LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.     

LACHESIS Restricts Gametic Cell Fate in the Female Gametophyte of Arabidopsis

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.     

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.     

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.     

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.     

Reconstructing the ancestral female gametophyte of angiosperms: Insights from Amborella and other ancient lineages of flowering plants

For more than a century, the common ancestor of flowering plants was thought to have had a seven-celled, eight-nucleate Polygonum-type female gametophyte. It is now evident that not one, but in fact three, patterns of female gametophyte development and mature structure characterize the common ancestors of the four most ancient clades of extant angiosperms: Amborella-type, Nuphar/Schisandra-type and Polygonum-type. The Amborella-type female gametophyte is restricted to a single extant species, Amborella trichopoda, and at maturity consists of eight cells and nine nuclei. Development of the Amborella-type gametophyte is essentially identical to the Polygonum-type except that there is an additional and asynchronous cell division at the micropylar pole prior to maturation that produces a third synergid and the egg cell. The Nuphar/Schisandra-type female gametophyte is four-nucleate and four-celled and at maturity contains a typical three-celled egg apparatus and a central cell with a single haploid polar nucleus. This type of gametophyte appears to be universal among extant members of the Nymphaeales (including Hydatellaceae) and Austrobaileyales. Based on explicit reconstruction of character distribution and evolution, the Polygonum-type female gametophyte is certain to be representative of the common ancestors of monocots, eudicots, magnoliids, Ceratophyllaceae, and Chloranthaceae. There are compelling biological reasons to suggest that the four-celled, four-nucleate female gametophyte (as found in Nymphaeales and Austrobaileyales) is ancestral among angiosperms, with transitions to Polygonum-type female gametophytes separately in the Amborellales and in the ancient angiosperm clade that includes all angiosperms except Amborella, Nymphaeales, and Austrobaileyales. Subsequent to the evolution of a seven-celled, eight-nucleate Polygonum-type female gametophyte in the Amborellales, we hypothesize that a peramorphic increase in egg apparatus cell number took place and led to the unique situation in which there are three synergids in Amborella trichopoda.     

Cell-cell communication during double fertilization

Double fertilization in flowering seed plants requires intercellular signaling events between many interacting partners. The four cell types of the seven-celled female gametophyte communicate with each other to establish and maintain their identity. They secrete signaling molecules to guide the male gametophyte and to mediate sperm cell discharge and transport towards the two female gametes (the egg and central cell). After fusion of the gametes, guidance signals have to be removed to prevent polyspermy, embryo and endosperm development is induced generating daughter cells or nuclear regions of a different fate, and cell death is induced in the surrounding ovular cells. Until recently, little was known about the molecular nature of the signaling molecules that are involved in these processes. Now, small secreted proteins and peptides have been identified as prime candidates mediating several of these communication events.     

CLO/GFA1 and ATO are novel regulators of gametic cell fate in plants

The formation of gametes is a key step in the life cycle of any sexually reproducing organism. In flowering plants, gametes develop in haploid structures termed gametophytes that comprise a few cells. The female gametophyte forms gametic cells and flanking accessory cells. During a screen for regulators of egg-cell fate, we isolated three mutants, lachesis (lis), clotho (clo) and atropos (ato), that show deregulated expression of an egg-cell marker. We have previously shown that, in lis mutants, which are defective for the splicing factor PRP4, accessory cells can differentiate gametic cell fate. Here, we show that CLOTHO/GAMETOPHYTIC FACTOR 1 (CLO/GFA1) is necessary for the restricted expression of egg- and central-cell fate and hence reproductive success. Surprisingly, infertile gametophytes can be expelled from the maternal ovule tissue, thereby preventing the needless allocation of maternal resources to sterile tissue. CLO/GFA1 encodes the Arabidopsis homologue of Snu114, a protein that is considered to be an essential component of the spliceosome. In agreement with their proposed role in pre-mRNA splicing, CLO/GFA1 and LIS co-localize to nuclear speckles. Our data also suggest that CLO/GFA1 is necessary for the tissue-specific expression of LIS. Furthermore, we demonstrate that ATO encodes the Arabidopsis homologue of SF3a60, a protein that has been implicated in pre-spliceosome formation. Our results thus establish that the restriction of gametic cell fate is specifically coupled to the function of various core spliceosomal components.     

Antipodal cells persist through fertilization in the female gametophyte of Arabidopsis

Key message

Extended antipodal life-span.

Abstract

The female gametophyte of most flowering plants forms four cell types after cellularization, namely synergid cell, egg cell, central cell and antipodal cell. Of these, only the antipodal cells have no established functions, and it has been proposed that in many plants including Arabidopsis, the antipodal cells undergo programmed cell death during embryo sac maturation and prior to fertilization. Here, we examined the expression of female gametophyte-specific fluorescent reporters in mature embryo sacs of Arabidopsis, and in developing seeds shortly after fertilization. We observed expression of the fluorescence from the reporter genes in the three antipodal cells in the mature stage embryo sac, and continuing through the early syncytial endosperm stages. These observations suggest that rather than undergoing programmed cell death and degenerating at the mature stage of female gametophyte as previously supposed, the antipodal cells in Arabidopsis persist beyond fertilization, even when the other cell types are no longer present. The results support the concept that the Arabidopsis female gametophyte at maturity should be considered to be composed of seven cells and four cell types, rather than the previously prevailing view of four cells and three cell types.     

