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This article describes the development of female gametophyte inSedum rupestreL. New embryological information about the processes of megasporogenesis and megagametogenesis provided in this paper expand the current knowledge about the embryology of the studied species.S.rupestreis characterized by monosporic megasporogenesis and the formation ofPolygonum–type embryo sac.The process of megasporogenesis is initiated by one megaspore mother cell, resulting in the formation of a triad of cells after meiosis and cytokinesis. The functional megaspore, which is located chalazally, is a mononuclear cell present next to the megaspore in the centre of the triad. Only one of the two non-functional cells of the triad is binucleate, which occur at the micropylar pole. In this paper, we explain the functional ultrastructure of the female gametophytic cells inS. rupestre. Initially, the cytoplasm of the gametophytic cells does not differ from each other; however, during differentiation, the cells reveal different morphologies. The antipodals and the synergids gradually become organelle-rich and metabolically active. The antipodal cells participate in the absorption and transport of nutrients from the nucellar cells towards the megagametophyte. Their ultrastructure shows the presence of plasmodesmata with electron-dense material, which is characteristic of Crassulaceae, and wall ingrowths in the outer walls. The ultrastructure of synergid cells is characterized by the presence of filiform apparatus and cytoplasm with active dictyosomes, abundant profiles of endoplasmic reticulum and numerous vesicles, which agrees with their main function—the secretion of pollen tube attractants. Reported data can be used to resolve the current taxonomic problems within the genusSedumser.Rupestria.

41 Pags.- 11 Figs. The definitive version is available at: https://link.springer.com/journal/497., Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen–ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo., This work was supported by Ministerio de Ciencia e Innovación (MICINN)-FEDER [AGL2006-13529-C02-01, AGL 12621-C02-01, AGL 2012–40239] and Gobierno de Aragón [group A43]. JIM antibodies distribution was partly supported by NSF grants [DBI-0421683, RCN 009281]. JML was supported by a FPI fellowship [BES-2007-16059] from MICINN.

Summary Signal-mediated regulation of protein trafficking is an elegant mechanism for controlling the delivery of molecules to a precise location for critical signaling events that occur over short time frames. During plant reproduction, the FERONIA receptor complex is critical for intercellular communication that leads to gamete delivery; however, the impact of the FERONIA signal transduction cascade on protein trafficking in synergid cells remains unknown. Live imaging of pollen tube reception has revealed that a key outcome of FERONIA signaling is polar accumulation of the MLO protein NORTIA at the filiform apparatus in response to signals from an arriving pollen tube. Artificial delivery of NORTIA to the filiform apparatus is sufficient to bypass the FERONIA signaling pathway and to promote interspecific pollen tube reception. We propose that polar accumulation of NORTIA leads to the production of a secondary booster signal to ensure that pollen tubes burst to deliver the sperm cells for double fertilization.

Calcium (Ca2+) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca2+ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca2+ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca2+-buffering protein, is able to bind Ca2+ reversibly, it can serve as a mobile store of easily releasable Ca2+ (so called an exchangeable Ca2+) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca2+ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca2+ are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca2+ storage and mobilization during sexual reproduction of angiosperms.

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L‐type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube reception in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube growth arrest at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants, a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell‐specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co‐receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female–male gametophyte interactions during plant fertilisation. Made available in DUO with special permission from EMBO Press.

