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Potassium antimonate was used to localize Ca2+ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca2+ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate.

The paper reports a comparative study of the female gametophyte and especially synergid structure in sexual and apomictic dandelions. We analyzed diploid sexually reproducing Taraxacum linearisquameum (2n = 2x = 16) and two triploids, T. alatum and T. udum (2n = 3x =24), with autonomous embryo and endosperm development. There were no observed differences in the organization of the mature megagametophyte between the examined species. Both meiotically reduced and diplosporous embryo sacs showed typical polarity of the egg apparatus cells, together with development of a filiform apparatus in the synergids, but immunocytochemical analyses indicated that microtubules form longitudinal brush-like bundles adjacent to the filiform apparatus in the synergids of the sexual T. linearisquameum. This arrangement of cytoskeletal elements is similar to the configuration described in other amphimictic plants. The synergids of the apomictic T. alatum and T. udum show a uncharacteristic and relatively weak cytoskeleton with no brush-like bundles. We discuss the role of synergids in autonomous apomicts.

The developmental stages of the maize embryo sac were correlated with the corresponding silk lengths of ear florets in the female inflorescence. The development of embryo sacs in the ovules of spikes occurs in a gradient pattern with the initiation of the embryo sac beginning at the base of the ear and progressing to the top. At the beginning of meiosis, the presence of conspicuous cortical microtubules coincides with the extensive elongation of the megasporocyte. The spindles at metaphase I and II align along the long axis of the megasporocyte leading to the linear alignment of the dyad and tetrad of megaspores. During megagametogenesis, micropylar and chalazal nuclei of the embryo sac undergo synchronized divisions and migration at the second and third mitosis. Radiate perinuclear microtubules are present during the interphase of the second and third mitosis, and inter-sister nuclear microtubules occur at the late four-nucleate embryo sac. The configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The fusion of the polar nuclei and the formation of a microtubule organizing center-like structure in the filiform apparatus occur right after the first division of the antipodal cells. The different patterns of organization of microtubules in the cells of the mature embryo sac reflect their structural adaptations for their future function.

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cell with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed “coronas”. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.

The micropylar exudate of Gasteria verrucosa (Mill.) H. Duval was studied using light and electron microscopic techniques. Ovules may contain micropylar exudate before stigma receptivity. During successive phases of stigma receptivity, the number of ovules with micropylar exudate and the amount of micropylar exudate per ovule increases. At the late phase of stigma receptivity, large amounts of micropylar exudate with a smooth to cauliflowerlike appearance were observed. Micropylar exudate is viscous and contains, among other components, proteins and carbohydrates. At all stages of the stigma investigated, ovules situated at the base of the ovary contain a larger quantity of micropylar exudate than those at the top. The appearance of micropylar exudate is related to the degree of development of the embryo sac and it originates primarily from the filiform apparatus. It is assumed that an uptake of water by the ovule initiates the outflow of micropylar exudate from the filiform apparatus into the micropyle. Both ovular pollen tube ingrowth and seed set mark the optimum pollination stage of the stigma, which for both events lies around the onset of stigma receptivity. When pollen tubes have reached the ovary, young micropylar exudate stimulates their growth rate. The presence of micropylar exudate seems to be a requirement for pollen tube penetration, and an interaction between the pollen tube and the micropylar exudate has been proposed. Possibly, the micropylar exudate serves as a nutritional source and, in an optimum condition, as an attractant for approaching pollen tubes.

The postanthesis synergids and zygote of Brassica campestris cv. Candle were examined using techniques of light, fluorescence, and electron microscopy. The pollen tube enters the degenerate synergid by way of the filiform apparatus. A degeneration of one of the two synergids occurs after anthesis and is independent of pollination. The first sign of synergid degeneration is a more intense staining of one of the synergids, followed by a loss of organelle membrane integrity. There is a disappearance of the plasma membrane and dictyosome cisternae; however, profiles of degenerate synergid mitochondria, plastids, and dilated endoplasmic reticulum remain along with dictyosome vesicles that contain periodic acid – thiocarbo-hydrazide – silver proteinate positive substances. The zygote, shortly after fertilization, is reduced in size and lacks the large micropylar vacuole characteristic of the mature unfertilized egg cell. Plastids and mitochondria are concentrated around the centrally located nucleus of the zygote, and dictyosomes, active in vesicle production, are located in the lateral and chalazal regions of the cell, adjacent to the cell wall. The lateral cell walls are periodic acid – Schiff's and Calcofluor positive, while the ampulliform chalazal tip of the cell is weakly periodic acid – Schiff s positive and Calcofluor negative. Microtubules, with the long axis perpendicular to the long axis of the zygote, are abundant in the ampulliform chalazal tip of the cell. Following fertilization the central cell becomes highly vacuolate. There is continuity between the zygote – central cell plasma membrane, the central cell vacuole tonoplast, and membranes of the central cell endoplasmic reticulum. Central cell wall projections, of the transfer cell type, are located in the lateral regions of the megagametophyte adjacent to the developing zygote cell and are positioned adjacent to the region of inner and outer integument starch. Key words: Brassica, ultrastructure, synergid, megagametophyte, pollen tube, zygote.

