POLLEN TUBE-SYNERGID INTERACTIONS IN PROBOSCIDEA LOUISIANICA (MARTINEACEAE)

The ultrastructure of the synergids of Proboscidea louisianica was investigated from just before fertilization until 48 hr after pollination. It was found that the cytoplasm of one synergid consistently begins to degenerate before arrival of the pollen tube at the embryo sac, and that it is always this synergid which receives the pollen tube tip and its discharge. The other synergid (persistent synergid) remained unchanged throughout the study period. Polysaccharide vesicles of pollen tube origin were observed fusing with the pollen tube wall as well as contributing to cell wall formation of the degenerate synergid. In one ovule (48 hr after pollination) two pollen tubes had entered and grown the length of the micropyle, but only the first tube penetrated the degenerate synergid and discharged normally. The second pollen tube was abutting against the persistent synergid, but had not entered or discharged. In another exceptional case (18 hr after pollination), a pollen tube had grown the length of the micropyle, but did not discharge, or enter either synergid. Both synergids of this ovule were observed to be completely intact. It is concluded that synergid and pollen tube cytoplasmic degeneration is the result of a very specific interaction between these two cells and that this degeneration is probably a prerequisite for normal pollen tube entrance and discharge into the embryo sac, and for male gamete transfer to the egg and central cell.     

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.     

Relatively high calcium is localized in synergid cells of wheat ovaries

Summary Using energy-dispersive X-ray microanalysis of mature, unpollinated wheat ovaries fixed by freeze-substitution, we show here that the synergid cells store a relatively high concentration of calcium. Based on our results and other indirect evidence, we suggest that supraoptimal levels of calcium in synergids may regulate: (1) correct orientation of the pollen tube, by forming a calcium gradient in the vicinity of the synergids, and (2) arrest and rupture of the pollen tube to release the sperm near the egg.     

CAPSELLA EMBRYOGENESIS: THE SYNERGIDS BEFORE AND AFTER FERTILIZATION

The ultrastructure and composition of the synergids of Capsella bursa-pastoris were studied before and after fertilization. The synergids in the mature embryo sac contain numerous plastids, mitochondria, dictyosomes and masses of ER and associated ribosomes. Each synergid contains a large chalazal vacuole, a nucleus with a single nucleolus and is surrounded by a wall. This wall is thickest at the micropyle end of the cell where it proliferates into the filiform apparatus. At the chalazal end of the cell the wall thins and may be absent for small distances. The pollen tube grows into one of the two synergids through the filiform apparatus and extends one-third the length of the cell before it discharges. Following discharge of the pollen tube, mitochondria and plastids of the tube can be identified in the synergid as can hundreds of 0.5 μ polysaccharide spheres liberated by the tube. The method by which the sperm or sperm nuclei enter the egg or central cell is not known although an apparent rupture was found in the wall of the egg near the tip of the pollen tube. The second synergid changes at the time the pollen tube enters the first synergid. These changes result in the disorganization of the nucleus and loss of the chalazal wall and plasma membrane. Eventually this synergid loses its identity as its cytoplasm merges with that of the central cell.     

Prepollination degeneration in mature synergids of pearl millet: an examination using antimonate fixation to localize calcium

Summary The pattern of degeneration in mature synergids of pearl millet prior to pollination was examined by transmission electron microscopy after conventional and antimonate fixation to precipitate loosely sequestered calcium (Ca). The extent of degeneration and the distribution of Ca in a synergid varied among samples, and also between some sister synergids. However, there seemed to be no difference between sister synergids in the total amount of precipitates present in each cell. Characteristic signs of degeneration in the antimonatefixed synergids were: a gap above the filiform apparatus with fibrillar material, precipitates, and fusing or disintegrating membranes; increased precipitates in the nucleus, nucleolus, and endoplasmic reticulum; fusion or collapse of a few vacuoles in the chalazal core with precipitates and flocculent material deposited nearby. The numerous mitochondria and proplastids in the micropylar portion of the synergid remained intact and mostly free of precipitates. The shape and content of the chalazal vacuoles appeared to be disparate and dependent on the fixation procedure as well as the extent of degeneration within a synergid. The results suggest that the sister synergids of pearl millet undergo autonomous degeneration apparently in two independent sequences that may be spatially and temporally separated. The vacuoles appear to be dynamic organelles that store Ca in association with some other material. A high concentration of Ca may be localized along or between the common wall of the two filiform apparatuses.On specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Fertilization inNicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

