被子植物胚柄研究进展

胚柄在胚胎发育过程中是一个暂时性的结构, 但却起着为胚体提供营养和生长调节因子的作用。相对胚体而言, 胚柄具有细胞类型单一、结构相对独立及发育时间短等特点, 其在胚胎发育的基因调控和细胞命运决定研究中具有独特的优势。该文从胚柄的形成及特点、胚柄在胚胎发育中的作用、信号传递系统对胚柄的影响以及与胚柄相关基因的功能等方面进行综述, 以期为研究胚柄在胚胎发育过程中所起的作用以及胚柄的命运决定提供参考信息。     

Histochemical Localization of Several Enzymes in Early Young Embryo of Pinus tabulaeformis

Histochemical localization of adenosine triphosphatase, acid phosphatase and peroxidase in young embryo of Pinus tabulaeformis has been studied. All cells of the embryo proper showed very intense lead phosphate disposite with ATP in the Wachs- tein Meisel medium and with B-glyceropbosphate in Gomori medium. In the suspensor 1–3 suspensor cells closed to embryo proper appear blackening with both the ATP and the B-glycerophosphate, and other cells of the suspensor showed less adenosine triphosphatase and B-glyeerophosphatase. In the young embryo the peroxidase distribution may vary with the developmental stages of the young embryo, for instance cone-shaped embryo proper showed dark blue, and which peroxidase occupies only 1–2 suspensor cells closed to embryo proper. When the young embryo developed further into columnus-shape, the peroxidase was mainly distributed in regions of both the posterior part of embryo proper and just derived suspensor cells. However the anterior part of embryo proper showed slight peroxidase. The experimental results indicate that all of these enzymes mentioned above are mainly present in both the embryo proper and the connecting end of the suspensor, and that these regions show great matabolic activity. From the above mentioned observation, the possible relationship between the young embryo and near surrounding tissue during the development of embryo is discussed.     

Diversity of plastid morphology and structure along the micropyle-chalaza axis of different Crassulaceae

The suspensor is a unique embryonic region that connects the embryo to the seed coat. In many angiosperms, the suspensor attains remarkably diverse morphological forms ranging from vesicular single-celled (Orchidaceae) to differentiated multicellular structures (Fabaceae). These variations may be specific to the genera and species of different families, and even members of a single family show a fair amount of diversity in suspensor morphology. Clear differences in the structure of plastids were observed due to type and phylogenetic relationship of angiosperm suspensors. In present study, diversity within suspensor plastids was evident in representatives of four Crassulaceae genera. In more closely related genera this difference was smaller, while in genera less related to each other, it was larger. In this family a decreasing gradation in the size and complexity of plastids from the basal cell to the chalazal suspensor cells and the embryo proper was found. In angiosperms, also a gradient in the size of nuclei and the degree of ploidy along the micropyle-chalaza axis embryo exists. Such a gradient can also be correlated with the gradient of plastids and the variation in plasmodesmata diameter along the micropyle-chalaza axis in the Crassulaceae embryo.     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein content

The role of the suspensor in the early development of the dicot embryo has not yet been defined. It has been described as merely an anchor and also as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor, early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos have been examined in tissue culture. It is known that Phaseolus embryos in culture at low osmotic potential will germinate precociously and that embryos in culture at high osmotic potential will either fail to grow or form callus. Optimum sucrose concentrations for continued, normal embryonic development of 0.2 mm and 0.5 mm P. vulgaris in tissue culture with Gamborg B5 medium were determined to be 12 and 6%, respectively. Protein content was examined in embryos and suspensors. Results showed that both 0.2- and 0.5-mm embryos required an attached suspensor for maximum protein content. Protein levels were substantially decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M restored the protein content to that of freshly excised embryos.     

The osmotic property and fluorescent tracer movement of developing orchid embryos of Phaius tankervilliae (Aiton) Bl

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.     

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.     