The synergids and other cells of mature, unpollinated pearl millet ovaries were investigated using: (1) freeze-substitution fixation in conjunction with scanning electron microscope observations and energy-dispersive X-ray microanalysis to localize total calcium (Ca) and other elements, and (2) antimonate precipitation to selectively localize loosely sequestered, exchangeable calcium (Ca++). In freeze-fixed ovaries, the synergid cells, ovary wall, nucellus, and other regions of the ovary displayed, respectively and relatively, extremely high, high, moderate, and low levels of Ca. In antimonate-fixed ovaries, Ca-containing antimonate precipitates exhibited similar distribution patterns. In ovaries fixed using the conventional 2% (w/v) antimonate in fixatives, the synergids were disrupted due to precipitate overload. In the ovary wall, precipitates were mainly located in the intercellular spaces. Some precipitates were observed at the micropyle and along the outer ovule integument, associated with diffuse extracellular material, and in the cell walls of nucellar cells proximal to the micropyle. Examination of precipitate distribution inside the synergids was possible in ovaries fixed using 0.5% (w/v) antimonate in the fixatives. Cytoplasmic organelles of all synergids examined exhibited variable states of disintegration. The amount of precipitates associated with the degenerated organelles appeared to be proportional to the degree of their degeneration. Distinct precipitates were localized in contiguous regions of the nucellar cells fused with the embryo sac, the micropylar half of the embryo sac wall, and the filiform apparatus. The results are discussed in relation to the involvement of Ca++ in mediating the functions of synergid cells during fertilization in angiosperms.

Embryological details pertaining to the development and structure of anther and ovule, sporogenesis, gametogenesis and gametophytes of 9 species of 7 genera belonging to Boraginaceae are described. Anther is tetrasporangiate with ‘Dicotyledonous type’ of wall development. The wall includes the epidermis, endothecium, single middle layer and secretory type tapetum. Cytokinesis is simultaneous. Tetrahedral, decussate and isobilateral tetrads are formed. Pollen grains are mostly dumb-bell shaped and 3-celled at shedding stage. Ovules are unitegmic, tenuinucellate and hemianatropous or anatropous. Megaspore tetrads are linear or ‘T’ shaped. The female gametophyte development conforms to Polygonum type except in Lycopsis and Anchusa where it is predominantly of Allium type. The mature embryo sac consists of two pear-shaped synergids with filiform apparatus, an egg cell, two polars and three ephemeral antipodals.

In flowering plants, germinated pollen grains form tubes that must directionally grow through the flower to deliver sperm cells to the egg cell. In the final phase of this journey, pollen tubes must enter the ovule through an opening known as the micropyle and grow toward and into one of the synergid cells located next to the egg cell. Synergid cells are thought to secrete a diffusible signal through a specialized cell wall structure called the filiform apparatus to guide pollen tubes, but the identity of these chemoattractants in several plant species has been elusive. Okuda et al. prepared a complementary DNA library from synergid cells in Torenia fournieri (bluewings or wishbone flower) and identified 16 transcripts that encoded a group of proteins that they named T. fournieri cysteine-rich polypeptides (TfCRPs). Immunofluorescence for TfCRP1 and TfCRP3 was detected only in synergid cells, at the surface of the filiform apparatus closest to the micropyle. T. fournieri pollen tubes grew toward beads that released TfCRP1 or TfCRP3 but not TfCRP2. Pollen tubes from a related species, Lindernia micrantha , did not grow toward the TfCRP1- and TfCRP3-releasing beads, which suggests that these chemoattractants are species specific. The authors renamed TfCRP1 and TfCRP3 LURE1 and LURE2, respectively. T. fournieri ovules that had been injected with antisense morpholino oligonucleotides against LURE1 or LURE2 attracted fewer pollen tubes compared with those injected with control morpholino oligonucleotides. Thus, the LUREs are one set of chemoattractants that guide growing pollen tubes in T. fournieri to their final destination. S. Okuda, H. Tsutsui, K. Shiina, S. Sprunck, H. Takeuchi, R. Yui, R. D. Kasahara, Y. Hamamura, A. Mizukami, D. Susaki, N. Kawano, T. Sakakibara, S. Namiki, K. Itoh, K. Otsuka, M. Matsuzaki, H. Nozaki, T. Kuroiwa, A. Nakano, M. M. Kanaoka, T. Dresselhaus, N. Sasaki, T. Higashiyama, Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458 , 357–361 (2009). [PubMed]

The female gametophyte contains two synergid cells that play a role in many steps of the angiosperm reproductive process, including pollen tube guidance. At their micropylar poles, the synergid cells have a thickened and elaborated cell wall: the filiform apparatus that is thought to play a role in the secretion of the pollen tube attractant(s). MYB98 regulates an important subcircuit of the synergid gene regulatory network (GRN) that functions to activate the expression of genes required for pollen tube guidance and filiform apparatus formation. The MYB98 subcircuit comprises at least 83 downstream genes, including 48 genes within four gene families (CRP810, CRP3700, CRP3730 and CRP3740) that encode Cys-rich proteins. We show that the 11 CRP3700 genes, which include DD11 and DD18, are regulated by a common cis-element, GTAACNT, and that a multimer of this sequence confers MYB98-dependent synergid expression. The GTAACNT element contains the MYB98-binding site identified in vitro, suggesting that the 11 CRP3700 genes are direct targets of MYB98. We also show that five of the CRP810 genes, which include DD2, lack a functional GTAACNT element, suggesting that they are not directly regulated by MYB98. In addition, we show that the five CRP810 genes are regulated by the cis-element AACGT, and that a multimer of this sequence confers synergid expression. Together, these results suggest that the MYB98 branch of the synergid GRN is multi-tiered and, therefore, contains at least one additional downstream transcription factor.

