or 0.45.
The cell number in the tunica layer (T) is equal to, or more likely greater, in cell number than that in C1. The number of T cells is greater than that of C1 in order for T to encompass C1, or T is composed of concentric elliptical rings of 27, 21, and 15 cells, for a total of 63 T cells with a lateral diameter of 12 cells and a vertical diameter of six cells, yielding an aspect ratio of 0.50. Fitting a smaller C2 in behind C1 gives elliptical rings of 20 and 14 cells, for a total of 34 C2 cells. For a smaller C3, rings of 16 and 6 fit best, for a total of 22 C3 cells. The number of cells in the founder population is 63 T + 49 C1 + 34 C2 + 22 C3, or 168 cells, of which 105 are in three layers of the corpus (Fig. 8F, G). The number of founder cells is ideally about 168, but in actuality the number could fluctuate considerably because of cell size: older cells are larger than younger cells. The absolute size of the founder group is, however, probably constant among leaves because the area of leaf insertion on the stem is fairly uniform.
If the founder group for a new leaf is composed of about 168 cells, then how does this value compare with the size of the stem from which it comes? The stem apex has a diameter of 16 cells, two of which are tunica cells, leaving 14 for the corpus, for a circumference of 14
, or about 44 cells (and about 16, or 50, T cells). With a decussate status, two leaves forming at a node will include 11 x 2, or 22, cells of the diameter, leaving 44 – 22, or 22 uncommitted cells. The next node will enlist another 22 cells or those not part of the first two leaves. Every pair of nodes will enlist (22 + 22)/44 cells, or all of the marginal corpus cells of the stem.
How does this size of the stem apex compare with the founder group for a new stem? The smallest green sector of a chimeric stem that gave rise immediately to an all-green branch is about 1/4. With the circumference of the stem apex of 44 T cells, then 1/4 of 44 cells is 11 cells for the width of the C1 founder group of a stem apex for a new branch. It appears that founder groups for new leaves and stems have about the same apparent cell number (ACN). The similarity of values, such as 11 cells for the diameter of the stem and leaf founder group by independent methods, should not be taken as an indication of the degree of accuracy of measurements leading to these calculated values. After all, fractional values of 1/2, 1/4, etc., are only approximations.
The number of founder cells for leaf initiation in maize is about 200, arranged in an overlapping arc, like a key ring (Poethig, 1984
), and in tobacco it is a patch of about 100 cells (Poethig and Sussex, 1985
). Scanlon, Schneeberhger, and Freeling (1996)
found that about 25 of the maize leaf founder cells are for leaf flank development and are initiated after the first 175 central leaf region cells are initiated; that is, there are at least two rounds of founder cell induction. Christiansen (1986)
found about eight cells give rise to a cotyledon in cotton, whereas Sussex et al. (1995)
identified about 25 founder cells in branch root initiation in radish and Arabidopsis. The leaf of cotton appears to come from about 100 founder cells (Dolan and Poethig, 1998
). Poethig (1984)
determined that the founder group in tobacco is four layers thick, based on types of periclinal chimeras examined. Poethig and Sussex (1985)
identified this founder group to be 13 cells wide, based on number of sector regions, and three cells high, based on the extent of chimeras. They estimated the total size of the founder group to be about 100 cells, but the number could also be calculated as 4 x 3 x 13, or 156, cells. Most recently Dolan and Poethig (1998)
estimated the leaf founder group in cotton to also be about 13 cells wide and 3 cells high. The founder group in juniper is not that different: it has a corpus width of 11 cells and 2 tunica cells, for total of 13, 6 tunical cells high, and a depth of 4 layers (T C1 C2 C3).
The variable fate of individual meristematic cells was first documented by Derman (1947)
in peach as a case of periclinal indeterminancy. The vascular tissue and pith usually come from the L3 cells but at times are derived from L2 cells. Anticlinal indeterminancy was noted by Jegla and Sussex (1989)
; different cells of the shoot meristem of the dry-seeded sunflower embryo give rise to different nodes with considerable overlap among plants. For example, cells of the second row of the meristem give rise to a range as extensive as nodes 3–14, while cells in the third row proliferate into a range as wide as nodes 12–26. Periclinal indeterminancy of cell fates exist in the variegated Hollywood juniper with respect to the locations of core chimeras. The smallest sectors appear to come from single founder cells, but no one set of fixed domains can be constructed to explain their locations. For instance, in Fig. 6, sectors in G, H, and I overlap. Also, the range of widths in each group of partial sectors (Fig. 7A) suggests an indeterminancy as to how large a sector each founder cell will eventually become. Coupled with this indeterminancy is that of the ACN for the founder group; the calculated number of 168 is only an approximation. The cell number of founder groups most likely varies in cell number.
The primary origin of these sectors in juniper is by cell replacement when an albino tunica cell with only achlorophyllous plastids at the summit of the apex divides periclinally to produce a daughter cell that is inserted into the green corpus (Hejnowicz, 1959
; Derman, 1960
; Podheim, 1971; Korn, 2001
). The occurrence of two layers in gymnosperm apices, as opposed to three layers in angiosperms (Tilney-Bassett, 1986
), provides straightforward analysis of periclinal chimeras. Cell replacement from tunica (L1) into corpus (L2) in gymnosperms is a single event, whereas replacement of a cell from L1 into L2 and then again from L2 into L3 in angiosperms is a two-event process in which the two events are independent of each other, leaving the origin of sectors as to when and where sectors originate uncertain. The chimeras studied here might be interpreted not as periclinal but anticlinal through normal green and mutant white plastid segregating during stem and leaf development. This interpretation does not hold because "sorting out" (Tilney-Bassett, 1986
) results in white spots that in gymnosperm leaves would be isolated streaks, whereas all chimeras observed here extend back into the stem. Hence, it can only be assumed that the initial chimeric event occurs in the stem apex and becomes a sectorial streak shared by the stem and one or more leaves. "Sorting out" in the stem apex would result in subsectors in shades of green different from those of cells with mixed green and white plastids. By contrast, secondary sectors arise in leaves and axillary branches by enlistment of one or more mutant cells, along with one or more wild-type cells, as the founder group for a new organ. Again, secondary sectors are neither isolated nor express shades of green coloration.
