Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.     

Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis

Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guidance signals from female cells are being identified; however, how pollen responds to those cues is largely unknown. Here, we show that two predicted cation/proton exchangers (CHX) in Arabidopsis thaliana, CHX21 and CHX23, are essential for pollen tube guidance. Male fertility was unchanged in single chx21 or chx23 mutants. However, fertility was impaired in chx21 chx23 double mutant pollen. Wild-type pistils pollinated with a limited number of single and double mutant pollen producing 62% fewer seeds than those pollinated with chx23 single mutant pollen, indicating that chx21 chx23 pollen is severely compromised. Double mutant pollen grains germinated and grew tubes down the transmitting tract, but the tubes failed to turn toward ovules. Furthermore, chx21 chx23 pollen tubes failed to enter the micropyle of excised ovules. Green fluorescent protein-tagged CHX23 driven by its native promoter was localized to the endoplasmic reticulum of pollen tubes. CHX23 mediated K(+) transport, as CHX23 expression in Escherichia coli increased K(+) uptake and growth in a pH-dependent manner. We propose that by modifying localized cation balance and pH, these transporters could affect steps in signal reception and/or transduction that are critical to shifting the axis of polarity and directing pollen growth toward the ovule.     

Pollen tubes enter neighbouring ovules by way of receptacle tissue,resulting in increased fruit-set in Sagittaria potamogetifolia Merr

Using fluorescence microscopy, deposition of pollen on stigmas and pollen tube growth in the gynoecium of Sagittaria potamogetifolia Merr., a monoecious species with an apocarpous gynoecium, were observed. The maximum rate of pollination averaged 83.9 +/- 4.7 %, and the number of pollen grains per stigma ranged from zero to 30. Pollen tubes grew through one stigma to the base of the ovary at almost the same speed, but generally only one of the pollen tubes then turned towards the ovule and finally entered the nucellus through the micropyle. The other pollen tubes grew through the ovary base and the receptacle tissue into ovules of adjacent carpels whose stigmas were not pollinated or which had been pollinated later. This phenomenon is termed pollen tube 'reallocation' by the authors. To verify the direct effect of the phenomenon on fruit set, artificial pollination experiments were conducted in which two or more pollen grains were placed onto only one stigma in each gynoecium; frequently more than one fruitlet was obtained from each flower treated. The reallocation of pollen tubes among pistils in the gynoecium could effect fertilization of ovules of unpollinated pistils and lead to an increase in sexual reproduction efficiency. It would, to some extent, also increase pollen tube competition among pistils of the whole gynoecium.     

Ovary and Gametophyte Development Are Coordinately Regulated by Auxin and Ethylene following Pollination

The differentiation and development of ovules in orchid flowers are pollination dependent. To define the developmental signals and timing of critical events associated with ovule differentiation, we have examined factors that regulate the initial events in megasporogenesis and female gametophyte development and characterized its progression toward maturity and fertilization. Two days after pollination, ovary wall epidermal cells begin to elongate and form hair cells; this is the earliest visible morphological change, and it occurs at least 3 days prior to pollen germination, indicating that signals associated with pollination itself trigger these early events. The effects of inhibitors of ethylene biosynthesis on early morphological changes indicated that ethylene, in the presence of auxin, is required to initiate ovary development and, indirectly, subsequent ovule differentiation. Surprisingly, pollen germination and growth were also strongly inhibited by inhibitors of ethylene biosynthesis, indicating that male gametophyte development is also regulated by ethylene. Detailed characterization of the development of both the female and male gametophyte in pollinated orchid flowers indicated that pollen tubes entered the ovary and grew along the ovary wall for 10 to 35 days, at which time growth was arrested. Approximately 40 days after pollination, coincident with ovule differentiation as indicated by the presence of a single archesporial cell, the direction of pollen tube growth became redirected toward the ovule, suggesting a chemical signaling between the developing ovule and male gametophyte. Taken together, these results indicate that both auxin and ethylene contribute to the regulation of both ovary and ovule development and to the coordination of development of male and female gametophytes.     

