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Structure and Development |
2The University Museum, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan; 3Department of Chemical and Biological Sciences, Japan Women's University, 2-8-1 Mejirodai, Tokyo 112-8681, Japan; 4Botanical Gardens, Graduate School of Science, The University of Tokyo, 3-7-1 Hakusan, Tokyo, 112-0001, Japan
Received for publication December 22, 2004. Accepted for publication April 15, 2005.
ABSTRACT
We studied the development and structure of the unusual trichotomous branching of Edgeworthia chrysantha. Three "branch primordia" are formed sequentially on the shoot apex of a main axis and develop into trichotomous branching. The branch primordia are clearly distinguishable from the typical axillary buds of other angiosperms; they develop much more rapidly than axillary buds, and the borders between the branch primordia and shoot apex of the main axis are anatomically unclear. Furthermore, at a later stage, leaves subtending the branch primordia produce typical axillary buds. These results suggest that the trichotomous branching in this species involves the division of the shoot apical meristem. Expression analysis of genes involved in branching or maintenance of the shoot apical meristem in this species should clarify the control mechanism of this novel branching pattern in angiosperms. We also observed the phyllotactic patterns in trichotomous branching and have related these patterns to the shoot system as a whole.
Key Words: Edgeworthia chrysantha • Japan • phyllotaxy • shoot apical meristem • Thymelaeaceae • trichotomous branching
Trichotomous branching is neither frequent nor rare among angiosperms (Kumazawa, 1979
). There are two major types of trichotomous branching, in which phyllotaxy is either whorled or spiral. In one type, three vegetative branches develop from the axillary buds of trimerous leaves (e.g., Nerium indicum, Fig. 1). In the other type, three axillary branches of spirally arranged leaves with extremely reduced internodes form shoots with trichotomous-like branching (e.g., Mallotus japonicus, Fig. 2). It is easy to understand the shoot organization of both types because subtending leaves or their scars remain at the branch bases.
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) and is well known for its trichotomous branching (the Japanese name of this species, "Mitsumata," means "trichotomous branching") (Clennett et al., 2002
). This trichotomous branching is unique in that two of the three branches are not subtended by leaves, although the remaining one has a subtending leaf (Fig. 3). Based on this morphology, Kumazawa (1979)
proposed that this form of trichotomous branching is produced by the following three processes: (1) abortion of the main shoot apex, (2) elongation of axillary buds in the last three leaves of the main shoot, and (3) adnation of the subtending leaves along the branches.
We recently presented a new interpretation of the shoot organization. We proposed that one of the trichotomous shoots is an extension of the main shoot and the others extend from axillary buds (Iwamoto et al., 2002
). This interpretation is based on the preliminary observation that the phyllotactic spiral direction (clockwise or counter clockwise) of one of the trichotomous shoots always corresponds to that of the main shoot, whereas the other trichotomous shoots have the reverse phyllotactic spiral direction.
In these previous studies, however, neither the development nor anatomy of this form of trichotomous branching was described in detail. This article aims to elucidate the precise development and anatomy of this enigmatic branching and to clarify this novel type of branching pattern in plants.
MATERIALS AND METHODS
Plant material
Living plants of Edgeworthia chrysantha Lindl. were collected at five sites in Japan: the Botanical Garden of the New Technology Development Foundation, Atami-shi, Shizuoka Prefecture; Josenji Temple, Yamato-shi, Kanagawa Prefecture; Yamauchi, Nikko-shi, Tochigi Prefecture; Japan Women's University, Bunkyo-ku, Tokyo; and the Botanical Gardens, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo. No significant developmental variation was observed among the materials collected from these five sites. Reference specimens are housed in the herbarium of the University Museum, The University of Tokyo (TI).
Methods of observation
For scanning electron microscopic (SEM) observations of shoot apical meristems, replicas of fresh material were cast in epoxy resin from silicone molds (Williams et al., 1987
) and materials fixed with FAA (50–70% absolute ethanol, 5% glacial acetic acid, and 5–7% formalin) were used. The fixed materials were dehydrated in a graded ethanol series, then the ethanol was replaced with isoamyl acetate. The samples were then dried with a critical point dryer (HCP-2, Hitachi, Japan). Both types of material were coated with Pt/Pd using a sputter coater (Ion Sputter E-1030, Hitachi, Japan) and observed with an S-2250N SEM (Hitachi, Japan) at 5 kV.
