Two-celled Proembryo Research Articles

The regulatory network and critical factors promoting programmed cell death during embryogenesis.

Programmed cell death (PCD) is essential for animal and plant development. However, the knowledge of the mechanism regulating PCD in plants remains limited, largely due to technical limitations. Previously, we determined that the protease NtCP14 could trigger PCD in the embryonic suspensor of tobacco (Nicotiana tabacum), providing a unique opportunity to overcome the limitations by creating synchronous two-celled proembryos with ongoing PCD for transcriptome analysis and regulatory factor screening. Here, we performed comparative transcriptome analysis using isolated two-celled proembryos and explored the potential regulatory network underlying NtCP14-triggered PCD. Multiple phytohormones, calcium, microtubule organization, the immunity system, soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins, long non-coding RNAs and alternative splicing are addressed as critical factors involved in the early stage of suspensor PCD. Genes thought to play crucial roles in suspensor PCD are highlighted. Notably, decreased antioxidant gene expression and increased reactive oxygen species (ROS) levels during suspensor PCD suggest a critical role for ROS signaling in the initiation of NtCP14-triggered PCD. Furthermore, five genes in the regulatory network are recommended as immediate downstream elements of NtCP14. Together, our analysis outlines an overall molecular network underlying protease-triggered PCD and provides a reliable database and valuable clues for targeting elements immediately downstream of NtCP14 to overcome technical bottlenecks and gain deep insight into the molecular mechanism regulating plant PCD.

Megasporogenesis, megagametogenesis, and embryogenesis in Dendrobium nobile (Orchidaceae).

The orchid reproductive strategy, including the formation of numerous tiny seeds, is achieved by the elimination of some stages in the early plant embryogenesis. In this study, we documented in detail the formation of the maternal tissues (the nucellus and integuments), the structures of female gametophyte (megaspores, chalazal nuclei, synergids, polar nuclei), and embryonic structures in Dendrobium nobile. The ovary is unilocular, and the ovule primordia are formed in the placenta before the pollination. The ovule is medionucellate: the two-cell postament and two rows of nucellar cells persist until the death of the inner integument. A monosporic eight-nucleated embryo sac is developed. After the fertilization, the most common central cell nucleus consisted of two joined but not fused polar nuclei. The embryogenesis of D. nobile is similar to the Caryophyllad-type, and it is characterized by the formation of all embryo cells from the apical cell (ca) of a two-celled proembryo. The only exception is that there is no formation of the radicle and/or cotyledons. The basal cell (cb) does not divide during the embryogenesis, gradually transforming into the uninuclear suspensor. Then the suspensor goes through three main stages: it starts with an unbranched cell within the embryo sac, followed by a branched stage growing into the integuments, and it ends with the cell death. The stage-specific development of the female gametophyte and embryo of D. nobile is discussed.

The First Stages of Liparis parviflora (Orchidaceae) Embryogenesis

Embryogenesis of the plant Liparis parviflora, a representative of the tribe Malaxideae (Orchidaceae), was studied. The multivariance of the first cell divisions in embryos belonging to different species and genera of the Malaxideae tribe was considered. All the cells of the embryo proper were shown to originate from the apical (ca) cell of the two-celled proembryo. The basal (cb) cell of the embryo does not undergo division over the entire embryogenesis. The suspensor remains unicellular but changes its shape upon development by forming several elongated lobes and is eliminated at later stages of the globular embryo. Thus, the embryonic development of this species generally obeys the Dienia type that was earlier described. It should be noted that the lobe-shape suspensor, firstly found in L. parviflora, had never been reported in any representative of the Malaxideae tribe.

The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two-celled proembryo stage.

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo invitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor invitro is highly similar to its undisturbed invivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both invivo and invitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two-celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo invivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

Formation of triploid plants via possible polyspermy

ABSTRACTPolyploidization is a common phenomenon in angiosperms, and polyploidy has played a major role in the long-term diversification and evolutionary success of plants. Triploid plants are considered as the intermediate stage in the formation of stable autotetraploid plants, and this pathway of tetraploid formation is known as the triploid bridge. As for the mechanism of triploid formation among diploid populations, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of polyploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic zygotes. In the study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells and monitored their developmental profiles. The two sperm nuclei and the egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos developed and regenerated into triploid plants. These results suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