油橄榄授粉受精和胚胎发育过程观察

该研究以油橄榄“鄂植-8”为材料,应用压片荧光观察花粉在柱头和花柱中的萌发及生长情况,采用石蜡制片法观察油橄榄的胚珠结构特点和授粉受精过程。结果表明：油橄榄为1子房2心室4胚珠,珠心较发达,由多层细胞构成,属于厚珠心椭圆型胚珠,胚珠直生;花开时花粉粒落到柱头上立刻萌发,随后花粉管在花粉通道中生长,再后进入子房经子房内表面,大部分花粉管不能到达胚珠,仅少数沿胎座生长经珠柄进入珠孔,释放2个精子,2个精核分别进入卵细胞和极核,并与卵核及极核相互融合;观察到合子中雌、雄性核仁融合的过程。授粉受精各阶段经历的时间为花粉落到柱头上立刻萌发;授粉后6d左右,花粉管长入胚珠的珠孔,随后释放精子;10 d左右完成授粉受精,30 d左右形成心型胚,40 d左右胚发育几乎成熟。在进行子房石蜡切片中,共观察到612个完整子房切片,发育完整的子房为97.53％,完成授粉受精的胚珠为15.52％。油橄榄在自然授粉下,约85％的胚珠授粉受精过程不能完成,在一定程度上影响其坐果。该研究结果为油橄榄授粉受精、高效生产等提供了理论依据。     

Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the beta-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.     

Cell–cell communications and molecular mechanisms in plant sexual reproduction

Sexual reproduction is achieved by precise interactions between male and female reproductive organs. In plant fertilization, sperm cells are carried to ovules by pollen tubes. Signals from the pistil are involved in elongation and control of the direction of the pollen tube. Genetic, reverse genetic, and cell biological analyses using model plants have identified various factors related to the regulation of pollen tube growth and guidance. In this review, I summarize the mechanisms and molecules controlling pollen tube growth to the ovule, micropylar guidance, reception of the guidance signal in the pollen tube, rupture of the pollen tube to release sperm cells, and cessation of the tube guidance signal. I also briefly introduce various techniques used to analyze pollen tube guidance in vitro.     

Fertilization recovery after defective sperm cell release in Arabidopsis

In animal fertilization, multiple sperms typically arrive at an egg cell to "win the race" for fertilization. However, in flowering plants, only one of many pollen tubes, conveying plant sperm cells, usually arrives at each ovule that harbors an egg cell. Plant fertilization has thus been thought to depend on the fertility of a single pollen tube. Here we report a fertilization recovery phenomenon in flowering plants that actively rescues the failure of fertilization of the first mutant pollen tube by attracting a second, functional pollen tube. Wild-type (WT) ovules of Arabidopsis thaliana frequently (～80%) accepted two pollen tubes when entered by mutant pollen defective in gamete fertility. In typical flowering plants, two synergid cells on the side of the egg cell attract pollen tubes, one of which degenerates upon pollen tube discharge. By semi-in vitro live-cell imaging we observed that fertilization was rescued when the second synergid cell accepted a WT pollen tube. Our results suggest that flowering plants precisely control the number of pollen tubes that arrive at each ovule and employ a fertilization recovery mechanism to maximize the likelihood of successful seed set.     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

The evolution of ovule number and flower size in wind-pollinated plants

In angiosperms, ovules are "packaged" within individual flowers, and an optimal strategy should occur depending on pollination and resource conditions. In animal-pollinated species, wide variation in ovule number per flower occurs, and this contrasts with wind-pollinated plants, where most species possess uniovulate flowers. This pattern is usually explained as an adaptive response to low pollen receipt in wind-pollinated species. Here, we develop a phenotypic model for the evolution of ovule number per flower that incorporates the aerodynamics of pollen capture and a fixed resource pool for provisioning of flowers, ovules, and seeds. Our results challenge the prevailing explanation for the association between uniovulate flowers and wind pollination. We demonstrate that when flowers are small and inexpensive, as they are in wind-pollinated species, ovule number should be minimized and lower than the average number of pollen tubes per style, even under stochastic pollination and fertilization regimes. The model predicts that plants benefit from producing many small inexpensive flowers, even though some flowers capture too few pollen grains to fertilize their ovules. Wind-pollinated plants with numerous flowers distributed throughout the inflorescence, each with a single ovule or a few ovules, sample more of the airstream, and this should maximize pollen capture and seed production.     

