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.     

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.     

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.     

An Efficient Method for Quantitative,Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.     

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.     

Synergids and filiform apparatus in the sexual and apomictic dandelions from section Palustria (Taraxacum, Asteraceae)

An evolutionary trend to reduce “unnecessary costs” associated with the sexual reproduction of their amphimictic ancestors, which may result in greater reproductive success, has been observed among the obligatory apomicts. However, in the case of the female gametophyte, knowledge about this trend in apomicts is not sufficient because most of the ultrastructural studies of the female gametophyte have dealt with amphimictic angiosperms. In this paper, we tested the hypothesis that, in contrast to amphimictic plants, synergids in apomictic embryo sacs do not form a filiform apparatus. We compared the synergid structure in two dandelions from sect. Palustria: the amphimictic diploid Taraxacum tenuifolium and the apomictic tetraploid, male-sterile Taraxacum brandenburgicum. Synergids in both species possessed a filiform apparatus. In T. brandenburgicum, both synergids persisted for a long time without any degeneration, in spite of the presence of an embryo and endosperm. We propose that the persistent synergids in apomicts may play a role in the transport of nutrients to the embryo.     

An electron microscopic study of the mature megagametophyte in Zea mays

With light microscopy maize megagametophytes stained with Alcian blue-periodic acid-Schiff (AB-PAS) reveal acid or neutral polysaccharides in various cell walls. Comparative fine structural studies were made of permanganate- or OsO₄-fixed material. Organelle distribution is random in the vacuolate and multinucleate antipodal cells; organelles are abundant; starch is scarce. Antipodal cell walls have large openings forming several syncytia. Some walls are papillate. In the central cell (primary endosperm cell) a thin peripheral layer of cytoplasm surrounds the large vacuole; organelle number is moderate; starch is abundant. The central cell wall is also papillate adjacent to the antipodals and around the egg apparatus. In the synergids organelle distribution is non-random; nuclei and numerous organelles occupy the micropylar cytoplasm of each synergid; vacuoles dominate the chalazal cytoplasm of these cells. The filiform apparatus stains with AB-PAS and is composed of both lightly and darkly stained amorphous material. In the egg, organelle distribution is perinuclear with vacuoles proximal to the micropyle; mitochondria are large, abundant and polymorphic; starch is abundant. Nucleolar diameter is five times greater in the central cell and egg than in the antipodal cells and ten times greater than in the synergids. Plasmodesmata occur in all cell walls within the gametophyte, but none appear in the gametophyte wall itself. It is suggested that the antipodals and synergids might be secretory, the latter probably being involved in pollen tube attraction, and that stored metabolites in the central cell and egg cytoplasm support rapid increase in metabolism following fertilization.     

粉叶小檗大孢子发生和雌配子体形成

采用石蜡切片方法对粉叶小檗（Berberis pruinosa Franch.)的大孢子发生和雌配子体形成过程进行了研究。主要结果如下：雌蕊1枚,子房单心皮,边缘胎座,2枚胚珠倒生,具双珠被,厚珠心,珠孔由内外两层珠被共同形成,呈“Z”字形;单孢原,位于珠心表皮下;直线形大孢子四分体,合点端的1个大孢子发育为功能大孢子,胚囊发育类型为蓼型;成熟胚囊中,2个极核在受精前融合为次生核;3个反足细胞不发达,较早退化;"品"字形卵器,其中助细胞发达且具丝状器。     

Structural and developmental variability in the female gametophyte of Griffithella hookeriana,Polypleurum stylosum,and Zeylanidium lichenoides and its bearing on the occurrence of single fertilization in Podostemaceae

Key message

Reduced female gametophyte.

