Incorporation of Exogenous Macromolecules and Impermeable Fluorescent Dyes into the Developing Proembryo in Triticum aestivum

Our recent work has uncovered that a direct communication is established between the developing wheat proembryo and the surrounding medium at the parietal area of the basal region, in which a few cells were undergoing structural deformation and disintegration. With fluorescence microscopy and electron microscopy a series of experiments were conducted by using cationized ferritin and lucifer yellow CH as tracers to investigate the above mentioned “direct communication” in relation to the absorption of external materials. Evidences obtained show that either the macromolecule, ferritin, or the fluorescent dye, lucifer yellow actually can enter into the proembryo cells through non-transmenbrane pathway. Ultrastructural studies confirm that the incorporation of these external chemicals is realized by traversing a special route (ectodesma-like plasmodesmata and open channels) at the boundary wall of the local area of the basal region.     

Transfer Cells in Suspensur and Endosperm during Early Embryogeny of Vigna sinensis

During early embryogeny, structural differentiation of the suspensor and endosperm can be observed with the formation of cells with wall ingrowths. In the early proembryo stage, wall ingrowths are seen only on the boundary walls of the embryo sac around the proembryo and at the chalazal end. Later, ingrowths appear in the outer walls of the basal suspensor cells and some wall ingrowths also begin to develop in the outer walls of cellular endospermic cells adjacent to the nucellar cap and the inner integumentary tissues. The suspensor appears to remain active throughout the differentiation stages. Two regions can be clearly distinguished in the suspensor: a basal region and a neck region. Wall ingrowths appear to form only in the cells of the basal region. During the development of the cellular endospermic sheath, its cell number and size both increase slightly. Later, these cells rapidly become separated from each other. Those endospermic cells that abut directly onto the integumentary tissues also develop wall ingrowths. In the region of the fluid endosperm, wall ingrowths are especially abundant in the boundary walls on the ventral side of the embryo sac. The possible pathway of nutrient flow to the developing embryo is discussed.     

小麦胚胎分化时营养吸收一种可能方式的细胞学论据

以光学与电子显微镜术结合荧光标记物示踪,对小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）胚胎分化时胚根鞘的形态结构特征的变化与营养吸收相联系进行了实验观察。发现在分化前期胚根鞘顶端的球状突起处和颈区朝向颖果背面一侧的特定位点上出现了结构的变更;外沿局部细胞的衰退瓦解导致这些区段内侧存活细胞分界壁上出现大量残存的胞间连丝。不透膜的荧光物质、分子量达４３ｋＤ的ＦＩＴＣｄｅｘｔｒａｎ可进入球状突起和颈区多处细胞,表明残存胞间连丝仍具有共质传输功能,是分化幼胚摄取外围介质中营养物质的一种方式。分化进程中颈区的短时期局部缺损与随后的修复以及衰退区周围多层细胞胞间通透性的显著增大,既有利于外源物质的适时与大量掺入和在细胞间的快速运转,并仍保持了胚根鞘的结构完整,与分化前期胚胎对养分的大量需求正相适应,显示出胚根鞘的颈区比之球状突起处在营养吸收上占有更重要的地位。     

Cytological Evidences of a Possible Mode of Nurture Absorption of the Differentiating Embryo in Triticum aestivum