She's the boss: signaling in pollen tube reception

In angiosperms, the sperm cells are carried within the pollen tubes (male gametophytes) to the female gametophyte so that double fertilization can occur. The female gametophyte exerts control over the male, with specialized cells known as synergids guiding the pollen tubes and controlling their behavior when they enter the female gametophyte so that the sperm cells can be delivered to the egg and central cell. Upon pollen tube arrival at the ovule, signal transduction cascades mediated by receptor-like kinases are initiated in both the synergid and the tip of the pollen tube, leading to synergid cell death and pollen tube rupture. In this review, we discuss the role of these receptors and of newly discovered members of the pollen tube reception pathway.     

Pollen tube reallocation in two preanthesis cleistogamous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae)

Pollen deposition on stigmas and pollen tube growth in two apocarpous species, Ranalisma rostratum and Sagittaria guyanensis ssp. lappula (Alismataceae), were examined with fluorescence microscopy. The reallocation of pollen tubes among pistils was observed in both species. The percentage of pollinated stigmas per flower was only 22.0% in R. rostratum and 51.0% in S. guyanensis, though the seed/ovule ratios are higher than 65% in both species. The number of pollen grains on each single stigma ranged from 0 to 96 in R. rostratum, and from 0 to 125 in S. guyanensis. When more than one pollen grain deposited on a stigma, all pollen tubes grew to the ovary, but only one of them turned towards the ovule and finally entered the nucleus. The other tubes grew through the receptacle tissue into ovules of adjacent carpels whose stigmas were unpollinated or pollinated later. The intercarpellary growth of pollen tubes could be a mechanism to increase the efficiency of sexual reproduction in an apocarpous gynoecium with low pollination on the pistils.     

Invvestigations on Early Embryogenesis of Actinidia chinensis Planch var. chinensis

This paper deals with early embryogenesis of Actinidia chinensis var. chinensis. 1. Ovary superior consists of 34—45 carpels. Each carpel contains 11–45 ovules. The ovule is uni-integument and tenuinucellar. The ovule is anatropous. The archesporium is formed by a single cell, and directly develops into megaspore mother cell. Sometimes the archesporium consists of 2–3 cells, but only one of them develops into megaspore mother cell and the others are degenerated. 2. The mature pollen grain is two-celled and the embryo sac belongs to olygonum type. In most embryo sacs two polar nuclei are fused before fertilization. One of the synergids was destroyed as the pollen tube penetrated into embryo sac the other one disappeared after fertilization. In most cases the antipodal cells became degenerated in fertilization process, only some remained until the first division of primary endosperm nucleus. 3. In Beijing area the double fertilization of Actinidia chinensis occurred 30–72 hours after pollination. In the fertilization one sperm fused with egg nucleus and the other sperm fused with the secondary nucleus as usual. The fusion of the secondary nucleus with sperm was in advance of the fusion of the egg nudeus. 4. The endosperm is cellular type.     

TICKET attracts pollen tubes and mediates reproductive isolation between relative species in Brassicaceae

In flowering plants, pollen tubes are attracted to the ovule by secreted peptides to release the sperm cells for double fertilization.This process is species-specific and acts as an important stage of reproductive isolation between species. Here we identified a cysteine-rich peptide TICKET2 in Arabidopsis thaliana and its orthologs in Arabidopsis lyrata and Capsella rebella that can attract the conspecific pollen tubes, but not the pollen tubes of relative species in Brassicaceae. Genetic knockout of the AtTICKET subclade compromised the pollen tube attraction efficiency. This study identified a new pollen tube attracting signal and shed light on the molecular basis of reproductive isolation.     