For anatomical observations, the materials fixed with FAA were dehydrated in a graded ethanol series and embedded in Leica Historesin (Leica, Germany) or Paraplast (Oxford, UK). Historesin blocks were cut into 2–3-µm-thick sections and stained with solutions of toluidine blue, safranin, and orange G (Jernstedt et al., 1992
). Paraplast blocks were cut into 7–10-µm-thick sections and stained with safranin, hematoxylin, and fast green FCF (Ruzin, 1999
).
RESULTS
Phenology of Edgeworthia chrysantha
In late summer or early autumn, a shoot apical meristem (SAM) of the main axis differentiates into a reproductive bud containing bracts and flowers and concurrently the axillary bud of the youngest leaf (i.e., the closest leaf to the SAM) develops into a lateral shoot. The SAM of this lateral shoot produces 4–5 immature leaves in late autumn to early winter, then ceases growing to form a winter bud. Older leaves formed on the original axis abscise before winter. The winter bud consists of only young leaves, lacking bud scales. Flowers bloom in late winter to early spring. Then the winter bud resumes growth and continues to produce new leaves on a new main axis. In other words, the original main axis is terminated by the flower bud, and the axillary bud produces the new main axis. The phyllotactic spiral direction of the lateral shoot is always reversed relative to that of the main axis.
In late spring to early summer, the shoot apex undergoes trichotomous branching after it has produced about 10 leaves. The formation of trichotomous branches is so rapid that the relationship of the three shoots cannot be distinguished by gross morphological observation. The phyllotactic spiral direction of one shoot always corresponds with that of the shoot before trichotomous branching, although the direction is always reversed in the other two shoots. After the first branching, another round of trichotomous branching sometimes occurs in late summer. Development of the first and second trichotomous branchings do not vary significantly.
Formation of trichotomous branching
Leaf primordia are numbered from the youngest to oldest (P1–P6). A vegetative SAM in E. chrysantha is covered with hairy young leaves (Fig. 4), that have a spiral phyllotactic pattern with a divergence angle of around 135° (Figs. 5–7).
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Subsequently, another branch primordium (A2) on the adaxial side of P2 is initiated (Fig. 9). Both branch primordia, A2 and A3, form the first leaf primordia, although the last branch primordium (A1) is still unclear at the axil of P1 (Fig. 10, arrowhead). At a slightly later stage, A1 becomes apparent, and the first leaf primordia on A2 and A3 also develop (Fig. 11). A1 becomes larger to form the first leaf primordium, and as a result, a trichotomous shoot is established (Fig. 12). By this stage, the second leaf primordia have already developed on A2 and A3. Note that the position of the first leaf primordium on A1, relative to the adaxial side of its subtending leaf, is reversed compared with the corresponding positions on A2 and A3 relative to their subtending leaves (Figs. 10–12). For instance, in Fig. 12, the first primordium on A1 develops on the left side relative to the adaxial side of its subtending P1 (clockwise phyllotaxy), whereas the first primordia on A2 and A3 develop on the right side relative to the adaxial sides of P2 and P3 (counter clockwise phyllotaxy).
After trichotomous branching has been established, each branch produces leaf primordia in sequence, and the older primordia develop hairs (Fig. 13). These branches elongate, producing new leaves (Fig. 14). P1 and P2 are shifted upward due to the elongation of portions below the insertion levels of P1 and P2 (Figs. 14, 15). In contrast, P3 usually remains at the branching point at this and later stages (Fig. 14).
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P2 P3, whereas shoots subtended by P2 and P3 have the reverse phyllotaxy (Fig. 14).
Developmental anatomy of shoot trichotomy
Before trichotomy, the SAM is rather flat, showing the tunica (two layered)–corpus configuration (Figs. 16, 17). Histologically, the SAM is composed of central and peripheral zones. The central zone consists of relatively large lightly stained cells, and the peripheral consists of small, densely stained cells (Figs. 16, 17). In E. chrysantha, the three youngest leaf primordia (P1– P3) do not develop any apparent axillary meristems (Fig. 17), and the fourth and fifth youngest leaf primordia (P4, P5) have distinct axillary meristems (Figs. 18–19). The axillary meristem is comprised of densely stained meristematic cells and is surrounded by lightly stained, somewhat differentiated cells (Fig. 19). However, there is no shell zone (procambial-cell-like elongated cells) demarcating the axillary meristem from the SAM, which is otherwise often visible in ordinary angiosperms (Esau, 1977
).