Development of Polyspermic Rice Zygotes.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In most animals and fucoid algae, polyspermy block occurs at the plasmogamy step. Because the polyspermy barrier in animals and in fucoid algae is incomplete, polyspermic zygotes are generated by multiple fertilization events. However, these polyspermic zygotes with extra centrioles from multiple sperms show aberrant nuclear and cell division. In angiosperms, polyspermy block functions in the egg cell and the central cell to promote faithful double fertilization, although the mechanism of polyspermy block remains unclear. In contrast to the case in animals and fucoid algae, polyspermic zygotes formed in angiosperms are not expected to die because angiosperms lack centrosomes. However, there have been no reports on the developmental profiles of polyspermic zygotes at cellular level in angiosperms. In this study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells, and monitored their developmental profiles. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle, as observed during mitosis of a diploid zygote. The two-celled proembryos further developed and regenerated into triploid plants. These findings suggest that polyspermic plant zygotes have the potential to form triploid embryos. Polyspermy in angiosperms might be a pathway for the formation of triploid plants, which can contribute significantly to the formation of autopolyploids.

Rape embryogenesis. V. Accumulation of lipid bodies

In embryo of winter rape var. Górczański lipid bodies have been observed in the light microscope starting from two-celled proembryo. Their number and size increase in the course of embryogenesis, especially since greening of endosperm and embryo. Lipid bodies, either single or in clusters, are present in all embryo cells, the clusters occur in various parts of the cytoplasm. During embryo maturation lipid bodies move and locate in a number of layers by the cell wall. At the same time their appearance change. Typical storage lipids originate. Lipid bodies are unevenly distributed within the embryo: their number and size decrease along the embryo axis from its part below cotyledons towards root apex. Moreover, they are histologically diversified: the biggest are located in epidermis and cortex, whereas the smallest -in central cylinder. In columella there are fewest lipid bodies.

The Fate of the Plant Embryo's Suspensor: Balancing Life and Death

The plant embryo is supported by a lifeline known as a suspensor that acts like an umbilical cord, connecting the embryo to surrounding tissues in the seed and ferrying in vital nutrients and growth factors. Although scientists have for a century known that the suspensor is a temporary organ, the molecular trigger for its demise was unknown. Now Meng-xiang Sun at Wuhan University in China and his colleagues have uncovered one key way this programmed cell death is controlled — a pair of proteins that antagonistically balance each other, with one spurring death and the other preventing it. Plant embryos are supported by a temporary organ called a suspensor that connects embryos to surrounding nutrient-providing tissues. The suspensor dies early in embryonic development due to the actions of a protein pair that antagonistically balance each ... After a plant egg cell is fertilized, it divides to become what is called a proembryo, which possesses two kinds of daughter cells with profoundly different fates. One, the smaller cell sitting at its tip, becomes the embryo, while the other, the larger cell resting at its base, develops into the suspensor that anchors the embryo to the parent plant. The suspensor, which occurs in a dazzling variety of shapes and sizes in the plant kingdom, is required only in the early stages of embryonic development — the disintegration of suspensor cells marks the earliest instance of programmed cell death in a plant's life. To help solve the mystery behind the suspensor's downfall, researchers experimented with tobacco plants, whose suspensors are relatively easy to study, made up as they are of just simple columns of about four cells that develop through a well-known, precisely timed sequence of cell divisions. The suspensor begins to die once the embryo reaches the 32-celled stage, with death beginning at its base and progressing up to its tip, with all the contents of its cells slowly digested from within. The scientists focused on a tobacco gene they named NtCYS. Genetic analysis revealed this gene is seen only in the basal cells that develop into suspensors — its activity is greatest during the eight- to 32-celled stage of the embryo, after which its levels decrease dramatically. The researchers discovered the natural onset of suspensor degeneration coincided with a steady decrease in NtCYS activity. When they artificially silenced NtCYS activity with interfering RNA molecules early on in plant development, premature cell death occurred in the basal cell lineage, which in turn killed off embryos. By contrast, overexpressing NtCYS profoundly delayed cell death in the suspensor. All in all, these findings revealed NtCYS suppresses cell death. NtCYS encodes a kind of protein known as a cystatin, which typically inhibit enzymes known as cysteine proteases. Genetic analysis revealed 20 genes encoding cysteine proteases that were active in the two-celled proembryo. Of those, NtCYS apparently interacted with six cathepsins, proteases linked with cell death; moreover, NtCYS was found in the same locations in the cell as one of those cathepsins named NtCP14, suggesting NtCYS targeted that enzyme. The scientists discovered that overexpressing NtCP14 triggered precocious cell death in the basal cell lineage, causing massive embryonic death, while silencing NtCP14 delayed suspensor cell death. In other words, whereas NtCYS is anti-death, NtCP14 is pro-death. It remains a puzzle as to what molecule or molecules naturally set off the rapid decrease of NtCYS activity that kills off suspensors. Since the ephemeral organ's programmed cell death starts at its base, the signal might come from cells at the micropyle, the opening through which the pollen tube penetrates into the ovule, the female part of the plant. Alternatively, the signal might come from the embryo itself, a possibility supported by the fact that suspensor programmed cell death always starts when the embryo reaches a very specific point in its life, its 32-celled stage. In addition to analyzing what controls the activity of NtCYS and NtCP14, future research can also pinpoint what they control. The investigators discovered that increasing NtCP14 activity in the two-celled proembryo correspondingly increased the activity of molecules similar to caspases, enzymes linked with programmed cell death in animals. Zhao P, Zhou X-M, Zhang L-Y, Wang W, Ma L-G, et al. (2013) A Bipartite Molecular Module Controls Cell Death Activation in the Basal Cell Lineage of Plant Embryos. doi: 10.1371/journal.pbio.1001655

