Pollen Tube Entry Research Articles

A Study on the Causes of Apomixis in Malus shizongensis

Apomixis is a unique reproductive process that produces fertile offspring without the combination of sperm and egg cells. This process perfectly reproduces maternal DNA, making it possible to fix heterosis during reproduction. Malus shizongensis is a newly discovered species that is closely related to Malus hupehensis Rehd. After de-male bagging, it was found that the fruit set rate reached 78.7%. Preliminary analysis indicated that M. shizongensis have apomictic reproductive characteristics. In this work, we employed paraffin sectioning and electron scanning microscopy to explore apomixis in M. shizongensis during the development of male–female gametes and embryo sacs. Stigma fluorescence assays showed that pollen germination was normal, but less pollen entered the ovaries. Additionally, analysis of anthers indicated the presence of dysplasia and paraffin sectioning revealed that the pollen mother cells were aborted due to abnormal disintegration of the tapetum layer. Taken together, our results indicate that the primary causes of apomixis in M. shizongensis are anther dysplasia and male gamete development failure, resulting in reduced pollen tube entry into ovaries and reduced reproduction of female gametes. In conclusion, this study provide a theoretical basis and technical supports for apple stock breeding and apple industry development.

Central-cell-produced attractants control fertilization recovery

Animal fertilization relies on hundreds of sperm racing toward the egg, whereas, in angiosperms, only two sperm cells are delivered by a pollen tube to the female gametes (egg cell and central cell) for double fertilization. However, unsuccessful fertilization under this one-pollen-tube design can be detrimental to seed production and plant survival. To mitigate this risk, unfertilized-gamete-controlled extra pollen tube entry has been evolved to bring more sperm cells and salvage fertilization. Despite its importance, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we report that, in Arabidopsis, the central cell secretes peptides SALVAGER1 and SALVAGER2 in a directional manner to attract pollen tubes when the synergid-dependent attraction fails or is terminated by pollen tubes carrying infertile sperm cells. Moreover, loss of SALs impairs the fertilization recovery capacity of the ovules. Therefore, this research uncovers a female gamete-attraction system that salvages seed production for reproductive assurance.

A Toolkit for Teasing Apart the Early Stages of Pollen-Stigma Interactions in Arabidopsis thaliana.

In hermaphroditic flowering plants, the female pistil serves as the main gatekeeper of mate acceptance as several mechanisms are present to prevent fertilization by unsuitable pollen. The characteristic Brassicaceae dry stigma at the top of pistil represents the first layer that requires pollen recognition to elicit appropriate physiological responses from the pistil. Successful pollen-stigma interactions then lead to pollen hydration, pollen germination, and pollen tube entry into the stigmatic surface. To assess these early stages in detail, our lab has used three experimental procedures to quantitatively and qualitatively characterize the outcome of compatible pollen-stigma interactions that would ultimately lead to the successful fertilization. These assays are also useful for assessing self-incompatible pollinations and mutations that affect these pathways. The model organism, Arabidopsis thaliana, offers an excellent platform for these investigations as loss-of-function or gain-of-function mutants can be easily generated using CRISPR/Cas9 technology, existing T-DNA insertion mutant collections, and heterologous expression constructs, respectively. Here, we provide a detailed description of the methods for these inexpensive assays that can be reliably used to assess pollen-stigma interactions and used to identify new players regulating these processes.

Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana

BackgroundIn the Brassicaceae, the early stages of compatible pollen-stigma interactions are tightly controlled with early checkpoints regulating pollen adhesion, hydration and germination, and pollen tube entry into the stigmatic surface. However, the early signalling events in the stigma which trigger these compatible interactions remain unknown.ResultsA set of stigma-expressed pseudokinase genes, termed BRASSIKINs (BKNs), were identified and found to be present in only core Brassicaceae genomes. In Arabidopsis thaliana Col-0, BKN1 displayed stigma-specific expression while the BKN2 gene was expressed in other tissues as well. CRISPR deletion mutations were generated for the two tandemly linked BKNs, and very mild hydration defects were observed for wild-type Col-0 pollen when placed on the bkn1/2 mutant stigmas. In further analyses, the predominant transcript for the stigma-specific BKN1 was found to have a premature stop codon in the Col-0 ecotype, but a survey of the 1001 Arabidopsis genomes uncovered three ecotypes that encoded a full-length BKN1 protein. Furthermore, phylogenetic analyses identified intact BKN1 orthologues in the closely related outcrossing Arabidopsis species, A. lyrata and A. halleri. Finally, the BKN pseudokinases were found to be plasma-membrane localized through the dual lipid modification of myristoylation and palmitoylation, and this localization would be consistent with a role in signaling complexes.ConclusionIn this study, we have characterized the novel Brassicaceae-specific family of BKN pseudokinase genes, and examined the function of BKN1 and BKN2 in the context of pollen-stigma interactions in A. thaliana Col-0. Additionally, premature stop codons were identified in the predicted stigma specific BKN1 gene in a number of the 1001 A. thaliana ecotype genomes, and this was in contrast to the out-crossing Arabidopsis species which carried intact copies of BKN1. Thus, understanding the function of BKN1 in other Brassicaceae species will be a key direction for future studies.

