Nord. J. Bot.

- Section of structural botany

A structural investigation of the ovule i n sugar beet, Beta vulgaris: Integuments and micropyle
P. Olesen and Lone Bruun

Olesen, P. & Bruun, L. 1990. A structural investigation of the ovule in sugar beet, Beta vulgaris: Integuments and micropyle. - Nord. J. Bot. 9: 499-506. Copenhagen. ISSN 0107-055X. The micropyle and the integuments of sugar beet (Beta vulgaris) ovules have been investigated by light and electron microscopy during differentiation and maturation of the ovule. The micropyle itself is formed by the inner integument which is surrounded by the outer integument at its base. The micropyle containts a fibrillar PAS+ substance and is often covered by a thin sheet or hymen. Both integuments are cuticle-covered thin sheets, each 2-few cell layers in thickness. In the outer integument an increase in starch accumulation occurs during ovule maturation and probably functions as nutrient storage for embryo development. The inner epidermis of the inner integument differentiates as the most conspicuous cell layer of the beet ovule. During growth and maturation of the ovule a system of small perinuclear vacuoles containing dense material increases steadily in these cells. At maturity this system fills up more than half of each cell and very dense material has accumulated in each vacuole. This vacuole content is highly refractive and contains tannins andfor polyphenols.

P. Olesen, Biotechnology Research Division, DANISCO AIS, P.O. Box 17, DK-1001 Copenhagen K , Denmark. - L. Bruun, Botanical Lab., Univ. of Copenhagen, Gothersgade 140, DK-1123 Copenhagen K , Denmark.

Introduction
Even though the integuments and the micropyle are some of the most distinct features of the angiosperm ovule, less attention has been paid to the ultrastructure and function in the reproduction processes of these parts of the ovule. As regards the integuments, only their number, initiation, testa development (ex. Bor & Bouman 1974; Bosewinkel 1980) and the integumentary tapetum has been treated in some detail (Kapil & Tiwari 1978). A detailed study on the inner epidermis of the inner integument (the fringe-layer) of cotton ovules and seeds has been made (Ryser et al. 1988). Tilton (1980) and Tilton & Lersten (1981) have described the ovule development in Ornithogalum and presented a review on selected papers on the micropyle and the integuments, respectively. In spinach, a species closely related to beets, Wilms (1980) made a comprehensive
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study of ovule structure and development and included a description of the integuments. Although sugar beet is an important socio-economic crop plant which has been subject to intensive breeding activities, not much information is available about its reproduction biology. Such data are highly needed for a successful implementation of biotechnological methods in the breeding of new beet varieties. Early light microscopical descriptions of the ovule in sugar beet have been made with little attention to the integuments and the micropyle (Heel 1925; Artschwager 1927). Recently a few studies have been published on structural aspects of sugar beet ovules, i.e., in vitro cultured ovules (Hosemans & Bossoutrot 1986) and some observations on fertilization (Shen et al. 1986). Again, these studies contain no new data related to the integuments and the micropyle of sugar beet. In this study the integuments and the micropyle from sugar beet ovules have been investigated by light-, scan-

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Figs 1 4 : Eera vulgaris. - Fig. 1. Light microscopical overview showing outer and inner integuments and micropyle ~ 2 5 (bar 0 = 50 pm). - Fig. 2. Cross section showing outer and inner integuments at the level of the egg cell ~ 3 6 0 (bar = 50 pm). - Fig. 3. Scanning electron micrograph of the micropyle showing the different lengths of the inner and outer integuments ~ 7 5 (bar 0 = 10 pm). - Fig. 4. Scanning electron micrograph of the micropyle covered with a sheet X950 (bar = 10 pm).

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Figs 5-8: Bern vulgaris. - Fig. 5. Cross section of the micropyle containing a fibrilar substance x 1 1 200 (bar = 1 pm). - Fig. 6. Cuticles covering the inner and outer integuments ~ 2 9 5 0 0 (bar = 1 pm). - Fig. 7. Cross section of the border between the inner integument and the micropyle at the level of the apex of the micropylar nucellus, note the accumulation of black tannin deposits in (bar = 1 pm). - Fig. 8. Details of outer integument, plastid with starch grains and the epidermis of the inner integument. ~ 3 0 0 0 outer cuticle ~ 2 9 5 0 0 (bar = 1 pm).

