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Structure and Development |
2Harvard Forest, Harvard University, Petersham, Massachusetts 01366 USA; and National Tropical Botanical Garden, 3530 Papalina Road, Kalaheo, Hawaii 96741 USA
Received for publication November 5, 2002. Accepted for publication February 21, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: fiber development • gelatinous fiber • Gnetales • Gnetum gnemon • reaction fiber • stem anatomy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This paper shows how these stem fibers are of three different kinds in terms of their development and have different diameters yet are all alike in having the cell wall features familiar in the tension wood fibers of dicotyledons. Apart from their extraxylary position and unique occurrence in a gymnosperm, the tension fibers of Gnetum are coenocytic, a condition that seems not to have been reported previously for tension wood fibers. Tension fibers also occur in leaves of Gnetum, but are not reported on in detail here.
Earlier observations and experiments have shown that reaction fibers occur in three positions in stems of Gnetum gnemon (Tomlinson, 2001
) and produce striking asymmetry in extraxylary tissues (Figs. 3–5). In both orthotropic and plagiotropic axes, narrow fibers are produced concentrically in the outer cortex of stems without regard to their orientation (i.e., they are obligate in their origin). Subsequently wider fibers can be developed eccentrically in the middle and inner cortex in an opportunistic manner as a gravimorphic response. This characteristically occurs on the upper side of leaning orthotropic axes or in plagiotropic axes also on the upper side either with increasing age or with displacement. In yet older axes, further narrow reaction fibers occur eccentrically in the outer secondary phloem to such an extent that they may virtually replace all phloem tissue. This results in three extraxylary regions with fibers on the upper side (Fig. 4, Co 1, Co 2, Phf), but only Co 1 fibers on the opposite side (Fig. 5, Co 1) at the same level. Clearly the sequence of developmental events is complex and requires detailed investigation.
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, 1996a
, b
; Carlquist and Robinson, 1995
). The latter implies a functional response to stimuli. "Tension wood fiber" cannot be used because the cells do not occur in secondary xylem. Evidence for the development of tension is largely circumstantial and is based on an association between fiber position and secondary stem re-erection. "Gelatinous fiber," as a descriptive term, refers to the distinctive unlignified and lamellate Sg layer of the secondary walls. In relation to these putative functions it seems appropriate to have some knowledge of the development of these reaction fibers in relation to shoot morphology as a whole. This study also provides information about the development of some other primary cell types in the stems of Gnetum, notably the laticifers, that are thought to be unique among gymnosperms (Behnke and Herrmann, 1978
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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For older stems and woody axes, freehand sections were made on a sliding microtome at 30–60 µm. Staining included 0.1% aqueous toluidine blue (general stain), iodine-potassium iodide (for starch), phloroglucinol and concentrated hydrochloric acid (for lignin), saturated alcoholic Sudan IV (for lipids), and 1% alcoholic chlorazol black (for pure cellulose). Maceration of developing internodes was done on small slices of material in concentrated HCl : water (1 : 3) in an oven at 55°C for 2–3 h. Older material was macerated as small slivers boiled for 2–3 min in 10% aqueous potassium hydroxide followed, after repeated washing in water, by 20% chromic acid (chromium trioxide) for 15–30 min. Macerated material was mounted and teased apart in glycerin : water (1 : 1) after repeated washing in water. It was found that careful maceration softened material sufficiently for cell separation while still retaining cell contents (e.g., Figs. 18–19). Nuclei could then be stained either in hot aceto-carmine, or iodine-potassium iodide, or acid fuchsin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Orthotropic shoots produce continuous sylleptic plagiotropic branches from primary buds, the decussate phyllotaxis resulting in four branch orthostichies and radial symmetry of trunk axes. Plagiotropic axes also have decussate phyllotaxis but become dorsiventral by continuous rotation of leaf pairs into the horizontal plane by internodal twisting and petiolar torsion. Higher-order branches are infrequently produced but always in the horizontal plane. Each leaf subtends a primary bud and one to two supernumerary serial buds, with the latter functioning as reserve buds, or in outer shoots as reproductive units. Reiterated axes on vegetative parts come from the reserve buds stimulated, either by damage to or tilting of axes, to develop by prolepsis and repeat the architecture of an orthotropic shoot (i.e., Roux's model). Growth of all axes is rhythmic but highly articulate because of the fairly constant extension of one internode at a time, with intervening rest or dormancy of the shoot apex. Infrequently, and then most often on orthotropic shoots, two or more internodes may extend without an intervening rest period. This articulated construction is related to bud morphology (Fig. 1). There are no bud scales, but the resting apex is protected by a pair of pouched petiole bases that produce a hemispherical cavity in which younger parts are enclosed. Presumed additional protection is provided by tufts of multicellular uniseriate hairs on the inner margin of the petiolar pouch. Resting buds include only two enclosed leaf pairs. The inner pair is represented only by each shallow leaf buttress, while the older pair is differentiated into distinct blade and petiole (Fig. 1). Extension of this pair proceeds in tandem with the development of a new leaf pair.
