ed in a hydrophilous plant, Ceratophyllum demersum L., with electron microscopy, The plant shows a character- istic feature in tetrad formation. A callose wall is ...
J. Plant Res. 108 : 205-208, 1995
Journal of Plant Research (~ by The Botanical Society of Japan 1995
Development of Structure-less Pollen Wall in Ceratophyllum demersum L. (Ceratophyllaceae) Masamichi Takahashi Department of Biology, Faculty of Education, Kagawa University Takamatsu, 760 Japan
Developmental process of structure-less exine is studied in a hydrophilous plant, Ceratophyllum d e m e r s u m L., with electron microscopy, The plant shows a characteristic feature in tetrad formation. A callose wall is not synthesized and exine initiation does not occur during the tetrad stage. After release of microspores, a trilaminar layer with two electron-dense lines is formed in the surface of each microspore. The trilaminar layer develops to a thin structure-less exine that is considered to consist of only an endexine. The unusual exine would be an adaptive feature for submersed pollination in fresh water. Key words : Ceratophyllaceae - - Ceratophyllum m Development m Pollen
The genus, Ceratophyllum, widely distributed in fresh water of the world, is a submersed, monotypic genus that constitutes a family of its own Ceratophyllaceae (Ceratophyllales) (Les 1988). The genus is characterized by rootless, monoecious herbs and hydrophilous pollination (Endress 1994). Ceratophyllum has recently attracted much interest, because nucleotide sequences analyses of the plastid gene rbcL suggested that the genus is located in an isolated primitive position and is a sister group to all other flowering plants (Les et al. 1991, Chase et al. 1993). Pollen of Ceratophyllum has been described as spheroidal and nonaperturate, with very thin and psilate exine (Erdtman 1972). Pettitt and Jermy (1975) showed a thin, structureless, and unsculptured exine with a fine fibrillar network in Ceratophyllum pollen with transmission electron microscopy (TEM). Les (1988) described, however, curious process of the outer wall of Ceratophyllum pollen with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The pollen wall structure of Ceratophyllum has not been ascertained. Many hydrophilous flowering plants have an extremely reduced exine or exine-less pollen. Pettitt (1981) showed that three hydrophilous plants in marine monocotyledons, Thalassia, Hatophita and Thalassodendron, have an extremely reduced exine. He also showed that a callose wall is not synthesized during meiotic prophase in pollen development of these marine angiosperms. In the present study, pollen wall structure and its
developmental process have been examined in the hydrophilous plant, Ceratophyllum demersum L., with transmission electron microscopy and field emission scanning electron microscopy. Materials and Methods Fresh anthers at various developmental stages were obtained from plants of Ceratophyllum demersum L., collected in Tsuda-cho, Kagawa Pref., Japan. Voucher specimens were deposited in the Biological Institute, Tohoku University (TUS). The anthers were fixed in a mixture of 1% paraformaldehyde and 3% glutaraldehyde in sodium cacodylate buffer (pH 7.4) for 2 days according to the procedure of Karnovsky (1965). The fixed anthers were kept in phosphate buffer (pH 7.4) for a week, and were then post fixed with 1% osmium tetroxide for 24 hr at 4C, and subsequently dehydrated in a graded ethanol series. Some of the anthers in the absolute ethanol were frozen with liquid nitrogen and freeze-cleaved on a TF-1 chamber (Tanaka 1981). After critical-point drying the freezecleaved anthers were sputter coated with platinum-palladium at ca. 5-8nm thick using an Eiko SR-2 ion sputtering apparatus and examined in a Hitachi S-800 field emission scanning electron microscope. The other fixed anthers were infiltrated with low viscosity resin (Kushida 1980). Thin sections (70-90 nm) were cut with a diamond knife on a Reichert Ultracut-N ultramicrotome and usually double-stained with 1% aqueous uranyl acetate (15 min.) followed by lead citrate (15 min.) (Reynolds 1963). Observations and photomicrography were done using a Hitachi H-7100EX transmission electron microscope. Results During pollen development in Ceratophyllum, a callose wall is not synthesized in the tetrad. Successive cytokinesis after meiosis results in the formation of tetrads in which four microspores are divided by a thin cell wall, not a thick callose wall (Fig. 1). Conformational or secretive changes in the plasma membrane were not seen during the brief tetrad stage. The tetrad period is very short, and the microspores are released into Ioculus immediately
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Figs. 1-9. Pollen development in Ceratophyllum demersum. 1. Freeze cleaved tetrad. Bar=lO ,urn. 2. Trilaminar layer in surface of released microspores. Bar---O.5/~m. 3. Duplicate trilaminar layers. Bar=O.1/zm. 4. Surface of young microspore. Bar=l/zm. 5. DeveloPing intine. Bar=O.2,~m. 6. Microspore cell surface. Bar=0.1 ~m. 7. Freeze cleaved pollen~ Bar=lO ,um. 8. Mature anther with four Ioculi. Bar=lOO,um. 9. Mature pollen wall. Bar=O.5/~m.
