Plasmodesmal Dynamics in Both Woody Poplar and Herbaceous Winter Wheat Under Controlled Short Day and in Field Winter Period

Electron microscopic observation revealed that poplar (Populus deltoides Marsh.) and winter wheat (Triticum aestivum L. cv. Seward 80004) plasmodesmatal structures significantly changed under short day (SD, 8 h light) and in winter period, and such changes differed also noticeably between these two woody and herbaceous plants. Under long day (LD, 16 h light), many plasmodesmata with strong stain appeared in the cell wall of both poplar apical buds and winter wheat young leaf tissues, and connections of cytoplasmic endoplasmic reticulum (ER) with the ER in some plasmodesmata were observed. In addition, the typical “neck type” plasmodesmata were observed in winter wheat young leaf tissues, and their central desmotubules (appressed-ER) could be clearly identified. Under SD, many poplar plasmodesmata showed only a partial structure in the cell wall and appeared to be discontinued; some plasmodesmata swelled in the mid-wall, forming the cavity, and no appressed-ER appeared. In winter wheat, however, no noticeable alterations of plasmodesmata occurred, and the plasmodesmatal structure essentially remained same as it was under LD. In winter period, poplar plasmodesmata had a similar morphology as those observed under SD, however, winter wheat manifested at least two types of significant plasmodesmatal alterations: one plugged by electron-dense materials and the other of reduced neck region compared to those under LD. The above dynamic difference of the two species plasmodesmata under SD and winter period revealed the difference of their dormancy development under those environmental conditions.     

Filament Disruption in Funaria Protonemata: Occlusion of Plasmodesmata

Plasmodesmata are occluded when Funaria chloronemata are fragmented by the development of tmema cells (TCs). The TC deposits a new wall layer along the cross wall toward the neighbouring non-sister cell (NC). This wall layer cuts off the plasmodesmata and its connection with the cross wall is soon lost. The plasmodesmata become isolated when the NC forms a new wall layer along the former cross wall. At the end of TC development, before its disintegration, the sister cell (SC) also deposits a new wall layer along the cross wall toward the TC, cutting off the plasmodesmata. For some time the plasmalemma of the plasmodesmata remains connected to the NC or the TC, whereas the desmotubule soon disappears. Relicts of the plasmalemma remain even after the isolation of the plasmodesmata and the disintegration of the TC. During the decay of the plasmodesmata, a cylinder of electron-dense material is frequently formed along the border of the plasmodesmatal channel. This may extend over the surface of the cell wall. Eventually, the plasmodesmatal channel is filled with wall material. Callose is only observed around functional plasmodesmata and does not seem to play a role in their occlusion.     

Studies on graft unions

Summary De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.     

PLASMODESMATA AND AN ASSOCIATED CELL WALL COMPONENT IN BARLEY ALEURONE TISSUE

A unique cell wall component has been observed in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). This wall component has been shown to be localized adjacent to the plasmalemma. Unlike the surrounding cell wall matrix it is resistant to “Onozuka” cellulase and remains intact during gibberellic acid-stimulated hydrolase release. After treatment of the tissue with gibberellic acid followed by digestion with “Onozuka” cellulase this resistant wall component can be isolated free of protoplast. Study of its surface features revealed the presence of numerous tubular extensions, 120 nm wide, connecting adjacent resistant walls. These tubes resembled light microscope images of plasmodesmata in size and appearance. E.M. sections of resistant walls showed the presence of unit membrane lining the inner surface of the wall tubes. It was concluded that the resistant wall constitutes a modified wall layer that is secreted uniformly across all plasmalemma surfaces, including those in the wall (plasmodesmata). The presence of wall tubes surrounding plasmodesmata enhances the apparent size of the plasmodesmata in the light microscope. This may account for previous inconsistencies in the literature between light and electron microscope determinations of plasmodesmata diameters.     

