Localisation pattern of homogalacturonan and arabinogalactan proteins in developing ovules of the gymnosperm plant Larix decidua Mill

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.     

Arabinogalactan proteins as molecular markers in Arabidopsis thaliana sexual reproduction

Some of the most important changes that occur in plants during sexual reproduction involve the transition from a sporophytic to a gametophytic type of development. In this paper, these changes were evaluated for Arabidopsis thaliana. The results obtained clearly show differences in the pattern of distribution of specific arabinogalactan protein (AGP) sugar epitopes, during anther and ovule development. AGPs are hydroxyproline-rich glycoproteins that are massively glycosylated and ubiquitous in plants. The molecular mechanism of action of AGPs is still unknown, mainly due to the difficulties posed by the complex saccharide chains. However, the complex structure of the sugar fraction of AGPs makes them a potential source of signalling molecules. The selective labelling obtained with AGP mAbs JIM8, JIM13, MAC207, and LM2, during Arabidopsis pollen and pistil development, suggests that some AGPs can work as markers for gametophytic cell differentiation. Specific labelling of the first gametophytic cells in the pistil, the strong labelling of the secretory cells of the embryo sac, the synergid cells, and the labelling of the integument micropylar cells, apparently outlining the pollen tube pathway into its final target, the embryo sac, have all been shown. In the anthers, the specific labelling of gametophytic cells, and of the male gametes that travel along the pollen tube, may indicate AGP epitopes acting as signals for the pollen tube to reach its final destiny. The specific labelling of cells destined to go into programmed cell death is also discussed.     

MICROPYLAR EXUDATE IN GASTERIA (ALOACEAE) AND ITS POSSIBLE FUNCTION IN POLLEN TUBE GROWTH

The micropylar exudate of Gasteria verrucosa (Mill.) H. Duval was studied using light and electron microscopic techniques. Ovules may contain micropylar exudate before stigma receptivity. During successive phases of stigma receptivity, the number of ovules with micropylar exudate and the amount of micropylar exudate per ovule increases. At the late phase of stigma receptivity, large amounts of micropylar exudate with a smooth to cauliflowerlike appearance were observed. Micropylar exudate is viscous and contains, among other components, proteins and carbohydrates. At all stages of the stigma investigated, ovules situated at the base of the ovary contain a larger quantity of micropylar exudate than those at the top. The appearance of micropylar exudate is related to the degree of development of the embryo sac and it originates primarily from the filiform apparatus. It is assumed that an uptake of water by the ovule initiates the outflow of micropylar exudate from the filiform apparatus into the micropyle. Both ovular pollen tube ingrowth and seed set mark the optimum pollination stage of the stigma, which for both events lies around the onset of stigma receptivity. When pollen tubes have reached the ovary, young micropylar exudate stimulates their growth rate. The presence of micropylar exudate seems to be a requirement for pollen tube penetration, and an interaction between the pollen tube and the micropylar exudate has been proposed. Possibly, the micropylar exudate serves as a nutritional source and, in an optimum condition, as an attractant for approaching pollen tubes.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis

Some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The specific labelling of the synergid cells and its filiform apparatus, which are the cells responsible for pollen tube attraction, and also the specific labelling of the micropyle and micropylar nucellus, which constitutes the pollen tube entryway into the embryo sac, are quite indicative of this role. We also discuss the possibility that AGPs in the sperm cells are probably involved in the double fertilization process.Key words: Arabidopsis, arabinogalactan proteins, AGP 6, gametic cells, pollen tube guidanceThe selective labelling obtained by us with monoclonal antibodies directed to the glycosidic parts of AGPs, in Arabidopsis and in other plant species, namely Amaranthus hypochondriacus,¹ Actinidia deliciosa² and Catharanthus roseus, shows that some AGP molecules or their sugar moieties are probably related to the guidance of the pollen tube into the embryo sac, in the final part of its pathway, when arriving at the ovules. The evaluation of the selective labelling obtained with AGP-specific monoclonal antibodies (Mabs) JIM 8, JIM 13, MAC 207 and LM 2, during Arabidopsis pollen development, led us to postulate that some AGPs, in particular those with sugar epitopes identified by JIM 8 and JIM 13, can be classified as molecular markers for generative cell differentiation and development into male gametes.Likewise, we also postulated that the AGP epitopes recognized by Mabs JIM 8 and JIM 13 are also molecular markers for the development of the embryo sac in Arabidopsis thaliana. Moreover, these AGP epitopes are also present along the pollen tube pathway, predominantly in its last stage, the micropyle, which constitutes the region of the ovule in the immediate vicinity of the pollen tube target, the embryo sac.³We have recently shown the expression of AGP genes in Arabidopsis pollen grains and pollen tubes and also the presence of AGPs along Arabidopsis pollen tube cell surface and tip region, as opposed to what had been reported earlier. We have also shown that only a subset of AGP genes is expressed in pollen grain and pollen tubes, with prevalence for Agp6 and Agp11, suggesting a specific and defined role for some AGPs in Arabidopsis sexual reproduction (Pereira et al., 2006).⁴Therefore we continued by using an Arabidopsis line expressing GFP under the command of the Agp6 gene promoter sequence. These plants were studied under a low-power binocular fluorescence microscope. GFP labelling was only observed in haploid cells, pollen grains (Fig. 1) and pollen tubes (Fig. 2); all other tissues clearly showed no labelling. These observations confirmed the specific expression of Agp6 in pollen grains and pollen tubes. As shown in the Figures 1 and ​and2,2, the labelling with GFP is present in all pollen tube extension, so probably, AGP 6 is not one of the AGPs identified by JIM 8 and JIM 13, otherwise GFP light emission would localize more specifically in the sperm cells.⁵ So we think that MAC 207 which labels the entire pollen tube wall (Fig. 3) may indeed be recognizing AGP6, which seems to be expressed in the vegetative cell. In other words, the specific labelling obtained for the generative cell and for the two male gametes, is probably given by AGPs that are present in very low quantities, apparently not the case for AGP 6 or AGP 11.Open in a separate windowFigure 1Low-power binocular fluorescence microscope image of an Arabidopsis flower with the AGP 6 promoter:GFP construct. The labelling is evident in pollen grains that are being released and in others that are already in the stigma papillae.Open in a separate windowFigure 2Low-power binocular fluorescence microscope image of an Arabidopsis ovary with the AGP6 promoter:GFP construct. The ovary was partially opened to show the pollen tubes growing in the septum, and into the ovules. The pollen tubes are also labelled by GFP.Open in a separate windowFigure 3Imunofluorescence image of a pollen tube growing in vitro, and labeled by MAC 207 monoclonal antibody. The labelling is evident all over the pollen tube wall.After targeting an ovule, the pollen tube growth arrests inside a synergid cell and bursts, releasing the two sperm cells. It has recently been shown that sperm cells, for long considered to be passive cargo, are involved in directing the pollen tube to its target. In Arabidopsis, HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to the ovules.⁶ The same could be happening with the AGPs identified in the sperm cells by JIM 8 and JIM 13. We are now working on tagging these AGPs and using transgenic plants aiming to answer to such questions.Pollen tube guidance in the ovary has been shown to be in the control of signals produced by the embryo sac. When pollen tubes enter ovules bearing feronia or sirene mutations (the embryo sac is mutated), they do not stop growing and do not burst. In Zea mays a pollen tube attractant was recently identified in the egg apparatus and synergids.⁷ Chimeric ZmEA1 fused to green fluorescent protein (ZmEA1:GFP) was first visible within the filiform apparatus and later was localized to nucellar cell walls below the micropylar opening of the ovule. This is the same type of labelling that we have shown in Arabidopsis ovules, using Mabs JIM 8 and JIM 13. We are now involved in the identification of the specific AGPs associated with the labellings that we have been showing.     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

Localization of arabinogalactan proteins in anther,pollen, and pollen tube of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Summary. Western blot analysis indicated the presence of two epitopes recognized by the anti-arabinogalactan protein antibodies JIM13 and LM2 and the absence of the JIM4 epitope in mature tobacco anthers. Immunoenzyme localization of arabinogalactan proteins (AGPs) with JIM13 showed that AGPs accumulate mainly at the early stages of anther development. AGP content and distribution were also investigated at the ultrastructural level in pollen tubes grown in vivo and in vitro. Abundant AGPs were present in the transmitting tissue of styles, and the AGP content of the extracellular matrix changed during pollen tube growth. In pollen tubes, immunogold particles were mainly distributed in the cell wall and cytoplasm, especially around the peripheral region of the generative-cell wall. β-D-Glucosyl Yariv reagent, which specifically binds to AGPs, caused slow growth of pollen tubes and reduced immunogold labeling of AGPs with JIM13 in vitro. These data suggest that AGPs participate in male gametogenesis and pollen tube growth and may be important surface molecules in generative and sperm cells. 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.     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca²⁺ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca²⁺ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate. Received: 14 August 1996 / Accepted: 9 October 1996     

Ovary signals for directional pollen tube growth

In angiosperms, the female gametophyte has a secluded life; it is protected by several concentric layers that envelop each other. The embryo sac is surrounded by the nucellus, which in turn is wrapped by the integuments forming the ovule, which is nested in the ovary. These wrappings are not hermetic, but contain little ”gates” the pollen tube must traverse on its way towards the embryo sac. Information is emerging that shows that the ovary and ovule provide signals orienting and directimng the pollen tube on the right course. There are three main bodies of evidence supporting this hypothesis. One relates to developmental changes in the female tissues and how they affect pollen tube growth. The second refers to defective ovule mutants, which induce defective pollen tube guidance. And the third relates to the possible molecules involved in this signalling. Here, information gathered along these three main lines of evidence is reviewed. All converge to the conclusion that different checkpoints exist all along the pollen tube pathway. These checkpoints provide active signalling that guides the pollen tube to its destination, the embryo sac. Received: 15 December 2000 / Accepted: 13 June 2001     

