Functional anatomy of the ovule in Genlisea with remarks on ovule evolution in Lentibulariaceae

The Lentibulariaceae are highly evolved and specialized carnivorous angiosperms displaying not only unusual morphology and embryology but also specific changes in the genome and chromosomes as large as bacterial chromosomes. Comparative study of the morphology and detailed anatomy of the ovule in the genera Genlisea, Utricularia, and Pinguicula should shed new light on the phylogeny of this family. The clade Genlisea + Utricularia is sister to the genus Pinguicula, which is considered the most primitive taxon within Lentibulariaceae. Thus we should expect the ovules of Genlisea to be more similar to those of the more closely related genus Utricularia than to Pinguicula. Surprisingly, the ovules of Genlisea retain characters (free funiculus, ES remaining in the ovule) in common with Pinguicula, presumably inherited from a common ancestor. Genlisea ovules have only one main character in common with subgenus Polypompholyx (Utricularia): a well-developed funiculus. There are differences between the ovules of the subgenera Genlisea and Tayloria. In subgenus Genlisea the micropyle tends to be closer to the funiculus and the ovule forms an unusual jacket-like nutritive tissue of integumental origin. The most specialized ovules in Lentibulariaceae evolved in the genus Utricularia. The special chalazal nutritive tissue in Genlisea and Utricularia is simply a hypostase.     

Synergids and filiform apparatus in the sexual and apomictic dandelions from section Palustria (Taraxacum, Asteraceae)

An evolutionary trend to reduce “unnecessary costs” associated with the sexual reproduction of their amphimictic ancestors, which may result in greater reproductive success, has been observed among the obligatory apomicts. However, in the case of the female gametophyte, knowledge about this trend in apomicts is not sufficient because most of the ultrastructural studies of the female gametophyte have dealt with amphimictic angiosperms. In this paper, we tested the hypothesis that, in contrast to amphimictic plants, synergids in apomictic embryo sacs do not form a filiform apparatus. We compared the synergid structure in two dandelions from sect. Palustria: the amphimictic diploid Taraxacum tenuifolium and the apomictic tetraploid, male-sterile Taraxacum brandenburgicum. Synergids in both species possessed a filiform apparatus. In T. brandenburgicum, both synergids persisted for a long time without any degeneration, in spite of the presence of an embryo and endosperm. We propose that the persistent synergids in apomicts may play a role in the transport of nutrients to the embryo.     

Some aspects of organization and histochemistry of the embryo sac ofScilla sibirica sato

Summary The present investigation deals with some of the organizational and histochemical aspects of the embryo sac ofScilla sibirica. Both the synergids and egg cell are invested by PAS-positive complete walls. The filiform apparatus comprises an elaborate system of fibrillar projections, showing extensive ramifications. The micropylar region of the embryo sac wall from where the filiform apparatus originates is composed of three distinct layers. On a histochemical basis it may be surmised that, unlike the egg cell, the synergids are metabolically very active. Two kinds of wall ingrowths (i) massive and highly branched very much akin to the filiform apparatus, and (ii) small tuberculate wall projections, are unique to the antipodal cells of S.sibirica. Small tuberculate projections have also been observed along the wall of the central cell adjacent to the nutrient-rich nucellar cells. The antipodals and the central cell show the presence of starch grains and abundant total proteins. All the cell types in the embryo sac ofS. sibirica are structurally so organized as to meet the requirements of its nutrition during pre- and postfertilization development. The presence of abundant PAS-positive granular substance in the cells of nucellar epidermis probably establishes a gradient which assists in the pollen tube growth.     

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.     

OCCURRENCE AND DEVELOPMENT OF A FILIFORM APPARATUS IN THE EGG OF PLUMBAGO CAPENSIS

Micropylar wall extensions in the egg of Plumbago capensis arise as small pegs of periodic acid-Schiff's-positive material soon after the egg is organized. These inward projections of egg wall increase in size, becoming extensive near anthesis. Some branching of the filiform apparatus occurs. In mature embryo sacs the micropylar portion, with the lowermost portion of the egg included, becomes entrenched in the nucellus. The possible significance of this “gametic transfer cell” is discussed in relation to the apparent absence of synergids from the reduced embryo sac of Plumbago.     