Figs 9-10: Beta vulgaris. - Fig. 9. Cuticle staining with Auramine 0: Slight staining of integument cuticles but strong reaction at nucellar surface ~4.50 (bar = 50 pm). - Fig. 10. Collapsed cell layers in the apex of the outer integument X7300 (bar = 1 pm). ning- (SEM), and transmission electron microscopy (TEM). The study is part of a research programme on the cellular and molecular biology of reproduction in sugar beet, Beta vulgaris L. (Bruun 1987; Nielsen & Olesen 1988; Bruun & Olesen 1988, 1989). Special emphasis is given to the cells of the inner epidermis of the inner integument and the importance of tannin deposition in this layer early in ovule development. as outdoors. Conditions and methods for fixation, embedding, and sectioning for light- and transmission electron microscopy were as previously described (Bruun 1987). 3.0 km sections were stained in periodic acidSchiff (PAS, Feder & OBrien 1968) for 30 minutes and in 1%Aninline Blue Black (ABB, Fischer 1968) in 7% acetic acid for 2 minutes for light microscopic detection of insoluble polysaccharides and proteins, respectively. For a better localization of tannins and polyphenols sections were double stained with ABB and 0.05% Toluidine Blue 0 (TBO) (5 minutes) in citrate-phosphate buffer (pH 4.4). 3.0 pm sections were also stained in 0.005% Auramine 0 in 0.1 M tris-buffer (pH 7.4) for fluorescence microscopy of cuticles. After photography
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Materials and methods

Male sterile varieties of Beta vulgaris were supplied by Maribo Seed (NS De Danske Sukkerfabrikker). Plants were grown in greenhouses throughout the year as well

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Figs 11-14, Development of the vacuole system in the inner epidermis of the inner integument of Beta vulgaris (all bars = 1 pm). Fig. 11. Young cell with perinuclear distribution of the vacuoles, note the heavy cuticle at the nucellar side. xY000. - Fig. 12. Later stage, but still perinuclear distribution and heavy cuticle at the nucellar side. x7500. -Fig. 13. Vacuole at the mature stage of ovule development x 11200. - Fig. 14. Vacuole system during early embryo and seed development X7500.

(excitation 450-490 nm. Kodak Ektachrome 400) the same sections were rinsed in distilled water and restained with PAS and ABB. Material for scanning electron microscopy (SEM) was fixed and dehydrated as described for TEM (Bruun 1987). After dehydration the material was dried in a critical point dryer (CPD, Polaron E 5000) and sputter coated with gold (Polaron E 3000). Specimens were viewed with a Jeol JMS - P15 SEM.

integument (01) consists of 2 cell layers: the outer epidermis (0.e.) and the inner epidermis (i.e.), except near the funiculus where it becomes multilayered (Figs 1,2). Also in the apical (micropylar) part of the apex, the 01 consists at least during some parts of the maturation process of more than 2 cell layers. In the mature stage a strongly PAS+ area occurs in a median layer at the apex (Fig. 1) which by TEM is resolved as an area of persistent cell walls formed by the collapse of degenerating cells (Fig. 10). The ultrastructure of the 0.e. and i.e. is very similar. The cytoplasm contains numerous plastids with a conspicuous accumulation of starch grains (Figs 2,8), little rough endoplasmic reticulum, many ribosomes and mitochondria and some lipid droplets. The cell walls are strongly PAS+ and consist of fibrillar material. Plasmodesmata occur in both periclinal and anticlinal walls. An increase in starch accumulation occurs during the maturation process and reaches its highest amount when the embryo sac is mature (Bruun 1987). After fertilization the starch gradually disappears, starting in the micropylar part of the 0.e. In neither outer nor inner integuments any traces of vascular tissue appears.

Results The outer

pears prominent with only little condensed heterochromatin. After fertilization and in senescing non-fertilized ovules an enlargement of the cells in the i.e. takes place and the cytoplasm appears more diluted (Fig. 14). At this stage some of the peculiar vacuoles are found also in the outer part of the cells (Fig. 14). In the light microscope the vacuole content appears strongly refractive and stains green with TBO and brownish/yellow with PAS/ABB which indicates the presence of tannins andlor polyphenols. Compared to cells of the 01 the 0.e. of the 11 consists of strongly vacuolated cells with only a few mitochondria in the thin parietal cytoplasmic layer. Shortly after fertilization (i.e. at early embryo development) this cytoplasm disappears followed by a complete collapse of the 0.e.