Embryogeny ofPhaseolus coccineus: Growth and microanatomy

Summary During early embryogeny, the development of the suspensor is rapid both in terms of size and fresh weight; structural differentiation can be observed as early as the proembryo stage with the formation of wall ingrowths. Ingrowths first appear in the outer wall of the suspensor cells adjacent to the integumentary tapetum, soon ingrowths begin to form in the inner suspensor cells as well. A basal-terminal gradation in nuclear size exists, with the largest nuclei in the basal suspensor cells. Cytologically, the suspensor cells appear to be very active, especially when the embryo reaches heart stage. Initially, the development of the embryo proper lags behind the suspensor, but its size and fresh weight increase rapidly as development proceeds. The volume of the liquid endosperm rises most rapidly during the late heart stage; and it is absorbed soon after. A cellular endospermic sheath surrounds the embryo, separating it from the liquid endosperm. Structural differentiation also occurs in the cellular endosperm cells with the formation of wall ingrowths in those cells that abut directly onto the integumentary tapetum. Both the suspensor and the cellular endosperm appear to remain active through the maturation of the seed. Storage bodies are formed in the cotyledons as well as in the embryonic axis. In the suspensor and the cellular endosperm, starch grains and lipid bodies can be found at the maturation stage.     

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.     

ABNORMAL DEVELOPMENT OF THE SUSPENSOR IN AN EMBRYO-LETHAL MUTANT OF ARABIDOPSIS THALIANA

Developmental arrest of the embryo proper in aborted seeds from mutant 50B, a recessive embryo-lethal mutant of Arabidopsis thaliana, was shown to be followed by abnormal growth of the suspensor. Each of the 12 aborted seeds examined in sectioned material contained an abnormally large suspensor and an embryo proper arrested at a preglobular stage of development. Analysis of serial sections revealed that mutant suspensors contained 15–150 cells whereas wild-type suspensors were composed of only six to eight cells. Development of the mutant endosperm continued to a late nuclear or early cellular stage even in the absence of further development of the embryo proper. These results suggest that the missing gene product in mutant 50B is required for development of the embryo proper but not for continued growth of the suspensor or endosperm tissue. The pattern of abnormal development observed in this mutant provides further evidence that continued growth of the suspensor during normal development is inhibited by the developing embryo proper and that the full developmental potential of cells in the suspensor is expressed only when this inhibitory effect is removed through a mutation or experimental treatment that is lethal only to cells of the embryo proper.     

An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae)

The suspensor is a specialized basal structure that differentiates early in plant embryogenesis to support development of the embryo proper. Suspensor differentiation in Arabidopsis is maintained in part by the TWIN1 (TWN1) gene, which suppresses embryogenic development in suspensor cells: twn1 mutants produce supernumerary embryos via suspensor transformation. To better understand mechanisms of suspensor development and further investigate the function of TWN1, we have characterized late-embryo and post-embryonic development in the twn1 mutant, using seedling culture, microscopy, and genetics. We report here that the twn1 mutation disrupts cotyledon number, arrangement, and morphology and occasionally causes partial conversion of cotyledons into leaves. These defects are not a consequence of suspensor transformation. Thus, in addition to its basal role in suspensor differentiation, TWN1 influences apical pattern and morphology in the embryo proper. To determine whether other genes can similarly affect both suspensor and cotyledon development, we looked for twinning in Arabidopsis mutants previously identified by their abnormal cotyledon phenotypes. One such mutant, amp1, produced a low frequency of twin embryos by suspensor transformation. Our results suggest that mechanisms that maintain suspensor identity also function later in development to influence organ formation at the embryonic shoot apex. We propose that TWN1 functions in cell communication pathways that convey local positional information in both the apical and basal regions of the Arabidopsis embryo.     