The cytoskeletal organization of the embryo sac of tobacco (Nicotiana tabacum L.) was examined at maturity and during synergid degeneration, pollen-tube delivery and gamete transfer using rapid-frozen, freeze-substituted and chemically fixed material in combination with immunofluorescence and immunogold electron microscopy. Before fertilization, the synergid is a highly polarized cell with dense longitudinally aligned arrays of microtubules adjacent to the filiform apparatus at the micropylar end of the cell associated with major organelles. The cytoskeleton of the central cell is less polarized, with dense cortical microtubules in the micropylar and chalazal regions and looser, longitudinally oriented cortical microtubules in the lateral region. In the synergid and central cell, F-actin is frequently found at the surface of the organelles and co-localizes with either single microtubules or microtubule bundles. Egg cell microtubules are frequently cortical, randomly oriented and more abundant at the chalazal end of the cell; actin filaments are associated with microtubules and the cortex of the egg cell. At 48 h after pollination and before the pollen tube arrives, the onset of degeneration is evident in one of the two synergids: the electron density of cytoplasmic organelles and the ground cytoplasm increases and the nucleus becomes distorted. Although synergids otherwise remain intact, the vacuole collapses and organelles degenerate rapidly after pollen-tube entry. Abundant electron-dense material extends from the degenerated synergid into intercellular spaces at the chalazal end of the synergid and between the synergids, egg and central cell. Rhodamine-phalloidin and anti-actin immunogold labeling reveal that electron-dense aggregates in this region contain abundant actin forming two distinct bands termed coronas. This actin is part of a mechanism in the egg apparatus which appears to precisely position and facilitate the access of male gametes to the egg and central cell for fusion.Abbreviations ES embryo sac - FA filiform apparatus - Mf microfilament - Mt microtubule - PT pollen tube - RF-FS rapid-freeze freeze-substitution - TEM transmission electron microscopy We thank Gregory W. Strout for technical assistance in the use of the RF-FS technique and Dr. Hongshi Yu for providing Fig. 1. This research was supported by U.S. Department of Agriculture grants 88-37261-3761 and 91-37304-6471. We gratefully acknowledge use of the Samuel Robert Noble Electron Microscopy Laboratory of the University of Oklahoma.     

莴苣助细胞发育过程中钙的分布研究

用焦锑酸盐沉淀法对莴苣助细胞中的钙分布进行了观察。结果表明,开花前3天刚形成的助细胞中的钙颗粒很少:开花前2天助细胞壁中的钙颗粒增加;开花前1天助细胞珠孔端细胞壁加厚,其中积累了许多钙颗粒:开花当天助细胞珠孔端的丝状器中聚集了大量的钙颗粒。授粉后1h时两个助细胞的结构和钙分布发生差异,一个呈退化状,其中的钙颗粒明显增多,另一宿存助细胞中的钙分布与授粉前相似。去雄不授粉1天后两个助细胞均保持完好,且两助细胞中的钙分布没有明显差异,表明由花粉管引起一个助细胞中钙含量增加进而导致了助细胞退化。退化助细胞在卵细胞与中央细胞之间形成一薄层。助细胞退化后不同部位的钙颗粒呈现出与受精作用密切有关的变化:授粉后1h时,钙主要聚集在近合点端部位;授粉后2.5h卵细胞即将受精,这时许多细小的钙颗粒主要聚集在卵细胞与中央细胞之间的薄层中;授粉后4h精、卵细胞已融合,这时退化助细胞合点端的钙颗粒明显减少,而在其珠孔端又聚集了较多的钙。上述助细胞中的钙含量变化与吸引花粉管进入胚囊和促使精卵细胞融合密切有关。     

Synergid degeneration in Nicotiana: a quantitative,fluorochromatic and chlorotetracycline study