The role of the integuments in pollen tube guidance in flowering plants.

In angiosperms, pollen tube entry into the ovule generally takes place through the micropyle, but the exact role of the micropyle in pollen tube guidance remains unclear. A limited number of studies have examined eudicots with bitegmic micropyles, but information is lacking in ovules of basal/early-divergent angiosperms with unitegmic micropyles. We have evaluated the role of the micropyle in pollen tube guidance in an early-divergent angiosperm (Annona cherimola) and the evolutionarily derived Arabidopsis thaliana by studying γ-aminobutyric acid (GABA) and arabinogalactan proteins (AGPs) in wild-type plants and integument-defective mutants. A conserved inhibitory role of GABA in pollen tube growth was shown in A.cherimola, in which AGPs surround the egg apparatus. In Arabidopsis, the micropyle formed only by the outer integument in wuschel-7 mutants caused a partial defect in pollen tube guidance. Moreover, pollen tubes were not observed in the micropyle of an inner no outer (ino) mutant in Arabidopsis, but were observed in homologous ino mutants in Annona. The similar distribution of GABA and AGPs observed in the micropyle of Arabidopsis and Annona, together with the anomalies from specific integument mutants, support the role of the inner integument in preventing multiple tube entrance (polytubey) in these two phylogenetically distant genera.

Ethylene signaling is critical for synergid cell functional specification and pollen tube attraction

ETHYLENE INSENSITIVE 3 (EIN3) is a key regulator of ethylene signaling, and EIN3-BINDING F-BOX1 (EBF1) and EBF2 are responsible for EIN3 degradation. Previous reports have shown that the ebf1 ebf2 double homozygous mutant cannot be identified. In this study, the genetic analysis revealed that the ebf1 ebf2 female gametophyte is defective. The pollination experiment showed that ebf1 ebf2 ovules failed to attract pollen tubes. In female gametophyte/ovule, the synergid cell is responsible for pollen tube attraction. Observation of the pEIN3::EIN3-GFP transgenic lines showed that EIN3 signal was over-accumulated at the micropylar end of ebf1 ebf2 female gametophyte. The overexpression of stabilized EIN3 in synergid cell led to the defect of pollen tube guidance. These results suggested that the over-accumulated EIN3 in ebf1 ebf2 synergid cell blocks its pollen tube attraction which leads to the failure of ebf1 ebf2 homozygous plant. We identified that EIN3 directly activated the expression of a sugar transporter, SENESCENCE-ASSOCIATED GENE29 (SAG29/SWEET15). Overexpression of SAG29 in synergid cells blocked pollen tube attraction, suggesting that SAG29 might play a role in ethylene signaling to repel pollen tube entry. Taken together, our study reveals that strict control of ethylene signaling is critical for the synergid cell function during plant reproduction.

Floral Biology and Embryological Studies are Important for Conservation of Threatened Plants Having Reproductive Bottlenecks:A Case Study of <i>Illicium griffithii</i> Hook. f. & Thomson

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.

Exocyst, exosomes, and autophagy in the regulation of Brassicaceae pollen-stigma interactions.

Brassicaceae pollen-stigma interactions have been extensively studied in Brassica and Arabidopsis species to identify cellular events triggered in the stigmatic papillae by pollen contact. Compatible pollinations are linked to the activation of basal cellular responses in the stigmatic papillae, which include calcium gradients, actin networks, and polarized secretion. The occurrence of these cellular events in stigmatic papillae coincides with the stages of pollen hydration and pollen tube entry into the stigmatic papillar cell wall. However, the form of the vesicle trafficking appears to differ between species, with vesicle-like structures detected in Arabidopsis species while exosomes were found to be secreted in Brassica species. Around the same timeframe, self-incompatible pollen recognition leads altered cellular responses in the stigmatic papillae to interfere with basal compatible pollen responses and disrupt regulated secretion, causing self-pollen rejection. Here, the literature on the changing cellular dynamics in the stigmatic papillae following pollination is reviewed and discussed in the context of other well-characterized examples of polarized secretion in plants.

RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis.

Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance.

High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen.

We have previously proposed that Exo70A1 is required in the Brassicaceae stigma to control the early stages of pollen hydration and pollen tube penetration through the stigmatic surface, following compatible pollination. However, recent work has raised questions regarding Arabidopsis thaliana Exo70A1's expression in the stigma and its role in stigma receptivity to compatible pollen. Here, we verified the expression of Exo70A1 in stigmas from three Brassicaceae species and carefully re-examined Exo70A1's function in the stigmatic papillae. With previous studies showing that high relative humidity can rescue some pollination defects, essentially bypassing the control of pollen hydration by the Brassicaceae dry stigma, the effect of high humidity was investigated on pollinations with the Arabidopsis exo70A1-1 mutant. Pollinations under low relative humidity resulted in a complete failure of wild-type compatible pollen acceptance by the exo70A1-1 mutant stigma as we had previously seen. However, high relative humidity resulted in a partial rescue of the exo70A1-1 stigmatic papillar defect resulting is some wild-type compatible pollen acceptance and seed set. Thus, these results reaffirmed Exo70A1's proposed role in the stigma regulating compatible pollen hydration and pollen tube entry and demonstrate that high relative humidity can partially bypass these functions.

Pollen tube entry into the synergid cell of Arabidopsis is observed at a site distinct from the filiform apparatus

In higher plants, the double-fertilization process begins with the successful delivery of two sperm cells to the female gametophyte. The sperms cells are carried by a pollen tube that upon arrival at the micropylar end of the female gametophyte, bursts, and discharges its content into one of two specialized cells called the synergid cells. At their micropylar ends, both synergid cells form a thickened cell wall with a unique structure called the filiform apparatus. The filiform apparatus is believed to play a major role in pollen tube guidance and reception. It has also been assumed that the pollen tube enters the receptive synergid cell through the filiform apparatus. Here, we show that in Arabidopsis ovules, the arriving pollen tube appears to grow beyond the filiform apparatus to enter the synergid cell at a more distant site, where the tube bursts to release its contents. Thus, fertilization in Arabidopsis might involve two spatially and temporally separable stages, recognition and entry, with the latter apparently not requiring the filiform apparatus.

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Double Fertilization and Embryogenesis of Eustoma grandiflorum

The double fertilization process and the development of endosperm and embryo were observed by microscopy in Eustoma grandiflorum. The flowers were fixed at various times after hand-pollination during stages of pollen tube extension, entry of pollen tubes into ovule, double fertilization, and early development of the embryo. Pollen grains initiated germination and penetrated the style 4 hours after pollination (HAP). Pollen tubes grew toward the ovule through the intercellular spaces along the solid style at 8 HAP. During pollen tube extension at 24 HAP, the generative cell divided into two sperm cells. Pollen tubes reached the ovary at 48 HAP, extended directly into the degenerating synergid cell and released the two sperm cells. One of the two sperm cells arrived adjacent to the egg cell at 60 HAP. Four to 5 days after pollination, a zygote was formed. The sperm nucleus adhered to the nuclear membrane of the secondary nucleus at 60 HAP. The primary endosperm nucleus was formed at 72 HAP. The zygote underwent the first mitosis 20 days after pollination. After the first divisions of the zygote, the apical cell divided transversely initially, and continued to develop to form the embryo proper; hence, the embryogenesis was of Solanad type. The mature seeds have a heart-shaped embryo. Only one spindle formed during the first mitosis of the zygote, and the fusion of male and female nucleoli took place before mitosis. The karyogamy was therefore of premitotic type.

EVALUATION OF PRE-FERTILIZATION BARRIERS BY OBSERVATION OF POLLEN TUBE GROWTH AND ATTEMPTS FOR OVERCOMING POST-FERTILIZATION BARRIERS IN INTERGENERIC HYBRIDIZATION BETWEEN ALSTROEMERIA AND BOMAREA BY OVULE CULTURE