The organization of isolated embryo sacs and eggs of Plumbago zeylanica was described before and after fertilization using microscopic cytochemistry and scanning electron microscopy. Major developmental events of fertilization, including preferential fertilization and early embryogenesis, are described in isolated embryo sacs. The two sperms, one unassociated with vegetative nucleus (Sua) and the other physically associated with the vegetative nucleus (Svn), fuse with nuclei of egg and central cell, respectively. The zygote divides asymmetrically to form a two-celled embryo, consisting of a massive suspensor occupying most ofthe micropylar portion of the embryo during early embryogenesis. Plastids are distributed in the perinuclear and micropylar regions of the egg cell and in cytoplasmic strands of the central cell before fertilization. Calcofluor white-positive fibrillar material in the filiform apparatus (presumed B- 1,4 linked glucans) was investigated using scanning electron microscopy. The egg of P. zeylanica can easily be divided into three cytologically distinct regions: 1) perinuclear cytoplasm, 2) lateral cytoplasm, and 3) micropylar cytoplasm. Cytological differences are evident in the organization of the cell walls, general degree ofvacuolization, and the distribution of heritable organelles, storage bodies, and microtubules. The present study supports the concept that the egg of P. zeylanica plays combined synergid and gamete functions. The mature embryo sac (ES) of Plumbago

Gynoecia ofPopulus deltoides, 48 to 60 h after pollination, were examined using transmission electron microscopy. The mature megagametophyte contains two synergids, an egg cell, a central cell with two unfused polar nuclei and three antipodals. Pollen tube passage between cells in the styloid, on the placenta and through the micropyle does not induce degeneration of surrounding cells. Pollen tube entry into the megagametophyte occurs through the degenerated synergid immediately adjacent to the filiform apparatus. The tube grows the length of the filiform apparatus 20–30 μm into the synergid, and then discharges its contents-two sperm cells, the vegetative nucleus, and pollen tube cytoplasm. Sperm cells appear progressively hypertrophied during their passage in the pollen tube. In the degenerate synergid, the sperm nucleus is located at one end of the cell and the remaining cytoplasm is diffuse, containing a large vacuole. Following transmission of sperm nuclei into the egg and central cell, one male nucleus fuses with the egg and the other fuses with the micropylar polar nucleus. Enucleate cytoplasmic bodies of apparent sperm origin were observed within the degenerated synergid. The gametic fusion mechanism in this plant appears to involve: (1) eccentric positioning of the nucleus within the sperm cell, (2) separation of cytoplasmic and nuclear portions of the sperm cell, and (3) fusion of the nuclear portion with the egg cell. This represents a previously undescribed mechanism by which male cytoplasmic organelles are eliminated from sperm cells prior to fusion.

SUMMARYMicro- and macrosporogenesis, as well as megagametogenesis and fertilization were studied by fluorescence microscopy in Cytinus hypocistis in order to get new data on the role played by callose in the reproduction process. Callose is present in the whole cell wall of the megasporocytes; the fluorescence decreases during the reductional division and disappears nearly completely before the equational division. Dyads, triads and tetrads show an intense callose fluorescence in the newly formed cell walls. In the megagametophyte the filiform apparatus exhibits the stronger fluorescence lasting till the entry of the pollen tube inside the embryo sac; it is no longer detectable after fertilization. The cytokinesis leading to the microspore formation is successive and the tetrads disposition is isobilateral. The role played by callose in the reproduction process is discussed.

SUMMARY An ultrastructural study on the embryo sac and the micropylar part of the nucellus, at the time of stigma receptivity, was carried out to reveal possible changes due to pollination. At the time of stigma receptivity, the development of the egg cell, central cell, antipodals and the micropylar nucellar cells of Gasteria have been completed. Only the synergids and the filiform apparatus are still differentiating. Frequently, anomalies in the morphology of the unfertilized embryo sac are observed. Comparative observations before and 2·5 h after cross-pollination did not show ultrastructural changes in the embryo sac and in the micropylar nucellar cells. The egg cell showed polarity. The nucleus and most of the cytoplasm lay in the micropylar half, while vacuoles filled most of the chalazal half. The two synergids did not differ from each other and a clear degeneration had not taken place. Based on the observed ultrastructural differences of the filiform apparatus and the synergid cytoplasm among the investigated ovules, and data of previous studies, a sequence of differentiation of the very large filiform apparatus was defined. The nucleus and most of the cytoplasm of the central cell were positioned near the degenerating antipodals. The ultrastructure of the micropylar nucellar cells indicates a secretory function of these cells and an involvement in the process of pollen tube acceptance.

The synergid cells of the female gametophyte play a role in many steps of the angiosperm fertilization process, including guidance of pollen tube growth to the female gametophyte. However, the mechanisms by which the synergid cells become specified and develop their unique features during female gametophyte development are not understood. We identified MYB98 in a screen for Arabidopsis thaliana genes expressed in the female gametophyte. MYB98 is a member of the R2R3-MYB gene family, the members of which likely encode transcription factors. In the context of the ovule, MYB98 is expressed exclusively in the synergid cells, and mutations in this gene affect the female gametophyte specifically. myb98 female gametophytes are affected in two unique features of the synergid cell, pollen tube guidance and the filiform apparatus, but are otherwise normal. MYB98 also is expressed in trichomes and endosperm. Homozygous myb98 mutants exhibit no sporophytic defects, including trichome and endosperm defects. Together, these data suggest that MYB98 controls the development of specific features within the synergid cell during female gametophyte development.

