Persistent Synergid Research Articles

Response to Zhang et al., 'do egg cell-secreted aspartic proteases promote gamete attachment?'

In angiosperms, the molecular mechanism controlling polytubey/polyspermy block and fertilization recovery was not unraveled until recently (Zhong et al., 2022), which can be described as follows: Five pollen tube-expressed RALF peptide ligands, perceived by the receptor complex composed of three receptors FER/ANJ/HERK1 at the septum, establish the polytubey block at the septum so that only one pollen tube is allowed to emerge from the septum to target the ovule for fertilization. Pollen tube rupture releases the two sperm cells and fertilization is completed within 20 minutes. This article is protected by copyright. All rights reserved.

F-actin regulates the polarized secretion of pollen tube attractants in Arabidopsis synergid cells.

Pollen tube attraction is a key event of sexual reproduction in flowering plants. In the ovule, two synergid cells neighboring the egg cell control pollen tube arrival via the active secretion of attractant peptides such as AtLURE1 and XIUQIU from the filiform apparatus (FA) facing toward the micropyle. Distinctive cell polarity together with longitudinal F-actin and microtubules are hallmarks of the synergid cell in various species, though the functions of these cellular structures are unclear. In this study, we used genetic and pharmacological approaches to indicate the roles of cytoskeletal components in FA formation and pollen tube guidance in Arabidopsis thaliana. Genetic inhibition of microtubule formation reduced invaginations of the plasma membrane but did not abolish micropylar AtLURE1.2 accumulation. By contrast, the expression of a dominant-negative form of ACTIN8 induced disorganization of the FA and loss of polar AtLURE1.2 distribution toward the FA. Interestingly, after pollen tube reception, F-actin became unclear for a few hours in the persistent synergid cell, which may be involved in pausing and resuming pollen tube attraction during early polytubey block. Our data suggest that F-actin plays a central role in maintaining cell polarity and in mediating male-female communication in the synergid cell.

Lack of ethylene does not affect reproductive success and synergid cell death in Arabidopsis

The signaling pathway of the gaseous hormone ethylene is involved in plant reproduction, growth, development, and stress responses. During reproduction, the two synergid cells of the angiosperm female gametophyte both undergo programmed cell death (PCD)/degeneration but in a different manner: PCD/degeneration of one synergid facilitates pollen tube rupture and thereby the release of sperm cells, while PCD/degeneration of the other synergid blocks supernumerary pollen tubes. Ethylene signaling was postulated to participate in some of the synergid cell functions, such as pollen tube attraction and the induction of PCD/degeneration. However, ethylene-mediated induction of synergid PCD/degeneration and the role of ethylene itself have not been firmly established. Here, we employed the CRISPR/Cas9 technology to knock out the five ethylene-biosynthesis 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes and created Arabidopsis mutants free of ethylene production. The ethylene-free mutant plants showed normal triple responses when treated with ethylene rather than 1-aminocyclopropane-1-carboxylic acid, but had increased lateral root density and enlarged petal sizes, which are typical phenotypes of mutants defective in ethylene signaling. Using these ethylene-free plants, we further demonstrated that production of ethylene is not necessarily required to trigger PCD/degeneration of the two synergid cells, but certain components of ethylene signaling including transcription factors ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1) are necessary for the death of the persistent synergid cell.

A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control.

Dimorphism of Synergids inAllium tuberosum(Amaryllidaceae)

In angiosperm fertilization, the pollen tube enters the female gametophyte via one of two synergids, in which the pollen tube ceases elongation and releases its contents, including two sperm cells. Whether the entrance of the pollen tube into a synergid occurs at random or is determined in advance is currently unclear. We examined two dimorphic synergids of Allium tuberosum (Amaryllidaceae). The smaller of the two cells becomes a degenerative synergid; the pollen tube enters this smaller cell and gains access to the female gametophyte for sperm discharge. The larger cell becomes a persistent synergid, which can persist for up to four days after anthesis. We further confirmed the dimorphism of the two synergids of A. tuberosum by isolating the egg apparatus from living, dissected ovule. The size difference between synergids increased with development. We also examined the calcium distribution in developing synergids, finding that the small synergid accumulated more calcium precipitates than the large one, ...

