STRUCTURE AND HISTOGENESIS OF THE EMBRYO OF ACER SACCHARINUM. I. EMBRYO SAC AND PROEMBRYO

Structure of the embryo sac and development of the proembryo of Acer saccharinum L. are described from paraffin sections. The embryo sac is monosporic and identical to the 8-nucleate Polygonum type in all respects. Cell, nuclear, and nucleolar sizes are constant within a narrow range and sharply distinctive for all components of the mature sac. Polar nuclei fuse before double fertilization. The longitudinal axis of symmetry of the egg, zygote, and proembryo is variously oriented with respect to the longitudinal axis of the embryo sac and is determined by the point of attachment of the presumptive egg cell to the sac wall. Subsequent development of the young embryo is responsive to aligning factors within the embryo sac and is collateral with the longitudinal axis of the sac. The first segmentation is transverse to the longitudinal axis of the zygote; the second and third are transverse in the basal cell and longitudinal in the apical cell. Descendants of ci form a short irregular suspensor; ca and m give rise to the apical and basal halves respectively of the embryo proper. The contribution of the proembryonic tiers to the older embryo differs in embryos of different initial orientation. Distribution and orientation of mitosis in the proembryo are shown in two accumulation maps.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Mutational analysis of morphogenesis of the maize embryo

Morphogenesis of the maize embryo is controlled by many genes. A group of 51 embryo-specific (emb) mutations representing at least 45 independent mutation events and many different gene loci have been isolated from active Robertson's Mutator stocks. The authors have reported previously that the embryo phenotype of 27 of these mutations, characterized by examining mature embryos in fresh dissection. The maximal development capacity of the 24 emb mutations are reported here which have not been reported previously. All result in retarded embryos that are morphologically abnormal. Three of the mutants are blocked during the first phase of morphogenesis, the period in which the basal-apical asymmetry is established and the embryo is regionalized into suspensor and embryo proper. Nineteen mutants are blocked during the second phase, the period in which radial asymmetry appears, the embryonic axis is established at a different angle than the original basal-apical axis of the zygote and the vegetative organ primordia of the adult plant make their first appearance. Two mutants are blocked or altered during the third phase, the period in which vegetative structures are elaborated. Some of the mutants affected in the first phase of morphogenesis may have defective mechanisms for establishing basal-apical asymmetry, including possibly the asymmetric distribution of morphogenic determinants. Similarly, some of the mutants affected in the second phase may be altered in the mechanisms establishing radial asymmetry and the origin of the meristems. Mutations of the first type may act as early as the first cell division when the zygote undergoes a transverse division, while mutations of the second type are likely to act during the proembryo and transition stages. Both types include mutations affecting embryo pattern formation. Mutations affecting the third phase of morphogenesis may identify genes regulating reiterative morphogenic processes of vegetative plant development and events of embryo maturation. This group of 24 mutations is like that reported previously in representing genes that are crucial to embryo morphogenesis.     

Confocal Microscopy Study of <Emphasis Type="Italic">Arabidopsis</Emphasis> Embryogenesis Using GFP:mTn

Embryogenesis in transgenic Arabidopsis plants with GFP:mTn, a chimeric fusion of soluble shifted green fluorescent protein and a mouse actin binding domain, was studied. Confocal laser scanning microscopy was used to determine patterns of formation and cellular responses during asymmetric cell division. Before such cells divide, the nucleus moves to the position where new cell walls are to be formed. The apical–basal axis of the embryo develops mainly at the zygote to octant stage, and these events are associated with asymmetric divisions of the zygote and hypophyseal cells. Formation of the radial axis is established from the dermatogen to the globular-stage embryo via tangential cell division within the upper tiers. Bilateral symmetry of the embryo primarily happens at the triangular stage through zig-zag cell divisions of initials of the cotyledonary primordia. All stages of embryogenesis are described in detail here.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.     

Signalling in plant embryos during the establishment of the polar axis.

