The synergid cell: attractor and acceptor of the pollen tube for double fertilization

In flowering plants, the egg cell is generally accompanied by two symmetrical cells, called synergid cells. As early as the 1870s, synergid cells were distinguished from egg cells and cooperation between synergid and egg cells was proposed; the term "synergid" is derived from the Greek "synergos," which means "working together." The accumulation of morphological and genetic data, and, more recently, the in vitro physiological analysis of the fertilization system of Torenia fournieri, have revealed that synergid cells work together with egg and central cells to accomplish double fertilization. This cooperation is of crucial importance in the attraction and acceptance of the pollen tube. In this review article, I focus on the physiological function and behavior of the synergid cell during the fertilization process. Received: December 20, 2001 / Accepted: December 27, 2001     

Callose in the walls of mature embryo sac ofTorenia fournieri

Summary During the course of a fluorescence microscopic investigation on the extra-ovular micropylar portion of the embryo sacs ofTorenia fournieri Lind. (Scrophulariaceae) a callosic wall was found which surrounded it almost completely until the time of anthesis. In addition, the walls of young synergids and the filiform apparatus also showed callosic fluorescence. Treatments with PAS reaction revealed a PAS-positive substance filling up the locular cavity. Our attempts to induce fluorochromasia by employing fluorescein diacetate failed, indicating the low permeability of the callosic wall around the embryo sac. It is assumed that the callose wall around the embryo sac isolates the latter from the contents of the locular cavity whereas the callose in the synergid walls may represent an intermediate stage in the maturation of these walls; the filiform apparatus is mainly composed of callose.     

蓝猪耳受精生物学研究进展

蓝猪耳(Torenia fournieri L.)胚囊半裸露,在光学显微镜下能清楚观察到卵细胞、助细胞及部分中央细胞的形态结构,有助于原位观察卵细胞在受精前后的变化状态,被认为是研究被子植物体内受精机理的一种模式植物。综述了蓝猪耳的受精机理:花粉管定向进入胚囊的方式与机理、钙在受精过程中的作用、受精前后胚囊细胞骨架的动态变化。简要介绍了离体受精技术在蓝猪耳受精生物学中的发展应用。根据前人对蓝猪耳的研究成果并结合我们的研究,指出蓝猪耳在受精生物学中的应用,特别是借助离体受精技术平台,将具有更大的研究前景。     

Symplastic communication between the central cell and the egg apparatus cells in the embryo sac of Torenia fournieri Lind. before and during fertilization

 Various membrane-impermeable, water-soluble fluorescent tracers with different molecular weights were microinjected into the central cell of the embryo sac of Torenia fournieri Lind. before and during fertilization. Before anthesis, there was high symplastic permeability between the central cell and the egg apparatus cells. In this stage, fluorescent tracers up to 10 kDa could pass from the central cell into the egg apparatus cells, whereas those with larger molecular weight remained in the central cell. As the embryo sac matured, symplastic permeability decreased such that 2 d after anthesis only tracers less than 3 kDa could spread from the central cell into the egg cell. There appeared to be no symplastic permeability between the primary endosperm and zygote after fertilization, since tracers as small as 521 Da could not pass into the zygote in about half of the microinjected embryo sacs. This is the first report of a change in cell-to-cell communication among the cells of the female germ unit before and after fertilization. Received: 16 December 1999 / Accepted: 4 February 2000     

钙和硼对蓝猪耳花粉萌发及花粉管生长的影响

研究了钙(Ca^2 )和硼(H3BO3)对蓝猪耳花粉萌发和花粉管生长的影响。结果表明：(1)在一定范围内Ca^2 几乎不影响花粉萌发频率,而主要影响花粉萌发速度和花粉管生长速度;低Ca^2 不利于花粉管生长,而高Ca^2 抑制花粉萌发速度和花粉管生长;在稍高于最适Ca^2 浓度的条件下,花粉管生长早期呈现波浪形。(2)硼明显影响花粉萌发频率及花粉管形态;花粉管生长必需硼,但不同浓度的硼对花粉管生长速度影响不明显;在高浓度硼条件下,较长时间内花粉管均呈现出波浪形。(3)Cooled-CCD动态跟踪观察进一步证实Ca^2 影响花粉管生长速度,而硼则不明显。     

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. Received: 11 November 1999 / Accepted: 31 January 2000     

Emerging data on pollen tube growth and fertilization in flowering plants, 1990–1995

Summary Since its discovery at the end of the last century, double fertilization remains of central interest in plant reproductive biology research. Although the sequence of events leading to fertilization is well known from cytological studies, the underlying mechanisms remain to be elucidated. This now seems feasible by the diversification and refinement of recently developed technologies presented in this review. The progress made during the last five years in understanding pollen tube guidance, discharge into the embryo sac, and gametic fusion are described. Future directions are also discussed.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Changes of calcium distribution in ovules ofTorenia fournieri during pollination and fertilization