During gamete delivery in Arabidopsis thaliana, intercellular communication between the attracted pollen tube and the receptive synergid cell leads to subcellular events in both cells culminating in the rupture of the tip-growing pollen tube and release of the sperm cells to achieve double fertilization. Live imaging of pollen tube reception revealed dynamic subcellular changes that occur in the female synergid cells. Pollen tube arrival triggers the trafficking of NORTIA (NTA) MLO protein from Golgi-associated compartments and the accumulation of endosomes at or near the synergid filiform apparatus, a membrane-rich region that acts as the site of communication between the pollen tube and synergids. Domain swaps and site-directed mutagenesis reveal that NTAs C-terminal cytoplasmic tail with its calmodulin-binding domain influences the subcellular localization and function of NTA in pollen tube reception and that accumulation of NTA at the filiform apparatus is necessary and sufficient for MLO function in pollen tube reception.During gamete delivery in Arabidopsis thaliana, intercellular communication between the attracted pollen tube and the receptive synergid cell leads to subcellular events in both cells culminating in the rupture of the tip-growing pollen tube and release of the sperm cells to achieve double fertilization. Live imaging of pollen tube reception revealed dynamic subcellular changes that occur in the female synergid cells. Pollen tube arrival triggers the trafficking of NORTIA (NTA) MLO protein from Golgi-associated compartments and the accumulation of endosomes at or near the synergid filiform apparatus, a membrane-rich region that acts as the site of communication between the pollen tube and synergids. Domain swaps and site-directed mutagenesis reveal that the NTA C-terminal cytoplasmic tail with its calmodulin-binding domain influences the subcellular localization and function of NTA in pollen tube reception and that accumulation of NTA at the filiform apparatus is necessary and sufficient for MLO function in pollen tube reception.

In angiosperms, pollen tube reception by the female gametophyte is required for sperm release and double fertilization. In Arabidopsis thaliana lorelei (lre) mutants, pollen tube reception fails in most female gametophytes, which thus remain unfertilized. LRE encodes a putative glycosylphosphatidylinositol (GPI)-anchored surface protein with a modified eight-cysteine motif (M8CM). LRE fused to citrine yellow fluorescent protein (LRE-cYFP) remains functional and localizes to the synergid plasma membrane-rich filiform apparatus, the first point of contact between the pollen tube and the female gametophyte. Structure-function analysis using LRE-cYFP showed that the role of LRE in pollen tube reception requires the M8CM, but not the domains required for GPI anchor addition. Consistently, LRE-cYFP-TM, where GPI anchor addition domains were replaced with a single-pass transmembrane domain, fully complemented the pollen tube reception defect in lre-7 female gametophytes. Ectopically expressed and delivered LRE-cYFP from pollen tubes could non-cell-autonomously complement the pollen tube reception defect in lre female gametophytes, only if they expressed FERONIA. Additionally, pollen tube-expressing LRE variants lacking domains critical for GPI anchor addition also rescued lre female gametophyte function. Therefore, LRE and FERONIA jointly function in pollen tube reception at the interface of the synergid cell and pollen tube.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Communication between the gametophytes is vital for angiosperm fertilisation. Multiple CrRLK1L-type receptor kinases prevent premature pollen tube burst, while another CrRLK1L protein, FERONIA (FER), is required for pollen tube burst in the female gametophyte. We report here the identification of two additional CrRLK1L homologues, HERCULES RECEPTOR KINASE 1 (HERK1) and ANJEA (ANJ), which act redundantly to promote pollen tube burst at the synergid cells. HERK1 and ANJ localise to the filiform apparatus of the synergid cells in unfertilised ovules, and in herk1 anj mutants a majority of ovules remain unfertilised due to pollen tube overgrowth, together indicating that HERK1 and ANJ act as female determinants for fertilisation. As in fer mutants, the synergid cell-specific, endomembrane protein NORTIA (NTA) is not relocalised after pollen tube reception; however, unlike fer mutants, reactive oxygen species levels are unaffected in herk1 anj double mutants. Both ANJ and HERK1 associate with FER and its proposed co-receptor LORELEI (LRE) in planta. Together, our data indicate that HERK1 and ANJ act with FER to mediate female-male gametophyte interactions during plant fertilisation.

The monosporic, tetranucleate embryo sac of Epilobium palustre (Onagraceae) develops from the micropylar megaspore in a linear tetrad. In mononucleate embryo sacs a peculiar chromatic structure associated with a nucleolus appears in the nucleus. This structure seems to be formed by fibrillar material and is not visible in the subsequant stages of development. A large amount of rough ER cisternae occurs in the late mononucleate stage, during the binucleate stage their contents become optically dense. It the early tetranucleate stage the amount of ER is small, it increases again in the developing synergids and central cell. Numerous amyloplasts present in the mononucleate embryo sac loose their starch grains and some are transformed into cup-shaped plastids or proplastids. They are passed on to each of the embryo sac cells. The growth of the pollen tube ceases immediately after the penetration through the filiform apparatus of a synergid. At the apex of the tube a pore is formed. At the last stages of the penetration the apical part of the pollen tube becomes separated by a transverse partition from the distal part of the tube. The contents of the both parts differ in their internal structure. The distal part contains cytoplasm with numerous organoids, while the apical part is mainly filled with spherical bodies.