The Tridimensional Morphology of Rice Embryo Development with Special Reference to the Scutellum and the Coleoptile

Using scanning electron microscopy and semi-thin plastic sections, the pattern of development of the rice ( Oryza sativa L. ) embryo from 2 days after pollination (DAP) to maturity was followed. ( 1 ) At 2 DAP, the young embryo was observed to consist of an embryo proper, a hypoblast and a suspensor. The trum-pet-shaped hypoblast was a transitional region situated between the suspensor and the embryo proper. To label the hypoblast as suspensor is incorrect. During this time, dorsiventrality was established, but a radicle was not yet differentiated. Therefore it is still referred to as a proembryo. (2) 3 ~ 5 DAP, the embryo underwent definite morphological and anatomical changes. In the young embryo at 3 DAP the scutellum and colcoptile appeared simultaneously directly from the proembryo. The coleoptile did not originate from the scutellmn. During these foremost 3 days, the coleoptile primordium underwent a special kind of morphological change and formed a young coleeptile having the shape of an inverted hollow cone. This process revealed the true mechanism of c61eeptile formation. Anatomical observation indicated that the embryo at 3 DAP began to differentiate procambium, ground meristem and root cap. At 4 DAP a dome-like growth cone and protoderm of radicle appeared. Then the shoot-root axis became established. At 5 DAP the plumule, hypocotyl and radicle were formed. (3) It was shown that the embryo of rice actually has two cotyledons: the scutellum (a part of the embryonic envelope) and the coleeptile (The scutellum being the lateral cotyledon, a part of outside cotyledon, and the coleoptile the apical cotyledon--the coleoptile may be considered to be a modified form of a cotyledon). This kind of structural arrangemem can be referred to as dimorphic cotyledon.     

Isolation and identification of substances inducing formation of tracheary elements in cultured carrot-root slices

Data are presented on the cytokinin status of seeds and seed components, at different stages of development in Phaseolus coccineus L., as determined with the soybean callus growth bioassay: A change in cytokinin types according to developmental stage occurred: from biologically very active less polar types (zeatin=Z) at early stages to more polar types (zeatin glucoside=Z₉G and zeatin riboside=Zr), with relatively low biological activity, at intermediate and late stages of seed development: When cytokinins were analyzed separately in embryos (embryo proper) and suspensors at two embryonic stages: heart-shaped (A) and middle cotyledonary embryos (stage B) respectively, it was found that: i) at stage A, the suspensor showed cytokinin activity at the level of Z, 2iPA (2-isopentenyladenosine) and Zr, whereas more polar cytokinins (Z₉G, Zr) were present in the embryo; ii) at stage B, when the embryo seems to become autonomous for cytokinin supply, there was a relative abundance of active cytokinins (Z, 2iPA) in the embryo to which Z₉G activity in the suspensor corresponded. It is concluded that the suspensor plays an essential role in embryogenesis by acting as a hormone source to the early embryo.Abbreviations GA gibberellic acid - 2iPA 2-isopentenyladenosine - Stage A heart-shaped embryo - siage B middle cotyledonary embryo - Z zeatin - Z₉G zeatin glucoside - Zr Zeatin riboside     

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.     

The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein synthesis

The role of the suspensor in the early development of the dicot embryo has been described as merely an anchor or, conversely, as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor we have examined protein synthesis in early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos in tissue culture. Protein synthesis was examined in embryos and suspensors. Our results showed that in 0.2-mm embryos virtually all protein synthesis was dependent on an attached suspensor. Maximum protein synthesis in 0.5-mm embryos was observed when embryos were cultured attached to the suspensor. The levels were moderately decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M elicited the same protein diversity and greater [35S]methionine incorporation than did the attached suspensor in 0.2-mm embryos. Embryos of 0.5 mm did not appear to be differentially responsive to various gibberellin concentrations.     

