Calcium changes in ovules and embryo sacs of Plumbago zeylanica L.

Calcium was localized in ovules of Plumbago zeylanica from 1 day before anthesis to 3 days after anthesis using potassium antimonate and transmission electron microscopy in pollinated and emasculated flowers. At 1 day before anthesis, embryo sacs (containing an egg cell, a central cell and zero to three accessory cells) appear mature and contain abundant calcium precipitates (ppts), in contrast to nucellar cells. At anthesis, the vacuoles of nucellar cells have enlarged, and micropylar cells, in particular, are heavily labeled with calcium ppts. As pollen tubes elongate through ovular tissues, ppts diminish in ovular cells and become concentrated in the pollen tube cell wall. After fertilization, the calcium ppts sharply diminish in fertilized ovules; in unfertilized ovules, calcium ppts remain abundant up to 3 days after anthesis (when unfertilized ovules are shed). The distribution of calcium in the ovule changes in apparent response to fertilization, suggesting that calcium content may be related to the attraction and receipt of the pollen tube. In contrast with conventionally-organized embryo sacs with synergids, Plumbago accumulates calcium in the egg cell. Received: 30 December 1999 / Revision accepted: 24 March 2000     

Calcium in the synergid cells and other regions of pearl millet ovaries

Summary The synergids and other cells of mature, unpollinated pearl millet ovaries were investigated using: (1) freeze-substitution fixation in conjunction with scanning electron microscope observations and energy-dispersive X-ray microanalysis to localize total calcium (Ca) and other elements, and (2) antimonate precipitation to selectively localize loosely sequestered, exchangeable calcium (Ca⁺⁺). In freeze-fixed ovaries, the synergid cells, ovary wall, nucellus, and other regions of the ovary displayed, respectively and relatively, extremely high, high, moderate, and low levels of Ca. In antimonate-fixed ovaries, Ca-containing antimonate precipitates exhibited similar distribution patterns. In ovaries fixed using the conventional 2% (w/v) antimonate in fixatives, the synergids were disrupted due to precipitate overload. In the ovary wall, precipitates were mainly located in the intercellular spaces. Some precipitates were observed at the micropyle and along the outer ovule integument, associated with diffuse extracellular material, and in the cell walls of nucellar cells proximal to the micropyle. Examination of precipitate distribution inside the synergids was possible in ovaries fixed using 0.5% (w/v) antimonate in the fixatives. Cytoplasmic organelles of all synergids examined exhibited variable states of disintegration. The amount of precipitates associated with the degenerated organelles appeared to be proportional to the degree of their degeneration. Distinct precipitates were localized in contiguous regions of the nucellar cells fused with the embryo sac, the micropylar half of the embryo sac wall, and the filiform apparatus. The results are discussed in relation to the involvement of Ca⁺⁺ in mediating the functions of synergid cells during fertilization in angiosperms.On Specific Cooperative Agreement 58-43YK-8-0026 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Changes of calcium distribution in ovules ofTorenia fournieri during pollination and fertilization

Summary Calcium distribution in ovules ofTorenia fournieri was studied by electron energy loss spectroscopy and transmission electron microscopic visualization of calcium antimonate precipitates. High calcium levels were found in the ovules ofT. fournieri. Calcium is situated mainly in extracellular regions before fertilization, including the surface of embryo sac, in the mucilage, and among the cells of the egg apparatus. Intracellular calcium was found only in the nucellar cells around the embryo sac and in the epidermis of the central axis and funiculus. After pollination, a labyrinthine structure (coralloid-like cell wall formation) develops on the micropylar surfaces of the egg apparatus that contain high levels of calcium. Calcium levels increase in the degenerating synergid after the penetration of the pollen tube. Calcium-antimonate precipitates are abundant in vacuoles of the disrupted synergid and pollen tube cytoplasm.Abbreviations EELS electron energy loss spectroscopy - EDX energy-dispersive X-ray microanalysis - LS labyrinthine structure     

Ovary-ovule transmitting tract in Crocus (Iridaceae), structure and calcium distribution