JAGGER, an AGP essential for persistent synergid degeneration and polytubey block in Arabidopsis

ABSTRACTA precise control of sperm cells delivery and fusion to the egg cell and the central cell is fundamental for the accomplishment of successful double fertilization in flowering plants. This is mostly regulated by female gametophyte egg and central cells, which control the timing of synergids cell degeneration. We recently identified an arabinogalactan protein, AGP4, named JAGGER, that impairs the persistent synergid degeneration, and consequently leads to the attraction of more than one pollen tube into one embryo sac, a situation termed polytubey. jagger mutants revealed an increased rate of polytubey and persistent synergids that do not degenerate. This persistent synergid, is, as we suggested, the cell responsible for attracting an extra pollen tube into the embryo sacs.

“Love Is Strong, and You're so Sweet”: JAGGER Is Essential for Persistent Synergid Degeneration and Polytubey Block in Arabidopsis thaliana

Successful double fertilization and subsequent seed development in flowering plants requires the delivery of two sperm cells, transported by a pollen tube, into the embryo sac of an ovule. The embryo sac cells tightly control synergid cell death, and as a result the polyspermy block. Arabinogalactan proteins are highly glycosylated proteins thought to be involved in several steps of the reproductive process. We show that JAGGER, Arabinogalactan Protein 4, is an important molecule necessary to prevent the growth of multiple pollen tubes into one embryo sac in Arabidopsis thaliana. In jagger, an AGP4 knockout mutant, the pistils show impaired pollen tube blockage as a consequence of the survival of the persistent synergid. JAGGER seems to be involved in the signaling pathway that leads to a blockage of pollen tube attraction. Our results shed light on the mechanism responsible for preventing polyspermy in Arabidopsis and for safeguarding successful fertilization of all ovules in one pistil, ensuring seed set and the next generation.

Three Cell Fusions during Double Fertilization

Fertilization of both egg and central cell is a major distinguishing feature of flowering plants. Now, Maruyama etal. report a third cell fusion event between the persistent synergid and the fertilized central cell shortly after double fertilization in Arabidopsis. This causes rapid dilution of pollen tube attractant(s), preventing polytubey.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Ultrastructure of Synergids in Sugar Beet

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.

Ethylene Signaling Is Required for Synergid Degeneration and the Establishment of a Pollen Tube Block

In flowering plants, sperm cells are delivered by pollen tubes, which are attracted by two egg-cell-adjoining synergids. Successful fertilization terminates pollen tube attraction; however, the underlying mechanisms are not understood. Here, we show that the process of fertilization activates an EIN3- and EIN2-dependent ethylene-response cascade necessary for synergid cell death and the concomitant establishment of a pollen tube block. Microinjection of the ethylene precursor ACC into the female gametophyte or constitutive ethylene response results in premature synergid disintegration. This indicates that the requirement of fertilization for synergid degeneration and associated establishment of a pollen tube block can be bypassed by mimicking a postfertilization ethylene burst. Surprisingly, the persistent synergid in ethylene-hyposensitive plants adopts the molecular profile and cell-cycle regime of the biparental embryo-nourishing tissue, suggesting that ethylene signaling prevents the formation of an asexual maternal endosperm fraction.

Ultrastructure of Synergid in Its Degenerative Process in Sugar Beet ( Beta vulgaris )

An experiment was conducted by using TEM to study the degenerative process of synergid in sugar beet, so as to provide more information for reproductive biology of angiosperm. The results were as follows: two synergids were similar in flower bud stage with large number of organelles. Both of them had developed filiform apparatus(FA) at micropyle end and lacked cell wall at chalazal end, showing obvious polarity. Then electron density in one synergid increased. On the other hand, vacuole membrane disappeared, membrane of mitochondrium, plastid and nuclear became illegible, which suggested cell degeneration began.But endoplasmic reticulum and Golgi bodies were still active. Complete degeneration of this synergid occurred before pollination.Number of organelles including mitochondrium, plastids and ribosomes gradually increased in the other synergid(persistent synergid). Mitochondria contained many tubular cristae and obvious DNA fibril. Plastids were irregular in shape and usually contained starch gains and thylakoid membranes. The metabolism of persistent synergid gradually enhanced until fertilization(about13 h after pollination). It began to degenerate when zygote had alveolate cell wall at the chalazal end and endosperm occurred,while complete degeneration and disappearance of FA took place at late stage of zygote. The results indicated 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 may absorb and transport nutrition for the development of embryo sac.

Ultrastructure of the fertilized embryo sac in the dwarf mistletoe Arceuthobium americanum (Viscaceae) and development of the caecum

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.

