Calcium changes in ovules and embryo sacs of Plumbago zeylanica L.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell. Received: 30 December 1999 / Revision accepted: 24 March 2000     

Three-dimensional organization and dynamic changes of the actin cytoskeleton in embryo sacs ofZea mays andTorenia fournieri

Summary Actin organization was observed inm-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs ofTorenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.     

Polarity of nuclear and organellar DNA during megasporogenesis and megagametogenesis in Plumbago zeylanica

Summary Megasporogenesis and megagametogenesis of Plumbago zeylanica were studied using isolated megasporocytes, megaspores, and embryo sacs labeled with Hoechst 33258 for nuclear and organellar (presumably plastid) DNA. Megasporogenesis conforms to the tetrasporic Plumbago type, producing a coenomegaspore with four megaspore nuclei. Organeller DNA is polarized in the micropylar end of the coenomegaspore and embryo sac, reflecting the site of egg cell formation. The three remaining nuclei are somewhat displaced to the chalazal pole, producing a variable number of accessory cells and a 4N secondary central cell nucleus. Ultimately, the mature embryo sac consists of two to five cells including an egg cell, a central cell, zero to two lateral cells, and zero to one antipodal cell depending on the degeneration of the lateral or chalazal nuclei during megagametogenesis.     

Fertilization in Torenia fournieri: Actin organization and nuclear behavior in the central cell and primary endosperm

Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus in the central cell. Our data also suggest that the dynamics of actin cytoskeleton may be responsible for the reorganization of the central cell and primary endosperm cytoplasm during fertilization.     

Fertilization in maize indeterminate gametophyte1 mutant

Summary. Mature embryo sacs of the maize mutant indeterminate gametophyte1 displayed different cellular patterns compared to those of the wild type. About 40% of the ig1 embryo sacs contained three or more synergids and two or more egg cells at the micropylar end. During fertilization in embryo sacs with two synergids, both of them frequently degenerated and were penetrated by two pollen tubes. 75% of the embryo sacs containing three or more synergid cells were penetrated by two or more pollen tubes, although most of them had only one degenerated synergid. Multiple fusions between the sperm cells and eggs frequently occurred in the same embryo sac, which subsequently generated multiple embryos. There were two or more central cells in about 33% of ig1 embryo sacs. The largest central cell was usually adjacent to the egg apparatus and contained two unfused polar nuclei, while those extra central cells located at the chalazal end usually had a single nucleus. Fertilization occurred only between the male gamete and the largest binucleate central cell. The extra central cells eventually degenerated after fertilization.Present address: GI Basic Research Center, Mayo Clinic, Rochester, Minnesota, U.S.A.Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, Peoples Republic of China.     

OCCURRENCE AND DEVELOPMENT OF A FILIFORM APPARATUS IN THE EGG OF PLUMBAGO CAPENSIS

Micropylar wall extensions in the egg of Plumbago capensis arise as small pegs of periodic acid-Schiff's-positive material soon after the egg is organized. These inward projections of egg wall increase in size, becoming extensive near anthesis. Some branching of the filiform apparatus occurs. In mature embryo sacs the micropylar portion, with the lowermost portion of the egg included, becomes entrenched in the nucellus. The possible significance of this “gametic transfer cell” is discussed in relation to the apparent absence of synergids from the reduced embryo sac of Plumbago.     

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a long (approx. 30 m) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (S_ua) is linked by plasmodesmata with the S_vn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The S_ua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.Abbreviations S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus 1=Russell and Cass (1981)     

Three-dimensional organization and dynamic changes of the actin cytoskeleton in embryo sacs ofZea mays andTorenia fournieri

Actin organization was observed in m-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs of Torenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.     