Guidance in vitro of the pollen tube to the naked embryo sac of torenia fournieri

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.     

NaCl effect on the distribution of wall ingrowth polymers and arabinogalactan proteins in type A transfer cells of Medicago sativa Gabès leaves

In most species of the Genlisea–Utricularia sister lineage, the organs arising directly after germination comprise a single leaf-like structure, followed by a bladder-trap/stolon, with the lack of an embryonic primary root considered a synapomorphic character. Previous anatomical work suggests that the most common recent ancestor of Utricularia possessed an embryo comprising storage tissue and a meristematic apical region minus lateral organs. Studies of embryogenesis across the Utricularia lineage suggest that multiple primary organs have only evolved in the viviparous Utricularia nelumbifolia, Utricularia reniformis, and Utricularia humboldtii within the derived Iperua/Orchidioides clade. All three of these species are specialized for growth as “aquatic epiphytes” in the tanks of bromeliads, with recent phylogenetic evidence suggesting the possibility that multiple primary organs may have evolved twice independently within this clade. The primary organs of viviparous Utricularia also possess epidermal surface glands, and our study suggests that these may function as root hairs for uptake of solutes from the external environment—a possible adaptation for the “aquatic–epiphytic” habitat.     

EMBRYO SAC LACKING ANTIPODAL CELLS IN ARABIDOPSIS THALIANA (BRASSICACEAE)

Ultrastructure of the embryo sac lacking antipodals in prefertilization stages in Arabidopsis thaliana has been examined 2 hr before and 5 hr after manual cross pollination. The cytoplasm of both synergids before fertilization is rich in ribosomes, mitochondria, and rough endoplasmic reticulum, and also contains several microbodies and spherosomes. The filiform apparatus includes electron-dense material and a fibrous part. Many cortical microtubules appear in the filiform apparatus area. One of the two synergids degenerates before fertilization. The synergids, the egg cell, and central cell have a rich cytoskeleton of microtubules; only the synergids appear to contain microfilaments. At the chalazal end, the antipodals are initially present but degenerate by the time of pollination in most embryo sacs in the starchless line studied. The embryo sac is completely surrounded by a wall containing an electron-dense layer, separating it from the nucellus, including the chalazal end. When the antipodals have degenerated, the electron-dense layer disappears at the chalazal end only, and the wall between the central cell and the nucellus is homogeneous. Between the central cell and nucellar cells no plasmodesmata are found. The membranes of both antipodal cells at the chalazal end of the embryo sac appear sinuous, like those of transfer cells. The central cell has plastids preferentially distributed around the nucleus, but the other organelles are randomly distributed. The central cell in the embryo sac and the adjacent chalazal nucellar cells show a transfer-cell function in the embryo sac after the antipodals degenerate.     

Ultrastructure of the Developing and Fertilized Embryo Sac of Amaranthus hypochondriacus L.

Amaranthus hypochondriacus embryo sac development was investigatedbefore and after fertilization. During the early stages of development,the young embryo sac displays three antipodal cells at the chalazalpole that degenerate very early in the maturation process, beforethe synergids and egg cell are completely differentiated. Themature embryo sac is composed only of the female germ unit.The synergid cells organize a filiform apparatus accompaniedby the presence of mitochondria and dictyosomes with numerousvesicles. The involvement of the synergids in transport andsecretory functions related to pollen tube attraction and guidance,are discussed. The egg cell is located at the micropylar polenear the synergids and displays exposed plasma membranes atthe chalazal pole. The fertilized egg cell does not exhibitmarked changes after fertilization except for the closure ofthe cell wall. The central cell is the largest cell of thisvery long embryo sac. The fused nucleus is close to the eggapparatus before fertilization and displays a remarkable chalazalmigration after gamete delivery. The ultrastructure of the centralcell cytoplasm and the numerous wall ingrowths around this cellsuggest an important role in nutrient transportation. Aftergamete delivery, the embryo sac displays electron dense bodiesthat aggregate within the intercellular space between the synergids,egg cell and central cell. These bodies, that appear in theembryo sac of several plants, are probably involved in gametedelivery for double fertilization. The possibility of biparentalinheritance of mitochondria in this plant is also discussed.Copyright 1999 Annals of Botany Company Amaranthus hypochondriacus, grain amaranth, embryo sac, fertilization.     

