A one‐step rectification of sperm cell targeting ensures the success of double fertilization

Successful fertilization in animals depends on competition among millions of sperm cells, whereas double fertilization in flowering plants usually involves just one pollen tube releasing two immobile sperm cells. It is largely a mystery how the plant sperm cells fuse efficiently with their female targets within an embryo sac. We show that the initial positioning of sperm cells upon discharge from the pollen tube is usually inopportune for gamete fusions and that adjustment of sperm cell targeting occurs through release and re-adhesion of one sperm cell, while the other connected sperm cell remains in stagnation.This enables proper adhesion of each sperm cell to a female gamete and coordinates the gamete fusions. Our findings reveal inner embryo sac dynamics that ensure the reproductive success of flowering plants and suggest a requirement for sperm cell differentiation as the basis of double fertilization.     

Centenary on S.G.Nawaschin's Discovery of Double Fertilization: Retrospects and Prospects

A review on the double fertilization in angiosperm is addressed at its centennial discovery by S.G. Nawaschen. Studies in the first 50 years mainly by light microscopy had defined this process of double fertilization as a general characteristic in angiosperms. In the later 50 years research works in this field have been greatly advanced on account of the developing new techniques especially the electron-microscopy. The topics in this review include: (1) The growth of pollen tube entering the embryo sac: role of the synergid in the pollen tube receiption and signals from the degenerated synergid. (2) The arrival of male gametes to female gametes: structure and function of the male germ unit, the function of cytoskeleton in the delivery of sperm cells. (3) Gametic fusion: the structure and function of the female germ unit, gametic membrane fusion, karyogamy, DNA contents in sperm and egg nuclei, the relationship between the karyogamy and cell cycle, sperm dimorphism and preferential fertilization, and spermegg recognition. Future directions for the research of double fertilization are also recommended.     

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。     

被子植物受精机制的研究进展

被子植物的受精是一个复杂而精巧的过程。花粉管到达子房,通过退化助细胞进入胚囊,释放出两个精细胞。原来在花粉管中相互联结的两个精细胞在退化助细胞中分开,一个与卵细胞融合,另一个与中央细胞融合,完成双受精。目前对双受精过程中有关雌、雄配子识别的机制还知之甚少。本文介绍了目前被子植物精、卵细胞融合前后的细胞周期变化、退化助细胞的功能、精细胞在退化助细胞中迁移的研究动态、精细胞的倾向受精和卵细胞的激活等被子植物受精生物学领域中的一些新的研究成果和发展趋势。     

Gamete attachment process revealed in flowering plant fertilization

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule (Fig. 1). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants.Open in a separate windowFigure 1.Illustration of the fertilization process in flowering plants. First, each pollen tube accesses an ovule containing egg and central cells. Next, the 2 sperm cells face the female gametes in the ovule after the pollen tube bursts. Finally, each sperm cell simultaneously fuses with either egg or central cell.     

Establishment of the male germline and sperm cell movement during pollen germination and tube growth in maize

Two sperm cells are required to achieve double fertilization in flowering plants (angiosperms). In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and are transported to the female gametes (egg and central cell) via the pollen tube. The two sperm cells arise from the generative pollen cell either within the pollen grain or after germination inside the pollen tube. While pollen tube growth and sperm behavior has been intensively investigated in model plant species such as tobacco and lily, little is know about sperm dynamics and behavior during pollen germination, tube growth and sperm release in grasses. In the March issue of Journal of Experimental Botany, we have reported about the sporophytic and gametophytic control of pollen tube germination, growth and guidance in maize.1 Five progamic phases were distinguished involving various prezygotic crossing barriers before sperm cell delivery inside the female gametophyte takes place. Using live cell imaging and a generative cell-specific promoter driving α-tubulin-YFP expression in the male germline, we report here the formation of the male germline inside the pollen grain and the sperm behaviour during pollen germination and their movement dynamics during tube growth in maize.Key words: male gametophyte, generative cell, sperm, pollen tube, tubulin, fertilization, maize     

烟草雌、雄配子DNA含量变化

采用显微分光光度法测定了烟草（Nieotiana tabacum）精细胞和卵细胞的DNA含量。烟草是二胞花粉,花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45h花粉管到达子房,在花粉管内的精细胞DNA含量为1C。当花粉管在退化助细胞中破裂,释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA含量接近2C。随着精细胞的到达及合成DNA,卵细胞也开始合成DNA,融合前的卵细胞DNA含量也接近2C。精、卵细胞融合后,合子DNA含量为4C。烟草雌、雄配子是在细胞周期的G2期发生融合,属于G2型。     