Identification of genes preferentially expressed in wheat egg cells and zygotes

KEY MESSAGE : Wheat genes differentially expressed in the egg cell before and after fertilization were identified. The data support zygotic gene activation before the first cell division in wheat. To have an insight into fertilization-induced gene expression, cDNA libraries have been prepared from isolated wheat egg cells and one-celled zygotes. Two-hundred and twenty-six egg cell and 253 zygote-expressed EST sequences were determined. Most of the represented transcripts were detected in the wheat egg cell or zygote transcriptome at the first time. Expression analysis of fourteen of the identified genes and three controls was carried out by real-time quantitative PCR. The preferential expression of all investigated genes in the female gametophyte-derived samples (egg cells, zygotes, two-celled proembryos, and basal ovule parts with synergids) in comparison to the anthers, and the leaves were verified. Three genes with putative signaling/regulatory functions were expressed at a low level in the egg cell but exhibited increased (2-to-33-fold) relative expression in the zygote and the proembryo. Genes with high EST abundance in cDNA libraries exhibited strong expression in the egg cell and the zygote, while the ones coding for unknown or hypothetical proteins exhibited differential expression patterns with preferential transcript accumulation in egg cells and/or zygotes. The obtained data support the activation of the zygotic genome before the first cell division in wheat.

Polar protein transport between apical and basal cells during tobacco early embryogenesis

KEY MESSAGE : We found that protein trafficking between apical and basal cell can be unidirectional, which reveals the different roles of the two cells in the cell-to-cell communication between them during early embryogenesis. In most angiosperm species, asymmetric zygote division results in an apical cell and a basal cell that have distinct cell fates. Much has been speculated about possible communication between these cell types in relation to their cell fate determination. Here, we report on the use of photoactivatable green fluorescent protein (PA-GFP) in tobacco to trace intercellular communication between apical and basal cells during early embryogenesis. We found that PA-GFP was transported between apical and basal cells of a two-celled proembryo, and that protein trafficking was unidirectional toward the apical cell, highlighting different cell communication roles. Further ultrastructural analysis showed numerous plasmodesmata in the walls connecting the apical and basal cells, which may provide channels for protein trafficking. Our data show a possible unique method of cell-to-cell communication between apical and basal cells during early embryogenesis.

Low-temperature treatment of tobacco ovaries improves the isolation, viability, and embryogenic potential of two-celled proembryos

The influence of low-temperature treatment of ovaries on the isolation and subsequent culture of two-celled proembryo in vitro was explored in tobacco. The efficiency of isolation of two-celled proembryos was significantly improved, by an average of 23.47 % after treatment of ovaries at 4°C for 5–24 h. Cell viability and polarity of two-celled proembryos, as assessed by organelle distribution, growth pattern, and cell wall component distribution, were all well maintained after cold treatment. Furthermore, in vitro cultivated two-celled proembryos divided normally, with timing paralleling that of two-celled proembryos isolated from fresh ovaries. Unexpectedly, the division frequency of two-celled proembryos was significantly improved by cold treatment. These results demonstrate that low-temperature treatment of tobacco ovaries was beneficial for two-celled proembryo manipulation in vitro, by not only improving isolation efficiency but also by promoting cell division and improving the potential for further cultivation.