甜菜无融合生殖单体附加系M14成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14（Betavulgaris,2n=18＋1）为实验材料,利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明：M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态：（1）极性正常的卵细胞,细胞核位于合点端,细胞质含有大量核糖体、线粒体、内质网等细胞器;（2）细胞核位于细胞中央;（3）细胞核位于珠孔端,且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近;未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显,细胞器的种类与数量多,呈现旺盛代谢活动特征,成为二倍体孢子无融合生殖过程中,卵细胞与次生核自发分裂的细胞学标志。     

甜菜无融合生殖单体附加系M14 成熟胚囊的超微结构特征

以甜菜无融合生殖单体附加系M14(Beta vulgaris, 2n=18+1)为实验材料, 利用电子显微镜技术对成熟胚囊及其超微结构进行研究。结果表明: M14成熟胚囊包括1个卵细胞、2个退化的助细胞、1个具有次生核的中央细胞和3-6个反足细胞。其卵细胞具有3种不同的形态: (1)极性正常的卵细胞, 细胞核位于合点端, 细胞质含有大量核糖体、线粒体、内质网等细胞器; (2)细胞核位于细胞中央; (3)细胞核位于珠孔端, 且后2种形态细胞器的种类与数量少。大多数胚囊中的2个助细胞在开花前已退化。中央细胞的次生核位于反足细胞附近; 未经受精自发分裂前的卵细胞与中央细胞的细胞核大、核仁明显, 细胞器的种类与数量多, 呈现旺盛代谢活动特征, 成为二倍体孢子无融合生殖过程中, 卵细胞与次生核自发分裂的细胞学标志。     

An embryological study ofPlatanthera bifolia (Orchidaceae)

Flowers ofPlatanthera bifolia were hand-pollinated and fixed in FPA₅₀ after 2, 5, 7, 14, and 21 days. Ovules, made transparent in Herr's clearing fluid, were investigated using confocal scanning laser microscopy. Pollination initiates the megasporogenesis. Two days after pollination dyads are frequent. Three days later most embryo sacs contain two nuclei. Seven days after pollination the embryo sacs are 4–8-nucleate and some are organized, and a week later all embryo sacs are organized and fertilization takes place. The embryo sac development follows thePolygonum type. Twenty-one days after pollination the egg nuclei have been fertilized and the embryo sacs contain 2- to many-celled embryos. A suspensor is formed during early stages of embryo development but degenerates later. Fertilization of the central nucleus does not lead to endosperm development.     

小麦受精过程中酸性磷酸酶的超微细胞化学定位

小麦（Ｔｒｉｔｉｃｕｍ ａｅｓｔｉｖｕｍ ）受精前成熟胚囊,除胚囊中央细胞的合点端细胞质中有酸性磷酸酶外,其余部位均未发现酸性磷酸酶。受精时期,以下部位存在酸性磷酸酶活性：卵细胞的细胞核内一部分染色质和细胞质中大部分线粒体;精、卵核融合时两核的核周腔内;退化助细胞合点端细胞质和一些液泡内;进入雌性细胞中的两个精核;胚囊各成员细胞的细胞壁及胚囊周围珠心细胞的细胞壁。二细胞原胚中未见有酸性磷酸酶。早期胚乳游离核染色质上有酸性磷酸酶。小麦受精过程酸性磷酸酶的分布特点可能与卵细胞生理状态的变化和细胞质中线粒体的改组、助细胞的退化、精核的生理状态以及精核与卵核的核膜融合等有关。     

Fertilization in Torenia fournieri: Actin organization and nuclear behavior in the central cell and primary endosperm

Studies of the living embryo sacs of Torenia fournieri reveal that the actin cytoskeleton undergoes dramatic changes that correlate with nuclear migration within the central cell and the primary endosperm. Before pollination, actin filaments appear as short bundles randomly distributed in the cortex of the central cell. Two days after anthesis, they become organized into a distinct actin network. At this stage the secondary nucleus, which is located in the central region of the central cell, possesses an associated array of short actin filaments. Soon after pollination, the actin filaments become fragmented in the micropylar end and the secondary nucleus is located next to the egg apparatus. After fertilization, the primary endosperm nucleus moves away from the egg cell and actin filaments reorganize into a prominent network in the cytoplasm of the primary endosperm. Disruption of the actin cytoskeleton with latrunculin A and cytochalasin B indicates that actin is involved in the migration of the nucleus     