Coleorhiza covered the radicle of differentiating wheat embryo and morphologically appeared as tapered tissue with an attached globular protrusion. In the early phase of differentiation a series of structural changes occurred at some local areas of the coleorhiza. By light and electron microscopic inspections combined with fluorescence labelling it was uncovered that cells of the peripheral layers of the globular protrusion and the neck region underneath gradually exhibited degeneration and disintegration while a lot of broken plasmodesmata still remained at the boundary wall of the inner surviving cells. Impermeable fluorescence probe 4.3 kD FITC- dextran could enter these living cells and continuously diffuse to their neighborhood. All these showed that a direct communication was estabhshed between the symplasm of coleorhiza and the external medium and the remaining plasmodesmata, though broken in one end, might function as symplasmic pathway for external material incorporation. Analyzing the above described results a possible mode of nurture absorption of differentiating embryo then could bo recognized; particularly, comparing with the globular pretmsion the limited area of the neck region towards dorsal side might play a more important role in nurture absorption of the young embryo. Temporal and partial disintegration of the neck region and consequential regeneration in situ and increase of plasmodesmatal conductivity in the surrounding surviving cell group were most favourable to the incorporation and intercellular translocation of the external material for the coleorhiza. A large quantity of nutrients intruding into the coleorhiza at proper instant through the transiently opened route would effectively coordinate with the need of embryo differentiation without leaving apparent destruction to the coleorhiza.     

Some speculations on the possible roles of the plasmodesmata in the control of differentiation

Most plasmodesmata are formed across the cell plate at cytokinesis. Most of them persist until the cell is mature. Depending upon the pattern of elongation of the cell in differentiation, the frequency of plasmodesmata per unit area will suffer dilution to a greater or lesser extent. This dilution effect is now well understood and results commonly in high concentrations of plasmodesmata across transverse walls which have undergone little elongation and low concentrations on the longitudinal walls.Apart from their obvious role in cell to cell communication it is now believed that some plasmodesmata may offer preferential sites from which endogenous wall lytic enzymes may attack some or all of the constituent polymers of the surrounding wall. The effects of the asymmetrical distribution of large numbers of plasmodesmata, leading to the asymmetrical penetration of the wall by lytic enzymes are described and a hypothesis concerning the later stages of cell differentiation is constructed. In addition the late stage differentiation of individual plasmodesmata based on the same proposed lytic action, is described and re-interpreted.     

Ultrastructural Changes of the Egg Apparatus Associated with Fertilization and Proembryo Development of Soybean, Glycine max (Fabaceae)

As part of a study involving pod retention in soybean, Glycinemax (L.) Merr., we investigated changes occurring in the eggapparatus of non-abscised flowers from the time immediatelypreceding fertilization through early embryogeny. Prior to theentry of the pollen tube into the embryo sac, one of the synergidsbegins to degenerate as evidenced by increased electron densityand a loss of volume. This cell serves as the site of entryfor the pollen tube. The cytoplasm of the second, or persistentsynergid, remains unaltered until after fertilization. Bothsynergids contain, in addition to a filiform apparatus, a singleunidentified inclusion of flocculent material located in thechalazal portion of each cell. The zygote can be distinguishedfrom the egg by its consistently narrow wall; and it dividesto form a proembryo, a mass of cells not yet differentiatedinto embryo proper and suspensor. The basal cells of the proembryoare more vacuolate than the apical ones, characteristic of thebasal vacuolation of both egg and zygote. Cells of the proembryoare connected to one another via plasmodesmata, and with theexception of the basal-most cell, are isolated symplasticallyfrom the surrounding endosperm. Wall ingrowths frequently occurin certain cells of the proembryo, notably those cells in contactwith the degenerate synergid and embryo sac wall. At a laterstage of ontogeny, by which time the globular embryo properhas become distinct from the suspensor, the wall ingrowths areconcentrated in the suspensor. Glycine max, soybean, embryogeny, synergids     

银杏套细胞发育的解剖学研究

银杏套细胞起源于近雄配子体表达的颈卵器母细胞周围细胞,中央细胞形成时,套细胞呈明显的一层,围绕颈卵器紧密排列,随着颈卵器的发育,套细胞体积逐渐地大,细胞质变浓厚,细胞质中脂滴增多,套细胞和中央细胞的接触壁开始出现局部加厚,在颈卵器发育的泡沫化阶段,套细胞和中央细胞的接触壁不均匀加厚较为显著,在较薄的区域可见胞间连丝,受精前,套细胞中液泡增多,脂滴迅速减少,造淀粉体增大,套细胞与卵细胞的接触壁的不均匀加厚非常明显,精核进入颈卵器以后,卵细胞与套细胞的接触壁和卵细胞的质膜之间形成一个薄厚不均的间隔层,受精卵分裂时,受精卵细胞与套细胞接触壁的凹陷处可见许多小泡和内质网,游离核期时,套细胞内出现大量小液泡,细胞内含物迅速消失,套细胞外形变长,胚胎长出颈卵器后,套细胞逐渐解体消失。     

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.     

