Planta

Planta 133, 179-189(1977)

9 by Springer-Verlag 1977

An Ultrastructural Study of Early Endosperm Development and Synergid Changes in Unfertilized Cotton Ovules William A. Jensen, Patricia Schulz, and Mary E. Ashton Department of Botany, University of California, Berkeley, CA 94720, USA

Abstract. Excised, unfertilized cotton (Gossypium hirsuture L.) ovules were cultured for 1-5 days postan-

thesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2-3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The m o r p h o l o g y of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the a m o u n t of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the p l a s m a l e m m a and the m e m branes of the R E R . The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac. Key words: E m b r y o sac - Endosperm tion - Gossypium -- Synergids.

Fertiliza-

Introduction

The technique of ovule culture offers new possibilities for obtaining data on the events associated with dou-

ble fertilization and early embryo and endosperm development in angiosperms. Beasley and Ting (1974) established culture methods for obtaining fiber development on isolated, unfertilized cotton ovules. They found that optimal fiber development was p r o m o t e d by the presence of certain phytohormones in the growth medium. The substitution, or partial substitution, of phytohormones for processes normally associated with fertilization shown by this work opens new opportunities for studying the physiological relationship of the pollen tube and sperm to the egg, synergids and central cell and to the formation of the zygote and endosperm. In this investigation unfertilized cotton ovules were cultured according to the methods of Beasley and Ting (1974) for 1-5 days postanthesis, and embryo-sac development was studied with the electron microscope. During this period endosperm development began and there were changes in one of the synergids. These and other changes in the unfertilized cultured ovules are reported here.

Materials and Methods

Glass-house culture of cotton plants (Gossypium hirsutum L., cultivat Acala SJ-l), preparation of culture media, and growth conditions were as described by Beasley and Ting (1973, 1974). Ovules were aseptically removed from ovaries on the day of anthesis. floated on the surface of 50 ml of liquid medium in 125-mi flasks, and cultured at 32~ C in constant darkness. The growth medium consisted of a basal nutrient solution (Beasley and Ting, 1973, Table 1, with 120 mM glucose in place of the reported 100 mM glucose +20 mM fructose) to which was added 0.5 gM gibberellic acid and 5.0 gM indole-3-acetic acid. Micropylar halves of ovules (integuments intact) were fixed in a 3% glutaraldehyde made up in 0.5 M cacodylate buffer, pH 6.8, for 24 h at room temperature. The integuments were removed after glutaraldehyde fxation, the ovules were washed in buffer, postfixed with 2% unbuffered osmium tetroxide overnight at 4~ C, dehydrated in a graded acetone series (1% uranyl nitrate was added to the 70% acetone), and embedded in Epon (Luft, 1961). Thin

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sections were cut with a diamond knife, stained with lead citrate (Reynolds, 1963),and observedwithaZeissEM 9A electronmicroscope.

much smaller numbers than observed in the cotton zygote (Jensen, 1968) and fertilized endosperm (Schulz and Jensen, 1977).

Results

Changes in the Synergids

Endosperm Development

On the day of anthesis the synergids are fully developed and appear to be ultrastructurally and histochemically indistinguishable from one another (Jensen, 1965). Synergids in unfertilized ovules remain unchanged in appearance during the first day of culture. On the 2nd day one of the synergids shows degenerative changes (Figs. 12, 13) which closely parallel in vivo changes induced by the presence of the pollen tube (Jensen and Fisher, 1968). The vacuoles in the degenerating synergid collapse and there is an overall increase in electron density of the cell. This is followed by an irregular darkening and thickening of the membranes surrounding mitochondria and plastids (Fig. 12), a swelling of the RER (Figs. 12, 13), and finally the disappearance of the membranes of the RER (Figs. 12, 13) and plasmalemma (Fig. 13). During these changes starch persists in the degenerate plastids and dictyosomes remain recognizable (Fig. 12). The second, or persistent, synergid remains unchanged in appearance after 3 days in culture (Fig. 14). Electron density does not increase, plastids and mitochondria look healthy, and the membranes of the plasmalemma and RER remain intact.