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

 Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus. Received: 20 January 1999 / Revision received: 12 July 1999 / Accepted: 17 August 1999     

FINE STRUCTURE AND COMPOSITION OF THE EGG APPARATUS BEFORE AND AFTER FERTILIZATION IN QUERCUS GAMBELII: THE FUNCTIONAL OVULE

A study of the egg apparatus of Quercus gambelii was made at both the light and the electron microscope levels. This investigation was concerned primarily with the changes that occur in these cells before and after the process of fertilization and what role, if any, is played by the synergids in this phenomenon. The synergids before fertilization are, on the basis of ultrastructure, healthy, intact, functional cells. They have numerous mitochondria, dictyosomes, endoplasmic reticulum, ribosomes, and a typical nucleus. A prominent filiform apparatus is present, but the cell wall only extends a short distance around the micropylar end of the cells. Just before fertilization, one of the synergids degenerates. This is the synergid that receives the pollen tube and its discharge, including both male gametes. Dictyosomes increase in number and activity in the other synergid (persistent synergid) after fertilization. Eventually a complete cell wall forms around both of the synergids. No plasmodesmata are present in these walls. The egg has numerous mitochondria, dictyosomes, endoplasmic reticulum, and ribosomes, both free in the cytoplasm and attached to the endoplasmic reticulum. Lipid bodies are characteristic of this cell. A cell wall is present only around the micropylar end of the egg. After fertilization, little change occurs in the zygote. The number and activity of the dictyosomes increase, apparently in correlation with cell wall formation. The number of lipid bodies increases. The zygote is approximately the same size as the egg. Plastids are scarce, and starch grains are typically absent from all cells of the egg apparatus. It is suggested that the synergids function in the secretion of chemotropic substances that guide the growth of the pollen tube. Comparisons are made between the egg apparatus of Quercus gambelii and that of the other plants studied thus far.     

云南油杉受精过程中新细胞质及蛋白泡的动态观察

云南油杉(Keteleeria evelyniana Mast)在受精前,精核与卵核周围的细胞质鞘不明显。受精后,合子核周围出现细密的新细胞质。应用孚尔根核染色法,可以较清晰地将新细胞质染出,呈现较弱的正反应,而合子的核质及受精前的精核与卵核染色极弱。卵细胞质及其中的蛋白泡均为负反应。原胚形成后,除上层外,其余几层细胞质内开始积累淀粉粒。此时胚原细胞核的孚尔根染色深度有所增加。幼胚形成后,在顶端的胚原细胞群中核的孚尔根染色反应已恢复正常。在原胚及幼胚胚原细胞质中也呈现很弱的正反应。在电镜下,胚原层细胞质及新细胞质中均含有核样电子致密小体或称作染色质小体,而原胚莲座层细胞质及四周套细胞质中的线粒体则不含这种核样小体。因此,大蛋白泡在卵核形成的早期数量不多,当合子形成时含量最高,而随着游离核的分裂进程,蛋白泡以及原卵质均逐渐地解体,在原胚形成后全部消解。     

ORCHID EMBRYOLOGY: MEGAGAMETOPHYTE OF EPIDENDRUM SCUTELLA FOLLOWING FERTILIZATION

The megagametophyte of Epidendrum scutella, an orchid, was examined with the electron microscope after the entrance and discharge of the pollen tube. The pollen tube enters the embryo sac by growing through the filiform apparatus of a synergid and discharges through a terminal pore into the degenerating cytoplasm of the synergid. The synergid nucleus appears pushed to one side by the discharge of the pollen tube. What is believed to be the remains of the vegetative nucleus has been found in the degenerate synergid, but no trace of the sperm cytoplasm has been seen. The zygote is approximately the same size as the egg. The ribosomes become grouped into polysomes. Both the egg and the zygote apparently completely lack dictyosomes. The polar nuclei partially fuse before fertilization, but fusion of the sperm nucleus with the polar nuclei does not occur and no endosperm is produced. Polysome formation occurs in the central cell and large amounts of tubular, smooth ER are seen. The antipodals remain following fertilization, undergoing ultrastructural changes similar to the central cell.     