The micropyle itself is formed by the inner integument (Figs 1, 3), and the outer integument is clearly seen around the basis of the I1 (Figs 3, 4). Often the micropyle entrance appears to be covered by a thin sheet or hymen (Fig. 4). Inside the micropyle a PAS+ substance (cf. Bruun & Olesen 1988,1989) is often seen and most probably corresponds to fibrillar material observed by TEM (Fig. 5). The characteristic appearance of the i.e. of the I1 as seen in Fig. 2 changes exactly where the micropyle begins. Here, cells of this layer become less vacuolated, bigger in size, and loose their dense vacuolar contents. This transition is clearly seen in a transverse section just touching the nucellar apex (Fig. 7) whereas at the level of the egg cell (Fig. 2) the characteristic vacuolar system containing tannins is still fully developed.
Cuticles are prominent features of both surfaces of 0 1 and I1 as well as of the surface of the nucellus (Figs 5-8). In this way the whole surface of the micropylar channel is covered by cuticles (Bruun & Olesen 1989 and Figs 5, 7). Although all the cuticles stain positively with Auramin 0, the cuticle covering the nucellar surface is by far the most conspicuous (Fig. 9).

The inner integument (11) consists like the 0 1 of 2 cell layers: the outer epidermis ( o x . ) and the inner epidermis (i-e.). As for the 0 1 the apex of the I1 contains an area of PAS+ cell wall remnants from degenerating cells. The plastids of 1 1 contain no o r only very few starch grains in i.e. All cell walls are PAS+ and early in the maturation process they appear curved. Plasmodesmata are found especially in the anticlinal walls. The two epidermal layers of the 1 1 are very different with the i.e. being by far the most conspicuous cell layer of beet ovules containing cells differing from all other cell types. In the i.e. an unusual vacuole system develops. During growth and maturation of the ovule (Figs 11-14), a system of small perinuclear vacuoles containing dense material increases steadily. At maturity this vacuolar system fills up a large part of each cell and dense material has accumulated in each vacuole (Figs 13, 14). At this stage the vacuoles are typically confined to the inner part of the cells with the large nucleus comprising the outer part (Fig. 13). The cytoplasm is well preserved with many mitochondria and a few plastids and dictyosomes (e.g., Fig. 13). The nucleus ap504