ELECTRON MICROSCOPIC OBSERVATIONS OF CULTURED CELLS AND PROTOPLASTS OF AMMI VISNAGA

Protoplasts were prepared from cultured cells of Ammi visnaga (Umbelliferae) by enzymatic digestion of the cell walls and examined microscopically. Staining of fresh protoplasts with Calcofluor and silver hexamine demonstrated the apparent absence of wall material. Protoplasts contained more cell organelles than the whole cells, particularly endoplasmic reticulum and associated polysomes. The plasmalemma of most protoplasts appeared smooth; some protoplasts were connected by structures resembling plasmodesmata. Multinucleates resulting from fusion were frequently observed.     

Endoplasmic Reticulum Forms a Dynamic Continuum for Lipid Diffusion between Contiguous Soybean Root Cells

Intercellular communication between plant cells for low molecular weight hydrophilic molecules occurs through plasmodesmata. These tubular structures are embedded in the plant cell wall in association with the plasmalemma and endoplasmic reticulum (ER). Transmission electron microscopy has provided strong evidence to support the view that both the ER and plasmalemma are structurally continuous across the wall at these sites. In experiments to be described, the technique of fluorescence redistribution after photobleaching was used to examine the lateral mobility and intercellular transport capability of a number of fluorescent lipid and phospholipid analogs. These probes were shown by confocal fluorescence microscopy to partition in either the ER or plasmalemma. Results from these measurements provide evidence for cell communication between contiguous cells for probes localized predominantly in the ER. In contrast, no detectable intercellular communication was observed for probes residing exclusively in the plasmalemma. It was of particular interest to note that when 1-acyl-2-(N-4-nitrobenzo-2-oxa-l,3-diazole)aminoacylphosphatidylcholine was utilized as a potential reporter molecule for phospholipids in the plasmalemma, it was quickly degraded to 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)aminoacyldiglyceride (NBD-DAG), which then appeared predominantly localized to the ER and nuclear envelope. This endogenously synthesized NBD-DAG was found to be capable of transfer between cells, as was exogenously incorporated NBD-DAG. Results from these investigations provide support for the following conclusions: (1) ER, but apparently not the plasmalemma, can form dynamic communication pathways for lipids across the cell wall between connecting plant cells; (2) the plasmodesmata appear to form a barrier for lipid diffusion through the plasmalemma; and (3) lipid signaling molecules such as diacylglycerol are capable of transfer between contiguous plant cells through the ER. These observations speak to issues of plant cell autonomy for lipid synthesis and mechanisms of intercellular signaling and communication.     

毛竹茎秆纤维细胞发育过程中ATP酶的超微细胞化学定位研究

采用磷酸铅沉淀技术,对毛竹茎秆纤维细胞发育过程中的ATP酶进行了超微细胞化学定位研究.在初生壁形成时期,大量的ATP酶的活性产物沉积在质膜、质膜内陷、运输小泡、胞间连丝等膜体系以及细胞核和各种细胞器上;在次生壁形成的初期,ATP酶在多泡小体和裂解的液泡膜上出现,凝聚并边缘化的染色质上仍然具有ATP酶活性;随着次生壁的逐渐加厚,在前四年中持续存在具有ATP酶活性的质膜内陷结构,以后消失;而在六年生纤维细胞的质膜、运输小泡、纹孔、胞间连丝和凝聚化的染色质上仍然发现有明显的ATP酶分布,并发现在染色质上ATP酶活性会随着凝聚程度的加深而增强.结果表明,ATP酶在毛竹茎秆纤维细胞壁的整个形成过程中发挥重要作用,而纤维细胞的次生壁形成过程是一个由核基因控制的主动的PCD过程;并证实毛竹茎秆纤维细胞的发育有别于其它木本植物纤维细胞的发育过程,这种纤维细胞是一种典型的长寿细胞.     