During the first 2 days of ovule culture the polar nuclei and the central cell cytoplasm change little from their appearance on the day of anthesis (Schulz and Jensen, 1977). The polar nuclei remain partially fused (Fig. 1) and are surrounded by cytoplasm that contains large, dense plastids with a few vesiculate lamellae, starch grains and osmiophilic granules (Fig. 2). Also present are numerous mitochondria that are less dense than plastids and have short, vesiculate cristae (Fig. 2). The rough endoplasmic reticulum (RER) occurs in long, single, dilated cisternae and there are moderate numbers of dictyosomes, lipid bodies and microbodies that contain dense cores (Fig. 2). Many single ribosomes, small polysomes and small vacuoles also appear in the central cell cytoplasm (Figs. 1, 2). The polar nuclei complete their fusion and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2-3 days in culture (Fig. 3). Precocius cell walls form between the nuclei on the 3rd day (Figs. 4, 5, 7, 8). Wall growth proceeds from the embryo-sac wall (Fig. 5) toward the large central vacuole of the embryo sac (Figs. 4, 7) by the apparent coalescence of dictyosome vesicles along a path paralleled by RER (Fig. 7). Some microtubules are perpendicularly oriented to the newly-forming wall (Fig. 7). The endosperm walls have an electron-dense middle lamella (Figs. 5, 8) and contain many membrane-bound vesicles (Figs. 5, 6, 8) that may be derived from the plasmalemma (Fig. 6). They also branch (Fig. 8) and have plasmodesmata (Fig. 4). Each diploid endosperm nucleus contains two nucleoli (Fig. 9) whereas the nuclei of fertilized triploid endosperm have three nucleoli (Schulz and Jensen, 1977). The nuclear envelope is lobed and in many places continuous with the RER (Fig. 9). The number and general morphology of the plastids, mitochondria, dictyosomes, microbodies and vacuoles are similar to those of the unfertilized central cell (Schulz and Jensen, 1977) and l-2-day cultured material (Figs. 4, 7, 8, 10). The RER remains in the form of single, dilated cisternae (Figs. 7, 8, 10) and there is little change in the amount of starch (Figs, 4, 8) and lipid (Fig. 9). Long, helical polysomes appear close to plastids and mitochondria (Fig. 11) but in

The Egg CeH The egg cell in unfertilized ovules remains highly vacuolate and structurally unchanged after 3 days in culture (Figs. 15, 16). It contains many mitochondria and starch-filled plastids and a substantial amount of reserve lipid. Single RER cisternae, some dictyosomes and microbodies, many small clusters of ribosomes and small vacuoles are also present. The egg cell does not shrink or undergo the structural changes characteristic of the cotton zygote (Jensen, 1968).

Discussion

This report of the experimental induction of endosperm development and pollen-tube-related synergid changes in unfertilized, cultured cotton ovules further demonstrates the potential for substituting phytohormones for the normal process of fertilization. The pattern of endosperm development without fertilization observed in this study differs in some ways from endosperm development after fertilization (Schulz

W.A. Jensen et al.: Endosperm Development in Unfertilized Cotton Ovules

Fig. 1. Unfertilized ovule cultured for one day showing partially fused polar nuclei, x 18,900 Fig. 2. Central cell cytoplasm in unfertilized ovule cultured for 1 day. (p plastid, M mitochondrion, Mb microbody), x 23,400

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Fig. 3. Several nuclei of the free nuclear stage of unfertilized endosperm development. Ovule cultured for 3 days. L lipid, x 3780

and Jensen, 1977). In addition to limited growth, the most striking differences include the precocious formation of endosperm cell walls and a lack of cytoplasmic differentiation. In fertilized cotton embryo sacs, endosperm wall formation occurs approximately 10 days after pollination, after many dozens of free nuclei are formed. In unfertilized endosperm wall formation occurs on the 3rd day of culture after only a half dozen or so free nuclei are formed. The pattern of wall growth in fertilized and unfertilized endosperm, however, is similar. Cell walls are initiated at the embryo-sac wall and grow toward the large

central vacuole of the embryo sac by apparant coalescence of dictyosome vesicles and with possible involvement of the RER. Similar patterns of in vivo endosperm wall formation have been observed in sunflower (Helianthus annuus L.) (Newcomb, 1973) and chickweed (Stellaria media L.) (Newcomb and Fowke, 1973). In unfertilized cotton endosperm there is no evidence of the prior formation of a plasmalemma along lines of wall formation like that reported in wheat endosperm (Mares, et al., 1975). Precocious endosperm wall formation in cultured ovules may be promoted by the availability of carbohydrates in