Sectors in both primary and secondary chimeras are established immediately. In UV- and X-ray-induced chimeras, the altered state undergoes phenotypic delay because of mitotic segregation (Korn, 1969
; Poethig, 1987
). This segregation complicates calculation of the number of founder cells that initiates an organ or a meristem (Poethig, 1987
). Unlike mutation, enlistment leads to immediate expression, permitting direct calculation of ACN as the reciprocal of the sector size. However, this reciprocal relationship does not hold for the primary origin of sectors by cell replacement because the replaced tunica daughter cell in the corpus proliferates into the entire corpus, with sectors expressing only transient states in the chimera (Korn, 2001
).
The determinant of the founder group of cells remains speculative. Poethig and Sussex (1985)
argue that some supracellular agent initiates leaf growth in tobacco because many cells have to be brought into the founder organization. In juniper the field of this supracellular agent is seen as a somewhat elliptical domain of competent cells. Its source could be an apical cell that determines the field of founder cells as well as inducing them to proliferate for the temporary apical phase of leaf growth. Leaf origin may then be similar to that of a stem with a regulatory apical cell (Korn, 2001
).
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Barlow P. W. 1978 The concept of the stem cell in the context of plant growth and development. In B. I. Lord, C. S. Potten, and B. J. Cole [eds.], Stem cells and tissue homeostasis, 87–113. Cambridge University Press, Cambridge, UK
Christianson M. L. 1986 Fate map of the organizing shoot apex in Gossypium. American Journal of Botany 73: 947-958[CrossRef][ISI]
Coe E. H. M. G. Neuffer 1978 Embryo cells and their destinies in the corn plant. In S. Subtelny and I. Sussex [eds.], The clonal basis of development, 113–129. Academic Press, New York, New York, USA
Derman H. 1947 Histogenesis of some bud sports and variations. Proceedings of the American Society of Horticultural Science 50: 51-73
Derman H. 1960 Nature of plant sports. American Horticultural Magazine 39: 123-173
Dolan L. R. S. Poethig 1998 Clonal analysis of leaf development in cotton. American Journal of Botany 85: 315-321[Abstract]
Hejnowicz Z. 1959 Eversporting periclinal chimeras. Recent Advances in Botany 2: 1446-1448
Irish V. F. I. M. Sussex 1992 A fate map of the Arabidopsis embryonic shoot apical meristem. Development 115: 745-753[Abstract]
Jegla D. E. I. M. Sussex 1989 Cell lineage patterns in the shoot meristem of the sunflower embryo in the dry seed. Developmental Biology 131: 215-225[CrossRef][ISI][Medline]
Johnson M. A. 1951 The shoot apex in gymnosperms. Phytomorphology 1: 188-204
Korn R. 1969 Induction and inheritance of morphological mutations in Cosmarium turpini Bréb. Genetics 65: 41-49[ISI]
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Nägeli C. K. 1858 Über das Wachsthum des Stammes und der Wurzel bei den Gefässpflanzen. Beiträge zur Wissenschaften Botanik 1: 1-156
Poethig R. S. 1984 Cellular parameters of leaf morphogenesis in maize and tobacco. In R. A. White and W. C. Dickinson [eds.], Contemporary problems in plant anatomy, 235–259. Academic Press, New York, New York, USA
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Poethig R. S. I. M. Sussex 1985 The cellular parameters of leaf development in tobacco: a clonal analysis. Planta 165: 170-184[CrossRef][ISI]
Pohlheim F. 1971 Untersuchungen der Sprossvariation der Cupressaceae. I. Nachweis immerspaltender Periklinalchimären. Flora 160: 264-293[ISI]
Ruth J. J. Klekowski O. Stein 1985 Impermanent initials of the shoot apex and diplontic selection in a juniper chimera. American Journal of Botany 72: 1127-1135[CrossRef][ISI]
Sacher J. A. 1955 Cataphyll ontogeny in Pinus lamberitiana. American Journal of Botany 42: 82-91[CrossRef][ISI]
Satina S. A. F. Blakeslee 1940 Periclinal chimeras in Datura stramonium in relation to the development of the leaf and flower. American Journal of Botany 27: 895-905[CrossRef][ISI]
Scanlon M. J. P. G. Schneeberger M. Freeling 1996 The maize mutant narrow sheath fails to establish leaf margin identity in a meristematic domain. Development 122: 1683-1691[Abstract]
Stewart R. N. H. Derman 1970 Determination of the number and mitotic activity of shoot apical initials by analysis of mericlinal chimeras. American Journal of Botany 57: 816-826[CrossRef][ISI]
Sussex I. M. J. A. Godoy N. M. Kerk M. J. Laskowski H. C. Nusmbaum 1995 Cellular and molecular events in a newly organizing lateral root meristem. Philosophical Transactions of the Royal Society of London, B, Biological Sciences 350: 39-43[CrossRef]
Tilney-Bassett R. A. E. 1986 Plant chimeras. Edward Arnold, London, UK
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