Extension of the shoot to produce internodes up to 15 cm long begins by extrusion of the older leaf primordial pair through the mouth of the petiolar pouch, followed by internodal extension and expansion that widens this cavity. On plagiotropic shoots the extending axis is floppy and pendulous but becomes rigid and self-supporting as tissues mature. When extension is complete, the apex again enters into a resting phase surrounded by the two pairs of leaf primordia. How this articulated shoot development produces a continuous vascular system remains unexplored.
Primary stem construction
The stem has a eustelic construction (Fig. 2), with about 45 primary vascular bundles in wider axes. In young axes the cortex is differentiated internally from the vascular cylinder by a conspicuous starch sheath that also includes a discontinuous series of irregularly stellate sclereids. Similar sclereids are isolated in the rest of the cortex and the medulla, with a concentration at the top of the internode immediately below the resting bud (Fig. 1, scl). A fascicular vascular cambium is a feature of each vascular bundle early in development (Figs. 20–21). It becomes a continuous cambial cylinder with the advent of an interfascicular cambium (e.g., Fig. 22). This results in a continuous secondary cylinder of xylem and phloem with very broad primary rays (Figs. 3–5). This paper first considers the maturation of the cortex in which the primary reaction fibers are present and then the outer phloem in which secondary reaction fibers develop.
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because differentiation involves dissolution of end walls between a longitudinal series of extending cells, although I did not observe such early stages. These authors also recognize laticifers by their characteristic cytoplasmic inclusions called spherule complexes that are said to have terpenoid-like contents. There is no evidence for apical intrusive growth because laticifers are always blunt-tipped (Fig. 11). Nevertheless, they are very long ranging to about 1 cm, so that even in macerated material complete laticifers cannot be measured precisely. Laticifers also occur diffusely in the medulla. Outer cortical fibers differentiate early (Fig. 7, Co 1) as elongated initials with thin primary walls. The subsequent production of a cellulosic secondary wall is artifactually always seen to be detached from the primary wall (Fig. 10). No evidence was found for the dissolution of transverse walls, as with laticifers, in these and other fibers. These outer fibers differentiate sufficiently early that their extension could be passive, i.e., without apical intrusive growth in contrast to later fibers.
In early internodal extension, middle and inner cortical cells appear uniform in transverse section (T.S.) except that outer cells include abundant chloroplasts (Fig. 6). As internodal extension proceeds, fiber initials continue to differentiate in a centripetal direction. Extension is in part passive because growth of fiber initials occurs during internodal elongation but apical intrusive growth also occurs, i.e., active extension. Fiber initials are recognizable in longitudinal section (L.S.) as elongated cells (Fig. 9, fi), which are highly vacuolated and with limited peripheral cytoplasm (Figs. 12–14 and 16). Cell walls remain thin but highly hydrophilic and refractive and stain intensely in safranin/alcian green preparations. As fiber differentiation proceeds, the cortex in transverse sections appears as a network of apparently empty cells (the cell contents are lost) among short undifferentiated parenchyma cells with normal cell contents (Fig. 2, co and Fig. 5, mc).