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The branch primordia continue to grow, mainly due to anticlinal cell divisions on their own flanks (Figs. 22, 23). At this stage, the central zone of the original SAM is retained, although its area has become much more reduced in size (Fig. 23). Its surface and several cell layers beneath it are continuous with those of the branch primordia. It seems that the marginal region of the central zone of the original SAM has been involved in developing the branch primordia. More developed branch primordia begin to produce their own leaf primordia from their SAMs (Fig. 24). Even at this stage, the central zone of the original SAM is still distinguishable in its original position, although its layered structure is not continuous with the well-developed branch primordia (Fig. 25).
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Structure of trichotomous branching
Transverse sections at various positions of the trichotomous branches are shown in Figs. 30–35. At the bottom of the trichotomous branching (level "a" in Fig. 30), there is the leaf scar of P3, which subtends one of the trichotomized shoots (Fig. 31). At the axil of P3, two vascular traces are observed (Fig. 31, white arrowheads). These can be specified as the vascular traces of the axillary bud of P3 (in detail, they are the vascular traces of the prophylls developed on the axillary bud). Each branch shoot becomes clearer at the middle position of the trichotomous conjunction (level "b" in Fig. 30; Fig. 32) and finally separates from the others (Fig. 33). At each axillary position, there are two vascular traces (Figs. 34, 35, white arrowheads), which can also be regarded as the vascular traces of the axillary buds of P1 and P2.
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DISCUSSION
Composition of trichotomous branching
The positions of branch primordia in E. chrysantha correspond to the axils of their subtending leaves, and the primordia develop in sequence according to the ages of the subtending leaves. Therefore, it could be interpreted that these branch primordia are the axillary buds of the subtending leaves. Our results, however, indicate that these primordia are clearly different from normal axillary buds in at least three points.
First, our SEM observations reveal that the branch primordia develop to become trichotomous shoots with great rapidity during branching. When the leaves subtending branches (P1– P3) are still primordia, the branch primordia develop shoots with some leaves. That is, the development of the subtending leaves and of the shoots arising from the branch primordia proceed in parallel. We observe that, in many woody species, the current year's axillary buds develop into sylleptic shoots (e.g., Iwamoto et al., 2001
), and also in E. chrysantha, after differentiating flower buds, the axillary bud of the youngest leaf develops sylleptically (see Results, Phenology of Edgeworthia chrysantha). However, the rapid development observed in E. chrysantha has not been reported in the axillary bud development of any other species except for Alstonia scholaris. In A. scholaris, "subterminal buds" in axils of scale leaves have a similar rapid development and are distinct from normal axillary buds (Mueller, 1985
). But as referred to hereinafter, most of the original SAM of E. chrysantha is involved in the branch primordia, while the original SAM of A. scholaris is so large that its central region is not involved in the subterminal buds. This might be related to the phyllotactic difference between the two species, i.e. alternate vs. whorled.
Second, the subtending leaves (P1–P3) develop normal axillary buds that are clearly distinguishable from the branch primordia. Although we could interpret these later-developing axillary buds as accessory buds of P1–P3 and the branch primordia as the main axillary buds, there are distinct developmental differences between the branch primordia and the axillary buds. It is difficult to believe that a branch primordium and an axillary bud, subtended by the same leaf, arise from the same axillary meristem. This suggests that we should identify the late-developing axillary buds as normal, typical axillary buds of P1–P3 and the branch primordia as different organs, quite distinct from them.
Third, anatomical observations indicate that the borders between an original shoot apex and the branch primordia are not distinguishable, and the abortion of the original SAM during trichotomous branching is not conspicuous. This suggests that the branch primordia are not normal axillary buds, but meristematic organs derived from the SAM or from a complex consisting of the SAM and the axillary meristems. Therefore, SAM division could be involved in the trichotomous branching of E. chrysantha. Although it is quite rare, some other studies have also confirmed or suggested the dichotomous branching of vegetative shoots by SAM division in angiosperms, in both monocotyledons (Flagellaria [Tomlinson, 1970
; Tomlinson and Posluszny, 1977a
, b
], Nypa [Tomlinson, 1971
], and Chamaedorea [Fisher, 1974
]) and dicotyledons (Mammillaria [Boke, 1976
]).
To clarify whether the branch primordia are derived from a part of the SAM and/or axillary meristems, studies of the gene expression involved in branching are imperative. LATERAL SUPPRESSOR (LAS) is a gene expressed in the axillary meristems of Arabidopsis thaliana, but not in SAMs, which indicates that this gene is a marker gene for the activity of axillary meristems (Greb et al., 2003
). Orthologous genes have been identified in tomato (Rossberg et al., 2001
) and rice (Tian et al., 2004
), and the cognate protein function is conserved among these three species (Schumacher et al., 1999
; Greb et al., 2003
; Li et al., 2003
). Accordingly, E. chrysantha presumably has an orthologous LAS gene, which could be a marker for axillary meristems. Analysis of this orthologous gene expression should elucidate the nature of the branch primordia and their differences and/or similarities to the axillary buds.