Summary Synergid degeneration was examined in the isolated embryo sac and egg apparatus of Nicotiana tabacum using quantitative cytology, fluorochromatic reaction (FCR) and chlorotetracycline (CTC). Most synergid degeneration occurs after pollen tubes (PT) arrive in the ovarian chamber between 42 and 48 h after pollination; synergid degeneration was precluded when PT were prevented from entering the ovary by stylar excision indicating that the signal that triggers synergid degeneration travels only relatively short distances in this plant. There was no evidence for any preferentiality between right or left synergids with regard to cell size or degeneration pattern. FCR staining confirms that synergid degeneration involves the loss of membrane integrity and is a reliable indicator of the onset of degeneration. CTC labeling of the degenerated synergid reveals that a concentrated reserve of membrane-bound calcium is present in the receptive synergid, possibly aiding in the attraction, arrest and discharge of the PT, releasing the sperms into the receptive ES.     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一,是大多数被子植物完成受精作用的关键环节之一。在受精过程中,助细胞吸引花粉管向雌配子体生长,并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外,雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚,但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果,尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果,并对助细胞的生殖功能进行了分析。     

The peroxin loss-of-function mutation abstinence by mutual consent disrupts male-female gametophyte recognition

In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1-4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two nonmotile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7-10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A), where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen-tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent (amc). Interestingly, pollen-tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte, resulting in pollen-tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophyte that is required for pollen-tube discharge.     

FINE STRUCTURE AND COMPOSITION OF THE EGG APPARATUS BEFORE AND AFTER FERTILIZATION IN QUERCUS GAMBELII: THE FUNCTIONAL OVULE

A study of the egg apparatus of Quercus gambelii was made at both the light and the electron microscope levels. This investigation was concerned primarily with the changes that occur in these cells before and after the process of fertilization and what role, if any, is played by the synergids in this phenomenon. The synergids before fertilization are, on the basis of ultrastructure, healthy, intact, functional cells. They have numerous mitochondria, dictyosomes, endoplasmic reticulum, ribosomes, and a typical nucleus. A prominent filiform apparatus is present, but the cell wall only extends a short distance around the micropylar end of the cells. Just before fertilization, one of the synergids degenerates. This is the synergid that receives the pollen tube and its discharge, including both male gametes. Dictyosomes increase in number and activity in the other synergid (persistent synergid) after fertilization. Eventually a complete cell wall forms around both of the synergids. No plasmodesmata are present in these walls. The egg has numerous mitochondria, dictyosomes, endoplasmic reticulum, and ribosomes, both free in the cytoplasm and attached to the endoplasmic reticulum. Lipid bodies are characteristic of this cell. A cell wall is present only around the micropylar end of the egg. After fertilization, little change occurs in the zygote. The number and activity of the dictyosomes increase, apparently in correlation with cell wall formation. The number of lipid bodies increases. The zygote is approximately the same size as the egg. Plastids are scarce, and starch grains are typically absent from all cells of the egg apparatus. It is suggested that the synergids function in the secretion of chemotropic substances that guide the growth of the pollen tube. Comparisons are made between the egg apparatus of Quercus gambelii and that of the other plants studied thus far.     

被子植物助细胞的发育和功能

助细胞是被子植物雌配子体的组成细胞之一, 是大多数被子植物完成受精作用的关键环节之一。在受精过程中, 助细胞吸引花粉管向雌配子体生长, 并接受花粉管长入细胞程序死亡助细胞中。接下来的花粉管停止生长和花粉管顶端破裂释放出2个精细胞的过程可能也依赖于助细胞。另外, 雄性生殖单位的解体、提供精细胞的运动轨道和启动精细胞合成DNA的生物学事件也可能与助细胞有关。助细胞的形态结构已研究得比较清楚, 但有关助细胞的发育机理和它在受精过程中的作用则仍不明确。近年来对助细胞的研究又取得了一些新结果, 尤其是一些分子生物学的研究结果为揭示助细胞的功能提供了重要线索。该文总结了这方面的研究成果, 并对助细胞的生殖功能进行了分析。     