Discussion
The physiology of ovules during formation of the female gametophyte, embryogenesis and seed development forms one of the big enigmas of plant developmental biology - especially when compared with the well known processes of seed germination. Still, the general ideas on ovule function are mainly derived from anatomical and histochemical data. The source of energy for embryo germination is well known as accumulated nutrients in the endosperm or parts of the embryo itself but typically very little is known about the nutritional aspects (source, pathways etc.) of embryo differentiation. In sugar beet the role of the endosperm in nutrient accumulation has been taken over by a specialized nucellus tissue, the perisperm (Artschwager 1927). As for Quercus (Mogensen 1973) and Spinuciu
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(Wilms 1980) the outer integument in the mature ovule of sugar beet has a high concentration of stored nutrients in the form of accumulated starch. In their early anatomical studies neither Heel (1925) nor Artschwager (1927) paid much attention to the structure of the integuments and the micropyle of sugar beet ovules. Artschwager (1927) observed starch in the 0 1 of sugar beet but did not confirm the disappearance of starch after fertilization. T h e decrease in starch after fertilization in sugar beet as well as in Quercw (Mogensen 1973) and Spinacia (Wilms 1980) may indicate that the starch is used as nutrient supply for the developing embryo. Contrary to what was observed in Quercus (Mogensen 1973), there seems in sugar beet to be no indication that the decrease of starch should begin at the chalazal end and continue progressively towards the apex. Here, in fact, the decrease of starch is first seen in the micropylar part. Nonetheless, the pathway of nutrients in the integuments of the sugar beet ovule must be from the 01 to the chalazal part of the integument and from here, crossing the window between the integuments, to the chalazal part of the nucellus and from here one way or another to the embryo sac or the embryo. A similar pathway has been suggested for Quercus (Mogensen 1973) and Spinacia (Wilms 1980) ovules. The presence of heavy cuticles around the integuments and the nucellus in sugar beet (Heel 1925; Artschwager 1927; Bruun & Olesen 1989) supports this view, because of the ability of cuticles to function as impermeable barriers. In contrast to Beta, Quercus (Mogensen 1973) and Spinacia (Wilms 1980), the 01 of Agave (Tilton & Mogensen 1979) only stores few nutrients. The endothelium or integumentary tapetum of the plant ovule normally originates from the i.e. of I1 (Kapil & Tiwari 1978; Tilton & Lersten 1981). The early differentiation of the i.e. of the I1 in sugar beet ovules indicates that this layer could be considered an endothelium. O n the other hand it is generally believed that only weakly crassinucellate or tenuinucellate ovules have an endothelium and that this normally has a nutritional function (Kapil & Tiwari 1978; Bouman 1984). Furthermore in sugar beet there is only little starch or lipid accumulation in the i.e. of the I1 but a high content of tannins which in fact are very difficult to metabolize. Therefore, as also suggested for the fringe-layer in cotton (Ryser et al. 1988). this layer in sugar beet does not represent a true endothelium but might well have unknown functions during ovule differentiation and seed development. Notwithstanding their widespread occurrence in plant tissues (often in strategic locations), very little information excists as to the functions of tannin and polyphenol inclusions. Since these molecules often are deposited in cells associated with defense reactions against predators and fungal and bacterial pathogens, they are normally considered as part of protection barriers (Swain 1979). In sugar beet ovules Artschwager (1927) showed that tannins are deposited in both the 0 1 (0.e.) and the I1
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(i.e.) during seed development but neither Heel (1925) nor Artschwager (1927) mentioned the conspicuous early deposit of tannins as showed in the present work. Identical structures are found in Beta rnaritirna ovules (Bruun, unpubl.) which rules out the possibility that tannin accumulation in this cell layer might be related to the intensive inbreeding of sugar beet. In Pelargoniurn x hortorurn a conspicuous tannin deposition also takes place in the i.e. of the I1 during embryo sac formation (Tsai et al. 1973), but n o comments were made on its potential functions. If the early development of tannin cells in the i.e. of the I1 should only function as a defense barrier against pathogens and predators, its deep localization inside the ovule is puzzling. For optimal protection one should expect such cells to develop in the 0 1 which also occurs in several species (Tilton & Lersten 1981; Bosewinkel & Bouman 1984). Being epidermal by nature, the tannin layer of the I1 displays obvious similarities to epidermal tanniferous inclusions found in many xero-halophytic species. Here, the tannin inclusions are considered to function in protection against both pathogens as well as damages to underlying tissues from strong exposure to UV-light. Being a halophytic or at least salt tolerant species, the tannin layer in the inner integument of sugar beet ovules might also function in the accumulation of high amounts of salt taken u p by the plant - and thus protecting the vital gynogenic tissue inside the ovule from excess salt stress. Taken together, it seems rather plausible that the special tannin layer of the I1 in sugar beet may have several, largely unknown functions and that its main function may well change from early stages of ovule differentiation to later stages of embryo and seed development where it takes part in the seed coat formation. Marked changes in function are known from other strategically positioned and tannin-contaning cell layers such as the endodermis in roots and bundle sheaths in leaves of certain grasses (cf. Bocher & Olesen 1978). From the present study there are no indications that tannin cells of the I1 should have anything to d o with neither the presence of PAS+ substances in the micropyle nor the precise guidance of pollen tubes towards the egg apparatus (Bruun & Olesen 1988, 1989). It is uncertain whether the thin sheet or hymen covering the micropyle entrance is related to the PAS+ substance, represents a novel structure, or simply is a fixation artifact. However, Tilton (1980) observed a similar structure covering the exostome of Ornithogalurn ovules and suggested that a function could be in keeping the putative chemotropic agents inside the micropyle. This might well be the case in sugar beet also
Acknowledgements - This work was supported partly by A / S De Danske Sukkerfabrikker and partly by the Danish Agricultural & Veterinary Research Counsil (grant no. 13-3965). Dr P. Steen and H. Olsen of Maribo Seed kindly provided plant material. The authors thank H. Fasting for valuable discussions

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on staining of tannins, Ruth B. Jakobsen and Lis M. Frederiksen for excellent technical assistance, and H. E. Jensen and G. Vestergaard-Hansen for photographic work.