CLAVATA3 (CLV3) is also a candidate gene with which to clarify the intrinsic character of trichotomous branching. CLV3 encodes a small, extracellular protein and is expressed at the meristem surface in A. thaliana (Fletcher et al., 1999
). The site of expression agrees with the central zone of the SAM, and analyses with mutants and transgenic plants imply that the expression zone enlarges when the SAM and its central zone expand (Fletcher et al., 1999
; Schoof et al., 2000
; Lenhard and Laux, 2003
). These results indicate that CLV3 should be a marker gene for the central zone of the SAM. Expression analysis of the orthologous CLV3 gene in E. chrysantha during trichotomous branching should provide an important insight, i.e., if trichotomous branching involves division of the SAM, as we infer, the expression zone of the CLV3 orthologue should become enlarged and subsequently divided into three zones, corresponding to the three central zones of the SAMs of the trichotomous shoots.
Phyllotaxy of trichotomous branching
Our observations reveal that there is no distinct morphological variation among the three developing branch primordia that comprise the trichotomous branching of E. chrysantha. This does not support the previous interpretation that one of the three shoots in trichotomous branching is the original main shoot itself and the other two shoots arise from axillary buds (Iwamoto et al., 2002
).
On the other hand, gross morphological and developmental observations confirm that the phyllotaxy of the youngest shoot of the trichotomous branching always corresponds to that of the main axis, whereas the other two older shoots show reversed phyllotaxy. This result can be reasonably explained if we assume that the youngest shoot is derived from the SAM and the others from axillary buds, because all lateral axillary shoots arising after flower initiation have reversed phyllotactic spiral directions relative to those of their main axes (see Results, Phenology of Edgeworthia chrysantha). Moreover, our results suggest that the division of the SAM may be involved in trichotomous branching. Although there is no exomorphological variation among the three branch primordia, there may be compositional differences, i.e., the youngest primordium retains the nature of the original SAM more than the other two primordia do, in response to an unknown mechanism. Some studies have referred to the relationship between the phyllotaxy of the main axis and that of the lateral axillary shoot (Tucker, 1963
; Gómez-Campo, 1970
; Kumazawa and Kumazawa, 1970
), but no one has yet correlated the meristematic characters of the shoots with their phyllotactic patterns. Gene expression analyses in E. chrysantha will elucidate this relationship between phyllotaxy and meristematic character.
Significance of trichotomous branching
Our study shows the novelty inherent in the trichotomous branching of E. chrysantha. This type of branching has never been reported in any other species and cannot be explained by the classical model of vegetative branching in angiosperms. The vegetative dichotomy exemplified by the division of the SAM is a similar aberration of the classical model (Fisher, 1976
). The reports of this type of branching have been limited to monocotyledons and a highly specialized dicotyledonous group, Mammillaria (Boke, 1976
). Therefore, this type of aberration has been considered exceptional and found only in specialized groups of angiosperms. Edgeworthia chrysantha, however, is neither a monocotyledonous nor a highly specialized species. A reevaluation of vegetative shoot organization among more examples of nonspecialized dicotyledonous species with trichotomous branching may lead to a new understanding of the branching system itself.
FOOTNOTES
1 The authors thank the Botanical Garden of the New Technology Development Foundation, Shizuoka, Japan, and Josenji Temple, Kanagawa, Japan, for support in collecting materials. Our thanks also go to Munetaka Sugiyama for helpful advice on the manuscript and Tomoko Yoshizawa for assistance in collecting plants. This study was partly supported by a Grant-in-Aid from the Japanese Ministry of Education, Science, and Culture (No. 09836026) and the New Technology Development Foundation. 