Summary Calcium distribution in ovules ofTorenia fournieri was studied by electron energy loss spectroscopy and transmission electron microscopic visualization of calcium antimonate precipitates. High calcium levels were found in the ovules ofT. fournieri. Calcium is situated mainly in extracellular regions before fertilization, including the surface of embryo sac, in the mucilage, and among the cells of the egg apparatus. Intracellular calcium was found only in the nucellar cells around the embryo sac and in the epidermis of the central axis and funiculus. After pollination, a labyrinthine structure (coralloid-like cell wall formation) develops on the micropylar surfaces of the egg apparatus that contain high levels of calcium. Calcium levels increase in the degenerating synergid after the penetration of the pollen tube. Calcium-antimonate precipitates are abundant in vacuoles of the disrupted synergid and pollen tube cytoplasm.Abbreviations EELS electron energy loss spectroscopy - EDX energy-dispersive X-ray microanalysis - LS labyrinthine structure     

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca²⁺ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca²⁺ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate. Received: 14 August 1996 / Accepted: 9 October 1996     

水稻双受精过程的共聚焦显微镜观察

(郑州大学离子束生物工程省重点实验室,郑州450052)摘要:首次利用核特异荧光染色与整体透明技术并利用激光扫描共聚焦显微镜(LSCM)对水稻双受精过程进行了观察。激光扫描共聚焦显微镜具有"组织与细胞CT"的功能,可以对整体组织进行扫描并构建三维结构。经荧光染色及透明处理后,在激光扫描共聚焦显微镜下经488nm激光激发,胚囊内各细胞的细胞核以及核仁发出明亮荧光,细胞核轮廓比较清晰,层次感强,并能清晰观察到胚囊内各细胞的结构特点和空间位置关系。不论是对开花前较小的成熟胚囊材料还是开花后较大的胚囊材料都取得了较好的观察效果。同传统的光学显微镜和荧光显微镜相比,其观察到的图像更清晰、更直观、更具立体感。     

Polar distribution dynamics of Con A binding sites in embryo sacs is temporally coupled by the fertilization process

The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.     

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.     

Overproduction and selective abortion of ovules based on the order of fertilization revisited

Given that seeds fertilized by slowly growing pollen are of low quality genetically, we theoretically reanalyzed the hypothesis that plants selectively abort ovules fertilized later to enhance the mean quality of resulting seeds. We assumed that both superior and inferior pollen exist, the superior pollen growing faster to fertilize ovules, resulting in seeds of higher quality than those of ovules fertilized by inferior pollen. We developed two models to determine the conditions under which selective abortion is favored. In the first model, ovules in one flower are fertilized by pollen grains that arrive at different times, with each visit bringing both fast- and slow-growing pollen. In the second model, ovules in two flowers are fertilized by all pollen grains that arrive at the same time. In the first model, we found that selective abortion based on the order of fertilization is never advantageous irrespective of the duration of the time lag between the two visits. Rather, random abortion is possibly favored. In the second model, although selective abortion based on the order of fertilization can be advantageous, the parameter region favoring it is rather restricted. This is because overproduction can be advantageous only if the quantity of the superior pollen is not limited in one flower but is limited in the other flower. In addition, the degree of overproduction was very low, implying that the merit of overproduction (increase in the number of superior seeds) is low compared to the cost of overproducing ovules. These results suggest that selective abortion of ovules based on the order of fertilization is not as advantageous as previously considered.     

The dynamics of calcium distribution in the synergid cells of wheat after pollination

Summary Sister synergids of wheat were examined for post-pollination calcium distribution using freeze-substitution fixation, scanning electron microscopy, and energy-dispersive X-ray microanalysis. At 15–30 min postpollination (before pollen-tube penetration and discharge), the sister synergids were similar in size and in elemental concentrations. Both synergids measured about the same as the egg and contained relatively high concentrations of calcium, potassium, and magnesium. The sister synergids remained identical even in an aberrant case where a pollen tube had penetrated, but not discharged into a synergid. After 30 min post-pollination (after pollen-tube discharge), typically one of the sister synergids was reduced in size and devoid of high concentrations of elements. The other synergid initially remained unaltered and only later underwent slight size reduction; however, it retained high concentrations of elements. The results indicate that high concentrations of calcium, potassium, and magnesium are rapidly lost from a synergid following pollen-tube discharge. A hypothesis is proposed to explain the regulation of entry by only a single pollen tube into an embryo sac.On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

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。     

A role for LORELEI,a putative glycosylphosphatidylinositol‐anchored protein,in Arabidopsis thaliana double fertilization and early seed development

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre‐5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)‐anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre‐5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre‐5/lre‐5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre‐5/lre‐5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre‐5/lre‐5 seeds. The lre‐5/lre‐5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI‐like‐GPI‐anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre‐5/lre‐5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.     