Differences in the initiation of the zygotic and parthenogenetic pathway in the Salmon lines of wheat: ultrastructural studies

The ultrastructure of the egg apparatus of the sexual (aestivum)-Salmon line (aS) and the isogenic but alloplasmic (kotschyi)-Salmon line (kS) of the Salmon system of wheat was studied by transmission electron microscopy 3 days before and during anthesis. Additionally, the zygotic stage of aS, 17 h after pollination, was included. Metabolic activity of egg cells from the sexual line aS was low 3 days before anthesis and increased dramatically after pollination and fertilization. This timing of increased activity was evident because of changes occurring in the egg cell nucleus and nucleolus, polysomes, endoplasmic reticulum and Golgi apparatus, and the completion of the cell wall around the zygote. In contrast to the sexual line, the egg cell of the parthenogenetic line showed high activity 3 days before anthesis. The metabolic and ultrastructural characters observed in the nucleus and cytoplasm of the kS line 3 days before and during anthesis corresponded with those of the isogenic sexual line aS during anthesis and 17 h after pollination, respectively. High metabolic activity observed in the persistent synergid of kS may be connected with the occurrence of additional embryos in seeds (twins) of this line.

Ultrastructure of the Maturing Egg Apparatus in Arundo formosana Hack. (Poaceae)

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 oil bodies increase; the amount of ER and dictyosomes decrease. During synergid maturation, the organelles gradually show a polarized distribution: the plastids are present only at the chalazal pole, and most of mitochondria and dictyosomes are distributed at the micropylar pole. After pollination, vacuolation usually occurs at the chalazal pole of the persistent synergid, and the degenerated synergid becomes filled with electron-dense materials. During egg-apparatus expansion, cell-wall materials are deposited and condense at the chalazal pole, forming electron-dense particles at maturity. Plasmodesmata are always present in the micropylar gametophytic cell walls of the egg apparatus.

Autecological studies on Audouinia capitata (Bruniaceae). 6. Nutritional aspects of the developing ovule

The present investigation provides a basis for studies about morphological and nutritional aspects involved in late abortion of embryos and nutrient-linked, pre-emergent mortality of seedlings in this threatened species. The nucellus is digested completely during megagametogenesis and the integument likewise during embryogenesis, except for a diminutive testa. Mass starch deposition is found in the mature embryo sac. Involvement of the persistent synergid in nutrition of the zygote and the 2-celled proembryo until the fifth week, is suggested. This function is taken over by the suspensor which initially channels integumentary reserves to the developing proembryo. From the eighth week, spherical protein bodies are evident in the endosperm. At this stage, the embryo proper is still surrounded by free nuclear endosperm, and the suspensor of the linear proembryo mobilizes, in addition to integumentary reserves, also adjacent cellular endosperm as well as the micropylar layer of cellular endosperm. The embryo proper of the more advanced stages mobilizes nutrients directly from the adjacent cellular endosperm. The seed enters dormancy at the heart-shape stage of embryo development. Die huidige ondersoek bied ’n grondslag vir ’n studie van morfologiese en voedingsaspekte wat betrokke is by laat aborsies van embrio’s en voedingsverwante, voor-opkoms-mortaliteit van saailinge in hierdie bedreigde spesie. Die nusellus word volledig verteer tydens megagametogenese. Die integument verdwyn gedurende embriogenese sodat net ’n onbeduidende saadhuid oorbly. Massastylselneerlegging kom in die volwasse embriosak voor. Daar word voorgestel dat die blywende sinergied in die voeding van die sigoot en die tweesellige proëmbrio betrokke is. Hierdie funksie word oorgeneem deur die suspensor wat aanvanklik integumentreserwes na die ontwikkelende proëmbrio kanaliseer. Sferiese proteïenliggaampies is vanaf die agste week in die endosperm sigbaar. In hierdie fase is die ware embrio nog steeds deur vrykernige endosperm omring. Bykomstig tot die integumentreserwes, mobiliseer die suspensor van die liniêre proëmbrio ook aangrensende sellulêre endosperm en die mikropilêre laag sellulêre endosperm. Die ware embrio van meer gevorderde fases mobiliseer nutriënte direk uit aangrensende sellulêre endosperm. Saadrus tree tydens die hartvormige fase van embrio-ontwikkeling in.