Pollen tube penetration and fertilization in Lilium longiflorum (Liliaceae)

The ultrastructure of the embryo sac, nucellus, and parts of the micropyle of Lilium longiflorum were studied both before and after pollen tube penetration to examine the interactions between ovule and pollen tube, using transmission electron microscopy and light microscopy. Before pollen tube penetration the egg cell and two synergids are similar. No filiform apparatus was detected and no synergid degeneration occurs prior to pollen tube penetration. The polar nuclei do not fuse until fertilization. No differences in embryo sac ultrastructure were detected between pollinated ovules unpenetrated by pollen tubes and unpollinated flowers of a comparable age. Shortly after the discharge of the pollen tube two enucleated cytoplasmic bodies with different ribosome densities were observed in the degenerated cytoplasm. These structures border both on the central cell and the egg cell as well as each other and are interpreted as remains of sperm cytoplasm after transmission of sperm nuclei. In the central cell both the sperm nucleus and the polar nuclei are associated with endoplasmic reticulum (ER). ER is thought to be a transport mechanism to achieve contact between the haploid polar nuclei and the sperm nucleus. In the egg cell sperm nucleus alignment is not visibly achieved by ER. The persistent cells of the egg apparatus and the central cell appear to become more metabolically active after pollen tube penetration. Pollen tube penetration already occurs despite the absence of a filiform apparatus and a low level of differences between the cells of the egg apparatus.     

东北地区野豌豆属VICIAL．物种生物学研究Ⅷ．幼苗形体结构动态和种间界限

在野外居群调查的启示下,本文以组件观点对柳叶野豌豆复合种和歪头菜幼苗亚单位的时序变化与开花关系进行了分析。结果发现在柳叶野豌豆复合种栽培居群中存在打破物种间形体结构特征的个体,即在复叶由一对小叶组成的植株就已开花而进入生殖时期。另外,在歪头菜的野生居群中发现由三或四枚小叶组成复叶的个体,因此,我们推测这种形体结构的变化可能暗示着柳叶野豌豆复合种和歪头菜有着共同的祖先。     

Structure of Embryo Sac Before and After Fertilization and Distribution of Transfer Cells in Ovules of Green Gram

The structure of embryo sac before and after fertilization, embryo and endosperm development and transfer cell distribution in Phaseolus radiatus were investigated using light and transmission electron microscopy. The synergids with distinct filiform apparatus have a chalazal vacuole, numerous mitochondria and ribosomes. A cell wall exists only around the micropylar half of the synergids. The egg cell has a chalazally located nucleus, a large micropylar vacuole and several small vacuoles. Mitochondria and plasrids with starch grains are abundant. No cell wall is present at its chalazal end. There are no plasma membranes between the egg and central cell in several places. The zygote has a complete cell wall, abundant mitochondria and plastids containing starch grains. Both degenerated and persistent synergids migh.t serve as a nutrient supplement to proembryo. The wall ingrowths occur in the central cell, basal cell, inner integumentary cells, suspensor cells and endosperm cells. These transfer cells may contribute to embryo nutrition at different developmental stages of embryo.     

Light microscopic detection of PAS-positive substances with thiosemicarbazide in freeze-substituted ovaries of Paspalum longifolium before pollination

Summary Localization of polysaccharides in the freeze-substituted, Eponembedded ovaries of Paspalum longifolium prior to pollination was carried out by periodic acid-Schiff's (PAS), periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-AgPr) and periodic acid-thiosemicarbazide-osmium (PA-TSC-OsO₄) reactions. The specificities of these three reactions were also studied. These three reactions are all effective for light microscopic demonstration of polysaccharides in the filiform apparatus, starch grains in the cells and PAS substance in the micropylar region. Nonspecific staining of the nucleoli of the egg and polar nuclei was observed in the PAS reaction. The PA-TSC-AgPr reaction is very specific for polysaccharides but its overall reaction takes a much longer period of time than the PAS reaction. The PA-TSC-OsO₄ reaction colors the cytoplasm and nuclei of most cells and therefore stains of the cell walls, especially those of the egg cell and synergids, do not stand out clearly. The synergid cytoplasm contains some amorphous polysaccharides and thus it colors even in PAS and PA-TSC-AgPr preparations. In the mature embryo sac, the egg and central cell as well as antipodals are vacuolated but the two synergids have no visible vacuoles under light microscope. Each synergid has a prominent filiform apparatus at the micropylar end, which stains intensely in all three preparations. The walls of the central cell and antipodals adjacent to the nucellar cells have many inward papillae which are also intensely stained in all three preparations. Starch grains are abundant in the ovary wall and usually absent in the nucellus and integuments. They are present in the egg, central cell and antipodals, but not in the two synergids.     