Synergid: a key link in fertilization of angiosperms

In over 80 % of the angiosperms, the female gametophyte is comprised of seven cells, two of which are the synergid cells. These cells are considered pivotal in assuring successful fertilization. The synergid cells direct pollen tube growth toward the female gametophyte, and facilitate the entrance of the tube into the embryo sac. Once the pollen tube enters the synergid cell, its growth is arrested, the tip of the tube breaks, and two sperm cells are released. This sequence of events is also synergid dependent. In addition, separation of the cells of the male germ unit, orientation of the two sperm cells in the degenerating synergid, and fusion of the egg and central cell with sperm cells may also be related to synergid cells. Synergid structure has been widely studied, but development and function of these cells during angiosperm fertilization remains elusive. Recent molecular approaches have provided an enhanced understanding of the role of synergid cells in fertilization. The present review summarizes the results of current studies regarding the role of synergids in angiosperm reproductive function.     

Ultracytochemical Localization of Calcium in Micropyle and Embryo Sac of Brassica napus Before and After Pollination

Potassiam antimonate was used to localize Ca2+ in the micropyle and embryo sac of Brassica napus L. before and after pollination. To identify the nature of the pyroantimonate deposits, energy-dispersive X-ray microanalysis (EDXA) was employed and the deposits were proved to contain calcium pyroantimonate. Image processing system was employed to measure the volume density and the diameter of the deposits. Before and after pollination, calcium was more abundant in the exostome and endostome as compared with the other regions of the integuments, and was concentrated at the apoplast system, i.e. the intercellular matrix of the micropyle canal and the cell wall. Before pollination, each of the two sister synergids accumulated more calcium than the other embryo sac cells. Although the mean diameter of the deposits in the synergid was only two-thirds as that in the egg cell and central cell, the volume density of the deposits in the synergid was about 2.5 times and 1.9 times as that in the egg cell and the central cell respectively. The filiform apparatus and the nucleus had the most abundant calcium within a synergid. After pollination both sister synergids degenerated conspicuously and were characterized by much more deposited calcium (about 2.4 times more than before); and the diameter of the deposits decreased dramatically, which was less than one-third as before. The relationship between calcium distribution and synergid degeneration as well as its functions was discussed.     

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.     

Development and function of the synergid cell

The synergid cells are located in the female gametophyte and are essential for angiosperm reproduction. During the fertilization process, a pollen tube grows into one of the synergid cells, ceases growth, ruptures, and releases its two sperm cells into this cell. The synergid cells produce an attractant that guides the pollen tube to the female gametophyte and likely contain factors that control arrest of pollen tube growth, pollen tube discharge, and gamete fusion. The synergid cells contain an elaborated cell wall at their micropylar poles, the filiform apparatus that likely plays a role in pollen tube guidance and pollen tube reception. Recent genetic, molecular, and physiological studies in Arabidopsis, maize, and Torenia have provided insights into synergid cell development and the control of pollen tube growth by the synergid cell.     

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir

Pollen of larch (Larix?×?marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix?×?marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.     

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.     

Observations on the Developmental Femal Gametophyte and the Process of Fertilization of Amcmum visllosum Lour.

The development of megarametophyte antl the process of fertnization are investigated in detail and the following results are obtained: 1. The development of the embryo sac conforms to Polygonmn type. The pollen tube reaches the base of style about 16 hrs., enters into the ovary, 16–18 hrs. and then it, to the embryo sae, 24–28 hrs. after pollination. 2. Pollezt grains are 2-celed at maturity. 3. When the pollen tube reaehes the mieropylar end, both synergids are intact; but their nueleolus become small. Pollen tube enter the synergid through the filiform apparatus and the synergid is degenerated. 4. Pollen tube discharges two sperms which move towards the egg cell and the secondary nucleus respectively. 5. One sprm fuses with the egg nucleus and another with the secondary nueleus. The proeess of syngamy is siniilar to that of secondary nucleus fusion. Primary endosperm nueleus s formed 30 hrs. after pollination. The zygote is formed 30–62 hrs. after pollination. 6. There is no distinct correlation between the syngamy and secondary nueleus fusion.