Perspective: the plant life cycle Reproduction in higher plants is relatively complex, involving a life cycle consisting of two phases, a diploid sporophyte phase and a haploid gametophyte phase, comprising what is called an alternation of generations . The prominent bodies of angiosperm trees, shrubs, perennials, and annuals as well as those of gymnosperms, ferns, sphenophytes, and lycophytes are sporophytes , having developed from fertilized egg cells (zygotes). The gametes which fused to form the zygotes, however, were produced by gametophytes , very small plant bodies, parasitic on the sporophytes in seed plants, but somewhat larger and free-living in pteridophytes (except in heterosporous species in which gametophytes when mature remain, at least in part, within the the walls of the spores from which they develop). The sporophyte in pteridophytes is dominant, and although dependent initially for its nutrition on the gametophyte, soon becomes independent. The gametophyte is much reduced in size but is free-living and either autotrophic or saprophytic. In seed plants, the sporophyte is also dominant and initially dependent on the gametophyte, but soon becomes independent. The gametophyte is greatly reduced, however, and parasitic on the sporophyte. In angiosperms it is exceptionally small, consisting in many taxa of only seven cells and eight nuclei, and can be observed only with a microscope. The life cycle of a vascular plant can be summarized as follows. The sporophyte produces specialized cells called sporocytes that undergo meiosis producing haploid spores . The spores germinate to form the gametophytes in which gametes are produced.

The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.

Pollen tube guidance precedes the double fertilization of flowering plants. Here, we report the identification of a small maize protein of 94 amino acids involved in short-range signaling required for pollen tube attraction by the female gametophyte. ZmEA1 is exclusively expressed in the egg apparatus, consisting of the egg cell and two synergids. Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. Transgenic down-regulation of the ZmEA1 gene led to ovule sterility caused by loss of close-range pollen tube guidance to the micropyle.

In flowering plants, the egg cell is generally accompanied by two symmetrical cells, called synergid cells. As early as the 1870s, synergid cells were distinguished from egg cells and cooperation between synergid and egg cells was proposed; the term "synergid" is derived from the Greek "synergos," which means "working together." The accumulation of morphological and genetic data, and, more recently, the in vitro physiological analysis of the fertilization system of Torenia fournieri, have revealed that synergid cells work together with egg and central cells to accomplish double fertilization. This cooperation is of crucial importance in the attraction and acceptance of the pollen tube. In this review article, I focus on the physiological function and behavior of the synergid cell during the fertilization process.

In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser cell ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.

In the pistil of Gasteria there is an activation by pollen coat proteins leading to water uptake of the pistil. This enhances the growth of pollen tubes of cross pollen. It also activates the ovule to attract the pollen tube. This may be a result of the exposition of the micropylar exudate with the attractant. The micropylar exudate attracts one or more pollen tubes. In vitro tubes may stop before they enter the micropyle which is marked by a swelling of the pollen tube tip. Thereafter they penetrate the funnel shaped micropyle.

The structure of the gynoecium and pollen tube pathway in unpollinated and pollinated carpels of Asclepias exaltata L. has been characterized. Pollen tubes penetrate a dry-type stigma, grow intercellularly in a core of solid tissue in the upper style, and subsequently traverse a hollow stylar canal to the ovary where they grow across the placental epithelium to the ovule micropyles. The fine structural characteristics of transmitting cells of the solid style, stylar canal, and placental epithelium indicate a secretory function. Extracellular secretions staining positively for proteins, insoluble carbohydrates, and arabinogalactans/arabinogalactan proteins are present in the solid style, hollow stylar canal, ovary, and micropyle. Micropylar exudate is present subtending the extended cuticle of the embryo sac adjacent to the filiform apparatus of the synergids, providing ultrastructural evidence for a secretion arising from the angiosperm embryo sac.

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cells with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed "coronas". This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.

The microtubular and actin cytoskeletons have been investigated during megagametogenesis in Arabidopsis thaliana using immunofluorescence labelling of isolated coenocytic and mature embryo sacs. We found both actin and microtubules (MTs) to occur in abundance throughout megagametogenesis and in all constituent cells of the mature embryo sac. During many stages, the patterns of distribution of these cytoskeletal elements are congruent and may prove to be co-aligned. Many changes in the arrays of MTs and microfilaments take place and indicate varying roles of the cytoskeleton in the different stages and cell types of megagametogenesis. Two major populations of MTs recur throughout embryo sac formation: (1) Elaborate nuclear-based networks are found during the two-nucleate and four-nucleate developmental stages as well as in the egg cell. These arrays may function in positioning the nuclei. (2) Cytoplasmic MTs in longitudinal orientation in the two-nucleate embryo sac, synergids and part of the egg cell, or in a reticulate pattern in the four-nucleate embryo sac, egg and central cell probably participate in organization of the cytoplasm. Synergid MTs converge at the filiform apparatus. Preprophase bands of MTs are absent throughout megagametogenesis but phragmoplast arrays occur during cellularization of the embryo sac. Well developed arrays of cortical MTs are restricted to the antipodal cells. A large concentration of MTs in the part of the egg cell adjacent to the synergids is well placed for being involved with sperm cell movement within the degenerative synergid. On the basis of the morphology of the cytoskeleton, we concur with views that the shape of megagametophyte is largely determined by the surrounding tissues, including the integumentary tapetum.