Unusual microtubular cytoskeleton of apomictic embryo sac of Chondrilla juncea L.

Summary. The mature apomictic embryo sac of Chondrilla juncea is highly vacuolated and demonstrates a polarization similar to that of the amphimictic gametophyte. The microtubule cytoskeleton of this embryo sac is uncharacteristic and relatively weak. The microtubules are positioned along cell walls and resemble cortical microtubules of somatic cells. They do not form the parallel, brushlike structures observed around the filiform apparatus of synergids in the amphimictic embryo sac. In the apomictic embryo sac, the microtubules of both the egg cell and the central cell develop a cortical-like structure, which is entirely different from the radial arrangement observed around the nuclei in the amphimictic embryo sac. Correspondence and reprints: Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Kraków, Poland.     

Embryogenesis of Polyembryonic Rice ApⅢ: Structural and Histochemical Studies of Egg Apparatus Around Fertilization

The structural and histochemical changes of the egg apparatus in the polyembryonic rice (Oryza sativa L.), ApⅢ with the highest frequence of additional embryos among the polyembryonic rice investigated, before and after fertilization were studied and compared with those of normal and other polyembryonic rices in a similar developmental period. A total of 2 932 ovules were observed and each of them contained only asingle embryo sac with a set of egg apparatus. Among 1 655 embryo sacs, there were 1 643 embryo sacs (99.27%) with one normal egg apparatus in each embryo sac, and only 12 embryo sacs (0.73%) from the remainder with 4 celled egg apparatus, i.e. two eggs and two synergids. Neither the numerous poly egg apparatus and egg like cells, nor the double set of embryo sacs each containing one egg apparatus and other abnormal egg apparatus in single ovary, which were reported by earlier investigators to have high frequency of embryo production in SB 1 and ApⅣ, were observed. The egg cell was located at the subterminal site of the micropylar end of embryo sac. The cytoplasm of egg cell was rich in protein materials and poly saccharide grains, which did not disappear until the division of zygote. The prominent nucleus was closely surrounded by protein and polysaccharide grains, which did not disappear until the division of zygote. No cytological difference was found between egg cells from the normal and abnormal egg apparatus. The two synergids were fully developed and situated at the upper most part of the micropylar end of the mature embryo sac. In most embryo sacs, the synergids were flask shaped with longer necks, and a widened cap shaped top, in close contact with the micropyle. The synergids had a well developed filiform apparatus. The characteristic appearance of the filiform apparatus as well as the cap neck region of synergids before and after pollen tube penetration were easily distinguishable from the egg cell. The structure, the stainability with Coomassie Brilliant Blue and PAS reaction, the process of accumulation, distribution and disapperance of the cytoplasmic protein materials and polysaccharide grains of the two synergids, the persistent and rarely the receptive synergids before and after pollen tube penetration, were closely similar to those of egg cell of the same developmental stage. In comparison with normal and other polyembryonic rice reported, the size of nucleus and nucleolus and their stainability also strongly resembled those of egg cell. Based on the results observed, the main conclusions are summarized as follows: (1) the additional embryos very frequently developed in the young and mature seed of polyembryonic rice ApⅢ were produced by one or two synergids of normal egg apparatus, rarely by 4 celled egg apparatus; (2) during fertilization, the synergids, in addition to the natural specific function of introducing pollen tube and transferring sperms to egg cell and central cell, could be closely associated with the potentiality to breed one or two additional embryos; and (3) as compared with that of normal or other polyembryonic rice it is firstly disclosed that in a few embryo sacs of ApⅢ, the cytoplasmic and nuclear structure, the active anabolism and catabolism of protein and polysaccharide materials and the delayed disorganization at the mid basal region of the receptive and persistent synergid still remained unchanged before the division of zygote. Such salient features could be the predisposition for the origin of additional embryos in ApⅢ.     