Are there symplastic connections between the endosperm and embryo in some angiosperms?—a lesson from the Crassulaceae family

It is believed that there is symplastic isolation between the embryo (new sporophyte) and the endosperm (maternal-parental origin tissue, which nourishes the embryo) in angiosperms. However, in embryological literature there are rare examples in which plasmodesmata between the embryo suspensor and endosperm cells have been recorded (three species from Fabaceae). This study was undertaken in order to test the hypothesis that plasmodesmata between the embryo suspensor and the endosperm are not so rare but also occur in other angiosperm families; in order to check this, we used the Crassulaceae family because embryogenesis in Crassulaceae has been studied extensively at an ultrastructure level recently and also we tread members of this family as model for suspensor physiology and function studies. These plasmodesmata even occurred between the basal cell of the two-celled proembryo and endosperm cells. The plasmodesmata were simple at this stage of development. During the development of the embryo proper and the suspensor, the structure of plasmodesmata changes. They were branched and connected with electron-dense material. Our results suggest that in Crassulaceae with plasmodesmata between the endosperm and suspensor, symplastic connectivity at this cell–cell boundary is still reduced or blocked at a very early stage of embryo development (before the globular stage). The occurrence of plasmodesmata between the embryo suspensor and endosperm cells suggests possible symplastic transport between these different organs, at least at a very early stage of embryo development. However, whether this transport actually occurs needs to be proven experimentally. A broader analysis of plants from various families would show whether the occurrence of plasmodesmata between the embryo suspensor and the endosperm are typical embryological characteristics and if this is useful in discussions about angiosperm systematic and evolution.

Comparative Transcriptional Analysis Reveals Differential Gene Expression between Asymmetric and Symmetric Zygotic Divisions in Tobacco

Asymmetric cell divisions occur widely during many developmental processes in plants. In most angiosperms, the first zygotic cell division is asymmetric resulting in two daughter cells of unequal size and with distinct fates. However, the critical molecular mechanisms regulating this division remain unknown. Previously we showed that treatment of tobacco zygotes with beta-glucosyl Yariv (βGlcY) could dramatically alter the first zygotic asymmetric division to produce symmetric two-celled proembryos. In the present study, we isolated zygotes and two-celled asymmetric proembryos in vivo by micromanipulation, and obtained symmetric, two-celled proembryos by in vitro cell cultures. Using suppression-subtractive hybridization (SSH) and macroarray analysis differential gene expression between the zygote and the asymmetric and symmetric two-celled proembryos was investigated. After sequencing of the differentially expressed clones, a total of 1610 EST clones representing 685 non-redundant transcripts were obtained. Gene ontology (GO) term analysis revealed that these transcripts include those involved in physiological processes such as response to stimulus, regulation of gene expression, and localization and formation of anatomical structures. A homology search against known genes from Arabidopsis indicated that some of the above transcripts are involved in asymmetric cell division and embryogenesis. Quantitative real-time PCR confirmed the up- or down-regulation of the selected candidate transcripts during zygotic division. A few of these transcripts were expressed exclusively in the zygote, or in either type of the two-celled proembryos. Expression analyses of select genes in different tissues and organs also revealed potential roles of these transcripts in fertilization, seed maturation and organ development. The putative roles of few of the identified transcripts in the regulation of zygotic division are discussed. Further functional work on these candidate transcripts will provide important information for understanding asymmetric zygotic division, generation of apical-basal polarity and cell fate decisions during early embryogenesis.

Transcription Profile Analysis Reveals That Zygotic Division Results in Uneven Distribution of Specific Transcripts in Apical/Basal Cells of Tobacco

BackgroundAsymmetric zygotic division in higher plants results in the formation of an apical cell and a basal cell. These two embryonic cells possess distinct morphologies and cell developmental fates. It has been proposed that unevenly distributed cell fate determinants and/or distinct cell transcript profiles may be the underlying reason for their distinct fates. However, neither of these hypotheses has convincing support due to technical limitations.Methodology/Principal FindingsUsing laser-controlled microdissection, we isolated apical and basal cells and constructed cell type-specific cDNA libraries. Transcript profile analysis revealed difference in transcript composition. PCR and qPCR analysis confirmed that transcripts of selected embryogenesis-related genes were cell-type preferentially distributed. Some of the transcripts that existed in zygotes were found distinctly existed in apical or basal cells. The cell type specific de novo transcription was also found after zygotic cell division.Conclusions/SignificanceThus, we found that the transcript diversity occurs between apical and basal cells. Asymmetric zygotic division results in the uneven distribution of some embryogenesis related transcripts in the two-celled proembryos, suggesting that a differential distribution of some specific transcripts in the apical or basal cells may involve in guiding the two cell types to different developmental destinies.

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical–basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical–basal axis for its first cleavage.