 The structure of and the calcium distribution in the ovary-ovule transmitting tracts of Crocus were examined by using conventional and antimonate fixation methods for electron microscopy, and fluorescence microscopy as well. The ovarian locules are bordered with epidermal cells and transmitting cells. The epidermal cells are extensively cutinized, and the cytoplasm is rich in the organelles and vesicles prevalently engaged in thickening the exposed wall. The transmitting cells have uncutinized walls with numerous labyrinthine ingrowths, and the cytoplasm is rich in organelles and vesicles engaged in the secretion of the extracellular matrix (e.m.). The micropylar tissue of the ovule consists of largely vacuolated cells with scarcely structured cytoplasm, the e.m. appears to be of gametophytic origin. Both the ovary and ovule e.m. have a glycosidic and proteinic nature, and are rich in loosely bound Ca²⁺ ions. In the embryo sac, Ca²⁺ ions are concentrated in one synergid. The results are discussed in relation to the guided growth of pollen tubes. Received April 9, 2002; accepted July 9, 2002 Published online: November 28, 2002 Addresses of the authors: Giuseppe Chichiriccò (e-mail: Chichiri@univaq.it), Dipartimento di Scienze Ambientali, University of L'Aquila, Via Vetoio, I-67100 L'Aquila, Italy. Anna Maria Ragnelli, Pierpaolo Aimola, Dipartimento di Biologia di Base ed Applicata, University of L'Aquila, Via Vetoio, I-67100 L'Aquila, Italy.     

莴苣助细胞发育过程中钙的分布研究

用焦锑酸盐沉淀法对莴苣助细胞中的钙分布进行了观察。结果表明,开花前3天刚形成的助细胞中的钙颗粒很少:开花前2天助细胞壁中的钙颗粒增加;开花前1天助细胞珠孔端细胞壁加厚,其中积累了许多钙颗粒:开花当天助细胞珠孔端的丝状器中聚集了大量的钙颗粒。授粉后1h时两个助细胞的结构和钙分布发生差异,一个呈退化状,其中的钙颗粒明显增多,另一宿存助细胞中的钙分布与授粉前相似。去雄不授粉1天后两个助细胞均保持完好,且两助细胞中的钙分布没有明显差异,表明由花粉管引起一个助细胞中钙含量增加进而导致了助细胞退化。退化助细胞在卵细胞与中央细胞之间形成一薄层。助细胞退化后不同部位的钙颗粒呈现出与受精作用密切有关的变化:授粉后1h时,钙主要聚集在近合点端部位;授粉后2.5h卵细胞即将受精,这时许多细小的钙颗粒主要聚集在卵细胞与中央细胞之间的薄层中;授粉后4h精、卵细胞已融合,这时退化助细胞合点端的钙颗粒明显减少,而在其珠孔端又聚集了较多的钙。上述助细胞中的钙含量变化与吸引花粉管进入胚囊和促使精卵细胞融合密切有关。     

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

 Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus. Received: 20 January 1999 / Revision received: 12 July 1999 / Accepted: 17 August 1999     

Prolongation of Embryo Sac Viability in Pear (Pyrus communis) Following Pollination or Treatment with Gibberellic Acid

Embryo sac development has been investigated in unpollinated,cross pollinated and gibberellic acid (GA₂) treated flowersof Pyrus communis L. While pollination and GA₃ treatments donot alter embryo sac development, they prolong embryo sac viability.In untreated unpollinated flowers, ovules degenerate between12 and 21 d after anthesis, while in cross pollinated and GA₃treated flowers this degeneration is postponed by about 10 d.Thus, in a cross pollinated flower this extends the period overwhich a successful fertilization can take place. This increasedperiod of viability is accompanied by an elongation of the embryosac itself. Elongation takes place two weeks prior to fertilizationin cross pollinated flowers. The extension of life span of embryo sacs following pollinationand treatment with gibberellic acid indicates that a stimulusinduced by ‘pollination’ could be mediated by GA₃Whatever its mechanism of operation, the prolongation of embryosac viability by pollination represents a selective advantage,in that the period at which the ovules are receptive to fertilizationmust be significantly extended. Embryo sac, gibberellic acid, Pyrus communis, pear, pollination     