Pearl millet synergids from unpollinated pistils at 1.5–2 and 2.5–3 days postmaturity (dpm) were examined using transmission electron microscopy following antimonate fixation to precipitate loosely-bound calcium (Ca). With increasing age of synergids, the gap above filiform apparatus (FA) and the coalesced vacuoles in midchalazal core extended and merged. The FAs became compressed and precipitates along their common wall were dispersed. The matrix material in numerous small chalazal vacuoles changed from dense to flocculent. Precipitates in vacuoles appeared mainly as clumps without or with a halo in the dense matrix and mostly finely distributed in the flocculent matrix. Eventually, vacuoles became free of both matrix and precipitates. Precipitates bound to nucleus, nucleolus, and micropylar cytoplasm increased initially, but then seemed to decrease, while the nucleus became disorganized and the nucleolus disappeared. Precipitates in the embryo sac wall and nucellar cells also increased initially, but then decreased. At very late stages, egg apparatus and ES lost structural integrity and lacked precipitates. Differences in degeneration and Ca levels of sister synergids were smaller at 1.5–2 dpm than at 2.5–3 dpm. A logical and correlative scheme of degenerative events and Ca distributional changes occurring in pearl millet synergids from maturity to 2.5–3 dpm is presented. The significance of results in pollen tube/sperm cell interactions with synergids and Ca is addressed.

This chapter deals with the study of the fertilization process in plants using micromanipulation techniques in both the experimental and the subsequent structural analytical phase of the experiments. Therefore we directly inject isolated sperm cells into egg cells and synergids. The main reasons to develop and use micromanipulation techniques are: firstly, to study the wound reaction of cells after injection with micropipettes of rather wide diameter, secondly to investigate the fate of isolated intra- and interspecific sperm cells after artificial injection into cells of the egg apparatus of intact ovules and thirdly, in case of a succesfull fertilization, obtaining interspecific hybrids. It must be mentioned that the third goal can be more easily reached by isolating not only the sperm cells ( = protoplasts), but also the egg cells, followed by (protoplast-)fusion of both cells (Kranz et al., 1991). However, in the latter case the fusion product ( = mainly the egg cell) has been taken out of the ovule, i.e. its natural environment for fertilization and embryogenesis, so that subsequent ultrastructural studies may reveal phenomena that are more the result of the in vitro situation than when working with intact ovules.

Publisher Summary This chapter focuses on megagametophytes and examines the female germ unit from four new perspectives: (1) the ultrastructure and cytochemistry of megagametophyte development and cellularization, (2) the organization and function of the female germ unit in reproduction, (3) the isolation and characterization of embryo sacs and their component cells, and (4) the involvement of the cytoskeleton in megagametophyte development and function. The development of the megagametophyte is divided into two connected developmental phases: (1) megasporogenesis, which entails the formation and maturation of the initial products of meiosis, followed by (2) megagametogenesis, which begins with the mitotic division of the meiotic products and continues through the cellularization and maturation of the megagametophyte. The female germ unit is comprised of the egg, two synergids, and the central cell. As such, it constitutes the minimum number of cells required to (1) receive the pollen tube, (2) cause the discharge of the sperm into the receptive portion of the female gametophyte, and (3) undergo double fertilization.

Using energy-dispersive X-ray microanalysis of mature, unpollinated wheat ovaries fixed by freeze-substitution, we show here that the synergid cells store a relatively high concentration of calcium. Based on our results and other indirect evidence, we suggest that supraoptimal levels of calcium in synergids may regulate: (1) correct orientation of the pollen tube, by forming a calcium gradient in the vicinity of the synergids, and (2) arrest and rupture of the pollen tube to release the sperm near the egg.

The embryo sac of Linum usitatissimum consists, as in most angiosperms, of the egg, two synergids, central cell and antipodals. In Linum the embryo sac is strongly polarized in the longitudinal axis, but rotationally random relative to the vascular bundles of the ovule and ovary. Synergids designated right or left based on their orientation relative to the egg can be distinguished unambiguously if viewed from one pole of the embryo sac — in this case, the chalaza was used as the reference point. The synergid most likely to degenerate is the left (60%), proximal (52.3%) or septal-facing (52.7%) synergid. The volume and the surface area of the filiform apparatus of the left synergid is significantly smaller than that of the right synergid. Synergid-degeneration patterns varied between individual plants, indicating genetic control; however, the preference for the proximal and septal-facing synergid, although weak, indicates the possibility of some physiological influence. The cotyledons appear to assume bilateral symmetry with respect to the ovule only once endosperm digestion has begun. As the cotyledons grow, the embryo rotates to occupy the widest part of the embryo sac, thus imposing bilateral symmetry between the embryo and seed; prior to that time, the early and heart-shaped embryos are randomly oriented.