Evidence for apical intrusive growth is demonstrated by the pointed tips, as seen in L.S. (Fig. 15), with the narrow intrusive tips seen as cells of small diameter in T.S., protruding within intercellular spaces (Fig. 8). The cytoplasm in these tips is densely stained with safranin (Fig. 15) but is not granular as in laticifers and with a gradual transition to the thin vacuolated cytoplasm proximally (cf. Figs. 15–16). Some evidence of polarity occurs because the dense tip cytoplasm is usually directed apically within the internode.
Fiber cytology
A conspicuous feature of fiber initials is their multinucleate condition that results from nuclear division without concomitant wall formation (Fig. 13, n). In young initials this is made evident by clusters of from two to six nuclei in each cell (Figs. 14 and 16, n), suggesting a rapid sequence of mitoses. However, division figures have rarely been observed. Fiber initials are therefore not articulated by breakdown of transverse septa, as is reported for laticifers. As fiber elongation proceeds, the nuclei, which are initially spherical (Fig. 18), become elongated within the narrow cell lumen and contrast with the nuclei of ground parenchyma cells. Nuclei also become dispersed throughout the very long fiber initial so that it is difficult to count their number. The maximum number observed was eight. Fiber initials can remain indefinitely in this incompletely differentiated state without developing a secondary wall.
Mature internode
When internodal elongation has ceased, the mature cortex consists of the outer series of fully differentiated narrow fibers interspersed among chlorophyllous ground tissue. Internally, there are idioblastic laticifers and long immature fibers with thin but refractive primary walls and sparse cell contents, the fibers interspersed among short ground parenchyma cells with relatively dense contents. The starch sheath represents the innermost layer of the cortex, together with an irregular series of stellate, often long-armed sclereids. There is a two- to five-celled layer of parenchyma within the sclereid layer and immediately outside the vascular tissue to which the topographic term "pericycle" might usefully be given (e.g., Figs. 25–26). Vascular differentiation is complete in the larger vascular bundles as phloem and xylem separated by the interfascicular cambium (Fig. 22). The developing and primary phloem (Figs. 20, 21), as described by Behnke and Paliwal (1973)
and Paliwal and Behnke (1973)
, includes alternate files of long sieve cells (range about 400–850 µm) and files of shorter phloem parenchyma cells (range about 80–210 µm). The parenchyma cells originate by transverse division within a longer procambial or cambial derivative and retain their nuclei (Fig. 23). Radial seriation of phloem cells begins in the developing vascular bundle as protophloem (pphl) and protoxylem (pxy) appear (Fig. 20). It continues in later stages (Fig. 21) so that the fascicular cambium has a very early origin. The protophloem is eventually represented by its crushed elements as an outer cap to the primary phloem (Fig. 22, cphl). Further changes in the cortex and outer phloem are determined by gravimorphic responses.
Late fiber maturation
Cortex
On the upper side of plagiotropic axes, and on orthotropic axes displaced from the vertical, maturation of fiber initials proceeds entirely as a localized response, with initials on the lower side continuing to remain undifferentiated (cf. Figs. 4 and 5). This eccentricity (Fig. 3) appears to be the source of tensions that maintain the horizontal position of plagiotropic axes and re-erect orthotropic axes. The response is therefore the major stimulus for final maturation of gelatinous or reaction fibers. Secondary wall development is completed as in the already mature outer fibers but with much larger resulting cell diameters. In transverse sections the Sg layer of the secondary wall is at first typically collapsed and withdrawn from the primary wall, but at maturity the layer is concentric and clearly multilamellate. The separation of secondary from primary wall is evident even as fibers mature (Fig. 10) and is striking in macerated fibers (Fig. 17). Maturation of cortical fibers is always centripetal, so that middle level fibers always mature before inner fibers.