CHEMOTROPIC ACTIVITY AND THE PATHWAY OF THE POLLEN TUBE IN LILY,

A study of the pollen tube pathway in Lilium leucanthum var. centifolium and in L. regale reveals that the entire pathway from stigma to ovule is lined with cytologically unique stigmatoid cells. Assays for chemotropic activity of tissues and exudates along the pathway of pollinated or unpollinated pistils showed that onset of chemotropic activity progressed basipetally (and, when pollinated, in advance of the pollen tubes), commencing at the stigma 3-5 days before anthesis and appearing in the ovules 1-2 days after anthesis. Activity persists about 10 days in ovules of pollinated pistils and for 14-16 days in ovules of non-pollinated pistils. Attempts to localize the source of the chemotropic factor showed that gynoecial tissues bearing stigmatoid cells are chemo-tropically active while slices of style or ovary wall lacking stigmatoid cells are inactive. When ovules were sliced transversely and the micropylar and chalazal halves assayed, only the micropylar half showed activity. We suggest that the ovules and the stigmatoid tissue along the pollen tube pathway are the sources of the chemotropic factor responsible for the directional growth of the pollen tube.     

从柱头到胚囊——花粉管发育研究进展

花粉管的生长和发育是植物有性生殖过程中重要环节。本文首先介绍了花粉管的结构及其脉冲生长方式。然后介绍了花粉粒在柱头上萌发、花粉管在花柱道中生长及其进入胚囊的一些特点,同时还介绍了雌蕊在花粉管生长发育所起的重要作用。     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

Silencing gene expression of the ethylene-forming enzyme results in a reversible inhibition of ovule development in transgenic tobacco plants

To study the role of ethylene in plant reproduction, we constructed transgenic tobacco plants in which the expression of a pistil-specific gene coding for the ethylene-forming enzyme 1-aminocyclopropane-1-carboxylate oxidase was inhibited. Flowers from transgenic plants showed female sterility due to an arrest in ovule development. Megasporogenesis did not occur, and ovules did not reach maturity. When pollinated, pollen tubes were able to reach the ovary but did not penetrate into the immature ovule in transgenic plants. Flower treatment with an ethylene source resulted in a functional recovery of ovule development and restored guidance of the pollen tube tip into the ovule micropyle that resulted in seed set. The recovery was abolished if inhibitors of ethylene action were present. These results demonstrate that the plant hormone ethylene is required during the very early stages of female sporogenesis and ultimately to enable fertilization.     

OVULE PACKAGING IN STOCHASTIC POLLINATION AND FERTILIZATION ENVIRONMENTS

The modular morphology of plants has important consequences for reproductive strategies. Ovules are packaged in discrete structures (flowers) that usually vary stochastically in pollen capture and ovule fertilization, because of the vagaries of pollen transfer by external agents. Different ovule packaging schemes may use limited reproductive resources more or less effectively, so that some number of ovules per flower may be optimal, given the prevailing probabilities of ovule fertilization. I derive a phenotypic model for ovule number per flower that maximizes the expected total ovule fertilizations on a plant when pollination and fertilization vary randomly among individual flowers. This model predicts that, except for small or inexpensive flowers, ovules should be “oversupplied” relative to the mean receipt of pollen tubes, so that pollen limitation of seed set should be common. Published data are congruent with this prediction. Additional hypotheses on the relation of ovule packaging to floral cost, plant size, and variance in pollen receipt are suggested by the model, but few data exist to evaluate these hypotheses.     

Pollen tube growth and early ovule development following self- and cross-pollination in<Emphasis Type="Italic"> Eucalyptus nitens</Emphasis>

Controlled self- and cross-pollinations were conducted on flowers of five mature Eucalyptus nitens trees. Levels of self-sterility of the trees ranged from 25.8 to 93.6%. Pollen tube numbers in styles and ovule penetration by pollen tubes was investigated 2 weeks after pollination by fluorescence microscopy. There were no significant differences between treatments in the number of pollen tubes present in styles or in the percentage of ovules penetrated by pollen tubes. Embryology of material harvested 2 and 4 weeks after pollination was investigated by bright-field microscopy. Fertilisation had taken place by 2 weeks after pollination with nearly every ovule showing evidence of fertilisation. Cross-pollination resulted in a greater proportion of healthy, developing ovules, at both 2 and 4 weeks after pollination, compared with self-pollination. The proportion of degenerating ovules increased from 2 to 4 weeks after pollination. The reduced ability of E. nitens to set self-pollinated seed compared with cross-pollinated seed appears to be controlled by a post-zygotic mechanism. Differences in ovule size may potentially assist in the identification of trees incapable of setting self-pollinated seed.