A Preliminary Observation on the Double Fertilization and Post-Fertilization Development of Ypsilandra thibetica Franch

Some investigations on the processes of double fertilization, endosperm development and embryogenesis, together with certain histo-chemical reactions in Ypsilandra thibetica Franch. have been made Their main features are: 1. The entry of pollen tube is porogamy and doable fertilization proceeds normally. It be longs to premitotic type. 2. The first division of the primary endosperm nucleus is followed by cell wall formation giving rise to a larger micropylar and a smaller chalazal chamber. But there is no further wall formation in the later nuclear divisions until the endosperm is nearly mature. Hence, it belongs to the Helobial type of development. Nuclear divisions tend to proceed faster in the micropylar than in the chalazal chamber and lately all freenuclei in both chambers become cellular when the proembryo consists of about four or eight cells. During the proembryogenesis some endosperm cells surrounding the proembryo degenerate gradually. 3. The zygote has a longer period of dormancy and slower rate of development than usual. When the seeds are mature, the embryos in them are not differentiated into organs yet. There are two types of embryo- genesis. Some are of the Caryophyllad type, while in others, the basal cell of the two-celled proembryo divides vertically so that two basal cells stand longitudinally parallel to each other forming the base of the suspensor. 4. The development of starch and accumulated protein grains in the process of double fertilization, embryogenesis and endosperm development were also observed.     

丫蕊花的双受精及胚和胚乳发育的初步观察

丫蕊花（Ypsilandra thibetica Franch.)为珠孔受精,进入胚囊的两个精子分别与卵细胞和中央细胞进行正常的双受精,其受精作用属有丝分裂前型,受精后的初生胚乳核立即分裂,其发育方式为沼生目型,到发育后期,由游离状态的胚乳核形成胚乳细胞时,珠孔室和合点室都形成胚乳细胞,合子的休眠期很长,而且胚的发育过程较为缓慢,种子成熟时胚尚无器盲的分化,本文还观察了以上发育过程中淀粉粒,蛋白质的动态。     

胞间连丝的次生形成和次生变化

本文介绍了胞间连丝次生形成和次生变化的研究进展。用统计特定细胞壁区段上胞间连丝数量与密度的变化,电镜观察嫁接组合中接穗与砧水间细胞壁上胞间连丝的形成等方法,证明了在植物生长发育过程中,存在着胞间连丝的次生形成。在某些特定部位,某一发育阶段,已形成的胞间连丝常会发生可逆的次生变化,这种变化和植物发育过程中的共质体隔离以及物质运输的调节有关。     

胞间连丝的次生形成和次生变化

本文介绍了胞间连丝次生形成和次生变化的研究进展,用统计特定细胞壁区段上胞间连丝数量与密度的变化,电镜观察嫁接组合中接穗与砧水间细胞壁上胞间连丝的形成等方法,证明了在植物生长发育过程中,存在着胞间连丝的次生形成。在某些特定部位,某一发育阶段,已形成的胞间连丝常会发生可逆的次生变化,这种变化和植物发育过程中的共质体隔离以及物质运输的调节有关。     

Distribution of Gibberellins A7 and A4 in Tobacco Proembryos Using Immunoelectron Microscopy