In the flower market there is a continuous demand for new cultivars of Alstroemeria as an ornamental flower. Many interspecific hybrids of Alstroemeria are utilized for commercial cultivars. However there is no report about intergeneric hybridization between Alstroemeria and Bomarea. We are investigating the possibility of creating intergeneric hybrids between Alstroemeria and Bomarea. Moreover, ovule culture conditions were examined for overcoming post-fertilization barriers after pollination of Bomarea pollen grains to A. aurea, A. pelegrina var. rosea and A. magenta. The frequency of pollen tube entry into ovules was compared in the pistil of A. aurea, A. pelegrina var. rosea and A. magenta after pollination of B. coccinea pollen grains. Pollen tubes in Alstroemeria ovaries were observed with aniline blue staining 48 hours after pollination. The frequencies of pollen tube entry into ovules were 0.3, 5.6 and 10.0% in A. aurea, A. pelegrina var. rosea and A. magenta, respectively. For ovule culture, pollinated ovaries were harvested 3 and 7 days after pollination, and the ovules were cultured on 2 g L -1 gellan gum-solidified MS medium with or without gibberellic acid, and supplemented with sucrose at different concentrations (30, 60, 80 or 100 g L -1 ). As a result, 3 plantlets were obtained in A. pelegrina var. rosea x B. coccinea cultured on MS medium supplemented with 80 g L -1 sucrose. Although pollen tubes of B. coccinea were reached to A. aurea ovules, this combination might have stronger pre-fertilization barriers than those of A. pelegrina var. rosea and A. magenta. It supposed to be different intensity of pre-fertilization barriers among species. Our data also suggested that sucrose concentration at 80 g L -1 in culture medium was effective to obtain progenies after intergeneric pollination. Confirmation of hybrid natures for the plantlets is now in progress.

Structure and Histochemistry of the Micropylar and Chalazal Regions of the Perisperm–endosperm Envelope of Cucumber Seeds Associated with Solute Permeability and Germination

The perisperm–endosperm (PE) envelope surrounding the embryo of cucumber (Cucumis sativus) acts as a barrier to apoplastic permeability and radicle emergence. The envelope consists of a single cell layer of endosperm whose outer surface is covered by noncellular lipid and callose-rich layers. We compared the structure and histochemistry of the radicle tip and chalazal regions of the envelope, because these regions differ in permeability. Seeds were treated with coumarin 151, a nonionic, fluorescent tracer with systemic activity. Treated seeds were imbibed and on seedcoat removal, the root tip area of the membrane-covered embryo accumulated the fluorescent tracer, but the tracer could not penetrate the envelope that bordered the cotyledons and chalazal region. The cone-shaped remnant of tissue opposite the micropylar region of the envelope was identified as nucellar tissue, the “nucellar beak.” The cuticular membrane and callose layer of the PE envelope were interrupted in the nucellar beak as well as in the chalazal region. Their role in permeability is apparently substituted by the presence of thick-walled suberized cells in the beak and chalaza. A canal was observed in the center of the nucellar beak that likely provided a conduit for the tracer to diffuse from the environment to the embryo. This canal was the remnant of pollen tube entry through the nucellus and was plugged with several cells, presumably residue of the suspensor. These cells degenerated just before cucumber seed germination. This remnant of the pollen tube canal presumably offers less mechanical resistance in the nucellar beak that might help facilitate radicle protrusion during germination. Cells of the outermost and basal regions of the nucellar beak as well as the walls of endosperm cells contained pectic material. Significant pectin methylesterase activity was found in the lateral and cap regions of the PE envelope long before seed germination. Lack of callose in the envelope at the radicle tip suggests that callose does not act as a barrier to radicle emergence during cucumber seed germination.

Self‐sterility in Ipomopsis aggregata (Polemoniaceae) is due to prezygotic ovule degeneration

Based on previous studies, extreme (>99%) self-sterility in scarlet gilia (Ipomopsis aggregata) appears to be involved in late-acting ovarian self-incompatibility (OSI). Here, we confirm this suggestion by comparing structural events that follow from cross- vs. self-pollinations of I. aggregata. Growth of cross- and self-pollen tubes in the style at 11 h and growth in the ovary at 24 h was equivalent. Nonetheless, by 24 h, cross-pollen effected a significantly higher percentage of both ovule penetration and fertilization. Ovules in self-pollinated flowers showed pronounced changes, including an absence of embryo sac expansion and reduced starch in the integument, by 11 h post-pollination, well before pollen tube entry into the ovary. In addition, the integumentary tapetum and adjacent 1-3 cell layers exhibited abnormal cell division, pronounced deposition of thick, pectin-rich cell walls, and cellular collapse. Ovules and embryo sacs from cross-pollinated flowers rarely showed such features. Developmental changes in ovules from self-pollinated flowers eventually resulted in integument and embryo sac collapse, a process not observed in ovules of unpollinated flowers. We suggest that OSI involves long-distance signaling between self-pollen or self-pollen tubes and carpel tissue that reduces availability of receptive ovules for fertilization before pollen tubes arrive in the ovary.