The development of the ovule, megaspore and megagametophyte in Saxifraga fortunei var. partita (Makino) Nakai was observed. The ovule is anatropous, bitegmic, and crassinucellate. Both integuments originate from the epidermis. The archesporium is considered to be multicellular. The primary sporogenous cell functions as the megaspore mother cell which forms a T-shaped tetrad. The chalazal member of the megaspore tetrad is functional and develops into a Polygonum-type embryo sac. In the pyriform synergids the filiform apparatus is observed, but any hook or indentations could not be recognized. The antipodal cells are detectable until the Helobial endosperm undergoes several nuclear divisions. Secondary multiplication of the nuclei or the cells of the antipodals could not be observed. THE FAMILY Saxifragaceae s.1.2 has been thought by many authors to consist of some heterogenous groups which might be divided into families. For example, Cronquist (1968) divided the traditional family Saxifragaceae into three families, the Hydrangeaceae, the Grossulariaceae, and the Saxifragaceae (sensu herbaceo), although he considered that his Saxifragaceae was still heterogenous and might be further divided. Hutchinson (1973) split it into 1 1 families and put Penthorum into Crassulaceae. Airy Shaw (1973), in his revision of Willis' "A Dictionary of the Flowering Plants and Ferns," gave 19 families which correspond to the Engler's Saxifragaceae. The development of the ovule and/or the embryo sac in the genus Saxifraga has been reported from time to time, e.g., by Vesque (1879) in S. ornata and S. ligulata, by Juel (1907) in S. granulata, by Pace (1912) in S. ligulata, S. sponhemica, S. cordifolia and S. crassifolia, by Schiirhoff (1925) in S. decipiens x granulata, by Chapman (1933) in S. virginiensis, by Harmsen (1939) in S. stellaris var. comosa, by Wiggins (1959) in S. hieracifolia and by Saxena (1964a) inS. diversifolia. In addition, there are some papers in which development is described incompletely. To 1 Received for publication 3 December 1980; revision accepted 2 April 1981. I am indebted to Dr. C. Kimura of the T6hoku University, Sendai, Japan, for his valuable criticism. 2 In this series of papers, as a matter of convenience, the delineation of the family Saxifragaceae s.l. is based on the Engler's system (Melchior, 1964). Major difference between Engler's Saxifragaceae and Bentham and Hooker's lies in the inclusion of the Cinoniaceae in the latter summarize these reports, the ovule in the genus Saxifraga is usually anatropous, unitegmic or bitegmic and always crassinucellate. The primary sporogenous cell is cut off from the single archesporial cell. It undergoes meiotic divisions to form a linear or less frequently a T-shaped tetrad. The chalazal member of the four megaspores is functional and gives rise to a Polygonum-type embryo sac. The synergids are hooked and exhibit the filiform apparatus in some species but neither the hook nor the filiform apparatus occur in others. The antipodals are mostly ephemeral and degenerate without further divisions but show secondary multiplication in S. diversifolia. This study, the first in a series of investigations, has been undertaken to furnish embryological data of the family Saxifragaceae s.l. and to contribute to a better understanding of the true relationships between the taxa within the family. The present paper deals with the development of ovule, megaspore and megagametophyte in S. fortunei var. partita (Makino) Nakai. MATERIALS AND METHODs-Many flower buds and flowers of Saxifraga fortunei var. partita were collected in Oct. 1975, from plants growing on wet rocks along one of the upper streams of the River Abu, called Ch6monkyo, Yamaguchi Prefecture, western Honshu, Japan. The materials, killed and fixed in FPA, were dehydrated and embedded in paraffin by the tertiary butyl alcohol method. Serial sections 7-12 ,um thick were cut and stained in Heidenhain's iron haematoxylin and fast

In Stipa elmeri Piper & Brodie ex Scribn., the pollen tube enters at the filiform apparatus of the degenerated synergid. The degenerated synergid has electron-dense cytoplasm in which organelles are not discernible. All other cells of the mature megagametophyte have nuclei, endoplasmic reticulum, plastids, mitochondria, dictyosomes, and vacuoles. Starch is found in the persistent synergid (in minute quantities), egg, and central cell. Lipids occur in the persistent synergid, central cell, and antipodals. The filiform apparatuses of the two synergids are hypothesized to perform different functions. In the degenerated synergid, the filiform apparatus serves to increase the surface area of the plasma membrane and thereby to offer a large area for pollen-tube-growth-directing compounds to diffuse out of the synergid. In the persistent synergid, the filiform apparatus is part of a suite of features which indicate that the persistent synergid is involved in the transference of materials into the megagametophyte. Another possible function of the persistent synergid is to aid in establishing the polarity of the egg. The pollen grain and tube have distinctive polysaccharide spheres that serve to delimit the pollen tube cytoplasm after discharge into the degenerated synergid. Associated with the degenerated synergid are bodies of dense materials as seen under electron microscopy, and bodies of RNA and protein as determined histochemically. These are probably the same thing and come from the degenerating synergid. The antipodals are the most cytologically active cells of the megagametophyte. They have some features which are characteristic of transfer cells and possibly function in the transference of materials into the megagametophyte. Other studies (Brink and Cooper 1944) have indicated that grass antipodals are involved in the control of endosperm development. The active cytoplasm of the antipodals may reflect the synthesis or transference of growth-controlling substances.

The ovule of Ornithogalum dubium is anatropic, bitegmic and crassinucellate. The integuments are biseriate. Starch grains occur in the outer integument, the funicule and in the intercellular space surrounding the embryo-sac. The embryo-sac development is of the Polygonum type. The synergids are conspicuous and contain a large filiform apparatus. Fertilization is porogamic and occurs 24 h after pollination. The pollen tube enters the egg apparatus in the region of an intercellular space between the micropylar ends of the synergids. Sperm cells in the pollen tube appear to be released through small protuberances of the pollen tube. Migration of the sperm cell to the egg cell is possibly through a gap in the wall between the egg cell and the receptive synergid. Changes in the synergids occur only after a pollen tube has entered the receptive synergid. Changes occurring in the persistent synergid suggest that this cell may be involved in the nutrition of the pro-embryo up to the eight-cell pro-embryo stage. Syngamy is of the post-mitotic type. The first division of the zygote is transverse. The basal cell gives rise to the multicellular suspensor, and the terminal cell to the embryo. The endosperm is of the Helobial type.