The ovules of Nicotiana tabacum var. macrophylla 8 days after pollination were fixed successively with 2% EDC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) and a mixture solution of paraformaldehyde and glutaraldehyde, then slightly postfixed in 0.5 % osmium solution. UI- trathin sections of 6-, 9- or 12- celled proembryos embedded in Epon 812 resin were stained in an anti-GA MAb and sheep anti-mouse IgG-colloidal gold (10 nm). This MAb specifically recognizes methyl esters of GA7 and GAn. Therefore, it could be used as a probe to localize GA7 and GAn in cells after EDC fixation. The 12-celled proembryo is composed of a 9-celled embryo and a 3-celled suspensor. Wide distribution of GA7 and GAn was observed in all proembryo cells and most organelles at subeellular level, including walls, plasmodesmata, plasma membrane, cytoplasmic matrix, mitochondria, plastids, vacuoles, endoplasmic reticulum and nuclei. Clusters of gold granules were found in nuclear envelope, nucleomatrix, nucleolus, chromosome, cytoplamic matrix. In a region composed of cytoplasmic matrix, a vacuole and a mitochondrium, such concentrated gold granules were particularly obviously observed. There appeared a gradient distribution of GA7 and Gan from embryo cells decreasingly to suspensor cells. GA7 and GAn could be translocated via intercellular walls, plasmodesmata and vesicles between embryo and suspensor. 1he authors suspect the direction of GA translocation in proembryo may be from suspensor to embryo. To author's knowledge, this is the first report to indicate subcellular and gradient distributions of bioactive gibberellins in plant proembryos.     

Osmotic Stress Decreases the Activity of ATPase Associated with the Root Cap Plasmodesmata in Zea mays

With light and electron microscopy the substructural change and the ATPase activity of corn (Zea mays L. ) root cap cells after short-term osmotic stress were studied. Some spoke-like fine strands originating from the departed periplasm and stretching towards cell wall could be observed even after plasmolysis. By observing the precipitation of ATPase activity product (lead phosphate) at plasma membrane and plasmodesmata, it was found that the fine strands were plasma membrane-lined channels surrounding the cytoplasm and that they still firmly connected to the plasmodesmata during plasmolysis. Compared with the control (unstressed), a sharp decrease of ATPase activity in the plasmodesmata of the stressed cells was observed. Inhibition of energy metabolism in these limited locales would affect the physiological activity, maybe including the regulation of permeability and the change of size exclusion limit (SEL) of plasmodesmata.     

FINE STRUCTURE OF THE ZYGOTE AND EARLY EMBRYO IN QUERCUS GAMBELII

This investigation begins with the late zygote and traces ultrastructural development to the late globular stage of the embryo. Two nucleoli and satellite nucleoli sometimes occur in the zygote nucleus. Mitochondria, dictyosomes, cytoplasmic ribosomes, rough ER, and lipid bodies are numerous in the zygote. Microbodies are occasionally seen. The cell wall becomes well developed before the first division. No plasmodesmata occur in the zygote wall. The basal cell of the proembryo and the suspensor cells of the later embryo have very dense cytoplasm with a high concentration of cytoplasmic ribosomes. The nuclei are very electron opaque. The terminal cell and the cells of the embryo proper have a fine structure similar to that of the zygote. Plastids increase in number, size, starch content, and amount of thylakoid lamellae as the embryo develops. Mitochondria are numerous and appear active at all stages. Dictyosome activity, ribosomal aggregation, and the amount of ER are highest during the late globular stage. Lipid bodies are present up to the early globular stage, then disappear. The inner cell walls of the embryo are thin and have many plasmodesmata. These walls begin to thicken at the late globular stage, and at this time the size of the embryo begins to show an increase over that of the zygote. The results show a corresponding increase in the amount and activity of the metabolic machinery as the development of the embryo progresses. Lipids are probably more important as a nutrient source in the zygote and early embryo; starch becomes more important in the late stages. Absorption of nutrient material into the embryo sac and developing embryo appears to be from the chalazal end.     