羊草受精作用及其胚与胚乳早期发育的观察

利用常规石蜡制片方法研究了羊草受精过程及胚与胚乳的早期发育,其主要结果为：(1)授粉后1h,花粉管破坏1助细胞,释放2精子。精子为眼眉状,难以区分其细胞质鞘;(2)授粉后1～2h,2个精子分别移向卵细胞与极核;(3)授粉后2～3h,精核分别贴附于卵细胞与极核核膜上;(4)授粉后3～10h,精核与卵核融合,并出现雄性核仁,形成合子;(5)授粉后3～4h,精核与极核融合,并出现雄性核仁,形成初生胚乳核,精核与极核的融合比与卵核融合快;(6)传粉后20h,合子分裂,合子的休眠期为10h左右;(7)传粉4h,初生胚乳核分裂,初生胚乳核没有休眠期;(8)羊草双受精作用属于有丝分裂前配子融合类型;(9)胚胎发育属于紫菀型,胚乳发育属于核型胚乳。     

Pre-treatment of cattle semen or oocytes with purified milk osteopontin affects in vitro fertilization and embryo development

This study was designed to investigate the effects of pre-incubating cattle spermatozoa or matured oocytes with purified osteopontin (OPN) from cattle milk on fertilization in cattle and embryonic development in vitro. There were two different experiments, semen from six mature Holstein bulls (Bos Taurus) was frozen with different concentrations of OPN (0, 1, 10, 100 μg/mL). Matured cattle oocytes were also pre-treated with OPN (0, 10, 100 μg/mL). In both experiments, pre-treated oocytes or frozen semen, was processed for in vitro fertilization and embryo development. Significantly more oocytes were fertilized when using frozen semen with 10 μg/mL OPN (bull 2 = 85 ± 4% and bull 5 = 78 ± 4%) than without OPN (bull 2 = 75 ± 4% and bull 5 = 69 ± 4%). Those bulls also had increase in cleavage and embryo development (bull 2 = 85 ± 3%, 41 ± 1.9%; bull 5 = 76 ± 2%, 37 ± 1.8%) compared with control (bull 2 = 75 ± 3%, 30 ± 2%; bull 5 = 68 ± 2%, 29 ± 2%). Incubating matured oocytes in 10 μg/mL OPN (87 ± 3%) and 100 μg/mL OPN (88 ± 3%) significantly increased fertilization than control (73 ± 3%). OPN also improve cleavage, and embryo development in treatments with 10 μg/mL OPN (82.7 ± 1.3%; 31.7 ± 1.4%) and 100 μg/mL OPN (85.8 ± 1.3%; 33.8 ± 1.5%) when compared with control (74.1 ± 1.3%; 24.2 ± 1.2%). These data suggest that both, spermatozoa from some bulls and oocytes may associate with OPN, suggesting a facilitory role on in vitro fertilization and embryo development.     

Root hair growth in Arabidopsis thaliana is directed by calcium and an endogenous polarity

Tip growth of plant cells has been suggested to be regulated by a tip-focused gradient in cytosolic calcium concentration ([Ca²⁺]_c). However, whether this gradient orients apical growth or follows the driving force for this process remains unknown. Using localized photoactivation of the caged calcium ionophore Br-A23187 we have been able to artificially generate an asymmetrical calcium influx across the root hair tip. This led to a change in the direction of tip growth towards the high point of the new [Ca²⁺]_c gradient. Such reorientation of growth was transient and there was a return to the original direction within 15 min. Root hairs forced to change the direction of their growth by placing a mechanical obstacle in their path stopped, reoriented growth to the side, and grew past the mechanical blockage. However, as soon as the growing tip had cleared the obstacle, growth returned to the original direction. Confocal ratio imaging revealed that a tip-focused [Ca²⁺]_c gradient was always centered at the site of active growth. When the root hair changed direction the gradient also reoriented, and when growth returned to the original direction, so did the [Ca²⁺]_c gradient. This normal direction of apical growth of Arabidopsis thaliana (L.) Heynh. root hairs was found to be at a fixed angle from the root of 85 ± 6.7 degrees. In contrast, Tradescantia virginiana (L.) pollen tubes that were induced to reorient by touch or localized activation of the caged ionophore, did not return to the original growth direction, but continued to elongate in their new orientation. These results suggest that the tip-focused [Ca²⁺]_c gradient is an important factor in localizing growth of the elongating root hair and pollen tube to the apex. However, it is not the primary determinant of the direction of elongation in root hairs, suggesting that other information from the root is acting to continuously reset the growth direction away from the root surface. Received: 22 April 1997 / Accepted: 14 May 1997