We use TEM to study the synergids in sugar beet, so as to provide more information for reproductive biology of angiosperm. Results were as follows: two synergids were similar in flower bud stage. Both of them show polarity with developed filiform apparatus (FA) at micropyle end and lacked cell wall at chalazal end. Then electron density in one synergid increased which suggested cell degeneration began. Complete degeneration finished before pollination. Organelles including mitochondrium, plastids and ribosomes gradually increased in the other synergid (persistent synergid). Metabolism of persistent synergid gradually enhanced. It began to degenerate when zygote had alveolate cell wall at the chalazal end, while completely degeneration and disappearance of FA took place at late stage of zygote. The results suggested that degeneration of one synergid in sugar beet must be triggered by other stimulation than pollination and pollen tube growth. As a transfer cell, persistent synergid transported nutrition for the development of embryo sac.

The egg apparatus of Spinacia was studied from the time the embryo sac reaches its maximal size to just before fertilization, i.e., until about 8-9 hours after pollination. At maturity each synergid has a large elongated nucleus and prominent chalazal vacuoles, Numerous mitochondria, plastids, dictyosomes, free ribosomes, rough endoplasmic reticulum (RER), and lipid bodies are present. The cell wall exists only around the micropylar half of the synergids and each cell has a distinct, striated filiform apparatus. In general, degeneration of one synergid starts after pollination. The egg cell has a spherical nucleus and nucleolus and a large micropylar vacuole. Numerous mitochondria, some plastids with starch grains, dictyosomes, free ribosomes, and HER are present. A continuous cell wall is absent around the chalazal end of the egg cell.

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.

A morphological, cytological and embryological investigation was conducted on the early development of embryo and endosperm in polyembryonic rice ( Oryza sativa L.) ApIII. We found that the percentage of single-, twin- and triple-seedlings from mature caryopses was 82.4–85.6%, 11.3–14.6% and 2.0–3.8%, respectively. From twin-seedlings one of the plants and in the triple-seedlings usually two seedlings were very weak having a mortality of about 50–60% and over 95%, respectively, and most of the dead seedlings were haploid. In addition, among 674 young caryopses the percentage of single-, double- and triple-embryo sacs was 77.60%, 18.10% and 4.30%, respectively. The development of both embryo and endosperm was observed in the embryo sacs of 653 young caryopses, while embryo development occurred in absence of endosperm in the embryo sacs of 21 ones. Furthermore, after pollen tube penetration, frequently one, rarely two, additional embryos arise via apogamy from the typical specialized synergid of normal egg apparatus with a collapsed cap-neck region that had a damaged filiform apparatus at the micropylar end. Finally, the formation of a strikingly degenerated endosperm cell region is a specialized functional structure that serves for the active transport of metabolites from endosperm to the developing embryo. Taken together, these results suggest that synergid apogamy seems to be the unique reason of polyembryony, poor growth and high mortality of many seedlings may be due to haploidy, and high genetic stability of polyembryony occurs in rice ApIII.

The content and distribution of the poly(A) RNA and splicing machinery element––TMG snRNA in cells of the Hyacinthus orientalis L. mature embryo sac, during the progamic phase and after fertilization, were investigated. Using fluorescence in situ hybridization and immunofluorescence methods we showed that in the mature unfertilized embryo sac strong signal indicating poly(A) RNA and TMG snRNA appeared only in synergids and antipodal cells. In synergids accumulation of poly(A) RNA was shown in cytoplasm around the filiform apparatus. In the egg cell and central cell of the embryo sac accumulation of poly(A) RNA was very low and only low pool of TMG snRNA was observed in the nuclei of these cells. During the progamic phase, dramatic changes in the accumulation and distribution of poly(A) RNA and TMG snRNA were observed in the synergids. In these cells a considerable decrease in accumulation of TMG snRNA in the nucleus and poly(A) RNA in the region of the filiform apparatus occurred. In the egg cell and central cell the content of polyadenylated transcripts and TMG snRNA was still low. After fertilization a drastic increase in polyadenylated transcripts and TMG snRNA content was observed in the cells which undergo a fusion with the sperm cells (the zygote and the fertilized central cells). In contrast, a progressive decrease in poly(A) RNA and splicing snRNAs accumulation was observed in degenerating antipodal cells and synergids.