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Fig. 4. Cellular endosperm in an unfertilized ovule cultured for 3 days. The endosperm is pulled away from the embryo-sac wall which is to the left and out of the picture. Endosperm cell-walls (arrows) originate at the embryo-sac wall and grow toward the central vacuole (CV) of the embryo sac. x 3150 Fig. 5. Origin of endosperm (E) cell-wails (CW) at the embryo-sac wall

(ESW) in an unfertilized ovule cultured for 3 days. x 11,700

Fig. 6. Cell wall of unfertilized, diploid endosperm containing membrane-bound vesicles that appear to originate from the plasmalemma (arrows). Ovule cultured for 3 days. x 51,300

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Fig. 7. Unfertilized, cellular endosperm showing growth of the cell wall (CW) toward the central vacuole (CV) of the embryo sac by the possible coalescence of dictyosome vesicles along a path paralleled by RER. Some microtubules (arrows) appear in a perpendicular orientation to the site of wall growth. Ovule cultured 3 days. Mb, microbody, x 46,800 Fig. 8. Unfertilized, cellular endosperm showing branched cell walls that contain membrane-bound vesicles. Ovule cultured 3 days. P starch-containing plastid, M mitochondrion, x 18,900

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Fig. 9. Nucleus of unfertilized, diploid endosperm containing two nucleoli. Ovule cultured for 3 days. • 5950 Fig. 10. Cytoplasm of unfertilized, cellular endosperm in an ovule cultured for 3 days. P plastid, M mitochondrion, D dictyosome, Mb microbody, R E R rough endoplasmic reticulum, Mt microtubule, N nucleus. • 26,100 Fig. 11. Helical polysome (arrow) in the cytoplasm of unfertilized, cellular endosperm. Ovule cultured 3 days. • 108,900

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Fig. 12. Dense cytoplasm of the degenerate synergid in an unfertilized ovule cultured for 2 days. Membranes of plastids (P) and mitochondria (M) are dark and thickened. Starch (S) persists in degenerate plastids. Membranes of the swollen R E R have disappeared but dictyosomes (D) remain recognizable, x 46,800 Fig. 13. Cytoplasm of the degenerate synergid shown in Figure 12. The membranes of the plasmalemma (arrows) and the swollen RER have disappeared, x 51,300

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Fig. 14. Cytoplasm of the persistent synergid in the same ovule as the degenerate synergid shown in Figure 12 and 13. There are no degenerative changes in plastids (P) and mitochondria (M) and the membranes of the plasmalemma (arrows), RER and vacuoles (V) are intact, x 23,400

the growth medium and thus reflect a mechanism for the storage of excess metabolites. A similar phenomenon could account for the maintenance of high levels of cytoplasmic starch and lipid that normally are depleted during the first 3 days of growth of fertilized endosperm (Schulz and Jensen, 1977). The lack of cytoplasmic differentiation is another striking difference in unfertilized endosperm when compared with fertilized endosperm. The cytoplasm of unfertilized endosperm looks much like that of the unfertilized central cell (Schulz and Jensen, 1977). There is no general increase in electron density or increased proliferation of R E R and dictyosomes like that which accompanies the early development of fertilized endosperm (Schulz and Jensen, 1977). Coupled with this is the absence of large numbers of helical polysomes and dense aggregates of granular material, both thought to be ribonucleoprotein in nature, which appear in the cytoplasm of the central cell shortly after fertilization (Schulz and Jensen, 1977). This last observation implicates a failure in R N A metabolism

as a possible cause for the lack of cytoplasmic differentiation and the premature arrest of development in unfertilized endosperm. A further note in support of this suggestion is the preliminary observation that the granular component of the nucleoli of unfertilized endosperm nuclei is, in some cases, reduced or entirely absent. The series of pollen-tube-induced degenerative changes that occur in one of the two cotton synergids is well documented (Jensen and Fisher, 1968) and is distinct from degenerative changes observed in other cell types, e.g., nucellar cells (Schulz and Jensen, 1971). The synergid changes in cultured ovules observed in this study are strikingly similar to those initiated by the pollen tube in vivo. Current data indicate that synergid degeneration is very important to pollen-tube penetration and may play a key role in directing pollen-tube growth (Jensen and Fisher, 1968). The potential for hormonal control of synergid changes in vitro makes possible a more controlled study of the relationship of synergid changes to the