The embryo sac ofNigella damascena, with wall ingrowths over entire surface and distinctly formed wandlabyrinthe at the poles, displays a well integrated model for short distance transport of nutrients. The degenerating nucellus creates an influx of metabolites into the embryo sac. The embryo sac wandlabyrinthe play a dual role—initially aid with the influx of metabolites into the embryo sac, but later participate in endosperm wall formation. The two synergid apices exhibit prominent filiform apparatus which function differently. One of the synergids degenerates immediately after fertilization where filiform apparatus helps in receiving the pollen tube. The other undamaged synergid persists along with filiform apparatus upto the octant embryo stage and this filiform apparatus probably facilitates short distance metabolite transport into the synergid. The antipodal cells show 3 types of wall ingrowths—(i) wall ingrowths at the antipodal-nucellus interface which are long, branched and spread fan-like into the antipodal cytoplasm, indicating the flow of metabolites into the antipodes from the nucellus, (ii) inter-antipodal wall-ingrowths that are small, papillate and present on both sides of the wall indicating the exchange of metabolites between the antipodes and (iii) wall labyrinths at the antipode-central cell interance which are very small and are directed into the antipode cytoplasm.

Ovule development, megasporogenesis and megagametogenesis in an aneuploid population of the arctic-alpine crucifer, Smelowskia calycina, were examined to test for the possibility of apomictic seed production. Whole mounts of ovules cleared in Herr's "4?2" clearing fluid were examined using Nomarski differential interference microscopy. The campylotropous ovule was bitegmic, with a micropyle formed by both integuments. The single archesporial cell of a crassinucellar nucellus functioned directly as a megasporocyte, dividing to form a linear tetrad of megaspores. The chalazal megaspore divided to form an 8-nucleate, 7-celled gametophyte of the Polygonum type, having hooked synergids with a well-developed filiform apparatus and polar nuclei that fused prior to fertilization. In the absence of any anomalous development indicative of agamospermy, seed production was assumed to be sexual.

Electron-microscopic examination of the embryo sac of soybean, Glycine max (L.) Merr. `Bragg' shows that each cell of the egg apparatus is enclosed by a wall that is thickest at the micropylar end and contains expanded wall areas interspersed between thin areas at the chalazal end. Expanded wall areas are much thicker in the egg cell than in synergids. A filiform apparatus (FA) at the micropylar end of each synergid appears continuous with the embryo sac wall. The surface area of the plasma membrane appressed to the FA is extensive, and abundant endoplasmic reticulum (ER) is associated with the FA. Many plasmodesmata are present in the wall between the egg cell and each synergid, suggesting that the synergids may contribute to egg cell nutrition. Numerous osmiophilic bodies in the egg and synergids indicate that some storage of reserves can occur in these cells. Abundant ER profiles within the egg indicate that it is a relatively active cell. There are few plasmodesmata between the central cell and two sy...

The reproductive system of alfalfa was studied for the presence of transfer cells. They were found in the tapetum of the anther, in the stylar canal, and along the ventral suture of the ovary wall. The cell wall of the filiform apparatus and the wall at the tip of the pollen tube resemble those of transfer cells.

The megagametophyte of Plumbagella micrantha, consisting of three cells: an egg, a central cell, and a single antipodal, represents the most structurally reduced female gametophyte of the flowering plants. In the absence of synergids, the pollen tube enters the megagametophyte through a filiform apparatus at the base of the egg, continues growth for 40 ,um through the egg-central cell wall, until reaching a region of strong curvature near the chalazal end of the egg. A terminal pollen tube aperture then forms, the sperm cells and vegetative nucleus are released between the egg and central cell, and the sperm undergo gametic fusion, thereby effecting double fertilization. The mode of fertilization in Plumbagella is therefore similar to that of Plumbago that has been described elsewhere. PLUMBAGELLA MICRANTHA, a monotypic species, possesses the most structurally simplified female gametophyte present in flower

The ultrastructural organization of the megagametophyte of Plumbago zeylanica, which lacks synergids, was examined in chemically and physically fixed ovules after entry of the pollen tube. Similar to angiosperms with conventionally organized megagametophytes, the pollen tube enters the ovule through a micropyle, formed by the inner integument, and approaches the female gametophyte by growing between nucellar cells. Unlike other described female gametophytes, however, continued pollen tube growth results in direct penetration of the base of the egg through cell wall projections forming a filiform apparatus and is completed between the egg and central cell without disrupting either of these cells' plasma membranes. A terminal pollen tube aperture forms when the pollen tube reaches an area of strong curvature near the summit of the egg; this results in the release of two sperm cells, the vegetative nucleus, and a limited amount of pollen cytoplasm. The formerly continuous chalazal egg cell wall is locally disrupted near the tip of the pollen tube and apparently is thus modified for reception of male gametes. Discharged pollen cytoplasm rapidly degenerates between the egg and central cell, but unlike pollen tube discharge in conventionally organized megagametophytes, it is unassociated with the degeneraton of any receptor cell within the female gametophyte. Sperm nuclei are transmitted, one to the egg and the other to the central cell, to effect double fertilization by nuclear fusion with their respective female reproductive nuclei. The vegetative nucleus and discharged pollen cytoplasm degenerate between the developing embryo and endosperm during early embryogenesis. The emerging concept that the egg of Plumbago possesses combined egg and synergid functions is supported by the present study and suggests that the megagametophyte of this plant displays a highly specialized egg apparatus composed exclusively of a single, modified egg cell.

SUMMARY The nucellus of immature crassinucellate ovules of Spinacia cannot be penetrated by pollen tubes, which cluster together in and around the micropyle. After maturation of the ovule, pollination activates the embryo sac, i.e. the synergids, to excrete substances which dissolve the middle lamellae of the nucellus tissue in front of the synergids. Eventually the nucellar cuticle near the micropyle becomes interrupted. At that developmental stage pollen tubes can penetrate into the nucellus, initially growing intercellularly, and subsequently following various pathways. More than one tube can reach the embryo sac, but only one pollen tube penetrates the degenerated synergid via the filiform apparatus. Sperm cells, vegetative nucleus and tube cytoplasm with many amyloplasts are discharged into the degenerated synergid. The vegetative nucleus and the sperm cells are pressed to the chalazal part of the synergid, of which the plasma membrane is degenerated previously. The discharge and the fusion processes of the sperm cells with the egg cell and central cell (7–9 hrs after pollination) are described and discussed. Apparently no or very few organelles of the sperm cells enter the female gametes.

