Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.     

天竺葵雌性生殖单位的超微结构

应用透射电镜研究了临近受精时天竺葵(Pelargonium hortorum Bailey)胚囊中的卵细胞、助细胞和中央细胞的结构。证明了卵细胞与助细胞以及助细胞与助细胞之间从合点端至珠孔端有很大的面积以质膜分界,仅珠孔端少部分以壁分隔。卵细胞与中央细胞之间同样缺乏细胞壁。在卵细胞的合点端,两质膜不同程度地分离形成宽窄相间的间隙。在间隙的絮状基质中存在小泡,这些小泡的产生似与卵和中央细胞中周质内质网的活动有关。推测小泡为多糖性质,可能为合子新壁的建造提供物质。卵细胞质中含巨大线粒体,质体和内质网也较丰富。基于超微结构的特征,可认为卵细胞具高度的生理合成活动的潜能。中央细胞极核位于珠孔端与卵器细胞毗邻,有利于在双受精作用中同时发生精细胞与卵细胞和精细胞与中央细胞核的融合。中央细胞的侧壁在珠孔端形成内突,具传递细胞的特点,表明这是雌配子体向孢子体摄取营养的重要部位。助细胞的细胞质含丰富的细胞器,这与多数植物中的相似,但具几个明显的特征,即核中存在微核仁,内质网形成圆球体或脂体,线粒体富集在丝状器的附近。传粉后花粉管进入胚囊之前,两个助细胞中一个退化。     

Ovule,Female and Male Gametophyte and Fertilization of Kingdonia uniflora Balfour F. et W. W. Smith

The structure of ovule, female and male gametophyte, double fertilization and the distrubution of starch grains during the fertilization have been studied. The main results are as follows: ( 1 ) Ovule The ovule is anatropous, unitegmic and tenuinucellate. The nucetlus appears cylindric, since megaspores and embryo sac development, its internal cells of nucellus become disorganized, so that only a single layer of epidermal cells remains toward the side of the micropyle, On the other hand, the integument is not as long as nucellus, as a result micropyle is not formed. And no vascular bundle is found in the integument. (2) Female gametophyte The mature embryo sac is slender and is composed of an egg cell, two synergids, a central cell and three antipodal cells. The egg cell is situated slightly away from the tip of embryo sac. Some of them contain starch grains. Synergids occupy the tip of embryo sac. Its wall at micropylar region appears irregular in thickenes and irregular in ingrowths to form the filiform apparatus. The centrateell is very large, and strongly vacuolated Two polar nuclei come to contact closely with each other, but not fuse, or to fuse into a large secondary nucleus before fertilization. The polar nuclei or the secondary nucleus are usually situated at the middle-lower position of the central cell or nearer to the chalazal end above the antipodal cell. It is different from egg cell, no starch grains are found here. In most embryo sacs three antipodal cells are found. They are not as large as those in other plants of Ranunculaceae. But six antipodal cells or the antipodal cell with two nuclei may rarely be found. Like synergid, the wall of them appears not only irregularly thickened, but clearly with irregular ingrowths. In a few antipodal cells the starch garins are usually found near the nucleus. By the end of fertilization, antipodal cells become disintegrated. (3) Male gametophyte Most pollen grains are two-celled when shedding, and rich in starch grains. A few of them contain single nucleus or three-celled. (4) The double fertilization The fertilization of Kingdonia unifiora Balfour f. et W, W. Smith is wholly similar to some plants of Ranunculaceae studied. First, the pollen tube penetrates a degenerating synergid. And the pollen tube discharges its contents with two sperm nuclei into the degenerating synergid cell. One of the two sperms fuses with the nucleus of the egg, and the other fuses with two polar nuclei or the secondary nucleus of the central cell. If one sperm nucleus at first fuses with one of the polar nuclei, and then the fertilized polar nuclei again fuses with other polar nucleus. Secondly, the fertilization of the polar nuclei or the secondary nuclei completes earlier than that of the egg. The primary endosperm nucleus begins to divide earlier than the zygote. It seems that one of the sperm nuclei come to contact with egg nucleus, the other has already fused with polar nuclei or the secondary nucleus. The zygote with a single nucleolus appears until the endosperm with 16–20 cell. Thirdly, before and after fertilization there are one to some small nucleoli in egg nucleus and polar nuclei or secondary nucleus. However they increase in quantity from the beginning of the fusion of male nucleis. These nucleoli quite differ from male nucleoli by their small size, and most of them disappear at the end of fertilization. It may be concluded that the small nucleoli increase in quantity is related to the fusion of male and female nuclei. In the duration of fertilization, in ovule starch distribution is in the basal region of integument. But in embryo sac, onlysome egg cells, or zygotes contain starch grains, a part of which was brought in by pollen tube. Sometimes the starch grains are found in some synergids and antipodal cells. No starch grains are found in the central cell.     