In this study, gynoeceum, development of megasporangium, megasporogenesis, megagametogenesis and female gametophyte of Leucojum aestivum were examined cytologically and histologically. Ovules of L. aestivum are of anatropous, bitegmic and crassinucellate type. Inner integument forms the micropyle. Archesporial cell develops directly into a megasporocyte. Embryo sac development is of bisporic Allium type. Filiform apparatus is observed in synergids. Polar nuclei fuse before fertilization to form secondary nucleus near the antipodals.

In flowering plants, signaling between the male pollen tube and the synergid cells of the female gametophyte is required for fertilization. In the Arabidopsis thaliana mutant feronia ( fer ), fertilization is impaired; the pollen tube fails to arrest and thus continues to grow inside the female gametophyte. FER encodes a synergid-expressed, plasma membrane–localized receptor-like kinase. We found that the FER protein accumulates asymmetrically in the synergid membrane at the filiform apparatus. Interspecific crosses using pollen from Arabidopsis lyrata and Cardamine flexuosa on A. thaliana stigmas resulted in a fer -like phenotype that correlates with sequence divergence in the extracellular domain of FER. Our findings show that the female control of pollen tube reception is based on a FER -dependent signaling pathway, which may play a role in reproductive isolation barriers.

The synergid cells within the female gametophyte are essential for reproduction in angiosperms. MYB98 encodes an R2R3-MYB protein required for pollen tube guidance and filiform apparatus formation by the synergid cells. To test the predicted function of MYB98 as a transcriptional regulator, we determined its subcellular localization and examined its DNA binding properties. We show that MYB98 binds to a specific DNA sequence (TAAC) and that a MYB98–green fluorescent protein fusion protein localizes to the nucleus, consistent with a role in transcriptional regulation. To identify genes regulated by MYB98, we tested previously identified synergid-expressed genes for reduced expression in myb98 female gametophytes and identified 16 such genes. We dissected the promoter of one of the downstream genes, DD11, and show that it contains a MYB98 binding site required for synergid expression, suggesting that DD11 is regulated directly by MYB98. To gain insight into the functions of the downstream genes, we chose five genes and determined the subcellular localization of the encoded proteins. We show that these five proteins are secreted into the filiform apparatus, suggesting that they play a role in either the formation or the function of this unique structure. Together, these data suggest that MYB98 functions as a transcriptional regulator in the synergid cells and activates the expression of genes required for pollen tube guidance and filiform apparatus formation.

The ultrastructural analysis using ultra-thin section and transmission electron microscope was carried out to characterize the features of the reproductive cells in aposporous initial cell (AIC)-derived embryo sac (AES), i.e. egg, synergid and polar cells, in ovule of facultative apomictic Panicum maximum. At anthesis, the first AES located in micropylar end consists of egg apparatus with 2 synergids and 1 egg cell, and the central cell with 1 nucleus. The other AESs contain less than four cells. The egg cell contains high dense of cytoplasm, and around the nucleus plastids and mitochondria are remarkable. There exist lipid bodies, few rough endoplasmic reticulum (rER), dictyosomes, and big vacuoles distributed in cytoplasm. The polar cell is occupied almost by one big vacuole, the lower dense cytoplasm than that of egg cell wraps 1 or 2 nuclei and is distributed around egg apparatus. Only in micropylar end of the AES the cell wall of the central cell with one nucleus exists with ingrowth of cell wall, indicating that this structure transports the nutrition for the embryo development. Synergid usually contains lowest dense of cytoplasm, and lipid bodies, plastid and mitochondria are distributed along the filiform apparatus in micropylar end. Filiform apparatus is located in the top place of the 2 synergids that is same to that of sexual embryo sac. Extreme rER and vacuoles distributed in chalazal end are the characteristics of the synergids. These vacuoles and rER might contain abundant inorganic substances that are absorbed as a part of nutrition for the developing embryo.