In brown algae fertilization takes place free from surrounding tissue layers. The cytoskeleton and transmembrane links to the cell wall are involved in establishing and stabilizing the polar axis and in determining the fate of cells in the early embryo. In seed plants, the egg cell and zygote exhibit apical basal polarity. Mutant studies suggest that axes of polarity of the early embryo depend on signalling between the apical and basal compartments, possibly involving auxin. Development of somatic cells into plant embryos involves extracellular matrix-derived arabinogalactan proteins. This suggests a role for the cell wall in plant embryogenesis.     

OBPC Symposium: Maize 2004 &; beyond-developmental and molecular genetics of embryogenesis in plants

Summary Embryo development is a very key phase in the life cycle of seed plants. At maturity, the embryo contains the complete machinery to elaborate the entire plant body. While the embryogenic process is an innate feature of the zygote, gametic and somatic cells can undergo embryogenesis under the appropriate culture conditions. Embryogenesis is a highly regulated process and the use of mutants, especially in Arabidopsis, has allowed the identification of genes regulating pattern formation during this process. The use of such mutants has revealed the eritical roles of auxin levels and transport in the establishment of embryo axis. Root and shoot apical meristem function and integrity, have been defined by examination of genes involved in their identity and function. Further knowledge of the molecular and biochemical aspects of zygotic embryogenesis should contribute to our understanding of the underlying regulatory pathways and networks and also provide critical insights into unique totipotent features of the plant cell.     

Establishment of cell polarity during early plant development

Cellular asymmetries have been proposed to play a role in plant embryogenesis. Genetic studies of Arabidopsis and other experimental approaches in several plant species have addressed the origins of cellular asymmetry in specific cases. Although zygote polarity, which precedes the formation of the apical—basal axis of the embryo, is normally aligned with that of the surrounding maternal tissue, isolated single somatic cells that give rise to embryos in culture appear to become polar in the absence of maternal factors. Gene expression patterns reveal the developmental consequences of cellular asymmetries occurring at later stages of embryogenesis. Genetic evidence suggests that these cellular asymmetries are established in response to as yet unidentified signals from adjacent cells.     

Polarity and signalling in plant embryogenesis

The establishment of the apical-basal axis is a critical event in plant embryogenesis, evident from the earliest stages onwards. Polarity is evident in the embryo sac, egg cell, zygote, and embryo-suspensor complex. In the embryo-proper, two functionally distinct meristems form at each pole, through the localized expression of key genes. A number of mutants, notably of the model genetic organism Arabidopsis thaliana, have revealed new gene functions that are required for patterning of the apical-basal axis. There is now increasing evidence that two particular modes of signalling, via auxin and cell wall components, play important roles in co-ordinating the gene expression programmes that define determinative roles in the establishment of polarity.     

Embryology ofCymbidium sinense:the Microtubule Organization of Early Embryos

InCymbidium sinense, the pattern of embryo development is unusualin that oblique cell divisions result in the formation of severalsuspensor cells prior to the development of the embryo proper.Characteristic changes in microtubular distribution can be foundwithin the zygote and the proembryo during their development.After fertilization, the ellipsoid-shaped zygote has randomlydistributed microtubules within its cytoplasm. As the zygotetakes on a more rounded appearance, microtubules organize intoa dense meshwork. Furthermore, microtubule bundles appear atthe chalazal region of the cell prior to the first mitotic divisionof the zygote. At the preprophase stage of mitosis, a preprophaseband of microtubules appears in the cytoplasm of the zygote.The zygote divides obliquely and unequally and gives rise toan apical cell and a slightly larger basal cell. Many randomly-alignedmicrotubules can be found in the cortex of the basal cell. Theincrease in the abundance of microtubules coincides with theisotropic expansion of the basal cell. The early division ofthe basal cell and subsequent division of the apical cell resultsin the formation of a four-celled embryo, of which three cellsnear the micropylar pole develop as suspensor cells. In thesuspensor cells, the microtubules tend to orient in the samedirection as the long axis of the cell. In addition, prominentmicrotubules can also be found near the adjoining cell wallsof the four-celled embryo. The terminal cell is highly cytoplasmicwith abundant microtubules within the cell. Subsequent divisionsof the terminal cell give rise to additional suspensor cellsand the embryo proper. In the mature embryo, five suspensorcells are usually present; one eventually grows through themicropyle of the inner integument and four grow towards thechalazal pole. The cortical microtubules of suspensor cellsredistribute from a longitudinal to a transverse direction asthey grow towards their respective poles.Copyright 1998 Annalsof Botany Company Embryogenesis, endosperm, microtubules, preprophase band, suspensor cells,Cymbidium sinense(Andr.) Willd.     