CHANGES IN MEGAGAMETOPHYTE STRUCTURE IN PAPAVER NUDICAULE L. (PAPAVERACEAE) FOLLOWING IN VITRO PLACENTAL POLLINATION

Papaver nudicaule placentae with attached ovules were dissected out of unpollinated gynoecia 1–3 days after anthesis, dusted with pollen, and cultured on modified Nitsch's growth medium at 23 C. Ovules were removed from expiants at 15, 24, 31, and 48 hr postpollination, fixed in GA-OsO₄, embedded in Spurr's resin and sectioned (1.0 μm) for light microscopy. Placentae, 15 hr after pollination, were fixed and processed for scanning electron microscopy. Pollen germinates within 1 hr. Although most pollen tube growth appears random, there is directional growth toward the micropyle. The crassinucellate ovule contains an embryo sac consisting of three antipodals, two polar nuclei, and an egg apparatus composed of two synergids and a polarized egg having a large chalazal vacuole and micropylar nucleus. Pollen tube access into the megagametophyte is through a degenerate synergid, with fertilization occurring between 24 and 31 hr after pollination. Zygote establishment is accompanied by polarity reversal in which the nucleus assumes the chalazal position subtended by a large micropylar vacuole. Fertilized ovules normally develop into germinable seeds.     

Ultrastructure of the Developing and Fertilized Embryo Sac of Amaranthus hypochondriacus L.

Amaranthus hypochondriacus embryo sac development was investigatedbefore and after fertilization. During the early stages of development,the young embryo sac displays three antipodal cells at the chalazalpole that degenerate very early in the maturation process, beforethe synergids and egg cell are completely differentiated. Themature embryo sac is composed only of the female germ unit.The synergid cells organize a filiform apparatus accompaniedby the presence of mitochondria and dictyosomes with numerousvesicles. The involvement of the synergids in transport andsecretory functions related to pollen tube attraction and guidance,are discussed. The egg cell is located at the micropylar polenear the synergids and displays exposed plasma membranes atthe chalazal pole. The fertilized egg cell does not exhibitmarked changes after fertilization except for the closure ofthe cell wall. The central cell is the largest cell of thisvery long embryo sac. The fused nucleus is close to the eggapparatus before fertilization and displays a remarkable chalazalmigration after gamete delivery. The ultrastructure of the centralcell cytoplasm and the numerous wall ingrowths around this cellsuggest an important role in nutrient transportation. Aftergamete delivery, the embryo sac displays electron dense bodiesthat aggregate within the intercellular space between the synergids,egg cell and central cell. These bodies, that appear in theembryo sac of several plants, are probably involved in gametedelivery for double fertilization. The possibility of biparentalinheritance of mitochondria in this plant is also discussed.Copyright 1999 Annals of Botany Company Amaranthus hypochondriacus, grain amaranth, embryo sac, fertilization.     

The Ultracytochemical Localzation of Acid Phosphatase Activity in Wheat During Fertilization

No acid phosphatase activity was observed in the mature embryo sac of wheat (Triticum aestivum) except the chalazal cytoplasm Of the central cell before fertilization. During fertilization, acid phosphataseactivity was observed in the following loci: part of chromatin of the egg nucleus and most of the mitochondria in the egg cytoplasm; the perinuclear spaces of the egg and sperm nuclei at the fusion of the egg and sperm nuclei; the chalazal cytoplasm and some vacuoles of the degenerated synergid; two sperm nuclei within the cytoplasm of female cells; the cell wall of each cell of the embryo sac and that of the nucellar cells surrounding the embryo sac. No acid phosphatase was observed in the two-celled proembryo. Dense enzyme reaction product was localized in the chromatin of the free nuclei at early stage of the endosperm. The characteristic of acid phosphatase distribution during fertilization may be associated with the physiological change of the egg Cell, the reorganization of mitochondria in the egg cell cytoplasm, the degeneration of one of the two synergids, the physiological state of the sperm nuclei and the nuclear membrane fusion of the egg and sperm nuclei.