5 Author for correspondence (e-mail: akitoshi{at}um.u-tokyo.ac.jp
)
LITERATURE CITED
Boke N. H. 1976 Dichotomous branching in Mammillaria (Cactaceae). American Journal of Botany 63: 1380-1384[CrossRef][ISI]
Clennett C. Y. Harvey G. Pearman 2002 Edgeworthia chrysantha: Thymelaeaceae. Curtis's Botanical Magazine 19: 19-27
Esau K. 1977 Anatomy of seed plants, 2nd ed. John Wiley & Sons, New York, New York, USA
Fisher J. B. 1974 Axillary and dichotomous branching in the palm Chamaedorea. American Journal of Botany 61: 1046-1056[CrossRef][ISI]
Fisher J. B. 1976 Development of dichotomous branching and axillary buds in Strelitzia (Monocotyledoneae). Canadian Journal of Botany 54: 578-592[ISI]
Fletcher J. C. U. Brand M. P. Running R. Simon E. M. Meyerowitz 1999 Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 283: 1911-1914
Gómez-Campo C. 1970 The direction of the phyllotactic helix in axillary shoots of six plant species. Botanical Gazette 131: 110-115[CrossRef]
Greb T. O. Clarenz E. Schafer D. Muller R. Herrero G. Schmitz K. Theres 2003 Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes and Development 17: 1175-1187
Huang S. C. 1999 Edgeworthia. In T. C. Ku [eds.], Flora republicae popularis sinicae, vol. 52(1), 389–393. Science Press, Beijing, P. R. China
Iwamoto A. K. Miyazaki J. Murata 2001 Variation in shoot organization in Prunus (Rosaceae) with special reference to Prunus ogawana. Journal of Japanese Botany 76: 329-338
Iwamoto A. J. Murata H. Ohba 2002 Structure of enigmatic trichotomous branching in Edgeworthia chrysantha. Journal of Plant Research 115: (Supplement) 136-137 (Abstract)
Jernstedt J. A. E. G. Cutter E. M. Gifford P. Z. Lu 1992 Angle meristem origin and development in Selaginella martensii. Annals of Botany 69: 351-363
Kumazawa M. 1979 Plant organography. Syokabo, Tokyo, Japan
Kumazawa M. M. Kumazawa 1970 Analytical studies on the anodic and cathodic positions of prophylls in some dicotyledonous plants. V. Periodic variation of prophyll position. Botanical Magazine, Tokyo 83: 242-248[ISI]
Lenhard M. T. Laux 2003 Stem cell homeostasis in the Arabidopsis shoot meristem is regulated by intercellular movement of CLAVATA3 and its sequestration by CLAVATA1. Development 130: 3163-3173
Li X. Y. Q. Qian Z. M. Fu Y. H. Wang G. S. Xiong D. L. Zeng X. Q. Wang X. F. Liu S. Teng F. Hiroshi M. Yuan D. Luo B. Han J. Y. Li 2003 Control of tillering in rice. Nature 422: 618-621[CrossRef][Medline]
Mueller R. J. 1985 Determinate branch development in Alstonia scholaris (Apocynaceae): the plagiotropic module. American Journal of Botany 72: 1435-1444[CrossRef][ISI]
Rossberg M. K. Theres A. Acarkan R. Herrero T. Schmitt K. Schumacher G. Schmitz R. Schmidt 2001 Comparative sequence analysis reveals extensive microcolinearity in the Lateral suppressor regions of the tomato, arabidopsis, and capsella genomes. Plant Cell 13: 979-988
Ruzin S. E. 1999 Plant microtechnique and microscopy. Oxford University Press, New York, New York, USA
Schoof H. M. Lenhard A. Haecker K. F. X. Mayer G. Jurgens T. Laux 2000 The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 100: 635-644[CrossRef][ISI][Medline]
Schumacher K. T. Schmitt M. Rossberg G. Schmitz K. Theres 1999 The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family. Proceedings of the National Academy of Sciences, USA 96: 290-295
Tian C. G. P. Wan S. H. Sun J. Y. Li M. S. Chen 2004 Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Molecular Biology 54: 519-532[CrossRef][ISI][Medline]
Tomlinson P. B. 1970 Dichotomous branching in Flagellaria indica (Monocotyledones). In N. K. B. Robson, D. F. Cutler, and M. Gregory [eds.], New research in plant anatomy, 1–15. Academic Press, London, UK
Tomlinson P. B. 1971 The shoot apex and its dichotomous branching in the Nypa palm. Annals of Botany 35: 865-879
Tomlinson P. B. U. Posluszny 1977a Features of dichotomizing apices in Flagellaria indica (Monocotyledones). American Journal of Botany 64: 1057-1065[CrossRef][ISI]
Tomlinson P. B. U. Posluszny 1977b Apical dichotomy demonstrated in the angiosperm Flagellaria. Science 196: 1111-1112
Tucker S. C. 1963 Development and phyllotaxis of the vegetative axillary bud of Michelia fuscata. American Journal of Botany 50: 661-668[CrossRef][ISI]
Williams M. H. M. Vesk M. G. Mullins 1987 Tissue-preparation for scanning electron microscopy of fruit surfaces. Comparison of fresh and cryopreserved specimens and replicas of banana peel. Micron and Microscopica Acta 18: 27-31
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