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Fig. 15. A portion of the egg cell in an unfertilized ovule cultured for 3 days. x 4860 Fig. 16. Enlarged portion of the egg cell in Figure 15 showing starch-containing plastids (P), mitochondria (M), dictyosomes (D), RER and microbodies (Mb). x 18,900

entrance and discharge of the pollen tube and other critical steps in the fertilization process. F r o m the results presented here it appears that endosperm development in the absence of fertilization has little, if any, effect on the egg cell. In fertilized cotton ovules the zygote shrinks to one-half the original volume of the egg cell and undergoes a series of structural changes during a 3-day period preceding

cell division (Jensen, 1968). Cell shrinkage increases the polarity of the zygote and has a pronounced effect on the pattern of early cell divisions and the normal development of the embryo. Ashley (1972) has shown that if the zygote of Hibiscus fails to shrink in size a haphazard pattern of cell divisions results that causes the embryo to abort. Jensen (1968) proposed a hypothesis to explain zygote shrinkage based on

W.A. Jensen et al. : Endosperm Development in Unfertilized Cotton Ovules

a presumed osmotic gradient induced by the initial rapid growth of the endosperm that would cause water to move from the vacuole of the zygote to the endosperm. Our data tend to discount this explanation of zygote shrinkage. There is also some preliminary evidence to indicate that it is the initial failure of the egg cell in cultured ovules that has a deleterious effect on the continued growth of the endosperm. Work is now in progress to combine in-vitro fertilization with the experimental modification of the embryo sac through ovule culture. This system offers new possibilities for studying biochemical interactions between the pollen tube and ovule, and for testing many of the hypotheses surrounding double fertilization and embryo and endosperm development. This research was supported by grants GB 3-1101 and BMS 7520246 from the National Science Foundation. We wish to thank Dr. C.A. Beasley (University of California, Riverside) for providing cultured material. Dr. Beasley's seeds were obtained from Dr. Hubert Cooper (USDA Cotton Research Station, Shafter, California). We also thank Bernice Lindner for technical assistance.

References Ashley, T. : Zygote shrinkage and subsequent development in some Hibiscus hybrids. Planta (Bed.) 108, 303-317 (1972) Beasley, C.A., Ting, I.P.: The effects of plant growth substances

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on in vitro fiber development from fertilized cotton ovules. Amer. J. Bot. 60, 130-139 (1973) Beasley, C.A., Ting, I.P.: Effects of plant growth substances on in vitro fiber development from unfertilized cotton ovules. Amer. J. Bot. 61, 188-194 (1974) Jensen, W.A. : The ultrastructure and histochemistry of the synergids of cotton. Amer. J. Bot. 52, 238-256 (1965) Jensen, W.A.: Cotton embryogenesis: the zygote. Planta (Berl.) 79, 346-356 (1968) Jensen, W.A., Fisher, D.B.: Cotton embryogenesis: the entrance and discharge of the pollen tube in the embryo sac. Planta (Berl.) 78, 158-183 (1968) Luft, J.H. : hnprovements in epoxy embedding methods. J. biophys. biochem. Cytol. 9, 409-427 (1961) Mares, D.J., Norstog, K., Stone, B.A. : Early stages in the development of wheat endosperm. I. The changes from free nuclear to cellular endosperm. Aust. J. Bot. 23, 311-326 (1975) Newcomb, W. : The development of the embryo sac of sunflower Helianthus annus after fertilization. Can. J. Bot. 51, 879-890 (1973) Newcomb, W., Fowke, L.D.: The fine structure of the change from the free-nuclear to cellular condition in the endosperm of chickweed, Stellaria media. Bot. Gaz. 134, 236-241 (1973) Reynolds, E.S. : The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Schulz, P , Jensen, W.A.: Capsella embryogenesis: the chalazal proliferating tissue. J. CelI Sci. 8, 201 227 (1971) Schulz, P., Jensen, W.A. : Cotton embryogenesis : the early development of the free nuclear endosperm. Amer. J. Bot., in press (1977)

Received 23 July; accepted 16 August 1976