烟草花粉萌发和花粉管生长期间柱头和花柱中的钙分布

烟草柱头表面有两层覆盖物,其中含有少量细小的钙颗粒.花粉落到柱头上后,储存在花粉外壁中的钙被释放到覆盖层中.当花粉管穿过覆盖层长入柱头细胞之间时,花粉管顶端的细胞壁中出现了大量的细小钙颗粒.开花后22 h观察时,在花柱引导组织中形成了钙的梯度分布:花柱上部引导组织中的钙较少,而下部连接子房处的花柱引导组织中含有较多的钙颗粒.去雄花开花后1 d时,花柱上部引导组织中的钙明显增多;3 d时,连柱头细胞中也出现了较多的钙颗粒.讨论了烟草花柱引导组织中钙梯度分布和花粉管生长的关系.     

Polar distribution dynamics of Con A binding sites in embryo sacs is temporally coupled by the fertilization process

The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.     

芒苞草形态学和胚胎学研究：Ⅱ.花药和胚珠发育的研究

芒苞草成熟胚珠为倒生型,薄珠心,双珠被。胎座为侧膜胎座向中轴胎座的过渡类型。胚囊发育为单孢蓼型。 成熟胚囊由印器,具二极核的中央细胞及三个反足细胞组成。助细胞呈倒梨形,极性不明显,珠孔端壁有角状的丝状器。中央细胞的二极核在受精前融合为次生核。 花药具二个小孢子囊,花药壁层为单子叶型,具分泌型绒毡层,小孢子母细胞减数分裂时,胞质分裂为连续型,四分体是左右对称式排列,成熟花粉粒为二细胞的。 在花药与胚珠发育过程中,多糖物质的消长是有规律的变化。     

Calcium changes and the response to methyl jasmonate in rice lodicules during anthesis

Summary. Potassium pyroantimonate precipitation was used to locate loosely bound calcium in rice (Oryza sativa L.) lodicules before and after anthesis, and flowering of panicles was accelerated by treatment with methyl jasmonate. From 1 day to 4 h before anthesis, the number of calcium precipitates in the cell walls and vacuole membranes decreased gradually, whereas they increased remarkably in the cytoplasm and nucleolus. At the beginning of anthesis, the number of calcium granules in lodicules reduced sharply, but there was a large accumulation of flocculent precipitates in the vacuoles. After anthesis, the flocculent precipitates decreased in number until they disappeared, whereas the granular precipitates started to accumulate once again. The rice florets treated with 2 mM methyl jasmonate were induced to open within 10–30 min and they then closed 0.5–1 h later. The nucleolus, cytoplasm, and vacuole membrane of the lodicule cells contained many calcium granules during flowering, although the cell walls lacked calcium. At 1 h after treatment, the number of calcium granules had decreased, while flocculent precipitates were regularly observed in the nondegenerated cells. At 6 h after treatment, calcium grains started to reappear in the cell walls. These changes in calcium precipitates before and after anthesis indicate that the opening and closing of florets correlates with the calcium level in lodicule cells. In addition, excised panicles, with florets judged to be nearing anthesis, were soaked in 2–200 mM EGTA solution for 2 min after treatment with 2 mM methyl jasmonate. The results indicate that EGTA had an antagonistic effect on the methyl jasmonate-induced floret opening in rice. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Distribution of calcium in the stigma and style of tobacco during pollen germination and tube elongation

Potassium antimonate was used to locate loosely bound calcium in the stigma and style of tobacco. The tobacco stigma is wet and covered by a thick layer of glycoprotein exudate at anthesis. The exudate contains abundant vesicles, which are densely labeled with calcium precipitates. When pollen grains arrive at the stigma, become hydrated, and as the pollen swells, Ca²⁺ precipitates accumulate at the aperture. Calcium precipitates that accumulate in pollen cytoplasm are initially concentrated within small vacuoles, but as germination proceeds these appear to fuse, forming prominent, densely labeled vesicles that preferentially accumulate near the proximal region of the growing tube. Although the stigma has abundant particles, few calcium precipitates are observed in the transmitting tissue from anthesis to 11 h after pollination. However, at 22 h after pollination, accumulation of calcium increases distally from the stigmatic interface with the transmitting tissue through the length of the style to the ovary. An examination of flowering plants with differing floral biology will be needed to understand the role of loosely bound calcium accumulation and its relationship to tissue-level changes in calcium uptake, maintenance of other calcium pools, including [Ca²⁺]_cyt, and in pollen and style maturation during the progamic phase.     