Structural characteristics of the mature embryo sac of Camellia oleifera

The ovule is the most important reproductive organ in the pistil of phanerogamae. Camellia oleifera (Theaceae) is an important woody plant producing edible oil in southern China, and its embryo sac structure has a positive effect on seed breeding. In this study, the microstructure, ultrastructure and three‐dimensional structure of the ovule and embryo sac of C. oleifera were observed and described based on a combination of advanced microscopy techniques (SEM, TEM, CLSM). The ovule comprises the inner and outer integument. Large quantities of secretions in the micropylar canal exit and may participate in the guidance of the entry of the pollen tube into the embryo sac. The synergids have a dense cytoplasm, abundant organelles, and strong polarity. Little cytoplasm is present in the egg cell, yet there are many vacuoles. The center of the cell is taken up by a large vacuole, and the cytoplasm is pushed towards the edges to form obvious cytoplasmic cords. The two polar nuclei are large and conspicuous. The antipodal cells degenerate to fulfill a nutritional function.     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde     

Postpollination-prezygotic ovular secretions into the micropylar canal inPseudotsuga menziesii (Pinaceae)

Ovular morphology was examined ultrastructurally inPseudotsuga menziesii to determine the effects of the ovule on pollen development. Vesicles containing lipid-like substances traverse cell walls of the inner epidemis of the integument and release their contents at the integument surface to form the integumentary membrane. A major aqueous secretion from the integument into the micropylar canal is proposed to occur by the movement of the integumentary membrane and its invaginations towards the center of the micropylar canal. The cellular degeneration of the nucellar apex results from the breakdown of vacuoles. After this degeneration, electron-dense substances move from the prothallial cells of the female gametophyte towards the nucellus, and many morphological changes in the nucellus, prothallial cells, and micropylar canal take place simultaneously. We interpret these changes to result from another major secretion from the prothallial cells. Egg cytoplasm appears to disorganize for a short time. Simultaneously, substantial amounts of electron dense-substances in the prothallial cells and lipid-like substances in surface cell walls of the female gametophyte move towards the nucellus as components of the third major secretion.     

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.     

Pollen movement in the micropylar canal ofLarix and its simulation

InLarix pollen captured by the ovule and rested at the distal end of the micropylar canal is transferred upward to the nucellus before it develops a pollen tube. This upward movement occurs after the canal is filled with secreted fluid, despite the fact that the pollen sinks in the fluid. We examined the mechanism of the movement based on the morphology of the canal and its simulation using pipettes. When a water column moves upward in a waxed pipette, suspended particles also move upward carried by the meniscus. InL. x eurolepis the inner surface of the integument lining the micropylar canal is coated by a cuticle layer. This layer is further coated by an integumentary membrane before the fluid is secreted. This membrane, however, becomes distorted or disappears during fluid secretion. The exposed cuticle and the degenerated hydrophilic nucellar apex may facilitate the movement of the meniscus toward the nucellus as in the simulated pipette. Pollen is interpreted to move by being carried by the meniscus when the fluid recedes.     

多花地宝兰胚囊和胚发育的细胞学研究

为探讨多花地宝兰(Geodorum recurvum)胚胎发育的系统分类学意义,采用石蜡制片法对多花地宝兰胚囊和胚的发育进行解剖学观察。结果表明,在开花前,多花地宝兰胚珠原基发育缓慢,开花授粉后胚珠原基快速发育成"树状二杈分枝结构",随后在"分枝结构"末端形成孢原细胞,开始胚囊发育。多花地宝兰的胚囊发育属于单孢蓼型胚囊,胚珠具有双层珠被。孢原细胞形成后,经过细胞膨大延长发育形成胚囊母细胞,胚囊母细胞经过减数分裂形成线性四分体,在珠孔端形成1个功能大孢子,功能大孢子经过3次有丝分裂形成8核胚囊。多花地宝兰的胚发育具有藜型和紫苑型两种方式。双受精完成后,多花地宝兰合子进行一次橫裂后形成基细胞和顶细胞;基细胞经过多次分裂形成细胞团,细胞团中的细胞向不同方向膨大延长形成多个胚柄细胞;顶细胞有两种分裂方式,一种是横裂形成藜型胚,一种是纵裂形成紫苑型胚。因此,推测多花地宝兰在兰科植物系统分类学上属于较为原始种。     

Truncation of a Protein Disulfide Isomerase,PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.     

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.     

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.