GABA,a new player in the plant mating game

Pollen tubes are guided through female tissues to deliver sperms to the embryo sac. A recent study reveals a GABA gradient along the pollen tube path, which, together with guidance defects in GABA-overaccumulating mutants, implies a role for GABA in regulating pollen tube growth and guidance.     

Guidance in vitro of the pollen tube to the naked embryo sac of torenia fournieri

The precise guidance of the pollen tube to the embryo sac is critical to the successful sexual reproduction of flowering plants. We demonstrate here the guidance of the pollen tube to the embryo sac in vitro by using the naked embryo sac of Torenia fournieri, which protrudes from the micropyle of the ovule. We developed a medium for culture of both the ovule and the pollen tube of T. fournieri and cocultivated them in a thin layer of solid medium. Although pollen tubes that had germinated in vitro passed naked embryo sacs, some pollen tubes that grew semi-in vitro through a cut style arrived precisely at the site of entry into the embryo sac, namely, the filiform apparatus of the synergids. When pollen tubes were unable to enter the embryo sac, they continuously grew toward the same filiform apparatus, forming narrow coils. Pollen tubes selectively arrived at complete, unfertilized embryo sacs but did not arrive at those of heat-treated ovules or those with disrupted synergids. These results convincingly demonstrate that pollen tubes are specifically attracted to the region of the filiform apparatus of living synergids in vitro.     

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.     

Ultrastructure of the micropyle and its relationship to pollen tube growth and synergid degeneration in sunflower

Summary Ultrastructural studies made on the micropyle of sunflower before and after pollination resulted in the following observations. (1) The micropyle is closed instead of a hole or canal. The inner epidermis of the integument on both sides of the micropyle is in close contact at the apex of the ovule. The boundary between the two sides consists of two layers of epidermal cuticle. (2) The micropyle contains a transmitting tissue. The micropyle is composed of an intercellular matrix produced by the epidermal cells of the integument. (3) The micropyle is asymmetrical, and is much wider on the side proximal to the funicle. On the funicle side the cells adjacent to the micropyle are similar to those of the transmitting tissue: they have large amounts of intercellular matrix and contain abundant dictyosomes, rough ER, and starch grains, and provide an appropriate environment for growth of the pollen tubes. The cells distal to the funicle are rich in rough ER and lipid bodies; they lack large intercellular spaces. (4) The micropyle is variable in the axial direction, i.e., it is much larger and more asymmetric at the level distal to the embryo sac than at a level close to the embryo sac. After pollination, one to four pollen tubes are seen in a micropyle. During their passage through the micropyle, most pollen tubes are restricted to the side proximal to the funicle. There is a greater tendency (81%) for the degenerate synergid to be located toward the funicle, i.e., at the same side as the pollen tube pathway. The data indicate a close relationship between micropyle organization, orientation of pollen tube growth, and synergid degeneration.     

Adhesion and guidance in compatible pollination

The mechanisms of compatible pollination are less studied than those of incompatible pollination and yet most of the angiosperms show self-compatibility. From the release of pollen from anthers to the penetration of the micropyle by the pollen tube tip, there are numerous steps where the interaction between pollen and the pistil can be regulated. Recent studies have documented some diverse ways in which pollen tubes carrying sperm cells are guided to the ovules through the pistil extracellular matrices of the transmitting tract. What is still missing is an understanding of pollen tube cell biology in vivo. A recent finding supports the role of the synergids in the crucial guidance cue for the pollen tube tip at the micropyle, but experimental evidence for other 'guidepost' cells in the pistil is still lacking. The fact that the pollen tube must first travel through the matrices of the stigma and style before it can respond to the cue from the ovule makes it likely that there is a hierarchy of signalling events in pollen-pistil interactions starting at the stigma and ending at the micropyle. On the pistil side, several model systems have been used in the discovery of molecules implicated in either physical or chemical guidance. In lily, which has a hollow style, adhesion molecules (pectin and SCA) are implicated in guidance. SCA alone is also capable of inducing pollen chemotropism in an in vitro assay, suggesting that this peptide plays a dual role in lily pollination: chemotactic in the stigma and haptotactic (adhesion mediated) in the style.     

