Synergid cell death in Arabidopsis is triggered following direct interaction with the pollen tube

During angiosperm reproduction, one of the two synergid cells within the female gametophyte undergoes cell death prior to fertilization. The pollen tube enters the female gametophyte by growing into the synergid cell that undergoes cell death and releases its two sperm cells within the degenerating synergid cytoplasm to effect double fertilization. In Arabidopsis (Arabidopsis thaliana) and many other species, synergid cell death is dependent upon pollination. However, the mechanism by which the pollen tube causes synergid cell death is not understood. As a first step toward understanding this mechanism, we defined the temporal relationship between pollen tube arrival at the female gametophyte and synergid cell death in Arabidopsis. Using confocal laser scanning microscopy, light microscopy, transmission electron microscopy, and real-time observation of these two events in vitro, we demonstrate that synergid cell death initiates after the pollen tube arrives at the female gametophyte but before pollen tube discharge. Our results support a model in which a signaling cascade triggered by pollen tube-synergid cell contact induces synergid cell death in Arabidopsis.     

Female gametophytic cell specification and seed development require the function of the putative Arabidopsis INCENP ortholog WYRD

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis, we identified the wyrd (wyr) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.     

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.     

Female Gametophyte Development in Higher Plants - Meiosis and Mitosis Break the Cellular Barrier

Abstract: Meiotic products in higher plants should undergo a determined number of mitotic cycles before differentiating gametes. This creates a unique meiosis-mitosis interface, traverse of which is an absolute requirement for gametophyte development. In the absence of cytokinesis during megasporogenesis - as seen in the bisporic and tetrasporic types - the haploid nuclei produced by meiosis are driven to undergo mitotic cycles within the same cell. Similarly, the last of the mitotic cycles leads to a unique type of cell wall formation resulting in cellularization of the coenocytic female gametophyte, creating a mitosis-cellularization interface. Cell cycle regulation in terms of the molecules that interface with these two key spatio-temporal developmental settings should be of interest to both cell and developmental biologists. High throughput techniques of functional genomics are required for both interpretation of female gametophyte evolution and success of the biotechnological initiatives of transferring apomixis-related genes to crop plants.     

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.     

Mitochondria-responsive signaling between egg and central cell controls their coordinated maturation

Recent studies suggest a complex regulatory network in female gametophyte of angiosperm. The cell-cell communication between female gametes was confirmed during their maturation and functional specialization. The mitochondria-responsive signaling may play a critical role in this process. Here, we briefly summarized the recent discussion on this topic and proposed a two-pathway’s mechanism for regulating coordinated development of the female gamete cells.     

Genetic analysis of female gametophyte development and function.

The female gametophyte is an absolutely essential structure for angiosperm reproduction. It produces the egg cell and central cell (which give rise to the embryo and endosperm, respectively) and mediates several reproductive processes including pollen tube guidance, fertilization, the induction of seed development, and perhaps also maternal control of embryo development. Although much has been learned about these processes at the cytological level, specific molecules mediating and controlling megagametogenesis and female gametophyte function have not been identified. A genetic approach to the identification of such molecules has been initiated in Arabidopsis and maize. Although genetic analyses are still in their infancy, mutations affecting female gametophyte function and specific steps of megagametogenesis have already been identified. Large-scale genetic screens aimed at identifying mutants affecting every step of megagametogenesis and female gametophyte function are in progress; the characterization of genes identified in these screens should go a long way toward defining the molecules that are required for female gametophyte development and function.     

Ovary and Gametophyte Development Are Coordinately Regulated by Auxin and Ethylene following Pollination

The differentiation and development of ovules in orchid flowers are pollination dependent. To define the developmental signals and timing of critical events associated with ovule differentiation, we have examined factors that regulate the initial events in megasporogenesis and female gametophyte development and characterized its progression toward maturity and fertilization. Two days after pollination, ovary wall epidermal cells begin to elongate and form hair cells; this is the earliest visible morphological change, and it occurs at least 3 days prior to pollen germination, indicating that signals associated with pollination itself trigger these early events. The effects of inhibitors of ethylene biosynthesis on early morphological changes indicated that ethylene, in the presence of auxin, is required to initiate ovary development and, indirectly, subsequent ovule differentiation. Surprisingly, pollen germination and growth were also strongly inhibited by inhibitors of ethylene biosynthesis, indicating that male gametophyte development is also regulated by ethylene. Detailed characterization of the development of both the female and male gametophyte in pollinated orchid flowers indicated that pollen tubes entered the ovary and grew along the ovary wall for 10 to 35 days, at which time growth was arrested. Approximately 40 days after pollination, coincident with ovule differentiation as indicated by the presence of a single archesporial cell, the direction of pollen tube growth became redirected toward the ovule, suggesting a chemical signaling between the developing ovule and male gametophyte. Taken together, these results indicate that both auxin and ethylene contribute to the regulation of both ovary and ovule development and to the coordination of development of male and female gametophytes.     

