Pollen Tube Arrival Research Articles

FERONIA controls pectin- and nitric oxide-mediated male-female interaction.

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.

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution.

Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

Pollen tube access to the ovule is mediated by glycoprotein secretion on the obturator of apple (Malus × domestica, Borkh).

Within the ovary, the obturator bridges the pathway of the pollen tube from the style to the ovule. Despite its widespread presence among flowering plants, its function has only been studied in a handful of species, and the molecules involved in pollen tube-obturator cross-talk have not been explored hitherto. This work evaluates the involvement of glucans and glycoproteins on pollen tube growth in the obturator of apple flowers ( Malus × domestica) . Pollen tube kinetics were sequentially examined in the pistil and related to changes occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate between changes in the obturator induced by pollen tubes from those developmentally regulated, both pollinated and unpollinated pistils were examined. Pollen tube growth rates were slow in the stigma, faster in the style and slow again in the ovary. The arrival of pollen tubes at the obturator was concomitant with the secretion of proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan proteins (AGPs). While some of these secretions - extensins and AGPs labelled by JIM13 - were developmentally regulated, others - AGPs labelled by JIM8 - were elicited by the presence of pollen tubes. Following pollen tube passage, all these glycoproteins were depleted. The results show a timely secretion of glycoproteins on the obturator surface concomitant with pollen tube arrival at this structure. The fact that their secretion is depleted following pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule. Remarkably, both the regulation of the secretion of the different glycoproteins, as well as their association with the performance of pollen tubes exhibit similarities with those observed in the stigma, in line with their common developmental origin.

Pollen Tube Discharge Completes the Process of Synergid Degeneration That Is Initiated by Pollen Tube-Synergid Interaction in Arabidopsis

In flowering plant reproduction, pollen tube reception is the signaling system that results in pollen tube discharge, synergid degeneration, and successful delivery of male gametes (two sperm cells) to the site where they can fuse with female gametes (egg cell and central cell). Some molecules required for this complex and essential signaling exchange have been identified; however, fundamental questions about the nature of the interactions between the pollen tube and the synergid cells remain to be clarified. Here, we monitor pollen tube arrival, pollen tube discharge, and synergid degeneration in Arabidopsis (Arabidopsis thaliana) wild type and in male and female gametophytic mutants that disrupt development and function of the gametophytes. By combining assays used previously to study these interactions and an assay that facilitates simultaneous analysis of pollen tube discharge and synergid degeneration, we find that synergid degeneration could be initiated without pollen tube discharge. Our data support the hypothesis that pollen tube-synergid contact, or signaling via secreted molecules, initiates receptive synergid degeneration. We also find that when pollen tubes successfully burst, they always discharge into a degenerated synergid. In addition to this pollen tube-dependent promotion of synergid degeneration, we also show that a basal developmental pathway mediates synergid degeneration in the absence of pollination. Our results are consistent with the model that a complex set of interactions between the pollen tube and synergid cells promote receptive synergid degeneration.

TURAN and EVAN Mediate Pollen Tube Reception in Arabidopsis Synergids through Protein Glycosylation

Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm–egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction.

Live imaging of calcium spikes during double fertilization in Arabidopsis.

Ca2+ waves and oscillation are key signalling elements during the fertilization process of animals, and are involved, for example, in egg activation. In the unique double fertilization process in flowering plants, both the egg cell and the neighbouring central cell fuse with a sperm cell each. Here we succeeded in imaging cytosolic Ca2+ in these two cells, and in the two synergid cells that accompany the gametes during semi-in vivo double fertilization. Following pollen tube discharge and plasmogamy, the egg and central cells displayed transient Ca2+ spikes, but not oscillations. Only the events in the egg cell correlated with the plasmogamy. In contrast, the synergid cells displayed Ca2+ oscillations on pollen tube arrival. The two synergid cells showed distinct Ca2+ dynamics depending on their respective roles in tube reception. These Ca2+ dynamics in the female gametophyte seem to represent highly specific signatures that coordinate successful double fertilization in the flowering plants.