Ultrastructural Studies on the Development of Oil Cells in Litsea pungens

The developmental process of oil cells in the shoot of Litsea pungens Hemsl. has been studied with transmission electron microscopy. According to the development of the three layers of cell wall, the developmental process could be divided into 4 stages. In stage 1, the cell wall consisted only of a primary (the outmost) cellulose layer, which might further be divided into two substages, the oil cell initial, and the vacuolizing oil cell. During this stage, there were some small electron translucent vesicles and dark osmiophilic droplets of variant sizes in the different-shaped plastids. It was observed that some dark and gray osmiophilic materials coalesced to vacuoles in the cytoplasm. In stage 2, a lamellated suberin layer accumulated inside the primary cellulose layer. In stage 3, a thicker and looser inner cellulose wall layer was formed gradually inside the suberin layer. Some dark osmiophilic droplets have been observed in this loose inner cellulose wall layer. The plasmodesmata were blocked up and became a special structure. Then, the big vacuole, which is the oil sac, was full of osmiophilic oil. In stage 4, the oil cell became matured and the cytoplasm disintegrated. The oil sac enveloped from plasmalemma was attached to the cupule, which was formed by the protuberance of the inner cellulose wall layer into the lumen. After the maturity of oil cell, the ground cytoplasm began to disintegrate and became electron opaque or exhibited in a disordered state, and the osmiophilic oil appeared light gray.     

木姜子油细胞发育的超微结构研究

利用超薄切片法和透射电镜研究了木姜子（Litsea pungens Hemsl.）油细胞的发育过程。油细胞3层细胞壁的发育可分为4个阶段,阶段1：油细胞仅有初生纤维素壁层,又可分为原始细胞和细胞 泡化两个时期。此阶段质体具透明小泡和黑色嗜锇物质,并与液泡融合。阶段2：木栓质化壁层的形成,片层状木栓质不断叠加在初生纤维素壁内侧,其细胞结构与前期相似,阶段3：内纤维素壁层的形成,较厚而松散的内纤维素壁层叠加在木栓质化壁层的内侧,在内纤维素壁层中可见黑色嗜锇物质,胞间连丝成为被阻塞的特化结构,此时大液泡被嗜锇油脂充满,成为油囊。阶段4：油细胞成熟及细胞质解体,杯形构造由内纤维素壁层向细胞腔内突起形成,油囊由液泡膜包被连接到杯形构造上,油呈浅灰色嗜锇状态,其细胞质和细胞器解体,变得电子不透明或呈杂乱状态。     

Ultrastructure ofAspergillus nidulans conidia and conidial lomasomes

Summary Lomasomes in the conidia ofAspergillus nidulans can be divided into at least two distinct structures. The first is a twice double membrane bound core of cytoplasmic origin. The outermost membrane of the lomasome becomes incorporated into the plasmalemma as it migrates to rest next to the cell wall. The second lomasome structure appears to be a triangle shaped series of tubules arranged in a parallel fashion. The wide end next to the cell wall connected to the plasmalemma and the opposite end to an element of the endoplasmic reticulum. The term membranosome has been coined to designate this lomasome structure with its function of plasmalemma extension. Various structures of the conidium such as wall, endoplasmic reticulum and the cytoplasmic matrix undergo changes from the conidial chain stage to the free or resting conidial stage. This suggests that after conidiation and before the resting stage, the conidium continues to mature.     

Transport of assimilates in the developing caryopsis of rice (Oryza sativa L.)