How females become complex: cell differentiation in the gametophyte

In contrast to animals, gametes in plants form a separate haploid generation, the gametophyte. The female gametophyte of flowering plants consists of just four different cell types that play distinct roles in the reproductive process. Differentiation of the distinct cell fates is tightly controlled and appears to follow regional cues that are arranged along a polar axis. Mutant analysis suggests that important aspects of gametophyte patterning are gametophytically regulated. Additionally, structural and molecular changes following misspecification indicate that the female gametophyte is a remarkably versatile structure with enormous respecification potential. Recently, new tools have been developed that open fascinating possibilities to access and analyze those processes that ultimately ensure successful fertilization.     

Analyzing female gametophyte development and function: There is more than one way to crack an egg

In flowering plants, gametes are formed in specialized haploid structures, termed gametophytes. The female gametophyte is a few-celled structure that integrates such diverse functions as pollen tube attraction, sperm cell release, gamete fusion and seed initiation. These processes are realized by distinct cell types, which ensure reproductive success in a coordinated manner. In the past decade, much progress has been made concerning the molecular nature of the functions carried out by the different cell types. Here, we review recent work that has shed light on female gametophyte development and function with a particular focus on approaches that have led to the isolation of genes involved in these processes.     

Mutants with aberrant numbers of gametic cells shed new light on old questions

In contrast to animals, plant gametes form in distinct haploid generations, termed gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg and central cell, which are flanked by accessory cells. The gametic cells differ with respect to morphology, molecular attributes and, importantly, their fate: whereas the egg cell, upon fertilisation, gives rise to the embryo, the central cell forms the endosperm. To ensure correct endosperm formation, not only the egg cell but also the central cell has to fuse with a sperm cell. The respective sperm cell pair is delivered by a single pollen tube. In some plant species, the two male gametes appear to express a different bias towards the female gametes. Such a preference consequently determines their respective contribution to either embryo or endosperm development. In Arabidopsis and many other species the sperm cells are indistinguishable and it has been discussed whether they possess an inherent preference for either of the female gametes. The recent isolation of mutants that form an aberrant number of either male or female gametes stimulates discussion, albeit with different results. Furthermore, some data indicate that the central cell is competent to initiate endosperm formation without a paternal contribution. These data support the theory that the endosperm is of gametophytic rather than sporophytic origin.     

Rapid and slow mechanisms for loss of cell adhesiveness during fertilization in Chlamydomonas

Although vegetative cells, gametes, and zygotes of the biflagellated alga Chlamydomonas bear flagella, only the flagella of mt+ and mt- gametes are adhesive. The molecules responsible for adhesiveness, mt+ and mt- agglutinins, are long rod-shaped glycoproteins displayed on the flagellar membrane. These flagellar agglutinins, which gametes use both as adhesion and signaling molecules during the early events of fertilization, are lost from the flagella during adhesion. Flagellar adhesiveness can be maintained, however, by recruitment and activation of preexisting, inactive agglutinins from the plasma membrane of the cell body (Hunnicutt et al, 1990, J. Cell Biol. 111, 1605-1616) unless the gametes of opposite mating types fuse to form zygotes. Upon cell fusion, flagellar adhesiveness is lost. In the studies presented here, we have employed an in vitro bioassay to measure agglutinins in both cell bodies and flagella at various times during gametogenesis, during fertilization, and after zygote-formation. By use of the bioassay, which can detect agglutinins that are functionally inactive in vivo, we found that vegetative cells are devoid of agglutinins. These adhesion molecules appear only after gametogenesis is underway with the cell body agglutinins appearing first and then the flagellar agglutinins. Surprisingly, 30 min after zygote formation, when the zygotes' flagella are no longer adhesive, the flagellar agglutinin activity detectable with the bioassay remains high. One interpretation of these results is that zygotes continue to recruit agglutinins from the cell body to the flagella, but cell fusion abrogates activation of the agglutinins. Within 45-90 min after fusion both the cell body and flagellar agglutinins are lost and can be detected in the medium. These mechanisms, which render the zygotes nonadhesive to other zygotes and unmated gametes, contribute to the Chlamydomonas equivalent of a block to polyspermy.     