Three MYB Transcription Factors Control Pollen Tube Differentiation Required for Sperm Release

In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization. Several female signaling components essential for pollen tube reception have been identified; however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes and degenerate upon pollen tube arrival. myb triple-mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate-active enzymes, and small peptides as candidate molecular mediators of pollen tube-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.

Relationship between double fertilization and the cell cycle in male and female gametes of tobacco

Nuclear DNA content of male and female gametes of tobacco was determined using 4′,6-diamindino-2-phenylindole and quantitative microfluorimetry. Pollen grains are released with generative cells containing 2C DNA. Mitotic division occurs in the pollen tube 8–12 h after germination. The resulting sperm cells have 1C DNA content during pollen tube elongation in the style. Sperm cells deposited in the degenerated synergid have a DNA content between 1C and 2C, indicating that sperm are in S-phase in the synergid. Concomitant with pollen tube arrival, the egg cell increases in DNA quantity from 1C to between 1C and 2C at 48 h after pollination. In the absence of pollination, S-phase in the egg cell is delayed by up to 36 h. Newly formed zygotes contain nuclear DNA concentrations of 4C at karyogamy and remain at 4C until zygote division. Tobacco displays cell fusion after the completion of S-phase, apparently during G2. Failure to achieve an optimized system for in vitro fertilization in Nicotiana may reflect the challenges of achieving cell cycle synchrony in gametes isolated from pollen tubes. Receptive gametes are presumably those that pass through the protracted S-phase, reaching G2 receptivity and cell cycle congruity before fusion.

Changes in actin organization in the living egg apparatus of Torenia fournieri during fertilization

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.

Actin coronas in normal and indeterminate gametophyte1 embryo sacs of maize

The actin cytoskeletal organization and nuclear behavior of normal and indeterminate gametophyte1 (ig1) embryo sacs of maize were examined during fertilization. After pollination, during degeneration of one of the synergids and before arrival of the pollen tube, the cytoskeletal elements undergo dramatic changes including formation of the actin coronas at the chalazal end of the degenerating synergid and at the interface between the egg cell and central cell. The actin coronas are present only for a limited period of time and their presence is coordinated with pollen tube arrival and fusion of the gametes; they disappear before the zygote divides. This allows us to estimate the frequency of fertilized ovules along the ear. Up to 88% of the ovules on an ear contain actin coronas in the embryo sacs when observed 16–19 h after pollination, indicating the high frequency of fertilizing kernels along the ear at this stage. In the ig embryo sacs, two or more degenerated synergids containing actin coronas at their chalazal ends receive multiple pollen tubes for gametic fusion and can consequently give rise to twin or polyembryos. These findings with the monocot maize are consistent with previous reports on the dicots Plumbago and Nicotiana, suggesting that the formation of actin coronas in the embryo sac during fertilization is a universal phenomenon in angiosperms and is part of a mechanism of interaction between gametic signaling and actin cytoskeleton behavior which appears to precisely position and facilitate the access of male gametes to the egg cell and central cell for fusion.

Pollen Tube Pathway in Chalazogamous Pistacia vera L.

Chalazogamy, the penetration of the pollen tube into the ovule via the chalaza instead of following the normal path through the micropyle, has been described in a number of unrelated species. The causes determining this behavior are unknown. We examined the pollen tube path from pollination to fertilization in Pistacia vera L., a chalazogamous species. While pollen germination and pollen tube growth in the style follow a pattern very similar to that observed in most plant species, pollen tube penetration into the ovule occurs via the funiculus. This is mediated through the formation of a ponticulus, a funicular protuberance that physically connects ovule and style. Once in the ponticulus, the pollen tube reaches the core of the funiculus, and there it takes a 90⚬ turn in direction to the nucellus, where it enters via the chalaza and grows bordering the embryo sac to penetrate through a synergid. While no specialized cells can be observed in the ponticulus, the central core of the funiculus, where the pollen tubes grow, very much resembles a transmitting tissue with cells rich in starch reserves. At anthesis the megagametophyte is immature: on pollen tube arrival, a mature embryo sac is present. Chalazogamy occurs in a consistent way in pistachio, leading to a successful fertilization. This event is associated with an immature stage at anthesis of both the obturator and the ovule with underdeveloped integuments that only partially surround the nucellus. Pollen tube penetration is supported by the ponticulus, a structure that fulfills the role of an obturator, and by the funicular transmitting tissue that conducts the pollen tube to the nucellus, as does the micropyle. The fostering of these functions by other structures giving an alternative pathway may have been responsible for making fertilization possible in this species.