Two Arabidopsis AGC kinases are critical for the polarized growth of pollen tubes

Reproduction of flowering plants requires the growth of pollen tubes to deliver immotile sperm for fertilization. Pollen tube growth resembles that of polarized metazoan cells, in that some molecular mechanisms underlying cell polarization and growth are evolutionarily conserved, including the functions of Rho GTPases and the dynamics of the actin cytoskeleton. However, a role for AGC kinases, crucial signaling mediators in polarized metazoan cells, has yet to be shown in pollen tubes. Here we demonstrate that two Arabidopsis AGC kinases are critical for polarized growth of pollen tubes. AGC1.5 and AGC1.7 are pollen-specific genes expressed during late developmental stages. Pollen tubes of single mutants had no detectable phenotypes during in vitro or in vivo germination, whereas those of double mutants were wider and twisted, due to frequent changes of growth trajectory in vitro . Pollen tubes of the double mutant also had reduced growth and were probably compromised in response to guidance cues in vivo . In the agc1.5 background, downregulation of AGC1.7 using an antisense construct phenocopied the growth defect of double mutant pollen tubes, providing additional support for a redundant function of AGC1.5/1.7 in pollen tube growth. Using the actin marker mouse Talin, we show that pollen tubes of double mutants had relatively unaffected longitudinal actin cables but had ectopic filamentous actin, indicating disturbed control of polarity. Our results demonstrate that AGC1.5 and AGC1.7 are critical components of the internal machinery of the pollen tube leading to polarized growth of pollen tubes.     

黄檗(芸香科)的珠孔塞和珠孔的形态发育及花粉管在子房室中的生长路径

利用扫描电子显微镜术和光学显微镜术研究了黄檗(Phellodendron amurense Rupr.)的珠孔塞和珠孔的形态发育和花粉管在雌蕊中的路径.黄檗胚珠的珠孔塞起源于珠柄.随着胚珠生长,珠孔塞逐渐增大,胚珠成熟时珠孔塞变得相当大并紧密地覆盖在珠孔上.当雌花进入可传粉期时,珠孔塞的形态发生很大变化,其表面细胞径向延伸,形成柱形、半乳突或乳突细胞.受精后,珠孔塞体积变小并逐步退化.花粉管在子房室中并非一定经过珠孔塞结构.花粉管是否经过珠孔塞取决于它们进入子房室的位置.我们不支持先前研究者关于珠孔塞主要充当对花粉管生长的机械作用的观点.我们对黄檗胚珠的珠孔的形态发育研究显示,在不同的生殖时期,珠孔的结构会发生变化,在传粉时期它的结构显示不对称性.黄檗珠孔塞和珠孔的发育与雌配子体发育存在密切关系.     

The phospholipid flippase ALA3 regulates pollen tube growth and guidance in Arabidopsis

Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6, and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance.

AMINOPHOSPHOLIPID ATPASE3 guides pollen tubes by regulating the distribution of anionic phospholipids to affect the precise localization of certain pollen-specific receptor kinases at pollen tubes.