Calcium changes during megasporogenesis and megaspore degeneration in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Potassium pyroantimonate was used to localize loosely-bound calcium in young ovules of lettuce (Lactuca sativa L.) during megasporogenesis to investigate the relationship between ionically available calcium and megaspore degeneration. At the megasporocyte (megaspore mother cell) stage, few calcium precipitates were located in the ovule. Following meiosis in the megasporocyte, a linear tetrad of four megaspores is formed, with three of the four megaspores degenerating from the micropylar end inward. Only the chalazal-most megaspore continues to develop, becoming the functional megaspore. A decrease in amount of calcium precipitates in the megaspore, particularly in the nucleus, precedes the breakdown of the micropylar megaspores, which subsequently undergo structural disintegration and loss of recognizable cellular features. A partial recovery of calcium precipitates occurs during later degeneration. The functional megaspore retains a consistently higher concentration of calcium precipitates during development, which is retained in the developing embryo sac. This, to our knowledge, is the first report related to calcium dynamics during megaspore degeneration, and may facilitate future research aimed at elucidating the mechanisms of megasporogenesis.     

Optimization of conditions for in situ hybridization and determination of the relative number of ribosomes in the cells of the mature embryo sac of maize

Summary We have initiated experiments to understand the molecular regulation of embryo sac development in flowering plants by a study of ribosome synthesis and accumulation. Because of the very small size of the embryo sac and the large volume of ovule tissue it is embedded in, in situ hybridization with nucleic acid probes is presently the only practical method for such molecular measurements on individual cells of the embryo sac. Methods of tissue preparation, sectioning and screening of ovules for embryo sac containing sections, in situ hybridization using a ribosomal mRNA probe, and staining were optimized. Relative densities of silver grains for individual cells of the mature maize (W22) embryo sac were determined from in situ hybridizations. The silver grain counts are directly related to the numbers of ribosomes. Volumes of individual cells were determined by confocal microscope image analysis, and this permitted the calculation of the relative total numbers of ribosomes in individual cells of the embryo sac and nucellus. The central cell has a volume 260 times that of a nucellar cell at the micropylar end of the ovule, 15 times that of the egg cell, 30 times that of a synergid, and 130 times the volume of an antipodal cell. The mature maize embryo sac has 20 or more antipodal cells. The central cell has approximately 200 times the number of ribosomes as are present in a nucellar cell, about 7 times as many ribosomes as are in the egg cell, 14 times as many ribosomes as in each synergid, and about 80 times the ribosome content of individual antipodal cells. The data are discussed with respect to the utilization of the ribosomes following fertilization in the early embryo and endosperm.     

水稻雌蕊与胚囊中钙的超微细胞化学定位

用焦锑酸盐沉淀法对水稻（OryzasativaL．）授粉前后雌蕊和胚囊中的钙进行了超微细胞化学定位。结果表明,柱头乳突细胞表面和花柱薄壁细胞中均含钙沉淀;开花前1d,整个胚囊中含钙较少,两个助细胞中钙分布无差异;临近开花时,1个助细胞已退化,其钙含量明显增加;开花后6h,胚囊已受精,退化助细胞中钙含量进一步增加;受精前卵细胞中钙主要分布在液泡中,核和胞质中较少;受精后,其钙含量明显增加,主要分布于核中。重点讨论了钙与助细胞退化和卵细胞激活的关系。     

Symplastic communication between the central cell and the egg apparatus cells in the embryo sac of Torenia fournieri Lind. before and during fertilization