Primary and secondary phloem
The centripetal process of fiber differentiation continues in the outer phloem and is initiated by expansion and elongation of cells adjacent to the crushed remains of the early primary phloem (Fig. 24). Such cells are initially short phloem parenchyma cells since these cells, unlike sieve cells, are the only elements capable of dedifferentiation (Fig. 23). Also these phloem fiber initials must extend by intrusive growth, i.e., actively as there is no internodal elongation within secondary tissues. The initials consequently progressively penetrate the remains of the early phloem (Figs. 24–26), which is finally obliterated (Fig. 27), and the mature fibers reach a length probably comparable to that of cortical fibers. Fiber differentiation proceeds as in cortical fibers with the establishment of a thick Sg layer, at first appearing convoluted in T.S. (Fig. 26, Phf), but later becoming rigid. Development of large numbers of fiber initials and their intrusive growth results in complete occlusion of the phloem (Fig. 27). Because of their large numbers, fibers toward the cambium are thus likely to be the products of secondary phloem parenchyma cells that have dedifferentiated. Where the process of fiber stimulation is long active, as in orthotropic axes that have been displaced and tied down, the contrast between upper and lower extraxylary tissues is pronounced (Figs. 3–5).
Fiber dimensions
The three topographic fiber types fall into three discrete diameter categories, with little overlap. Mean value (N = 20) is 21 µm for Co 1 fibers (range 15–20 µm); 51.5 µm for Co 2 fibers (range 42.5–67 µm); and 27 µm for Phf fibers (range 22.5–32.5 µm). In transverse view the range appears to be greater because of the tapering ends of fibers (e.g., Fig. 27, Co 2). As indicated earlier, because macerated fibers are brittle and not easily separated, fiber length is difficult to measure precisely. Representative values for the few complete cells measured (N = 10) give a range of 6.5–14 mm for Co 1 fibers, 4–19 mm for Co 2 fibers, and 4–18 mm for Phf fibers. More precision would be misleading because fiber length is partly determined by internode length, which is variable within an order of magnitude on different parts of the tree. The quoted values are certainly minimal.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 1943
), but there are no reports of coenocytic stages specifically in tension wood fibers of dicotyledons, despite the large amount of literature on the subject. Emphasis has been directed more toward the nature of the cell wall and its variability (e.g., Prodhan et al., 1995
). The most striking feature is the distinctive and almost wholly cellulosic G-layer (Sg) of the secondary wall, with its shallow helical microfibrillar organization and weak attachment to the primary and outer secondary wall layer (S1). These features are found in the gelatinous fibers of Gnetum and are suggestive of cell type homologies between Gnetales and dicotyledons. However, there are unique features in the way in which gelatinous fibers develop in Gnetum because three cell types can be recognized within the continuum of centripetal fiber differentiation in extraxylary tissues. Their progressive development is charted in Fig. 28, which is complicated by the frequency of alternative pathways. The first category (Fig. 28, Fiber type Co 1) includes outer gelatinous fibers that are initiated in elongating internodes and do not show a gravimorphic response. They complete their maturation to form a concentric layer that is of presumed mechanical importance. Such fibers are therefore obligate in their development. Otherwise stem mechanical tissues consist only of the limited amount of primary xylem and sclereids with an idioblastic distribution. The latter largely form a discontinuous layer immediately outside the stele that is gradually completed by differentiation of cubical sclereids in thicker older stems.
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These developmental events continue in Gnetum within primary but largely secondary phloem cells (Fig. 28, Fiber type Phf) that dedifferentiate from existing parenchyma cells into fibers under the same continued gravimorphic stimulus. Fiber determination is again an entirely opportunistic process, differing on upper and lower sides of the same axis. Such gelatinous fibers elongate entirely by intrusive growth. Because the later-formed ones can be derived from cambial initials, i.e., are secondary in origin, they are more directly comparable to tension wood fibers of dicotyledons. The process of secondary fiber development in phloem tissues of Gnetum can be so prolonged that virtually the entire phloem sector becomes converted into gelatinous fibers and presumably loses most of its transport capacity (Fig. 27). Transport then becomes the function of the phloem in the lower stem sector in which there is no transformation, the phloem tissue retaining the characteristic features described by Behnke and Paliwal (1973)
and Paliwal and Behnke (1973)
. These authors make no indication of the presence of fibers in the phloem of Gnetum, presumably because they studied either younger stems not yet capable of any gravimorphic response or only erect orthotropic shoots.