During the angiosperm (flowering-plant) life cycle, double fertilization represents the hallmark between diploid and haploid generations [1]. The success of double fertilization largely depends on compatible communication between the male gametophyte (pollen tube) and the maternal tissues of the flower, culminating in precise pollen tube guidance to the female gametophyte (embryo sac) and its rupture to release sperm cells. Several important factors involved in the pollen tube reception have been identified recently [2-6], but the underlying signaling pathways are far from being understood. Here, we report that a group of female-specific small proteins, early nodulin-like proteins (ENODLs, or ENs), are required for pollen tube reception. ENs are featured with a plastocyanin-like (PCNL) domain, an arabinogalactan (AG) glycomodule, and a predicted glycosylphosphatidylinositol (GPI) anchor motif. We show that ENs are asymmetrically distributed at the plasma membrane of the synergid cells and accumulate at the filiform apparatus, where arriving pollen tubes communicate with the embryo sac. EN14 strongly and specifically interacts with the extracellular domain of the receptor-like kinase FERONIA, localized at the synergid cell surface and known to critically control pollen tube reception [6]. Wild-type pollen tubes failed to arrest growth and to rupture after entering the ovules of quintuple loss-of-function EN mutants, indicating a central role of ENs in male-female communication and pollen tube reception. Moreover, overexpression of EN15 by the endogenous promoter caused disturbed pollen tube guidance and reduced fertility. These data suggest that female-derived GPI-anchored ENODLs play an essential role in male-female communication and fertilization.

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.

Post-fertilization changes in the seven-celled embryo sac of the parasitic angiosperm Arceuthobium americanum Nutt. ex Engelm. were investigated with light, fluorescence, and electron microscopy. Two embryo sacs, of which only one is fertilized, are found within the reduced ategmic ovule (the placental–nucellar complex). All cells of the fertilized embryo sac developed thickened cellulosic and (or) hemicellulosic bounding walls and, aside from the degenerative synergid, acquired ultrastructure reflective of metabolically active cells. The filiform apparatus became thickened in the persistent synergid, and a similar structure developed in the antipodals. Unlike the changes observed in the zygote and central cell, those in the persistent synergid and antipodals were unusual, as these cells regularly degenerate after fertilization in a typical angiosperm; therefore, in A. americanum, they likely play a role in providing nutrition to the zygote and (or) central cell. A pouch-like outgrowth (caecum) initiated from the central cell near the primary endosperm nucleus and, after vacuolar expansion and intercellular growth, reached the base of the placental–nucellar complex in three days. The observation of mitochondria clusters within the central cell, caecum, and adjacent placental–nucellar complex cells suggests the caecum functions in embryo sac expansion and haustorial nutrient absorption.Key words: Arceuthobium, caecum, cell walls, fertilized embryo sac, mistletoe, ultrastructure.

Information on reproductive biology of threatened plant species could be useful for conservation, particularly when the species fails to perpetuate in nature due to regeneration failure. In flowering plants, the domain of reproductive biology includes structural details of reproductive units such as flower or inflorescence, formation of viable gametes, pollination dynamics, role of pollinators, pollen–pistil interactions as determined through compatibility, breeding system and mating strategies, fertilization and embryogeny, seed development, dispersal and germination. The importance of reproductive biology in species conservation has been demonstrated through a case study of Illicium griffithii Hook f. & Thomson, a threatened plant species from Arunachal Pradesh. I. griffithii (Illiciaceae) is a member of the ANITA clade and a representative taxon of the three most basal angiosperms. The flowers of I. griffithii are obligate xenogamous, and hence compatible pollen grains only germinate on the stigmatic papillae. Esterase and phosphatase enzymatic activities are absent in most stigmatic surfaces. Therefore, stigmatic receptivity is absent in many carpels. The ovules are anatropous and bitegmic with a four-celled/four-nucleate structure of embryo sac at maturity. Antipodals and filiform apparatus are absent. Presence of mucilage cells in the embryo sac facilitates the entry of pollen tubes into it. Embryolike organization of the endosperm is present, with one pole globular having smaller cells and the other pole roughly filamentous with larger cells resembling that of a suspensor. Extragynoecial compitum is not observed in I. griffithii . The flowers are brooding sites for the midges and the young ovules are eaten by larvae of the midges. Only 10% of the flowers mature into 13-seeded fruits and the carpels of the remaining 90% of the flowers have seeds ranging from 1 to 5. The seedling survival rate is only 7%. In addition to the above-mentioned reproductive bottlenecks, the species is also being over-harvested. It is now categorized as endangered. The findings of the present study on reproductive biology of I. griffithii should help in improving its conservation status.