烟草雌、雄配子DNA 含量变化

采用显微分光光度法测定了烟草( Nicotiana tabacum) 精细胞和卵细胞的DNA 含量。烟草是二胞花粉, 花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45 h 花粉管到达子房, 在花粉管内的精细胞DNA 含量为1C。当花粉管在退化助细胞中破裂, 释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA 含量接近2C。随着精细胞的到达及合成DNA, 卵细胞也开始合成DNA, 融合前的卵细胞DNA 含量也接近2C。精、卵细胞融合后, 合子DNA 含量为4C。烟草雌、雄配子是在细胞周期的G2 期发生融合, 属于G2 型。     

Attraction and transport of male gametes for fertilization

 Two capabilities are critical in attracting and transporting male gametes for fertilization: (1) the pollen tube must locate, enter and discharge its contents at the correct site within the female gametophyte, and (2) once inside the embryo sac, the non-motile male gametes must be transported to the egg and the central cells for double fertilization. This review summarizes current information about evidence for communication between embryo sac and pollen tube and the means by which the non-motile male gametes are transported from the aperture of the pollen tube to the site of gamete fusion. Received: 6 June 1996 / Revision accepted: 9 July 1996     

Female control of male gamete delivery during fertilization in Arabidopsis thaliana

Fertilization in both animals and plants relies on the correct targeting of the male gametes to the female gametes. In flowering plants, the pollen tube carries two male gametes through the maternal reproductive tissues to the embryo sac, which contains two female gametes. The pollen tube then releases its two male gametes into a specialized receptor cell of the embryo sac, the synergid cell. The mechanisms controlling this critical step of gamete delivery are unknown. Here, data based on the new sirène (srn) mutant of Arabidopsis thaliana provide the first evidence for female control over male gamete delivery. Live imaging of fertilization shows that wild-type pollen tubes do not stop their growth and do not deliver their contents in srn embryo sacs.     

GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization

The double fertilization process in angiosperms is based on the delivery of a pair of sperm cells by the pollen tube (the male gametophyte), which elongates towards an embryo sac (the female gametophyte) enclosing an egg and a central cell. Several studies have described the mechanisms of gametophyte interaction, and also the fertilization process - from pollination to pollen tube acceptance. However, the mechanisms of gamete interaction are not fully understood. Cytological studies have shown that male gametes possess distinct cell-surface structures and genes specific to male gametes have been detected in cDNA libraries. Thus, studies of isolated gametes may offer clues to understanding the sperm-egg interaction. In this study, we identified a novel protein, designated GCS1 (GENERATIVE CELL SPECIFIC 1), using generative cells isolated from Lilium longiflorum pollen. GCS1 possesses a carboxy-terminal transmembrane domain, and homologues are present in various species, including non-angiosperms. Immunological assays indicate that GCS1 is accumulated during late gametogenesis and is localized on the plasma membrane of generative cells. In addition, Arabidopsis thaliana GCS1 mutant gametes fail to fuse, resulting in male sterility and suggesting that GCS1 is a critical fertilization factor in angiosperms.     

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。     

Maternal Control of Male-Gamete Delivery in Arabidopsis Involves a Putative GPI-Anchored Protein Encoded by the LORELEI Gene

In Angiosperms, the male gametes are delivered to the female gametes through the maternal reproductive tissue by the pollen tube. Upon arrival, the pollen tube releases the two sperm cells, permitting double fertilization to take place. Although the critical role of the female gametophyte in pollen tube reception has been demonstrated, the underlying mechanisms remain poorly understood. Here, we describe lorelei, an Arabidopsis thaliana mutant impaired in sperm cell release, reminiscent of the feronia/sirène mutant. Pollen tubes reaching lorelei embryo sacs frequently do not rupture but continue to grow in the embryo sac. Furthermore, lorelei embryo sacs continue to attract additional pollen tubes after arrival of the initial pollen tube. The LORELEI gene is expressed in the synergid cells prior to fertilization and encodes a small plant-specific putative glucosylphosphatidylinositol-anchored protein (GAP). These results provide support for the concept of signaling mechanisms at the synergid cell membrane by which the female gametophyte recognizes the arrival of a compatible pollen tube and promotes sperm release. Although GAPs have previously been shown to play critical roles in initiation of fertilization in mammals, flowering plants appear to have independently evolved reproductive mechanisms that use the unique features of these proteins within a similar biological context.     

Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

Double Fertilization in Gnetum gnemon: The Relationship between the Cell Cycle and Sexual Reproduction

Gnetum gnemon, a nonflowering seed plant and member of the Gnetales, expresses a rudimentary pattern of double fertilization that results in the formation of two zygotes per pollen tube. The process of double fertilization in G. gnemon was examined with light and fluorescence microscopy, and the DNA content of various nuclei involved in sexual reproduction was quantified with 4[prime],6-diamidino-2-phenylindole microspectrofluorometry.Male and female gamete nuclei pass through the synthesis phase of the cell cycle and increase their DNA content from 1C to 2C before fertilization. Each of the two zygotes found in association with a pollen tube is diploid and contains the 4C quantity of DNA at inception. Based on these results as well as previous studies of nuclear DNA content in plant sperm, eggs, and zygotes, three fundamental and distinct patterns of gamete karyogamy among seed plants can be circumscribed: (1) G1 karyogamy, in which male and female gametes contain the 1C quantity of DNA throughout karyogamy and the zygote undergoes DNA replication; (2) S-phase karyogamy, in which gamete nuclei initiate fusion at 1C but pass through the S phase of the cell cycle before completely fusing; and (3) G2 karyogamy, in which male and female gamete nuclei pass through the S phase of the cell cycle before the onset of fertilization. Our results show definitively a pattern of G2 karyogamy in G. gnemon.     

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.     

Occurrence of unique three-celled megagametophyte and single fertilization in an aquatic angiosperm-Dalzellia zeylanica (Podostemaceae-Tristichoideae)

Angiosperms are characterized by the occurrence of double fertilization. However, Podostemaceae is considered an exception with the presence of only single fertilization (syngamy) though two male gametes are formed conventionally. To determine the cause for the failure of double fertilization in Dalzellia zeylanica (Gardn.) Wight, we closely tracked the movement of the male gametes from the time of pollen tube initiation to the time of entry into the megagametophyte to affect fertilization. We report for the first time, the presence of a novel type of three-nucleate/three-celled mature megagametophyte consisting of two synergids and an egg cell in D. zeylanica. Therefore, of the two male gametes formed in this plant, one fuses with the egg cell resulting in syngamy, whereas the other male gamete eventually degenerates due to the absence of its partner i.e. single polar nucleus of the central cell that degenerates prior to the entry of the pollen tube into the synergid. The present work not only highlights the highly reduced nature of megagametophyte but also the occurrence of single fertilization resulting in sperm selection in D. zeylanica.     

Polyspermy barriers in plants: from preventing to promoting fertilization

Barriers to polyspermy (fertilization of a female gamete by more than one sperm) are essential to successful reproduction in a wide range of organisms including mammals, echinoderms, fish, molluscs, and algae. In animals and fucoid algae, polyspermy results in early death of the zygote due to transmission of extra centrioles from the sperm and consequent disruptions to the mitotic spindle. Accordingly, a variety of mechanisms have evolved to prevent penetration of an egg by more than one sperm, or more than one sperm nucleus from fusing with an egg nucleus. The evolution of internal fertilization has also provided an opportunity to limit the number of sperm that gain access to each egg, as occurs in the mammalian female reproductive tract. Polyspermy and polyspermy barriers in plants have received much less attention. Plants lack centrioles and therefore, polyspermy would not be expected to cause lethal aberrant spindle organization. However, we find evidence from cytological, genetic and in vitro fertilization studies for polyspermy barriers in plants. Angiosperms, like mammals, are internally fertilized, and exert a high level of control over the number of sperm that have access to each female gamete. In particular, regulation of pollen tube growth ensures that in general only two sperm enter each embryo sac, where one fertilizes the egg and the other the central cell. Despite this 1:1 ratio of sperm to gametes within the embryo sac, angiosperms still require a mechanism to ensure that each female gamete is fertilized by one and only one sperm. Here, we present evidence suggesting that a polyspermy block on the egg may be part of the mechanism that promotes faithful double fertilization.     

Synergid: a key link in fertilization of angiosperms

In over 80 % of the angiosperms, the female gametophyte is comprised of seven cells, two of which are the synergid cells. These cells are considered pivotal in assuring successful fertilization. The synergid cells direct pollen tube growth toward the female gametophyte, and facilitate the entrance of the tube into the embryo sac. Once the pollen tube enters the synergid cell, its growth is arrested, the tip of the tube breaks, and two sperm cells are released. This sequence of events is also synergid dependent. In addition, separation of the cells of the male germ unit, orientation of the two sperm cells in the degenerating synergid, and fusion of the egg and central cell with sperm cells may also be related to synergid cells. Synergid structure has been widely studied, but development and function of these cells during angiosperm fertilization remains elusive. Recent molecular approaches have provided an enhanced understanding of the role of synergid cells in fertilization. The present review summarizes the results of current studies regarding the role of synergids in angiosperm reproductive function.