CAPSELLA EMBRYOGENESIS: THE EGG,ZYGOTE, AND YOUNG EMBRYO

The ultrastructure and composition of the egg, zygote, and young embryo of Capsella bursa-pastoris were examined. The egg is a highly polarized cell; one-half to one-third of the micropylar end is filled with a large vacuole while the chalazal end contains the nucleus and much of the cytoplasm of the cell. The wall which surrounds the cell is incomplete at the chalazal end. Ribosomes fill the cytoplasm and show little or no aggregation into polysomes. The structure of the nucleolus suggests that ribosomes are not being produced. Following fertilization and the formation of the zygote, the cell decreases slightly in volume as the large central vacuole becomes smaller. The zygote soon increases in size as the small chalazal vacuoles present before fertilization begin to enlarge. The dictyosomes become active and a continuous wall forms around the zygote. Aggregation of the ribosomes begins and numerous polysomes are formed. Before division of the zygote all plasmodesmata between the zygote and the surrounding cells are lost. The first division of the zygote is unequal as a result of its marked polarity. A large basal cell and a small terminal cell are produced. The basal cell appears to contain more protein, RNA, carbohydrate, and cell organelles than the terminal cell. Ribosomal aggregation is even more pronounced at this stage. Starch accumulates in the plastids. Numerous plasmodesmata are present between the terminal and basal cells but there are no connections between the endosperm or other cells. The basal cell divides next to give rise to a three-celled linear embryo consisting of the basal cell, the suspensor cell, and the terminal cell. The terminal cell stains more intensely for protein and RNA as a result of increased numbers of ribosomes. Starch in all the cells is about equal and reaches a maximum in the embryo at this stage.     

The puromycin-sensitive aminopeptidase PAM-1 is required for meiotic exit and anteroposterior polarity in the one-cell Caenorhabditis elegans embryo

In the nematode Caenorhabditis elegans, sperm entry into the oocyte triggers the completion of meiosis and the establishment of the embryonic anteroposterior (AP) axis. How the early embryo makes the transition from a meiotic to a mitotic zygote and coordinates cell cycle changes with axis formation remains unclear. We have discovered roles for the C. elegans puromycin-sensitive aminopeptidase PAM-1 in both cell cycle progression and AP axis formation, further implicating proteolytic regulation in these processes. pam-1 mutant embryos exhibit a delay in exit from meiosis: thus, this peptidase is required for progression to mitotic interphase. In addition, the centrosomes associated with the sperm pronucleus fail to closely associate with the posterior cortex in pam-1 mutants, and the AP axis is not specified. The meiotic exit and polarity defects are separable, as inactivation of the B-type cyclin CYB-3 in pam-1 mutants rescues the meiotic exit delay but not the polarity defects. Thus PAM-1 may regulate CYB-3 during meiotic exit but presumably targets other protein(s) to regulate polarity. We also show that the pam-1 gene is expressed both maternally and paternally, providing additional evidence that sperm-donated gene products have important roles during early embryogenesis in C. elegans. The degradation of proteins through ubiquitin-mediated proteolysis has been previously shown to regulate the cell cycle and AP axis formation in the C. elegans zygote. Our analysis of PAM-1 requirements shows that a puromycin-sensitive aminopeptidase is also required for proteolytic regulation of the oocyte to embryo transition.