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.

The yellow passion fruit, Passiflora edulis f. flavicarpa, is one of the most important Brazilian fruit crops. It is an allogamous, diploid, and self-incompatible species. It has hermaphrodite, solitary flowers, located in the leaf axils and protected by leaf bracts. The flower has an androgynophore, which is a straight stalk supporting its reproductive parts. There are usually five anthers, located at the tip of each of the five filaments. The ovary is borne just above the filaments, at the top of the androgynophore; there are three styles that are united at their base, and at the top there are three stigmas. The objective of this research was to observe embryo sac development in yellow passion flowers. Ovaries at different stages of development were fixed in FAA (formalin, acetic acid and alcohol solution), hydrated, stained with Mayer’s hemalum, and dehydrated. Ovules were cleared by using methyl salicylate, mounted on slides, and observed through a confocal scanning laser microscope. The yellow passion fruit ovule is bitegmic, crassinucellate, and anatropous, and its gametophyte development is of the Polygonum type. After meiosis, functional megaspores under go three successive mitotic divisions, resulting in an eight-nucleate megagametophyte: the egg apparatus at the micropylar end, two polar nuclei at the cell center, and three antipodals at the chalazal end. The egg apparatus is formed by an egg cell and two synergids, each with a filiform apparatus. The mature embryo sac has an egg cell, two synergids, two polar nuclei, and three antipodes, as has been described for most angiosperms.

Changes in actin organization in the living egg apparatus of Torenia fournieri from anthesis to post-fertilization have been investigated using microinjection and confocal microscopy. Our results revealed that the actin cytoskeleton displays dramatic changes in the egg apparatus and appears to coordinate the events of synergid degeneration, pollen tube arrival and gametic fusion during fertilization. Synergid degeneration occurs after anthesis and is accompanied by actin fragmentation and degradation. The actin cytoskeleton becomes organized with numerous aggregates in the chalazal end of the degenerating synergid, and some of the actin infiltrates into the intercellular gap between synergids, egg and central cell, forming a distinct actin band. An actin cap is present near the filiform apparatus after anthesis and disappears after pollen tube arrival. In the egg cell, actin filaments initially organize into a network and after pollination become fragmented into numerous patches in the cortex. These structures, along with the actin in the degenerating synergid and intercellular spaces form two distinct actin coronas during fertilization. The actin coronas vanish after gametic fusion. This is the first report of changes in actin organization in the living egg apparatus. The reorganization of the actin cytoskeleton in the egg apparatus and the presence of the actin coronas during fertilization suggest these events may be a necessary prelude to reception of the pollen tube and fusion of the male and female gametes.

Amaranthus hypochondriacus embryo sac development was investigated before and after fertilization. During the early stages of development, the young embryo sac displays three antipodal cells at the chalazal pole that degenerate very early in the maturation process, before the synergids and egg cell are completely differentiated. The mature embryo sac is composed only of the female germ unit. The synergid cells organize a filiform apparatus accompanied by the presence of mitochondria and dictyosomes with numerous vesicles. The involvement of the synergids in transport and secretory functions related to pollen tube attraction and guidance, are discussed. The egg cell is located at the micropylar pole near the synergids and displays exposed plasma membranes at the chalazal pole. The fertilized egg cell does not exhibit marked changes after fertilization except for the closure of the cell wall. The central cell is the largest cell of this very long embryo sac. The fused nucleus is close to the egg apparatus before fertilization and displays a remarkable chalazal migration after gamete delivery. The ultrastructure of the central cell cytoplasm and the numerous wall ingrowths around this cell suggest an important role in nutrient transportation. After gamete delivery, the embryo sac displays electron dense bodies that aggregate within the intercellular space between the synergids, egg cell and central cell. These bodies, that appear in the embryo sac of several plants, are probably involved in gamete delivery for double fertilization. The possibility of biparental inheritance of mitochondria in this plant is also discussed.