Identification of genes expressed in the Arabidopsis female gametophyte

The angiosperm female gametophyte typically consists of one egg cell, two synergid cells, one central cell, and three antipodal cells. Each of these four cell types has unique structural features and performs unique functions that are essential for the reproductive process. The gene regulatory networks conferring these four phenotypic states are largely uncharacterized. As a first step towards dissecting the gene regulatory networks of the female gametophyte, we have identified a large collection of genes expressed in specific cells of the Arabidopsis thaliana female gametophyte. We identified these genes using a differential expression screen based on reduced expression in determinant infertile1 (dif1) ovules, which lack female gametophytes. We hybridized ovule RNA probes with Affymetrix ATH1 genome arrays and validated the identified genes using real-time RT-PCR. These assays identified 71 genes exhibiting reduced expression in dif1 ovules. We further validated 45 of these genes using promoter::GFP fusions and 43 were expressed in the female gametophyte. In the context of the ovule, 11 genes were expressed exclusively in the antipodal cells, 11 genes were expressed exclusively or predominantly in the central cell, 17 genes were expressed exclusively or predominantly in the synergid cells, one gene was expressed exclusively in the egg cell, and three genes were expressed strongly in multiple cells of the female gametophyte. These genes provide insights into the molecular processes functioning in the female gametophyte and can be used as starting points to dissect the gene regulatory networks functioning during differentiation of the four female gametophyte cell types.     

Bridging the generation gap: flowering plant gametophytes and animal germlines reveal unexpected similarities

Alternation of generations underpins all plant life histories and is held to possess important adaptive features. A wide range of data have accumulated over the past century which suggest that alternation from sporophyte to gametophyte in angiosperms includes a significant phase of 'informational reprogramming', leaving the founder cells of the gametophyte developmentally uncommitted. This review attempts to bring together results from these historic studies with more recent data on molecular and epigenetic events which accompany alternation, gametophyte development and gametogenesis in angiosperms. It is striking that most members of the other principal group of multicellular eukaryotes – the animals - have a completely different a life history: animals generate their gametes directly from diploid germlines, often set aside early in development. Nevertheless, a comparison between animal germlines and angiosperm gametophyte development reveals a number of surprising similarities at the cytological and molecular levels. This difference in life history but similarity in developmental process is reviewed in the context of the very different life strategies adopted by plants and animals, and particularly the fact that plants do not set aside diploid germlines early in development.     

Maternal Gametophyte Effects on Seed Development in Maize

Flowering plants, like placental mammals, have an extensive maternal contribution toward progeny development. Plants are distinguished from animals by a genetically active haploid phase of growth and development between meiosis and fertilization, called the gametophyte. Flowering plants are further distinguished by the process of double fertilization that produces sister progeny, the endosperm and the embryo, of the seed. Because of this, there is substantial gene expression in the female gametophyte that contributes to the regulation of growth and development of the seed. A primary function of the endosperm is to provide growth support to its sister embryo. Several mutations in Zea mays subsp. mays have been identified that affect the contribution of the mother gametophyte to the seed. The majority affect both the endosperm and the embryo, although some embryo-specific effects have been observed. Many alter the pattern of expression of a marker for the basal endosperm transfer layer, a tissue that transports nutrients from the mother plant to the developing seed. Many of them cause abnormal development of the female gametophyte prior to fertilization, revealing potential cellular mechanisms of maternal control of seed development. These effects include reduced central cell size, abnormal architecture of the central cell, abnormal numbers and morphology of the antipodal cells, and abnormal egg cell morphology. These mutants provide insight into the logic of seed development, including necessary features of the gametes and supporting cells prior to fertilization, and set up future studies on the mechanisms regulating maternal contributions to the seed.     

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.     

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.