渗透胁迫引起玉米根冠处胞间连丝的ATP酶活性下降

利用光镜和电镜研究了短期渗透胁迫下玉米(Zea mays L.)根冠区超微结构和ATP酶活性变化。经历了质壁分离后,在根冠细胞仍然可以观察到许多从“撤退”的周质出发向细胞壁辐射的纤丝。利用ATP酶活性产物(磷酸铅)在原生质膜和细胞壁处沉积的特点,发现这些纤丝是质膜围绕原生质而形成的管状结构;在质壁分离过程中,这些纤丝依然与胞间连丝相连。与对照(未胁迫)相比,受渗透胁迫细胞胞间连丝处ATP酶活性明显下降。能量代谢在局部区段的抑制会影响胞间连丝的生理活性,可能包括胞间连丝的扩散调节能力和分子扩散上限的改变。     

The occurrence of plasmodesmata-like structures in a non-division wall

Summary The fine structure of the interspecific junction in the periclinal chimeraCytisus adami has been investigated. This non-division wall shows the occurrence of pits of normal morphology. The pit membrane is partially penetrated by structures closely resembling plasmodesmata. These plasmodesmata, however, do not show direct continuity across the wall. Connections where they are seen appear to be established by a process of bridging between two half-plasmodesmata. The results are discussed with reference to the reversibility of the differentiation of plasmodesmata and their mode of formation.     

Investigation on Sexual Reproductive Cycle in Torreya grandis

Before May the first, the ovular primordium of Torreya grandis has differentiated. From this early moment the primordium look like the parabolic form and it is surrounded by many pairs of scales, of which a pair of the inner scales are lying at the same level as the primordium of ovule. About May the first of the second year, the differentiation of the various tissues in the ovule has essentially completed. And the fertilization takes place from the end of August to the beginning of September. After overwintering, the proembryo developes into a young embryo in April of the third year, and at the last stage both the seed and the embryo become mature from September to November. In the Taxaceae, the embryogenesis is similar in Amentotaxus,Austrotaxus, Taxus, and Pseudotaxus; their proembryos form cell wall all at the stage of 16-free nuclei and simple polyembryony is common among them. In Torreya, however, the cell wall of proembryo appear at the stage of 4 or 8-free nuclei, and cleavage polyembryony is its feature. On the basis of our observation, the sexual reproductive cycle of Torreya grandis seems to have two important features, one of which is rather long (31 moths from ovular primordium to seed maturity; about 4 months from pollination to fertilization and 7-8 months for development of proembryo). The state of the long sexual cycle in Amentotaxus and Austrotaxus is different from each other; in the former development of young embryo lasts 10-11 months, and in the latter the interval between pollination and fertilization is 13.5 months. The second feature of the sexual cycle in Torreya grandis is over two winters: development of the sporogenous cells in the first, and the proembryo development in the second. From the point of view of phylogenesis, some primitive characters are present in the sexual cycle of Torreya grandis although a specialized feature of the embryogenesis occurs in some degree.     

芡实绒毡层细胞发育的超微结构变化

芡实（ Ｅｕｒｙａｌｅｆｅｒｏｘ Ｓａｌｉｓｂ） 绒毡层细胞在小孢子母细胞时期, 质体出现明显的变形期,细胞中二核常相互贴近或呈嵌合状态, 细胞壁间层中胞间连丝发达。减数分裂期, 绒毡层细胞壁融解消失, 胞间连丝断离, 细胞间发育出现不同步现象。质体开始积累淀粉, 部分质体呈空泡状, 并出现质体膜内陷, 这与液泡具相似的功能。四分体时期, 绒毡层细胞内部结构开始解体。单核小孢子时期, 绒毡层细胞解体消失, 使小孢子后期发育的营养来源受到影响,作者认为这是生产上成熟花粉囊中花粉粒少而且发育不正常的主要原因之一。