Changes in the distribution of iron during the embryo sac formation of Hemerocallis middendorffii var. esculenta were cytochemically examined. Iron was detected as Prussian blue products of various forms. The reaction products were not observed in the early stages of the megaspore, but they appeared in some cells in the chalazal region of the nucellus. The degenerating nucellar cells around the megaspore also had a number of small granular reaction products.In the mature embryo sac, the egg contained a number of small granular products around the nucleus, and a few products were observed under the filiform apparatus of the synergids in some preparates. The degenerating antipodals and the central cell contained a cluster of granular products or the products of the complicate shape. The reason for the difference in the distribution of iron between Hemerocallis and Lilium is still unknown.

The egg apparatus of Nicotiana tabacum was studied from just before flower opening until 92 h after pollination. Each synergid has a large nucleus and nucleolus and a prominent chalazal vacuole. Numerous mitochondria, plastids with starch, dictyosomes, free ribosomes, and rough endoplasmic reticulum (ER) are present. A cell wall exists only around the micropylar half of the synergids, and each cell has a distinct filiform apparatus. Numerous conspicuous cell-wall evaginations located just chalazal to the hook region extend laterally from the synergids into the central cell. The egg has a chalazally located nucleus with a single nucleolus and a large micropylar vacuole. With the exception of dictyosomes, the same cytoplasmic organelles are present in the egg as in the synergids, but in much lesser amounts. A continuous cell wall is not present around the chalazal end of the egg. Before pollination, numerous large lipid bodies are localized in the outer integumentary cells. Near the time of fertilization ma...

A Wandlabrinthe develops around the micropylar end of the central cell at the time of ovule maturation and fertilization in soybean, Glycine max (L.) Merr. Wandlabrinthe development occurs during morphogenesis of the central cell as it is transformed from being one of the seven cells of the megagametophyte (prefertilization state) to becoming the embryo sac after syngamy (postfertilization state). The Wandlabrinthe circumvallates the synergids by forming a ridge of cell wall material that projects into the central cell toward the zygote and the synergids at the level of the filiform apparatus. Degenerating nucellar cells are adjacent to, and contiguous with, the Wandlabrinthe, and cells in this region of both integuments are rich in starch reserves. The Wandlabrinthe most likely functions in translocating nutrients from degenerating nucellar cells into the central cell/embryo sac for endosperm and embryo nutrition.

There is little information on the fate of embryo sacs in plant ovules if pollination is prevented. In this study embryo sacs from watermelon were observed over a 13 day period following flowering with (a) normal pollination, (b) non-pollination and (c) induction of parthenocarpic fruit development with naphthalene acetic acid. Following pollination, and prior to fertilization approximately 2 days later, the embryo sacs completed development and consisted of two synergids with prominent filiform apparatus, an egg cell, a central cell with two polar nuclei and three antipodal cells. Sperm nuclei were observed within the embryo sac at 2 days and by 4 days the endosperm was proliferating. In the non-pollination treatment the embryo sac was still intact after 4 days although the antipodal nuclei were becoming hard to distinguish. By 7 days only the two synergids and the egg cell were still well defined, the polar nuclei appeared in some preparations to be fused, and the antipodals had degenerated. By 10 days the embryo sac was a structure-less watery mass. In parthenocarpic fruit the fate of the embryo sac was similar to that in non-pollinated fruit except that final breakdown was delayed past 10 days. Maturity of the majority of embryo sacs in an ovary appeared to be contemporaneous with penetration of the pollen tube, and on the basis of the anatomical results it seems possible that embryo sacs could be fertilized up to 2 days beyond the normal time.

Abnormalities in avocado (Persea americana) ovules at blossoming are explained by demonstrating their possible origin at some stage of ovule development. Degeneration of the embryo sac at the micropylar site may occur at the four-nucleate stage or at egg-apparatus organization, prior to or after the filiform apparatus has formed. The time of occurrence of degeneration decisively determines the appearance of the degenerate remnants. The absence of an embryo sac and the presence of a double embryo sac originate at the earliest stages of the nucellar primordium; a third abnormality, compartmentalization, is assumed to originate at the megaspore tetrad stage.

The present investigation deals with some of the organizational and histochemical aspects of the embryo sac ofScilla sibirica. Both the synergids and egg cell are invested by PAS-positive complete walls. The filiform apparatus comprises an elaborate system of fibrillar projections, showing extensive ramifications. The micropylar region of the embryo sac wall from where the filiform apparatus originates is composed of three distinct layers. On a histochemical basis it may be surmised that, unlike the egg cell, the synergids are metabolically very active. Two kinds of wall ingrowths (i) massive and highly branched very much akin to the filiform apparatus, and (ii) small tuberculate wall projections, are unique to the antipodal cells of S.sibirica. Small tuberculate projections have also been observed along the wall of the central cell adjacent to the nutrient-rich nucellar cells. The antipodals and the central cell show the presence of starch grains and abundant total proteins. All the cell types in the embryo sac ofS. sibirica are structurally so organized as to meet the requirements of its nutrition during pre- and postfertilization development. The presence of abundant PAS-positive granular substance in the cells of nucellar epidermis probably establishes a gradient which assists in the pollen tube growth.