Fertilization in Nicotiana tabacum: ultrastructural organization of propane-jet-frozen embryo sacs in vivo

Ovules of Nicotiana tabacum L. were cryofixed with a propane-jet freezer and freeze-substituted in acetone to examine technique-dependent changes in pre- and post-fertilization embryo sacs using rapidly frozen material. Freezing quality was acceptable in 10% of the embryo sacs in the partially dissected ovules, with ice-crystal damage frequently evident in vacuoles and nuclei. One of the two synergids begins to degenerate before pollen-tube arrival in cryofixed material, with breakdown of the plasma membrane and large chalazal vacuole delayed until the penetration of the pollen tube. Early synergid degeneration involved characteristic increases in cytoplasmic electron density and the generation of cytoplasmic bodies to the intercellular space through “pinching-off”. Upon pollen-tube arrival, the male gametes are released through a terminal aperture into the degenerate synergid. Sperm cells undergo morphological alteration before gametic fusion: their mitochondrial electron density increases, the endoplasmic reticulum dilates, cytoplasm becomes finely vacuolated and the surrounding pollen plasma membrane is lost, causing the sperm cells and vegetative nucleus to dissociate. Discharge of the pollen tube results in the formation of numerous enucleated cytoplasmic bodies which are either stripped or shed from sperm cells and pollen-tube cytoplasm. Two so-called X-bodies are found in the degenerate synergid after pollen-tube penetration: the presumed vegetative nucleus occurs at the chalazal end and the presumed synergid nucleus near the micropylar end.

Cytoskeletal organisation and modification during pollen tube arrival, gamete delivery and fertilisation in Plumbago zeylanica.

The cytoskeletal organisation of the isolated embryo sac and egg cells of Plumbago zeylanica was examined before, during and after pollen tube penetration into the embryo sac to determine the potential involvement of microtubules and actin filaments in fertilisation. Material was singly and triply stained using Hoechst 33258 to localise DNA, fluorescein isothiocyanate (FITC)-labelled anti-alpha-tubulin to detect microtubules and rhodamine-phalloidin to visualise F-actin. Microtubules in the unfertilised egg cell are longitudinally aligned in the micropylar and mid-lateral areas, aggregating into bundles near the filiform apparatus. In the perinuclear cytoplasm of the egg cell, microtubules become more or less randomly aligned. F-actin bundles form a longitudinally aligned mesh in the chalazal cytoplasm of the egg cell. In the central cell, microtubules and F-actin are distributed along transvacuolar strands and are also evident in the perinuclear region and at the periphery of the cell. During pollen tube penetration, sparse microtubule bundles near the pathway of the pollen tube may form an apparent microtubular 'conduit' surrounding the male gametes at the delivery site. Actin aggregates become organised near the pathway of the pollen tube and at the delivery site of the sperm cells. Subsequently, actin aggregates form a 'corona' structure in the intercellular region between the egg and central cell where gametic fusion occurs. The corona may have a role in maintaining the close proximity of the egg and central cell and helping the two sperm cells move and bind to their target cells. The cytoskeleton may also be involved in causing the two nuclei of the egg and central cell to approach one another at the site of gametic fusion and transporting the two sperm nuclei into alignment with their respective female nucleus. The cytoskeleton is reorganised during early embryogenesis.