 Various membrane-impermeable, water-soluble fluorescent tracers with different molecular weights were microinjected into the central cell of the embryo sac of Torenia fournieri Lind. before and during fertilization. Before anthesis, there was high symplastic permeability between the central cell and the egg apparatus cells. In this stage, fluorescent tracers up to 10 kDa could pass from the central cell into the egg apparatus cells, whereas those with larger molecular weight remained in the central cell. As the embryo sac matured, symplastic permeability decreased such that 2 d after anthesis only tracers less than 3 kDa could spread from the central cell into the egg cell. There appeared to be no symplastic permeability between the primary endosperm and zygote after fertilization, since tracers as small as 521 Da could not pass into the zygote in about half of the microinjected embryo sacs. This is the first report of a change in cell-to-cell communication among the cells of the female germ unit before and after fertilization. Received: 16 December 1999 / Accepted: 4 February 2000     

CHANGES IN MEGAGAMETOPHYTE STRUCTURE IN PAPAVER NUDICAULE L. (PAPAVERACEAE) FOLLOWING IN VITRO PLACENTAL POLLINATION

Papaver nudicaule placentae with attached ovules were dissected out of unpollinated gynoecia 1–3 days after anthesis, dusted with pollen, and cultured on modified Nitsch's growth medium at 23 C. Ovules were removed from expiants at 15, 24, 31, and 48 hr postpollination, fixed in GA-OsO₄, embedded in Spurr's resin and sectioned (1.0 μm) for light microscopy. Placentae, 15 hr after pollination, were fixed and processed for scanning electron microscopy. Pollen germinates within 1 hr. Although most pollen tube growth appears random, there is directional growth toward the micropyle. The crassinucellate ovule contains an embryo sac consisting of three antipodals, two polar nuclei, and an egg apparatus composed of two synergids and a polarized egg having a large chalazal vacuole and micropylar nucleus. Pollen tube access into the megagametophyte is through a degenerate synergid, with fertilization occurring between 24 and 31 hr after pollination. Zygote establishment is accompanied by polarity reversal in which the nucleus assumes the chalazal position subtended by a large micropylar vacuole. Fertilized ovules normally develop into germinable seeds.     

Ultracytochemical Localization of Calcium in Micropyle and Embryo Sac of Brassica napus Before and After Pollination

Potassiam antimonate was used to localize Ca2+ in the micropyle and embryo sac of Brassica napus L. before and after pollination. To identify the nature of the pyroantimonate deposits, energy-dispersive X-ray microanalysis (EDXA) was employed and the deposits were proved to contain calcium pyroantimonate. Image processing system was employed to measure the volume density and the diameter of the deposits. Before and after pollination, calcium was more abundant in the exostome and endostome as compared with the other regions of the integuments, and was concentrated at the apoplast system, i.e. the intercellular matrix of the micropyle canal and the cell wall. Before pollination, each of the two sister synergids accumulated more calcium than the other embryo sac cells. Although the mean diameter of the deposits in the synergid was only two-thirds as that in the egg cell and central cell, the volume density of the deposits in the synergid was about 2.5 times and 1.9 times as that in the egg cell and the central cell respectively. The filiform apparatus and the nucleus had the most abundant calcium within a synergid. After pollination both sister synergids degenerated conspicuously and were characterized by much more deposited calcium (about 2.4 times more than before); and the diameter of the deposits decreased dramatically, which was less than one-third as before. The relationship between calcium distribution and synergid degeneration as well as its functions was discussed.     

Callose in the walls of mature embryo sac ofTorenia fournieri

Summary During the course of a fluorescence microscopic investigation on the extra-ovular micropylar portion of the embryo sacs ofTorenia fournieri Lind. (Scrophulariaceae) a callosic wall was found which surrounded it almost completely until the time of anthesis. In addition, the walls of young synergids and the filiform apparatus also showed callosic fluorescence. Treatments with PAS reaction revealed a PAS-positive substance filling up the locular cavity. Our attempts to induce fluorochromasia by employing fluorescein diacetate failed, indicating the low permeability of the callosic wall around the embryo sac. It is assumed that the callose wall around the embryo sac isolates the latter from the contents of the locular cavity whereas the callose in the synergid walls may represent an intermediate stage in the maturation of these walls; the filiform apparatus is mainly composed of callose.     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsis binata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料.龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%.胚囊发育属大黍型.多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%.胚珠内没有有性胚囊的发育.胚的发生有两种类型:(1)早发生胚(74%),开花前1～2 d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2～3 d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚.存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%.胚乳由极核不经受精自发分裂产生.