Actin organization was observed in m-maleimidobenzoic acid N-hydroxysuccinimide ester(MBS)-treated maize embryo sacs by confocal laser scanning microscopy. The results revealed that dynamic changes of actin occur not only in the degenerating synergid, but also in the egg during fertilization. The actin filaments distribute randomly in the chalazal part of the synergid before fertilization; they later become organized into numerous aggregates in the chalazal end after pollination. The accumulation of actin at this region is intensified after the pollen tube discharges its contents. Concurrently, actin patches have also been found in the cytoplasm of the egg cell and later they accumulate in the cortical region. To compare with MBS-treated maize embryo sacs, we have performed phalloidin microinjection to label the actin cytoskeleton in living embryo sacs of Torenia fournieri. The results have extended the previous observations on the three-dimensional organization of the actin arrays in the cells of the female germ unit and confirm the occurrence of the actin coronas in the embryo sac during fertilization. We have found that there is an actin cap occurring near the filiform apparatus after anthesis. In addition, phalloidin microinjection into the Torenia embryo sac has proved the presence of intercellular actin between the cells of the female germ unit and thus confirms the occurrence of the actin coronas in the embryo sac during fertilization. Moreover, actin dynamic changes also take place in the egg and the central cell, accomplished with the interaction between the male and female gametes. The actin filaments initially organize into a distinct actin network in the cortex of the central cell after anthesis; they become fragmented in the micropylar end of the cell after pollination. Similar to maize, actin patches have also been observed in the egg cortex after pollination. This is the first report of actin dynamics in the living embryo sac. The results suggest that the actin cytoskeleton may play an essential role in the reception of the pollen tube, migration of the male gametes, and even gametic fusion.

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.

A protocol for isolating viable eggs in Plumbagozeylanica by mechanical dissection is reported. The optimum solution for isolation was 0.8 M mannitol + 10 mM MOPS + 10 mM CaCl2, (pH 4.5–5.0) with an osmolality of 860–940 mmol/kg. Eggs retain their viability for at least 24 h. Isolated eggs were true protoplasts without cell walls and could tolerate osmolality of 437 mmol/kg to 965 mmol/kg. Observation of the isolated eggs using transmission electron microscopy indicated that they were well preserved and reflected the ultrastructure of physiologically active cells, displaying features similar to those of in vivo egg cells. Notable differences include the absence of a filiform apparatus and the accumulation of dense particles in the plastids, which was most conspicuous in egg cells that were damaged during isolation.

After cell compartmentation, the young embryo sac of Arundo formosana consists of seven cells: the egg, two synergids, the central cell with two polar nuclei, and three antipodal cells. All three cells of the newly formed egg apparatus surrounded by incomplete cell walls initially have similar cellular contents. However, as the egg apparatus develops, the egg and the synergids gradually differentiate in cellular contents prior to the initiation of the filiform apparatus. The egg develops many starch-containing plastids. The synergids become rich in rough endoplasmic reticulum (ER) and dictyosomes. These differences are retained throughout maturation. The micropylar common wall of the two synergids produces many cell-wall ingrowths, which consitute the filiform apparatus. Endoplasmic reticulum and dictyosomes are involved in filiform-apparatus formation. During maturation, the egg shows some changes: the nucleus migrates toward the cell center; the degree of vacuolation and the number of starch grains and ...

A scanning electron and light microscopical study of the multi-ovular ovaries of Trachyandra saltii (Baker) Oberm. var. saltii showed that the locules of open flowers contained bitegmic, arillate ovules, obliquely arranged on axile placentae with the micropyles of most ovules pointing towards the septa. In the uncurved embryo sac the synergids with strongly PAS-positive filiform apparatus and the overgrown central cell nucleus were the most noticeable elements. The antipodals were still present in the chalazal extension of the embryo sac into the hypostase. At anthesis the ovules were hemitropous. It is suggested that, ontogenetically, the development of an aril on the ventral side of the ovule has prevented anatropy in the Asphodelaceae.