IN A NUTSHELL Background: In flowering plants, pollen tube guidance regulates the rapid growth and timely targeting of the pollen tube to the ovule in the pistil during sexual reproduction, when signaling between the male and female gametophytes occur. The small peptide-RLK signaling module is essential for the interaction between the male and female gametophyte. Certain members of the pollen-specific receptor kinase (PRK) family have different subcellular localization patterns in Arabidopsis pollen tubes and play critical roles in pollen tube growth and guidance. However, the molecular mechanisms that regulate and maintain the polar localization of PRKs at the pollen tube tip are still unknown. Question: We were interested in exploring how Arabidopsis P4-ATPase (aminophospholipid ATPase, ALA) precisely regulates pollen tube guidance and maintains the polar localization patterns of PRK6 and PRK3. How plant ALA family members regulate pollen tube guidance has not yet been documented. Findings: The loss of ALA3 function not only caused sluggish pollen tube growth and aberrant ovule targeting but also affected the polar localization patterns of several PRKs at the pollen tube tip. Members of the PRKs family can directly interact with anionic phospholipids such as phosphatidylserine (PS), and the capacity of PRK3/6 to bind anionic phospholipids is crucial for both their polar localization and physiological functions. ALA3 establishes and maintains the polar distribution of PS, which influences secretory vesicles-mediated polar trafficking at the pollen tube tip to affect the distribution of PRK3 and PRK6. On the other hand, PS might also directly recruit PRK3 and PRK6 to the pollen tube tip and sustain their localization. Next steps: The localization of PRKs is a complex, finely regulated process. The C-termini of PRKs may also affect their polar distribution. More research is required to reveal how the C-terminus domain precisely controls the localization of PRKs.     

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。     

The evolution of fertilization modes independent of the micropyle in Fagales and `pseudoporogamy'

In contrast to a majority of angiosperms showing porogamous fertilization, several fagalean families such as Betulaceae and Casuarinaceae are known to show chalazogamy, where fertilization is effected by a pollen tube passing through the chalaza instead of the micropyle. Our developmental study of pollen-tube growth in pistils of Myrica rubra (Myricaceae, Fagales) further shows that pollen tubes reached the nucellus before the micropyle is formed by the integument. Since fertilized ovules appeared as if the pollen tube had passed through the micropyle for fertilization, we propose the new term `pseudoporogamy' to this mode. By mapping diverse modes of fertilization, dependent or independent of the micropyle, onto a phylogenetic tree of Fagales, it appears that fertilization mode evolved from porogamy to chalazogamy and then further from chalazogamy to pseudoporogamy. Possible reasons for the evolution of fertilization modes independent of the micropyle in Fagales are discussed.     

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.     

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.     

Attractive and repulsive interactions between female and male gametophytes in Arabidopsis pollen tube guidance

Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.     

Floral Viability and Pollen Tube Growth in Vicia faba L.

Durations of stigmatic receptivity, pollen viability and pollen tube growth were investigated in the largely entomophilous faba bean. Stigmas were examined for deposited and germinated pollen, and growth of pollen tubes was investigated using aniline blue-induced fluorescence. Entry of a pollen tube into a micropyle was found to be a reliable indicator of fertilization. After anthesis, stigmas remained receptive to pollination for six days, and pollen viable for five. Pollen tubes took up to three days to reach the ovules furthest from the stigma. Inspection of ovular development within pods showed that the incidence of fertilization had been accurately determined in the flowers.     

钙在被子植物受精过程中的作用

近年来,花粉管中的钙信号和生理功能的研究取得了明显的进展,同时在雌蕊系统中有关钙分布的研究也初步显示了其时、空特征与被子植物的受精作用密切相关。该文总结了花粉萌发和花粉管生长过程中外源钙和内源钙的作用机制,结合雌蕊组织中钙分布的特征,进一步探讨了钙在被子植物受精过程中的功能。     

鹤顶兰花粉管在子房中的生长途径

运用扫描电镜对鹤顶兰(Phaiustankervilliae(Aiton)Bl.)花粉管在子房内的生长途径进行了观察。结果表明:花粉管在子房中的生长途径可以分为3个阶段:(1)沿子房壁轴向生长阶段,从授粉开始至大孢子母细胞四分体时期,花粉管经过合蕊柱到达子房,经由胎座基部沿子房壁轴向生长;(2)沿子房径向生长阶段,二核胚囊之后,花粉管在胚珠之间穿梭,以径向生长为主;(3)朝珠孔定向生长阶段,胚囊成熟时,花粉管朝珠孔定向生长进入胚囊。实验结果说明花粉管的定向生长受胚珠的分子信号调控。     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style