Assimilates entering the developing rice caryopsis traverse a short-distance pathway between the terminal sieve elements of the pericarp vascular bundle and the aleurone layer. The ultrastructure of this pathway has been studied. Sieve elements in the pericarp vascular bundle are smaller than their companion cells.The sieve elements show few connections with surrounding vascular parenchyma elements but are connected to companion cells by compound plasmodesmata. Companion cells, in turn, are connected to vascular parenchyma elements by numerous compound plasmodesmata present in wall thickenings. Assimilates leaving the sieve element — companion cell complex must laterally traverse cells of the pigment strand before they come into contact with the aleurone layer. The pigment strand cells have modified inner walls made up of a suberin-like material. This material may act as a permeability barrier isolating the apoplast from the symplast of the pigment strand. The walls of the pigment strand cells are traversed by numerous plasmodesmata. Water may be conducted to the endosperm through the isolated cell-wall system of the pigment strand while assimilates possibly move via plasmodesmata. High frequencies of plasmodesmata occur at the junction between the pigment strand and the nucellus and also between adjacent cells of the nucellus. By contrast, plasmodesmata are absent between the nucellus and the aleurone layer and also between the nucellus and the seed coat. A predominantly circumferential and symplastic transport pathway is likely between the pigment strand and nucellus. In view of the total absence of plasmodesmata between the nucellus and the aleurone layer assimilates entering the endosperm may have to cross the plasmalemma of the nucellus. It is possible that constraints to the flow of assimilates may occur in the short-distance pathway between the terminal sieve element — companion cell complexes and the endosperm, and this is discussed.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Guard cell wall: immunocytochemical detection of polysaccharide components

The composition of guard cell walls in sugar beet leaves (Beta vulgaris L.) was studied by using histochemical staining and immunocytochemical detection of cell wall antigens. The findings were compared with those in the walls of epidermal and mesophyll cells. Probing of leaf sections with monoclonal antibodies against pectins, terminal fucosyl residues linked alpha-(1-->2) to galactose, beta-(1-->3)-glucans and arabinogalactan-proteins revealed several specific features of guard cells. Pectic epitopes recognized by JIM7 were homogeneously distributed in the wall, whereas pectins recognized by JIM5 were not found in the walls themselves, but were abundant in the cuticular layer. Large amounts of molecules bearing terminal fucose were located predominantly in ventral and lateral guard cell walls. Much smaller amounts were detected in dorsal walls of these cells, as well as in the walls of pavement and mesophyll cells. Conspicuous accumulation of these compounds was observed in the vicinity of the guard cell plasmalemma, whereas labelling was scarce in the areas of the wall adjacent to the cell surface. The presence of callose clearly marked the ventral wall between the recently formed, very young guard cells. Callose also appeared in some mature walls, where it was seen as punctate deposits that probably reflected a specific physiological state of the guard cells. Large amounts of arabinogalactan-proteins were deposited within the cuticle, and smaller amounts of these proteoglycans were also detected in other tissues of the leaf. The histochemical and immunocytochemical structure of the guard cell wall is discussed in the light of its multiple functions, most of which involve changes in cell size and shape.     

A new interpretation of plasmodesmatal ultrastructure

Summary It is shown that simple, unbranched, plasmodesmata between young xylem ray cells of willow have no direct intercellular continuity apart from the plasmalemma which limits the cytoplasm and lines the plasmodesmatal canal. Each plasmodesma is traversed by a 200 Å diameter tubule (the desmotubule) which has a wall with probably 11 subunits arranged around a central cavity through which runs a 40 Å diameter rod. This rod is connected to the inside of the tubule wall, by fine filaments. At the ends of each plasmodesma the plasmalemma and cell wall are closely appressed to the tubule, thus precluding direct continuity between the cytoplasm of adjacent cells. Through the central part of the plasmodesmata the tubule is separated from the plasmalemma by a 90–100 Å wide gap. Cytoplasmic microtubules in the same tissue have a diameter of approximately 250 Å and a wall probably composed of 13 subunits: both desmotubules and cytoplasmic microtubules therefore have a centre-to-centre subunit spacing of about 47 Å. It is suggested that the desmotubules are not microtubules but may be nuclear spindle fibres which become trapped in the wall during cell plate formation. The endoplasmic reticulum, while closely approaching the plasmodesmata, is not continuous across them. It is thought most unlikely that the endoplasmic reticulum traverses plasmodesmata, as the dimensions of the central tubule — found here as well as by other workers — are smaller than those which would be expected to allow a stable molecular configuration in a unit membrane. The plasmalemma, where it lines the plasmodesmatal canal, appears to have particulate subunits in the outer opaque layers and the presence of these subunits may be attributable to the need for stability in membranes arranged about so small a radius.     