Expressed sequence-tag analysis of tobacco sperm cells reveals a unique transcriptional profile and selective persistence of paternal transcripts after fertilization

Transcript analysis of male gametes of Nicotiana tabacum was conducted to gather gene expression data regarding the specialization of male germ cells and transmission of paternal transcripts during fertilization. We constructed a tobacco sperm cell cDNA library yielding 1,864 expressed sequence tags representing 1,050 clusters; 37.2% of these clusters have no homologs in GenBank, and 42% did not match any functionally classified protein. A comparative analysis of tobacco sperm transcripts with those of Arabidopsis and maize confirms that some genes are conserved in sperm specialization, while some are distinct to tobacco germline cells. Using reverse transcription-PCR (RT-PCR) of selected transcripts, we evaluated expression of sperm-obtained sequences in vegetative tissue, isolated egg cells, zygotes, and two-celled proembryos, identifying sperm cell-specific transcripts as potential markers for fertilization analysis. We further confirmed that two clusters of sperm transcripts were detected in zygotes about 10h after fertilization, offering new examples of apparently paternally transmitted transcripts that may be involved in egg cell activation and/or early embryogenesis.

Positional relationship between the gamete fusion site and the first division plane in the rice zygote.

In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell that possess different cell fates. This first division of the zygote is a putative step in the formation of the apical–basal axis of the proembryo. The gamete fusion activates the egg, and the gamete fusion site on the zygote has been reported to provide a possible cue for subsequent zygotic development and/or embryonic patterning in animals and plants. In this study, the gamete fusion site on the rice zygote was labelled by in vitro fertilization of a rice egg cell with a fluorescence-stained sperm cell. The positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage was monitored using a fixed culture of the fusion site-labelled zygote until the two-celled proembryo stage. The results indicate that gamete fusion sites exist on two-celled proembryos with no relation to the position of the first division plane, and that the gamete fusion site on the rice zygote does not function as a determinant for positioning the zygote division plane.

Localization of arabinogalactan proteins in egg cells, zygotes, and two-celled proembryos and effects of -D-glucosyl Yariv reagent on egg cell fertilization and zygote division in Nicotiana tabacum L.

Arabinogalactan proteins (AGPs) have been implicated in a variety of plant development processes including sexual plant reproduction. As a crucial developmental event, plant sexual reproduction generally occurs inside an ovule embedded in an ovary. The inaccessibility of the egg cells, zygotes, and embryos has hindered our understanding of the importance of AGPs in the early events involving fertilization, zygotic division, and early embryogenesis. In this study, the well-established in vitro zygote and ovary culture systems, together with immunofluorescence and immunogold labelling techniques, were employed to investigate the role of AGPs in the early events of sexual reproduction in Nicotiana tabacum. Dramatic changes in AGP content during ovule development were evidenced by western blotting. Subcellular localization revealed that AGPs are localized in the plasma membrane, cell wall, and cytoplasm of pre- and post-fertilized egg cells, and cytoplasm and vacuoles of two-celled proembryos. Abundant AGPs were detected in unfertilized egg cells; however, the level of AGPs substantially decreased in fertilized egg cells. Polar distribution of AGPs in elongated zygotes was observed. The early two-celled proembryos just from zygote division displayed accumulation of AGPs at a low level, while in the elongated two-celled proembryos at the late stage, the AGP content clearly increased. Provision of betaGlcY, a synthetic phenylglycoside that specifically binds AGPs, to the in vitro cultures of isolated zygote and fertilized ovaries increased abnormal symmetrical division of zygotes. In the culture of pollinated but unfertilized ovaries, addition of betaGlcY resulted in arrest of fertilization of the egg cells, but had no effect on fertilization of the central cells. The possible roles of AGPs in fertilization, zygotic division, and proembryo development are discussed.

The transcript composition of egg cells changes significantly following fertilization in wheat (Triticum aestivum L.)

Here, we report the transcript profile of wheat egg cells and proembryos, just after the first cell division. Microdissected female gametophytes of wheat were used to isolate eggs and two-celled proembryos to construct cell type-specific cDNA libraries. In total, 1197 expressed sequence tags (ESTs) were generated. Analysis of these ESTs revealed numerous novel transcripts. In egg cells, 17.6% of the clustered ESTs represented novel transcripts, while 11.4% novel clusters were identified in the two-celled proembryo. Functional classification of sequences with similarity to previously characterized proteins indicates that the unfertilized egg cell has a higher metabolic activity and protein turnover than previously thought. Transcript composition of two-celled proembryos was significantly distinct from egg cells, reflecting DNA replication as well as high transcriptional and translational activity. Several novel transcripts of the egg cell are specific for this cell. In contrast, some fertilization induced novel mRNAs are abundant also in sporophytic tissues indicating a more general role in plant growth and development. The potential functions of genes based on similarity to known genes involved in developmental processes are discussed. Our analysis has identified numerous genes with potential roles in embryo sac function such as signaling, fertilization or induction of embryogenesis.