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。     

Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance

Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.     

Differentiation of generative and vegetative cells in angiosperm pollen

 Cellular differentiation of a generative and a vegetative cell is an important event during microspore and pollen development and is requisite for double fertilization in angiosperms. The generative cell produces two sperm cells, or male gametes, whereas the vegetative cell produces an elongated pollen tube, a gametophytic cell, to deliver the male gametes to the embryo sac. For typical differentiation of the gametic and gametophytic cells, cell polarity, including nuclear positioning, must be established prior to microspore mitosis and be maintained during mitosis. Microtubules are closely involved in the process of asymmetric cell division. On the other hand, alteration of the chromatin composition seems to be responsible for the differential gene expression between the generative and vegetative cells. Cytoplasmic regulatory molecules, which affect chromatin configuration, are postulated to be unequally distributed to the two cells at the asymmetric cell division. Thus, typical differentiation of the cells is accomplished by a cellular mechanism and a molecular mechanism, which might be independent of each other. These results are discussed in relation to one model that accounts for the different fates of generative and vegetative cells during sexual plant reproduction. Received: 3 September 1996 / Revision accepted: 23 September 1996     

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     

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.     

Development of male gametes in flowering plants

The male gametes of angiosperms consist of two sperm cells within a pollen grain or a pollen tube. They are derived from a single generative cell, which is formed as the smaller cell by unequal cell division in the microspore after meiosis. Limited information is available about these male gametic cells, beyond observations by electron microscopy, because each is surrounded by the cytoplasm of a larger vegetative cell. Recently, large quantities of generative cells and sperm cells have been isolated from pollen grains or pollen tubes of various plant species, and their physiological, biochemical and molecular characterization is now possible. Although almost all the available results are still preliminary, it is evident that the male gametic cells are peculiar in terms both of cell structure and composition. For example, they are rich in axial microtubules which maintain the spindle-like shape of each cell. However, they lack plastids which are DNA-containing cytoplasmic organelles. Biochemical characterization of their proteins indicates the presence of male gamete-specific polypeptides. These findings suggest, not unexpectedly, the possibility of male gamete-specific gene expression and of a strict genetic mechanism that controls the formation of male gametes.     

Mechanisms for independent cytoplasmic inheritance of mitochondria and plastids in angiosperms

The inheritance of mitochondria and plastids in angiosperms has been categorized into three modes: maternal, biparental and paternal. Many mechanisms have been proposed for maternal inheritance, including: (1) physical exclusion of the organelle itself during pollen mitosis I (PMI); (2) elimination of the organelle by formation of enucleated cytoplasmic bodies (ECB); (3) autophagic degradation of organelles during male gametophyte development; (4) digestion of the organelle after fertilization; and (5)—the most likely possibility—digestion of organellar DNA in generative cells just after PMI. In detailed cytological observations, the presence or absence of mitochondrial and plastid DNA in generative cells corresponds to biparental/paternal inheritance or maternal inheritance of the respective organelle examined genetically. These improved cytological observations demonstrate that the replication or digestion of organellar DNA in young generative cells just after PMI is a critical point determining the mode of cytoplasmic inheritance. This review describes the independent control mechanisms in mitochondria and plastids that lead to differences in cytoplasmic inheritance in angiosperms.     

She's the boss: signaling in pollen tube reception

In angiosperms, the sperm cells are carried within the pollen tubes (male gametophytes) to the female gametophyte so that double fertilization can occur. The female gametophyte exerts control over the male, with specialized cells known as synergids guiding the pollen tubes and controlling their behavior when they enter the female gametophyte so that the sperm cells can be delivered to the egg and central cell. Upon pollen tube arrival at the ovule, signal transduction cascades mediated by receptor-like kinases are initiated in both the synergid and the tip of the pollen tube, leading to synergid cell death and pollen tube rupture. In this review, we discuss the role of these receptors and of newly discovered members of the pollen tube reception pathway.     

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.     

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。     

The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception

Reproduction in angiosperms depends on communication processes of the male gametophyte (pollen) with the female floral organs (pistil, transmitting tissue) and the female gametophyte (embryo sac). Pollen-pistil interactions control pollen hydration, germination and growth through the stylar tissue. The female gametophyte is involved in guiding the growing pollen tube towards the micropyle and embryo sac. One of the two synergids flanking the egg cell starts to degenerate and becomes receptive for pollen tube entry. Pollen tube growth arrests and the tip of the pollen tube ruptures to release the sperm cells. Failures in the mutual interaction between the synergid and the pollen tube necessarily impair fertility. But the control of pollen tube reception is not understood. We isolated a semisterile, female gametophytic mutant from Arabidopsis thaliana, named feronia after the Etruscan goddess of fertility, which impairs this process. In the feronia mutant, embryo sac development and pollen tube guidance were unaffected in all ovules, although one half of the ovules bore mutant female gametophytes. However, when the pollen tube entered the receptive synergid of a feronia mutant female gametophyte, it continued to grow, failed to rupture and release the sperm cells, and invaded the embryo sac. Thus, the feronia mutation disrupts the interaction between the male and female gametophyte required to elicit these processes. Frequently, mutant embryo sacs received supernumerary pollen tubes. We analysed feronia with synergid-specific GUS marker lines, which demonstrated that the specification and differentiation of the synergids was normal. However, GUS expression in mutant gametophytes persisted after pollen tube entry, in contrast to wild-type embryo sacs where it rapidly decreased. Apparently, the failure in pollen tube reception results in the continued expression of synergid-specific genes, probably leading to an extended expression of a potential pollen tube attractant.     

Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1

In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K⁺-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K⁺ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K⁺ influx. We further suggest that K⁺ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.     

被子植物受精作用的分子和细胞生物学机制

被子植物双受精包括精-卵、精子-中央细胞两个融合过程。由于双受精深藏于母体组织中进行, 长期以来一直是植物有性生殖研究中的难点。近年来, 随着各种植物配子体cDNA文库的构建, 各种离体研究系统的建立和突变体分析的兴起, 极大地推动了被子植物受精作用研究的快速发展, 增进了人们对被子植物受精过程的分子和细胞生物学机制的深入了解。本文着重讨论受精作用的若干重要发育事件, 包括受精前卵器细胞对花粉管向胚珠定向生长的近距离引导信号, 精子的靶向运动,精、卵细胞相互作用和配子融合后卵细胞的激活与中央细胞发育的启动等。     

被子植物受精作用的分子和细胞生物学机制

被子植物双受精包括精-卵、精子-中央细胞两个融合过程。由于双受精深藏于母体组织中进行,长期以来一直是植物有性生殖研究中的难点。近年来,随着各种植物配子体cDNA文库的构建,各种离体研究系统的建立和突变体分析的兴起,极大地推动了被子植物受精作用研究的快速发展,增进了人们对被子植物受精过程的分子和细胞生物学机制的深入了解。本文着重讨论受精作用的若干重要发育事件,包括受精前卵器细胞对花粉管向胚珠定向生长的近距离引导信号,精子的靶向运动,精、卵细胞相互作用和配子融合后卵细胞的激活与中央细胞发育的启动等。     

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。     

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.     

Haustorial Role of Pollen Tubes

In angiosperms, the pollen tube is siphonogamous and its mainfunction is to carry the male gametes for double fertilization.In some taxa, as in Cucurbitaceae, the tube branches after enteringthe ovule, prior to fertilization. The tube may even swell andform a bulla. During post-fertilization development of the ovule,a portion of the tube may persist in the micropyle, or in theembryo sac, or in both, sometimes even in the micropyle of themature seed. Haustorial function has been presumed in a numberof taxa. In Grevillea, following fertilization, the pollen tube branchesat the micropyle, and the branches grow intercellularly intothe ovarian tissue where further branching occurs. A haustorialrole of the pollen tube is presumed from circumstantial evidence.In gymnosperms (for example, Cycas, Zamia and Ginkgo) the pollentube is nonsiphonogamous, arises from the distal (upper) poleof pollen grain, and grows laterally in the apical region ofthe nucellus. The tube branches in Cycas and Ginkgo but remainsunbranched in Zamia. These pollen tube branches are enucleate,and are not concerned with the transport of male gametes forfertilization. However, the haustorial role has been well documented.In Podocarpus, the pollen tube is siphonogamous and arises fromthe proximal (lower) pole of pollen grain. After traversingthe nucellus, the tube forms a bulla at the point of contactwith the female gametophyte, and several branches originatefrom the bulla. The pollen tube branches grow along the innersurface of the nucellus and the outer surface of the femalegametophyte. The haustorial role of the pollen tube branchesis uncertain. Procedures for convincingly demonstrating thehaustorial role of pollen tubes are discussed. Angiosperms, gymnosperms, pollen tube, bulla, fertilization, haustorial role     

花粉管生长和极性引导的孢子体和配子体控制研究进展

双受精是被子植物特有的生殖方式,精细胞只有通过花粉管穿过花柱才能到达子房、胚珠受精。花粉管在母本组织中的生长和引导包括孢子体控制(sporophytic control)和配子体控制(gametophytic control)两个连续的过程,现已克隆出不同阶段花粉管生长和引导的基因,通过分析其表达调控揭示出花粉管生长和引导的分子机制。该文就近年来国内外有关花粉管生长和极性引导的调控机制研究进展进行综述,并对禾本科(Poaceae)和十字花科(Brassicaceae)植物花粉管引导的异同点进行了比较分析。