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed. Received: 7 March 2000 / Revision accepted: 15 June 2000     

Isolation of gametes and central cells from Oryza sativa L.

In vitro fertilization system of higher plants has been well established using maize gametes and central cells, which can produce embryos and endosperms. In the present study, procedures for isolating gametes and central cells from rice (Oryza sativa L. cv. Nipponbare), a model plant, are reported with the goal of establishing rice in vitro fertilization system. Egg cells and central cells were isolated by manual manipulation of enzyme-treated unpollinated ovules, and an alternative direct isolation method for egg cells that does not use enzymatic treatment was also established. Fluorescent visualization of the granular structures in the cytoplasm of isolated egg cells and the nucleoli in two polar nuclei of isolated central cells suggest that these cells are reliable gametes and central cells. For sperm cell isolation, the contents of rice pollen grains were released by osmotic pressure-induced bursting of the grains. In addition, electrofusion with isolated gametes was successfully conducted.     

Invvestigations on Early Embryogenesis of Actinidia chinensis Planch var. chinensis

This paper deals with early embryogenesis of Actinidia chinensis var. chinensis. 1. Ovary superior consists of 34—45 carpels. Each carpel contains 11–45 ovules. The ovule is uni-integument and tenuinucellar. The ovule is anatropous. The archesporium is formed by a single cell, and directly develops into megaspore mother cell. Sometimes the archesporium consists of 2–3 cells, but only one of them develops into megaspore mother cell and the others are degenerated. 2. The mature pollen grain is two-celled and the embryo sac belongs to olygonum type. In most embryo sacs two polar nuclei are fused before fertilization. One of the synergids was destroyed as the pollen tube penetrated into embryo sac the other one disappeared after fertilization. In most cases the antipodal cells became degenerated in fertilization process, only some remained until the first division of primary endosperm nucleus. 3. In Beijing area the double fertilization of Actinidia chinensis occurred 30–72 hours after pollination. In the fertilization one sperm fused with egg nucleus and the other sperm fused with the secondary nucleus as usual. The fusion of the secondary nucleus with sperm was in advance of the fusion of the egg nudeus. 4. The endosperm is cellular type.     

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.     

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.     

Fertilization and Histochemical Investigation on Pulsatilla chinensis (Bung) Regel

Fertilization and variation of protein and starch grains in Pulsatilla chinensis (Bung) Regel have been studied at light microscopic level with histochemical test. Based upon the observations, the main conclusions are summarized as follows: The mature pollen grains are two-celled in which the generative cell shows the stronger protein staining than the vegetative cell. And vegetative cells are full of starch garins. When the pollen tube enters into the embryo sac, one synergid is destroyed, or in a few cases synergids are intact. Occasionally two synergids are disorganized as pollen tube penetrates. However, most of the remaining syuergids break down during fertilization, only in a few cases it remains till early stage of embryo development. The contents discharged by the pollen tube consist of two sperms, which stain intensely blue with protein dyes, a great amount of protein and starch grains. Mature female gametophyte (embryo sac) consists of an egg apparatus, central cell, which has a huge secondary nucleus, and antipodal apparatus which retain in course of fertilization. A few of embryo sac contain two sets of egg apparatus, a central cell with two huge secondary nuclei and two sets of antipodal apparatus. In some nucleoli of the central cell the comb-like structure pattern may be detected clearly. There are 1–2 small nucleoli in some egg cells and central cells. All the cells in embryo sac show protein positive reaction. According to the different shades of the color in cells, its may be arranged in the following order: antipodal cells, synergids, central cell and egg cell. Only a few small starch grains are present near nuclei of central cell and egg cell before fertilization, but no starch grains remain in most of the central cell, the synergids and antipodal cells. The fertilization is of the premitotic type. The fusion of the sexual nuclei progresses in the following order: 1, sperms approach and lie on the egg nucleus and secondary nucleus; 2, sperm chromatin sinks themselves into female nucleus, and male nucleolus emerges with the sperm chromosome; and 3, male nucleoli fuse with the nucleoli of egg nucleus and central cell nucleus, and finally forming the zygote and the primary endosperm cells respectively. Nevertheless, as it is well known, the fertilization completes in central cell obviously earlier than that in egg cell. Though it has been explained in cereals and cotton, in Pulsatilla chinensis the main reason is that nucleolar fusion of the male and female nucleoli in egg nucleus is slower than that in secondary nucleus. And the dormancy of the primary endosperm nucleus is shorter than that of the zygote. In the process of fertilization, histochemical changes are considerably obvious in the following three parts: 1, from the begining of fusion of male and female nuclei to form zygote and primary endosperm cell, Protein staining around female nucleus appears to increase gradually; 2, no starch grains are detected in embryo sac. Though only starch grains are carried in by pollen tube, they are completely exhausted during this period; and 3, near completion of fertilization starch grains appear again in zygote, however, not yet in primary endosperm nucleus till its dividing for the first time. The present study reveals that antipodal cells and synergids seem to play a significant role in nutrition of the embryo sac during the fertilization.     