The function of reaction fibers in Gnetum is still imprecisely understood because only an association between the presence of fibers with an eccentric distribution and a self-erecting response that seems to be dependent on induced tensions is observed. This is a common critique in this aspect of plant anatomy as a reverse explanation might suggest that induced tensions are the cause and not the effect of reaction fibers, as reviewed in Wilson and Archer (1977)
. Nevertheless, there is clear evidence that tension wood fibers are contractile agents in the establishment of the initially free-hanging roots of Ficus, as described by Zimmerman et al. (1964)
. Here, once a stem-attached aerial root with entirely primary vascular organization is planted in a pot, tension wood fibers are developed in newly formed secondary wood. The pot becomes lifted off the ground, i.e., the tension develops only after tension wood fibers are differentiated. Furthermore, this reaction wood is developed concentrically in the root. Such circumstantial evidence implicates reaction fibers in the development of tension. Therefore, fibers of this type, all with the same characteristic wall ultrastructure, can reasonably be expected to perform similar functions.
The observations on Gnetum demonstrate the complexity of reaction tissue formation in plants. In this genus there are different reaction responses in different parts of the same plant (e.g., the contrast both between orthotropic and plagiotropic axes and upper or lower stem sectors) and different responses to the same stimulus in different tissue regions (fibers differentiated under three contrasted developmental regimes). Gnetum may provide a suitable experimental object for elucidating the mechanisms that control cell differentiation and cell wall formation in plants.
There is also now evidence of a cell type in a gymnosperm whose apparent homologue was previously thought to be unique to angiosperms. This raises the evolutionary question whether the similarity is the result of shared ancestry or remarkable convergence.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Behnke H.-D. G. S. Paliwal 1973 Ultrastructure of phloem and its development in Gnetum gnemon, with some observations on Ephedra campylopoda. Protoplasma 78: 305-319[CrossRef][ISI]
Carlquist S. 1994 Wood and bark anatomy of Gnetum gnemon. Botanical Journal of the Linnean Society 116: 203-211[CrossRef]
Carlquist S. 1996a Wood, bark and stem anatomy of New World species of Gnetum. Botanical Journal of the Linnean Society 120: 1-19[CrossRef]
Carlquist S. 1996b Wood and bark anatomy of lianoid Indo-Malesian and Asiatic species of Gnetum. Botanical Journal of the Linnean Society 121: 1-24[CrossRef]
Carlquist S. A. A. Robinson 1995 Wood and bark anatomy of the African species of Gnetum. Botanical Journal of the Linnean Society 118: 123-137[CrossRef]
Esau K. 1938 The multinucleate condition of fibers of tobacco. Hilgardia 11: 427-434
Esau K. 1943 Vascular differentiation in the vegetative shoot of Linum. III. The origin of the bast fibers. American Journal of Botany 30: 579-586[CrossRef][ISI]
Hallé F. R. A. A. Oldeman P. B. Tomlinson 1978 Tropical trees and forests: an architectural analysis. Springer Verlag, Berlin, Germany
Paliwal G. S. H. D. Behnke 1973 Light microscopic study of the organization of phloem in the stem of Gnetum gnemon. Phytomorphology 23: 183-193
Prodhan A. K. M. J. Ohtani R. Funada H. Abe K. Fukazawa 1995 Ultrastructural investigation of tension wood fibers in Fraxinus mandshurica Rupr. var. japonica Maxim. Annals of Botany (London) 75: 311-317
Tomlinson P. B. 2001 Reaction tissues in Gnetum gnemon: a preliminary report. Journal of the International Association of Wood Anatomists 22: 401-413
Wilson B. F. R. R. Archer 1977 Reaction wood: induction and mechanical action. Annual Review of Plant Physiology 28: 23-43[ISI]
Zimmermann M. H. A. B. Wardrop P. B. Tomlinson 1964 Tension wood in aerial roots of Ficus benjamina L. Wood Science and Technology 2: 95-104
This article has been cited by other articles:
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  P. B. Tomlinson and J. B. Fisher Development of nonlignified fibers in leaves of Gnetum gnemon (Gnetales) Am. J. Botany, March 1, 2005; 92(3): 383 - 389. [Abstract] [Full Text] [PDF]
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