References
Artschwager, E. 1927. Development of flowers and seed in the sugar beet. - J. Agr. Res. 34: 1-25. Bor, J. & Bouman, F. 1974. Development of ovule and integuments in Euphorbia milii and Codiaeum variegatum. Phytomorphology 24: 280-296. Bouman, F. 1984. The ovule. - In: Johri, B. M. (ed.), Embryology of angiosperms. Springer, Berlin, Heidelberg, pp. 123-157. Bosewinkel, F. D. 1980. Development of ovule and testa of Linum usitatissimum L. - Acta Bot. Neerl. 29: 17-32. - & Bouman, F. 1984. The seed: Structure. -In: Johri, B. M. (ed.), Embryology of angiosperms. Springer, Berlin Heidelberg, pp. 567-610. Bruun, L. 1987. The mature embryo sac of the sugar beet, Beta vulgaris: A structural investigation. - Nord. J. Bot. 7: 543511. - & Olesen. P. 1988. A structural investigation of the ovule in sugar beet, Beta vulgaris: The degenerated synergid and the micropylar nucellus. - In: Cresti, M. et al. (eds), Sexual reproduction in higher plants. Springer, Berlin, p. 462. - & Olesen, P. 1989. A structural investigation of the ovule in sugar beet, Beta vulgaris: The micropylar nucellus. - Nord. J. Bot. 9: 81-87. Bocher. T. W. & Olesen, P. 1978. Structural and ecophysiological pattern in the xero-halophytic C, grass, Sporobolus rigens (Tr.) Dew. - Kgl. Dan. Vid. Selsk. Biol. Skr. 22,3:
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Kapil, R. N. & Tiwari, S. C. 1978. The integumentary tapeturn. - Bot. Rev. 44: 457-490. Mogensen, H. L. 1973. Some histochemical, ultrastructural, and nutritional aspects of the ovule of Quercus gambelii. Amer. J. Bot. 60: 48-54. Nielsen, J. E. & Olesen, P. 1988. Isolation of sperm cells from trinucleate pollen of sugar beet (Beta vulgaris). - In: Wilms, H . J. & Keijzer, C. J. (eds), Plant sperm cell as tools for biotechnology. Pudoc, Wageningen, pp. 111-123. Ryser, U., Schorderet, M., Jauch, U. & Meier, H. 1988. Ultrastructure of the Fringe-layer, the innermost epidermis of cotton seed coats. - Protoplasma 147: 81-90. Shen, J., Li, H., Han, X. & Wang, P. 1986. Observations on fertilization in sugar beet. - Acta Bot. Sin. 28: 251-255. Swain, T. S. 1979. Tannins and lignins. - In: Rosenthal, G . A. & Janzen, D. H. (eds), Herbivores, their interaction with secondary plant metabolites. Academic Press, New York, pp. 657-675. Tilton, V. R. 1980. The nucellar epidermis and micropyle of Ornithogalum caudatum (Liliaceae) with a review of these structures in other taxa. - Can. J. Bot. 58: 1872-1884. - & Lenten, N. R. 1981. Ovule development in Ornithogalum caudatum (Liliaceae) with a review of selected papers on angiosperm reproduction. I. Integuments, funiculus, and vascular tissue. - New Phytol. 88: 439457. - & Mogensen, H. L. 1979. Ultrastructural aspects of the ovule of Agave parryi before fertilization. - Phytomorphology 29: 338-350. Tsai, A. H., Harney, P. M. & Peterson, R. L. 1973. Megasporogenesis and megagametogenesis in Pelargonium x hortorum. - Can. J. Bot. 51: 607-612. Wilms, H. J. 1980. Development and composition of the spinach ovule. - Acta Bot. Nearl. 29: 243-260.

Feder, N . & OBrien, T. P. 1968. Plant microtechnique: Some principles and new methods. - Amer. J. Bot. 55: 123-142. Fischer, D. B. 1968. Protein staining of ribboned epon sections for light microscopy. - Histochemie 16: 92-96. Heel, Jr., J. P. D. van. 1925. Onderzoekingen over de ontwikkeling van den zaadknop en van het zaad bij Beta vulgaris L. - Thesis, Naarden. (Unpubl.). Hosemans. D. & Bossoutrot, D. 1986. In vitro culture of unpollinated beet (Beta vulgaris L.) ovules of male sterile and male fertile plants and induction of haploid plants. -In: Chapmann, G. P. et al. (eds), Experimental manipulation of ovule tissues, Longman Inc., New York, pp. 79-87.

Abbreviations
micropyle outer integument I1 inner integument ES embryo sac F funiculus MNu micropylar nucellus SG = starch grain Nu = nucellus
M
01
= = = = = =

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