Identification and overcoming barriers between Brassica rapa L. em. Metzg. and B. nigra (L.) Koch crosses for the resynthesis of B. juncea (L.) Czern.

Brassica juncea (2n = 36, AABB) is an amphidiploid derived from its diploid progenitor species B. rapa (2n = 20, AA) and B. nigra (2n = 16, BB). Resynthesis of B. juncea by exploiting the wider gene pool of the present day diploids offers a powerful tool for obtaining novel genetic variation. Hybridization between the two species is difficult because of the presence of crossability barriers. Barriers to hybridization were identified using Aniline Blue Fluorescence (ABF) method. Crosses were classified as having pre- or post-fertilization barriers based on whether less than or more than one per cent ovules showed pollen tube entry. Of the 23 crosses studied, seven crosses all with B. rapa as the female parent showed pre-fertilization barriers. Only 0 to 0.3 per cent ovules were fertilized in these crosses. The remaining 16 crosses, four with B. rapa as the female parent and 12 with B. nigra as the female parent, showed post-fertilization barriers. In crosses with pre-fertilization barriers use of bud pollination and stump pollination ensured or increased fertilization. These two simple techniques were used in combination with ovary-ovule culture to ensure recovery of hybrids. In crosses with post-fertilization barriers, ovary-ovule culture alone helped to obtain hybrids. Hybrids were obtained at higher frequency with the use of direct ovule culture compared to ovary-ovule culture. The F1 plants obtained in vitro were multiplied on MS medium + 2 mg/l Kn + 0.2 mg/l IAA. These F1 plants were confirmed as true hybrids through morphology, leaf isozymes and cytology. In all 17 hybrids were obtained of which three were amphidiploids.

Cell Wall Reactive Proteins in the Coat and Wall of Maize Pollen: POTENTIAL ROLE IN POLLEN TUBE GROWTH ON THE STIGMA AND THROUGH THE STYLE

The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055-49064). The other protein of 70 kDa was purified to homogeneity and shown to be a beta-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known beta-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank trade mark data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX3CX10-15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat beta-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance.

The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception.

Reproduction in angiosperms depends on communication processes of the male gametophyte (pollen) with the female floral organs (pistil, transmitting tissue) and the female gametophyte (embryo sac). Pollen-pistil interactions control pollen hydration, germination and growth through the stylar tissue. The female gametophyte is involved in guiding the growing pollen tube towards the micropyle and embryo sac. One of the two synergids flanking the egg cell starts to degenerate and becomes receptive for pollen tube entry. Pollen tube growth arrests and the tip of the pollen tube ruptures to release the sperm cells. Failures in the mutual interaction between the synergid and the pollen tube necessarily impair fertility. But the control of pollen tube reception is not understood. We isolated a semisterile, female gametophytic mutant from Arabidopsis thaliana, named feronia after the Etruscan goddess of fertility, which impairs this process. In the feronia mutant, embryo sac development and pollen tube guidance were unaffected in all ovules, although one half of the ovules bore mutant female gametophytes. However, when the pollen tube entered the receptive synergid of a feronia mutant female gametophyte, it continued to grow, failed to rupture and release the sperm cells, and invaded the embryo sac. Thus, the feronia mutation disrupts the interaction between the male and female gametophyte required to elicit these processes. Frequently, mutant embryo sacs received supernumerary pollen tubes. We analysed feronia with synergid-specific GUS marker lines, which demonstrated that the specification and differentiation of the synergids was normal. However, GUS expression in mutant gametophytes persisted after pollen tube entry, in contrast to wild-type embryo sacs where it rapidly decreased. Apparently, the failure in pollen tube reception results in the continued expression of synergid-specific genes, probably leading to an extended expression of a potential pollen tube attractant.

Reproductive biology of Polypleurum stylosum (Podostemaceae)

Floral biology, pollination, pollen–pistil interaction and fruit development were studied in Polypleurum stylosum var. stylosum and var. laciniata growing attached to rocks in rivers and in the laboratory. Floral development and morphology are similar in both varieties but there are differences in flower bud size, number of pollen grains, ovules, seeds and ovule : seed ratio. Both varieties are self-compatible and predominantly self-pollinated. The inter-locking of sex organs within a flower is an adaptation for self-pollination although cross-pollination cannot be ruled out. Germination of pollen on the stigma and entry of pollen tubes into the ovules have been observed. Fruits appear mature in an incredibly short period of 4–5 days after pollination.