Structure-less Exine in Ceratophyllum after the meiotic cytokinesis. After release of microspores, a trilaminar layer that consists of two electron-dense lines on either side of a light zone develops on the surface of each microspore (Fig. 2). The trilaminar layers are partly duplicated and detached from the outer layer (Fig. 3). Subsequently, the trilaminar layer transforms into a skin-like membranous exine, 13-15nm thick, on the surface of developing microspores (Fig. 4). A white line remains underneath the membranous exine. After microspore mitosis, an intine is formed evenly underneath the membranous exine by the accumulation of fibrous cellulose (Figs. 5, 6). The cytoplasm includes many Golgi bodies, lipid droplets, and mitochondria. Mature pollen grains are spheroidal, 20-22/~m in diameter, that include two spermatozoid cells, a vegetative nucleus, amyloplasts, lipid droplets, and a few mitochondria (Fig. 7). The anthers have four Ioculi including pollen grains (Fig. 8). The pollen grains are surrounded by degenerated tapetal remnants, but their exine does not show any apparent sculpture or protrusions. The mature exine is extremely reduced, skin-like, and structure-less (Fig. 9). The intine, evenly 0.5/zm thick, consists of cellulosic fibrous materials (Fig. 9). Cytoplasmic channels are not observed in the intine.
Discussion In most angiosperms, exine patterns are formed within a callose wall during the tetrad stage, mediated by the microspore plasma membrane (Takahashi 1989, Takahashi and Skvarla 1991a, 1991b). Pollen development in Ceratophyllum is different from the sequence of exine formation in most angiosperms. A callose wall is not deposited during the pollen mother-cell stage and after the meiotic cytokinesis, and no indication for exine formation was recognized during the tetrad stage. Little or no deposition of a callose wall in the tetrad has been shown in submarine hydrophilous flowering plants (Ducker et al. 1978, Pettitt 1981). A characteristic feature of the tetrad is the conspicuous absence of an outer callose wall, and only a very thin callose deposition on the inner walls in Thalassia and Thalassodendron (Pettitt 1981). The callose wall is considered to play an effective role in the establishment of the exine pattern, as reviewed by Bhandari (1984). The absence of a callose wall in pollen development of the submarine flowering plants and Ceratophyllum suggests that the callose wall could be expected to play a role in exine development of other angiosperms. The present study showed that a membranous exine is formed by deposition of the trilamellar layer after the release of microspores in Ceratophyllum. Lamellations of trilamellar units have been observed in endexine development of many angiosperms after release of microspores, as Epiloboium (Rowley 1987-1988), Faffugium (Takahashi 1989a), Caesalpinia (Takahashi 1989b), and Oenothera (Takahashi and Skvarla 1990). The membranous exine of
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Ceratophyllum pollen corresponds to endexines of other angiosperms in developmental process and conformation. The extremely reduced exine is structure-less without ornamentation. The curious process of exine described by Lea (1988) would be a kind of artifacts by dehydration, because of the extremely reduced exine. Ducker et al. (1978) reviewed the studies on the adaptation of the pollen of aquatic plants for submarine pollination. The same thin and structure-less exines with Ceratophyllum pollen are recognized in pollen of Najas and Halodule (Pettitt and Jermy 1975). Pollen of Amphibois antarctica has no exine, and only intine occupies the pollen wall (Ducker et ah 1978). Martinsson (1993) showed that the exine in some Callitriche species (Callitrichaceae) tend to be reduced in relation to the submergence of pollination. Although the genus Ceratophyllum has attracted systematic attention because the sequence analysis of the gene rbcL suggested the genus as the most primitive taxon in the angiosperms (Chase et ah 1993), the reduced membranous exine would be due to the adaptation for submersed pollination in fresh water. It would be improbable that the thin structure-less exine of Ceratophyllum pollen represents a plesiomorphic character state in angiosperms. The author thanks Prof. Hiroshi Tobe (Kyoto Univ.) and Dr. John R. Rowley (Stockholm Univ.) for valuable comments and suggestions on the manuscript. This study was partly supported by grants from Ministry of Education, Science and Culture Japan (05454031 to M.T., 05304009 and 06454031 to Hiroshi Tobe).
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(Received March 6, 1995 : Accepted April 5, 1995)