Abstract

Angiosperms are characterized by the phenomenon of double fertilization with Podostemaceae as an exception that appears to extend to the entire family. Our earlier work demonstrated the cause of failure of double fertilization and ascertained the occurrence of single fertilization in Dalzellia zeylanica (Tristichoideae, Podostemaceae). In continuation with this work, three more members, i.e., Griffithella hookeriana (Tul.) Warming, Polypleurum stylosum (Wight) Hall, and Zeylanidium lichenoides (Kurz) Engl. (Podostemoideae), have been investigated in the present work. We studied the ontogenetic development of female gametophyte and tracked the path of the two sperm cells from the time of their formation in the pollen tube through their entry into the synergid and gamete fusion. We report the occurrence of a remarkably reduced 3-nucleate, 3-celled mature female gametophyte consisting of an egg cell and two synergids in all the three genera. Interestingly, the central cell is formed during female gametophyte development, but exhibits a species-specific, limited life span, and eventually degenerates prior to the entry of the pollen tube into the synergid, resulting in a failure of double fertilization. Sperm dimorphism on the basis of fluorochrome stainability has been recorded in Z. lichenoides. Further, morphogenetic constraints on the part of male (sperm selection, functional reductionism) and female gametophyte (structural reductionism, inaccessibility of central cell) presumably ensure the failure of double fertilization in these species. Thus, loss of double fertilization in this family is likely a derived condition.     

The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate

Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gβ, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca²⁺ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.     

Development of the Female Gametophyte in Hydrobryum griffithii (Podostemaceae)

The development of the female gametophyte in Hydrobryum griffithiiis of the Apinagia type. The chalazal megaspore nucleus of thetwo-nucleate embryo sac completely degenerates, and only themicropylar megaspore nucleus contributes to the four nucleipresent in the organized embryo sac. The female gametophyteconsists of two synergids, an egg and a haploid central cell.The latter degenerates before the entry of the pollen tube andthere is only syngamy. The nucellar cells below the embryo sacorganize into a nucellar plasmodium.     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.     

Megasporogenesis, female gametophyte development and embryonic development of Ambrosia L. in China

The megasporogenesis, female gametophyte development and embryonic development of Ambrosia artemisiifolia L. and Ambrosia trifida L. of genus Ambrosia L. in China were studied using conventional paraffin section technology and optical microscopy. The results show that both A. artemisiifolia L. and A. trifida L. have a bilobed pistil stigma, two carpels, one chamber, basal placenta, unitegmic, tenuinucellate, anatropous ovule, and well-developed integumentary tapetum. Megaspore mother cells are directly developed from archesporial cells originated from the nucellar cells under the nucellar epidermis and further undergo meiosis to form linear tetrads. The megaspore at the chalazal end develops into a functional megaspore and the other three megaspores are degraded. The development of embryo sac is monosporic type. After three consecutive mitosis, mononucleate embryo sac becomes a mature embryo sac with two synergids and one egg cell at the micropylar end, a central cell at the center and three antipodal cells at the chalazal end. Most antipodal cells are mononucleate or binucleate, only few are trinucleate. The embryonic development process contains four stages: globular embryo, heart-stage embryo, torpedo-stage embryo and mature embryo. The development of endosperm is cellular type.     

Actin coronas in normal and indeterminate gametophyte1 embryo sacs of maize

 The actin cytoskeletal organization and nuclear behavior of normal and indeterminate gametophyte1 (ig1) embryo sacs of maize were examined during fertilization. After pollination, during degeneration of one of the synergids and before arrival of the pollen tube, the cytoskeletal elements undergo dramatic changes including formation of the actin coronas at the chalazal end of the degenerating synergid and at the interface between the egg cell and central cell. The actin coronas are present only for a limited period of time and their presence is coordinated with pollen tube arrival and fusion of the gametes; they disappear before the zygote divides. This allows us to estimate the frequency of fertilized ovules along the ear. Up to 88% of the ovules on an ear contain actin coronas in the embryo sacs when observed 16–19 h after pollination, indicating the high frequency of fertilizing kernels along the ear at this stage. In the ig embryo sacs, two or more degenerated synergids containing actin coronas at their chalazal ends receive multiple pollen tubes for gametic fusion and can consequently give rise to twin or polyembryos. These findings with the monocot maize are consistent with previous reports on the dicots Plumbago and Nicotiana, suggesting that the formation of actin coronas in the embryo sac during fertilization is a universal phenomenon in angiosperms and is part of a mechanism of interaction between gametic signaling and actin cytoskeleton behavior which appears to precisely position and facilitate the access of male gametes to the egg cell and central cell for fusion. Received: 15 May 1998 / Revision accepted: 17 August 1998