Subcellular localization of ubiquitin in plant protoplasts and the function of ubiquitin in selective degradation of outer-wall plasmodesmata in regenerating protoplasts

Immunocytochemical localizations in Vicia faba L. protoplasts and cultures of regenerating Solanum nigrum L. protoplasts support former observations that in plant cells ubiquitin occurs within the cytoplasm, the nucleus, the chloroplasts and at the plasmalemma, but not within the vacuole or the cell wall. Immunoresponses were also observed within mitochondria and associated with the endoplasmic reticulum, which is in accordance with previous findings on animal cells. Moreover, the tonoplast membrane system was found to be labelled. For regenerating S. nigrum protoplasts, evidence is given that ubiquitin plays a role in selective degradation even of whole subcellular structures. Most of the discontinuous plasmodesmata formed in the newly deposited outer cell walls during the early stages of culture disappear later on, except for those near the periphery of division walls or of non-division walls, which are probably used for the formation of continuous cell connections during further culture. Outer-wall plasmodesmata which are destined to disappear show high immunoreactivity to ubiquitin antibody, but no conspicuous immunolabelling was observed with the remaining plasmodesmata. Thus, the selective disintegration of whole plasmodesmatal structures is obviously regulated by ubiquitination of plasmodesmatal proteins. A model for the mechanism of degradation of outer-wall plasmodesmata during extension growth of the cell wall is presented.Dedicated to Professor Dr. Andreas Sievers on the occasion of his retirementThis work was supported by grants to R. K. (Deutsche Forschungsgemeinschaft) and to M. S. (Bennigsen-Foerder Preis des Landes Nordrhein-Westfalen). We thank Dipl.— Biol. Kirsten Leineweber for help with the V. faba protoplast isolation and Dr. Olaf Parge, Institut für Psychologie und Sozialforschung, Kiel, Germany, for giving assistance with the statistical analysis.     

鹤顶兰胚囊发育过程中Ca~（2＋）分布的超微细胞化学定位（英文）

用焦锑酸盐沉淀法对鹤顶兰（Phaius tankervilliae）胚囊发育过程中的Ca2＋状态进行超微细胞化学定位。观察结果发现：功能大孢子时期,珠孔端的胚囊壁上开始出现小颗粒的Ca2＋沉淀,但功能大孢子细胞内未见明显的Ca2＋标记;四核胚囊时期胚囊壁上的Ca2＋沉淀明显增多,液泡膜上有Ca2＋沉淀出现,珠孔处的Ca2＋沉淀颗粒较大;成熟胚囊时期,胚囊壁上的Ca2＋沉淀进一步增多,且胚囊内Ca2＋分布明显增多,且极性明显,珠孔端助细胞、卵细胞比合点端反足细胞有更多的Ca2＋沉淀。鹤顶兰成熟胚囊内Ca2＋积累的来源有：（1）在胚囊成熟前主要由珠被细胞、珠细胞通过胞间连丝向胚囊运输;（2）以沉淀有大量Ca2＋的小泡形式跨过胚囊壁进入胚囊。     

LEAF OF SPINACIA OLERACEA (SPINACH): ULTRASTRUCTURE,AND PLASMODESMATAL DISTRIBUTION AND FREQUENCY,IN RELATION TO SIEVE-TUBE LOADING