Alisma embryogenesis: The development and ultrastructure of the suspensor

Summary The development of the suspensor (consisting of a basal cell and a few chalazal cells) inAlisma plantagoaquatica andA. lanceolatum was investigated using cytochemical methods, light and electron microscopy. The basal cell becomes differentiated during the first three days of embryo development. As a result of endopolyploidization the volume of the nucleus rapidly increases, as does the quantity of chromatin it contains and the size of the nucleolus. As basal cell grows, its cytoplasm increases in volume and the number of organelles increase, and wall ingrowths begin to form on the walls at the micropylar pole of the cell. The full development and functioning of the suspensor occurs during the next three days. The enormous basal cell then attains its maximum degree of differentiation: its nucleus reaches a ploidy of 256n or 512n, the micropylar transfer wall is fully developed, as is the cytoplasm, rich in proteins, ribonucleic acids (RNA) and organelles, particularly dictyosomes and long cisternae of the rough endoplasmic reticulum. The chalazal suspensor cells joining the embryo proper to the basal cell also become differentiated. In the seven-day embryo the suspensor begins to degenerate which coincides with the cellularization of the endosperm at the micropylar pole of the embryo sac. The senescence of the suspensor involves the degradation of the nucleus, increasing cytoplasmic vacuolization, and a distinct decrease in protein and RNA content, first in the basal cell, then in the chalazal suspensor cells. Analysis of the development and ultrastructure of the basal suspensor cell suggests that it plays the role of an active metabolic transfer cell, translocating nutrients from the maternal tissues via the chalazal suspensor cells to the growing embryo proper.     

Identification of a factor that complementarily inhibits somatic embryogenesis with vanillyl benzyl ether in Japanese larch

In Japanese larch (Larix leptolepis Gordon), a well-developed suspensor forms during somatic embryogenesis. The suspensor is the essential tissue for development of the embryo proper. In high-cell-density culture, the embryogenic cells proliferate, but no somatic embryos form because suspensor development is suppressed. Previously, we identified vanillyl benzyl ether (VBE) as a novel factor suppressing suspensor development from the high-cell-density conditioned medium (HCM), but the inhibitory effect of VBE was weaker than that of HCM added. Therefore, this study attempted to identify another inhibitory factor in the culture medium. Induction of somatic embryos was performed in a medium containing both VBE and a fraction of each chromatogram extracted from the culture medium. Results of the bioassay showed that a fraction had strong inhibitory activity with VBE, but weak activity without it. By physicochemical analyses of the fraction, 4-[(phenylmethoxy)methyl]phenol was identified as an inhibitory factor of larch somatic embryogenesis.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

A Model for Seed Transmission of a Plant Virus: Genetic and Structural Analyses of Pea Embryo Invasion by Pea Seed-Borne Mosaic Virus

Pea seed-borne mosaic virus (PSbMV), a seed-transmitted virus in pea and other legumes, invades pea embryos early in development. This process is controlled by maternal genes and, in a cultivar that shows no seed transmission, is prevented through the action of multiple host genes segregating as quantitative trait loci. These genes control the ability of PSbMV to spread into and/or multiply in the nonvascular testa tissues, thereby preventing the virus from crossing the boundary between the maternal and progeny tissues. Immunocytochemical and in situ hybridization studies suggested that the virus uses the embryonic suspensor as the route for the direct invasion of the embryo. The programmed degeneration of the suspensor during embryo development may provide a transient window for embryo invasion by the virus and could explain the inverse relationship between the age of the mother plant for virus infection and the extent of virus seed transmission.     

油菜裂外壁小孢子胚在分子水平上具有胚体/胚柄分化

早期合子胚取材困难, 难以开展相关研究。前人的工作表明, 油菜(Brassica napus)裂外壁小孢子胚胎发生系统能够较好地模拟合子胚的分化模式, 因而可替代早期合子胚胎作为研究材料。但目前尚缺乏该胚胎发生系统中胚胎具有胚体/胚柄分化的分子水平的证据。该文首次证明了油菜WOX家族基因能够用于标记胚体/胚柄的分化过程, 利用胚柄标记基因BnWOX8的表达模式, 从分子水平上证明了带胚柄的裂外壁小孢子胚的确存在胚体/胚柄的分化。研究结果为充分利用油菜裂外壁小孢子胚胎发生系统, 解决早期胚胎取材困难的问题奠定了坚实的基础。同时, 建立了活体激光切割分离特定细胞的技术, 结合用于少量细胞RNA提取的活体特异细胞RNA提取技术, 为鉴定少量特异分化细胞的基因表达模式提供了一个可行且明确的解决方案。