EMBRYO SAC DEVELOPMENT IN SOYBEAN: THE CENTRAL CELL AND ASPECTS OF FERTILIZATION

Development of the soybean central cell and events associated with fertilization are described. Central cell development involves the simultaneous formation of numerous multigrain amyloplasts, development of wall ingrowths, reduction of the cell's major vacuole, and nuclear migration. As these processes reach completion, a phase of lytic activity begins. This activity ultimately results in both the breakdown of most multigrain amyloplasts in the central cell and the reestablishment of the cell's large vacuole. While these processes occur, the egg and synergid cells continue development. Examination of the egg apparatus after plasmogamy but prior to karyogamy reveals several changes. Whereas the synergid containing the pollen tube is degenerate and the distribution of organelles in the persistent synergid has changed, the egg cell with a free sperm nucleus exhibits higher levels of metabolite storage and changes in its wall structure. The walls of the newly formed sporophyte are similar to those of egg apparatus cells (Folsom and Cass, Canadian Journal of Botany 68: 2135–2147 [1990]) in that they seem to lack a middle lamella. This layer becomes apparent only in the two‐celled proembryo. While these changes occur in the egg, zygote, and proembryo, synergids undergo different degradative pathways.

Ultrastructure and Cleavage Pattern of Rice Proembryos

Up to 12 h after anthesis (HAA), the first cell division in rice zygotes occurred with the formation of a slightly oblique transverse wall, causing the partition of the zygote into an apical cell and a basal cell. At 18 HAA, the partition in the apical cell was brought about by the presence of a longitudinal wall and in the basal cell by a transverse wall, which resulted in the formation of a four-celled embyro. At 24 HAA, the middle cell of the four-celled embryo was partitioned by a longitudinal wall, and the basal cell was again partitioned by a slightly oblique transverse wall. At 30 HAA, the apical and middle regions had four cells each with the further formation of longitudinal walls, so that the embryo became ten-celled. After that, cell division was directed at random. Well-developed cytoplasm existed around nuclei in proembryo cells. The cup-shaped plastids containing starch grains, the cup-shaped mitochondria, and the stratiform type of rough endoplasmic reticula were frequently observed. Many vacuoles were seen in the peripheral cytoplasm. The basal cells were more vacuolated than the cells at the upper side of the proembryo. At 18 HAA, the persistent synergid completely degenerated, involving electron dense materials. The endosperm became cytoplasm rich around the proembryo, especially after the embryo had started to increase in size.

Ultrastructure of the developing embryo sac of sunflower (Helianthus annuus) before and after fertilization

The ultrastructure of the embryo sac of the sunflower (Helianthus annuus) was investigated before and after fertilization. In the young embryo sac, walls were observed that completely surrounded the egg, synergids, and the central cell. However, as maturation continued, the extent of the wall changed. By the time the embryo was mature, the chalazal portion of the walls of the egg and synergids had disappeared so these cells have a plasma membrane to plasma membrane contact. This is also true for the central cell, which has plasma membrane contact with the egg and synergids. However, the chalazal and lateral walls of the central cell become considerably thicker at this time. Before the entry of the pollen tube, the synergid that is located toward the placenta degenerates. After fertilization, a wall forms over the chalazal portion of the zygote and the persistent synergid. The endosperm appears to play an active role in this process, contributing substantial amounts of wall material. However, the wall covering the chalazal portion of the zygote is not complete by the time the zygote divides. In the proembryo, ribosome density increases and lipid bodies decrease in number. The suspensory cell has autophagic vacuoles that encircle some of the organelles. Our results support the concept that the egg, synergids, and central cell form a single functional unit, the female germ unit. Key words: sunflower, ultrastructure, embryo sac, female germ unit.

Ultrastructure of egg apparatus of rice (Oryza sativa) after anthesis.

A light and transmission electron microscope study was made on the disposition of an egg cell and two synergids in Oryza sativa ovules. After anthesis, one of the synergids is rather persistent while the other is degenerating with collapse of the plasma membrane and penetration of ground plasma into the space between the plasma membranes of the egg cell and the central cell. Spherical vesicles in degenerating synergids closely resemble polysaccharide particles in intact pollen grains. Singular cisternae in the central cells tightly associate with the plasma membrane which borders on the degenerating synergids. There are osmiophilic deposits in the narrow interspace between the plasma membranes of the egg cell and the persistent synergid or the central cell. The egg cell has a nucleus with two nucleoli. The persistent synergid has a malformed nucleus forced on a pocket portion of central cell. The portion is occupied by a large number of cell wall ingrowths. It is postulated from the cyto-histological features of ovaries that nutrition into the egg cells is supplied through transmembranal, symplastic and apoplastic routes.