甜菜无融合生殖单体附加系M14雌配子体的发生与发育

利用常规石蜡制片法,对甜菜单体附加系M14(Beta vulgarisL.VV 1C、2n=18 1)雌配子体的发生与发育进行了研究,结果表明:二倍体孢子生殖雌配子体为韭型(Allium odorum-type)和蝶须型(Antennaria-type),有性生殖雌配子体为蓼型(Polygonum-type)。韭型和蝶须型的大孢子母细胞发育成为二倍体雌配子体,蓼型大孢子母细胞形成单倍体的雌配子体。在二倍体孢子生殖雌配子体发育过程中,出现发育迟缓,胚囊败育等情况,正常发育的雌配子体只有25%。讨论了二倍体孢子生殖雌配子体发生与发育特点。     

Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1

In Arabidopsis thaliana, the female gametophyte is a highly polarized structure consisting of four cell types: one egg cell and two synergids, one central cell, and three antipodal cells. In this report, we describe the characterization of a novel female gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac. The eostre phenotype is caused by misexpression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac. It is known that BELL-KNAT proteins function as heterodimers whose activities are regulated by the Arabidopsis ovate family proteins (OFPs). We show that the phenotypic effect of BLH1 overexpression is dependent upon the class II knox gene KNAT3, suggesting that KNAT3 must be expressed and functional during megagametogenesis. Moreover, disruption of At OFP5, a known interactor of KNAT3 and BLH1, partially phenocopies the eostre mutation. Our study indicates that suppression of ectopic activity of BELL-KNOX TALE complexes, which might be mediated by At OFP5, is essential for normal development and cell specification in the Arabidopsis embryo sac. As eostre-1 embryo sacs also show nuclear migration abnormalities, this study suggests that a positional mechanism might be directing establishment of cell fates in early megagametophyte development.     

Nuclear behavior, cell polarity, and cell specification in the female gametophyte

In flowering plants, the haploid gamete-forming generation comprises only a few cells and develops within the reproductive organs of the flower. The female gametophyte has become an attractive model system to study the genetic and molecular mechanisms involved in pattern formation and gamete specification. It originates from a single haploid spore through three free nuclear division cycles, giving rise to four different cell types. Research over recent years has allowed to catch a glimpse of the mechanisms that establish the distinct cell identities and suggests dynamic cell–cell communication to orchestrate not only development among the cells of the female gametophyte but also the interaction between male and female gametophytes. Additionally, cytological observations and mutant studies have highlighted the importance of nuclei migration- and positioning for patterning the female gametophyte. Here we review current knowledge on the mechanisms of cell specification in the female gametophyte, emphasizing the importance of positional cues for the establishment of distinct molecular profiles.     

The Gametophytes and Fertilization in Pseudotaxus

The development of the gametophytes and fertilization of Pseudotaxus chienii Cheng has been investigated. Pollination first occurred on April 17 (1964). The pollen grains shed at the uninucleate stage and germination on the nucellus is almost immediate. The pollen tubes approached the freenucleate female gametophyte about May 5. The spermatogenous cell is continuously enlarging with the growth of the pollen tube and two unequal sperms are formed after its division. Occasionally the small sperm may divide further into two smaller ones. During pollination the megaspore mother cell is in meiosis and 3 or 4 megaspores are formed. Generally 2 or 3 megaspores at the micropylar end are going to degenerate while the chalaza] megaspore is rapidly enlarging. After 8 successive simultaneous divisions of the functional megaspore 256 free nuclei are resulted and they are evenly distributed at the bulge of the famale gametophyte. Then the wall formation follows. Sometimes there are more than two, even as many as 5–6 gametophytes developed within a single ovule. The archegonial initials become differentiated at the apical end of the female gametophyte. They are usually single and apical, rarely lateral in position. The number of the archegonia vary from 3 to 7, usually 4–6. There are 2–8 neck cells in each archegonium which is surrounded by a layer of jacket cells. The central cell divided about May 20–26 (1964) and the division of the central cell gives rise to the egg and the ventral canal nucleus, the latter being degenerated soon. There are many proteid vacuoles near the nucleus of the matured egg. The fertilization took place about May 23–26 (1964). At first, the pollen tube discharges its contents into the egg, then the larger sperm fuses with the egg nucleus in the middle part of the archegonium. At the same time the male cytoplasm also fuses with the female cytoplasm and a layer of densely-staining neocytoplasm is formed around the fused nucleus. The smaller sperm, tube nucleus and sterile cell usually remain in the cytoplasm above the egg nucleus for some time. Based upon the observations of the development of the gametophytes and fertilization the authors conclude that Pseudotaxus is more close related to Taxus than any other member of Taxaceae.