Minor veins and contiguous tissues of the Spinacia oleracea leaf were analyzed by electron microscopy to determine the characteristics of the component cells and the structure, distribution, and frequency of plasmodesmata between the various cell types of the leaf. Mesophyll and bundle-sheath cells contain components typical of photosynthetic cells although the latter cell type contains smaller chloroplasts and fewer mitochondria and microbodies than the mesophyll cells. In addition, the mesophyll cells contain numerous invaginations of the plasmalemma bordering the chloroplasts and evaginations of the outer membrane of the opposing chloroplast envelope. In places, these membranes appear continuous with each other. The minor veins consist of tracheary elements, xylem parenchyma cells, sieve-tube members, companion and phloem parenchyma cells, and other cells simply designated vascular parenchyma cells. The companion and phloem parenchyma cells are typically larger than the sieve-tube members with the companion cells containing a much denser cytoplasm that the phloem parenchyma. Cytoplasmic connections occur along all possible routes from the mesophyll to the sieve-tube members and consist of either simple or branched plasmodesmata between parenchymatic elements or pore-plasmodesmata between the sieve-tube members and parenchyma cells. The highest frequency of plasmodesmata occurs between the sieve-tube members and companion cells, although the value is essentially the same as between the various parenchymatic elements of the phloem. Compared to several previously studied species, the frequency of plasmodesmata between cell types of the spinach leaf is low. These results are discussed in relation to apoplastic vs. symplastic solute transport and sieve-tube loading in this species.     

Ultrastructural characterization of apospory in Panicum maximum

The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.     

SOME ULTRASTRUCTURAL OBSERVATIONS ON ENDODERMAL CELL DEVELOPMENT IN ZEA MAYS ROOTS

The fine structure of primary, secondary, and tertiary stages of Zea endodermal cell development was investigated. The casparian strip formed in situ in the anticlinal walls and remained at a fixed point relative to the endodermis-pericycle boundary. The only protoplasmic structure that had a constant spatial association with the developing strip was the plasmalemma. Plasmodesmata appeared to be more numerous on the tangential walls than on radial walls; only rarely were they located in the casparian strip. The suberized lamella developed on inner and outer tangential walls before it appeared on the radial walls. No cytoplasmic organelles were found to have any particular spatial association with this layer. The suberized lamella was about 0.04 μm thick except near plasmodesmata and along the adaxial margin of the casparian strip, where it was thicker. Occasionally it failed to form along the abaxial margin of the strip. The adherent affinity between plasmalemma and casparian strip was lost after the strip was covered by suberized lamella. The secondary wall became asymmetrically thickened by differential deposition of successive lamellae. A thin layer of secondary wall material extended across the floor of each pit. Pit cavities often contained mitochondria, and plasmodesmata were restricted to the pits. The plasmodesmata were constricted where they entered the thin layer of secondary wall material and where they penetrated the suberized lamella. The various stages of cell development tended to be asynchronous. No passage cells were observed. Endodermal cell development in Zea closely resembles that described for barley.     

EMBRYO SAC LACKING ANTIPODAL CELLS IN ARABIDOPSIS THALIANA (BRASSICACEAE)

Ultrastructure of the embryo sac lacking antipodals in prefertilization stages in Arabidopsis thaliana has been examined 2 hr before and 5 hr after manual cross pollination. The cytoplasm of both synergids before fertilization is rich in ribosomes, mitochondria, and rough endoplasmic reticulum, and also contains several microbodies and spherosomes. The filiform apparatus includes electron-dense material and a fibrous part. Many cortical microtubules appear in the filiform apparatus area. One of the two synergids degenerates before fertilization. The synergids, the egg cell, and central cell have a rich cytoskeleton of microtubules; only the synergids appear to contain microfilaments. At the chalazal end, the antipodals are initially present but degenerate by the time of pollination in most embryo sacs in the starchless line studied. The embryo sac is completely surrounded by a wall containing an electron-dense layer, separating it from the nucellus, including the chalazal end. When the antipodals have degenerated, the electron-dense layer disappears at the chalazal end only, and the wall between the central cell and the nucellus is homogeneous. Between the central cell and nucellar cells no plasmodesmata are found. The membranes of both antipodal cells at the chalazal end of the embryo sac appear sinuous, like those of transfer cells. The central cell has plastids preferentially distributed around the nucleus, but the other organelles are randomly distributed. The central cell in the embryo sac and the adjacent chalazal nucellar cells show a transfer-cell function in the embryo sac after the antipodals degenerate.