A PERIODIC ACID-SCHIFF'S SUBSTANCE RELATED TO THE DIRECTIONAL GROWTH OF POLLEN TUBE INTO EMBRYO SAC IN PASPALUM OVULES

The presence of a periodic acid-Schiff's (PAS) substance at the micropylar portion of the ovules of Paspalum orbiculare and three other species of Paspalum has been revealed by improved cytological techniques. The substance is located in the distal portion of the outer integument, especially at its edges, and in the micropyle, which is formed by the inner integument. A study of the origin of this substance in P. orbiculare indicates that it is produced from the cellular material of the dissolved cells in situ. The PAS substance is highly water soluble and may contain some protein. The final directional growth of the pollen tube into the filiform apparatus of the degenerated synergid is related to this substance. The role of the synergids in its formation is discussed.     

Flower palate ultrastructure of the carnivorous plant <Emphasis Type="Italic">Genlisea hispidula</Emphasis> Stapf with remarks on the structure and function of the palate in the subgenus <Emphasis Type="Italic">Genlisea</Emphasis> (Lentibulariaceae)

In the genus Genlisea as well as in its sister genus Utricularia, the palate probably plays a key role in providing the colour, mechanical and olfactory stimuli to attract insect pollinators and to guide them to the generative structures and the nectary spur. However, information about the micro-morphology of the palate of Genlisea is scarce. This study aims to examine the structure of the palate in Genlisea hispidula in detail as well as the palate from other five species from the subgenus Genlisea. In particular, its aim is to ascertain whether these palates function as an area for the osmophores in the flower or whether they produce nectar. We showed that the palate in all of the species that were examined was the glandular type and that it had capitate, glandular trichomes, which had a similar general architecture across the species that were examined. No nectar secretion was observed on the palates. The ultrastructure of the palate trichomes showed that the palate glandular trichomes most probably function as scent glands that produce an olfactory stimulus for flower pollinators.     

Structure,ultrastructure, and histochemistry of the pollen tube pathway in the milkweed Asclepias exaltata L.

The structure of the gynoecium and pollen tube pathway in unpollinated and pollinated carpels of Asclepias exaltata L. has been characterized. Pollen tubes penetrate a dry-type stigma, grow intercellularly in a core of solid tissue in the upper style, and subsequently traverse a hollow stylar canal to the ovary where they grow across the placental epithelium to the ovule micropyles. The fine structural characteristics of transmitting cells of the solid style, stylar canal, and placental epithelium indicate a secretory function. Extracellular secretions staining positively for proteins, insoluble carbohydrates, and arabinogalactans/arabinogalactan proteins are present in the solid style, hollow stylar canal, ovary, and micropyle. Micropylar exudate is present subtending the extended cuticle of the embryo sac adjacent to the filiform apparatus of the synergids, providing ultrastructural evidence for a secretion arising from the angiosperm embryo sac.     

CAPSELLA EMBRYOGENESIS: THE SYNERGIDS BEFORE AND AFTER FERTILIZATION

The ultrastructure and composition of the synergids of Capsella bursa-pastoris were studied before and after fertilization. The synergids in the mature embryo sac contain numerous plastids, mitochondria, dictyosomes and masses of ER and associated ribosomes. Each synergid contains a large chalazal vacuole, a nucleus with a single nucleolus and is surrounded by a wall. This wall is thickest at the micropyle end of the cell where it proliferates into the filiform apparatus. At the chalazal end of the cell the wall thins and may be absent for small distances. The pollen tube grows into one of the two synergids through the filiform apparatus and extends one-third the length of the cell before it discharges. Following discharge of the pollen tube, mitochondria and plastids of the tube can be identified in the synergid as can hundreds of 0.5 μ polysaccharide spheres liberated by the tube. The method by which the sperm or sperm nuclei enter the egg or central cell is not known although an apparent rupture was found in the wall of the egg near the tip of the pollen tube. The second synergid changes at the time the pollen tube enters the first synergid. These changes result in the disorganization of the nucleus and loss of the chalazal wall and plasma membrane. Eventually this synergid loses its identity as its cytoplasm merges with that of the central cell.