CAPSELLA EMBRYOGENESIS: THE SYNERGIDS BEFORE AND AFTER FERTILIZATION

The ultrastructure and composition of the synergids of Capsella bursa-pastoris were studied before and after fertilization. The synergids in the mature embryo sac contain numerous plastids, mitochondria, dictyosomes and masses of ER and associated ribosomes. Each synergid contains a large chalazal vacuole, a nucleus with a single nucleolus and is surrounded by a wall. This wall is thickest at the micropyle end of the cell where it proliferates into the filiform apparatus. At the chalazal end of the cell the wall thins and may be absent for small distances. The pollen tube grows into one of the two synergids through the filiform apparatus and extends one-third the length of the cell before it discharges. Following discharge of the pollen tube, mitochondria and plastids of the tube can be identified in the synergid as can hundreds of 0.5 μ polysaccharide spheres liberated by the tube. The method by which the sperm or sperm nuclei enter the egg or central cell is not known although an apparent rupture was found in the wall of the egg near the tip of the pollen tube. The second synergid changes at the time the pollen tube enters the first synergid. These changes result in the disorganization of the nucleus and loss of the chalazal wall and plasma membrane. Eventually this synergid loses its identity as its cytoplasm merges with that of the central cell.     

Pollen tube penetration and fertilization in Lilium longiflorum (Liliaceae)

The ultrastructure of the embryo sac, nucellus, and parts of the micropyle of Lilium longiflorum were studied both before and after pollen tube penetration to examine the interactions between ovule and pollen tube, using transmission electron microscopy and light microscopy. Before pollen tube penetration the egg cell and two synergids are similar. No filiform apparatus was detected and no synergid degeneration occurs prior to pollen tube penetration. The polar nuclei do not fuse until fertilization. No differences in embryo sac ultrastructure were detected between pollinated ovules unpenetrated by pollen tubes and unpollinated flowers of a comparable age. Shortly after the discharge of the pollen tube two enucleated cytoplasmic bodies with different ribosome densities were observed in the degenerated cytoplasm. These structures border both on the central cell and the egg cell as well as each other and are interpreted as remains of sperm cytoplasm after transmission of sperm nuclei. In the central cell both the sperm nucleus and the polar nuclei are associated with endoplasmic reticulum (ER). ER is thought to be a transport mechanism to achieve contact between the haploid polar nuclei and the sperm nucleus. In the egg cell sperm nucleus alignment is not visibly achieved by ER. The persistent cells of the egg apparatus and the central cell appear to become more metabolically active after pollen tube penetration. Pollen tube penetration already occurs despite the absence of a filiform apparatus and a low level of differences between the cells of the egg apparatus.     

YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis

Background  

In flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.     

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.     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一,是大多数被子植物完成受精作用的关键环节之一。在受精过程中,助细胞吸引花粉管向雌配子体生长,并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外,雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚,但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果,尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果,并对助细胞的生殖功能进行了分析。     

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。     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一, 是大多数被子植物完成受精作用的关键环节之一。在受精过程中, 助细胞吸引花粉管向雌配子体生长, 并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外, 雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚, 但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果, 尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果, 并对助细胞的生殖功能进行了分析。     

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。