The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.     

被子植物质体遗传的细胞学研究

植物细胞质遗传涉及细胞质中含DNA的两种细胞器——质体和线粒体从亲代至子代的传递。相对来说线粒体遗传的研究远不及质体的多,这可能是线粒体这种细胞器缺乏合适的表型突变体之故。高等植物质体遗传的研究历史可追溯到本世纪初在杂交试验中对叶色遗传的非孟德尔定律的发现,Baur在马蹄纹天竺葵(Pelargonium zonale)中从叶色突变体(白化体)的杂交遗传分析,发现了双亲质体遗传;而Correns在紫茉莉(Mirabilis jalapa)中则发现了单亲母本质体遗传(见Kuroiwa)。此后,对质体基因组突变性状遗传分析的研究,大量的资料说明了在被子植物中存在双亲质体遗传和单亲母系质体遗传两种类型,而后一种占大多数,仅少数是比较有规律的为双亲质体遗传或偶尔是双亲质体遗传。几十年来应用遗传分析的方法对被子植物质体遗传的研究,着重于揭示不同植物种质体的遗传是单亲母系或是双亲质体传递,以及探索杂种核基因对质体传递方式的影响。     

Elimination of plastids during spermatogenesis and fertilization in the plant kingdom

Ultrastructural and genetic investigations involving diverse species of plants have demonstrated that plastids may be transmitted either biparentally or maternally during sexual reproduction. In species in which plastid transmission is maternal, elimination of plastids from the paternal parent may occur in a number of ways: exclusion from the male gamete during spermatogenesis, loss from the motile sperm, exclusion during fertilization, or degradation within the zygote. These diverse ways in which maternal inheritance of plastids is achieved suggest that this inheritance pattern may have evolved independently many times in response to different selective pressures in different phyletic lineages.     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. II. FERTILIZATION AND PROEMBRYO DEVELOPMENT

Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) ovules were used to study male gamete formation, insemination of the egg, and free nuclear and cellular proembryo development. Two male nuclei form as the pollen tube either reaches the megaspore wall or as it enters the archegonial chamber. No cell wall separates them. They are contained within the body-cell cytoplasm. A narrow extension of the pollen tube separates the neck cells and penetrates the ventral canal cell. The pollen tube then releases its contents into the egg cytoplasm. The two male gametes and a cluster of paternal organelles (plastids and mitochondria) migrate within the remains of the body-cell cytoplasm toward the egg nucleus. Microtubules are associated with this complex. The leading male gamete fuses with the egg nucleus. The zygote nucleus undergoes free nuclear division, but the cluster of paternal organelles remains discrete. Free nuclei, paternal and maternal nucleoplasm, maternal perinuclear cytoplasm, and the cluster of paternal organelles migrate en masse to the chalazal end of the archegonium. There, paternal and maternal organelles intermingle to form the neocytoplasm, the nuclei divide, and a 12-cell proembryo is formed. The importance of male nuclei or cells, the perinuclear zone, and large inclusions in cytoplasmic inheritance are discussed in the Pinaceae and in other conifer families. This completes a two-part study to determine the fate of paternal and maternal plastids and mitochondria during gamete formation, fertilization, and proembryo development in Douglas fir.     

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination.The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote.Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.     

Cytoplasmic inheritance in green algae: patterns,mechanisms and relation to sex type

Cytological and genetic investigations of two major groups of green algae, chlorophyte and streptophyte green algae, show a predominance of uniparental inheritance of the plastid and mitochondrial genomes in most species. However, in some crosses of isogamous species of Ulva compressa, these genomes are transmitted from mt⁺, mt⁻, and both parents. In species with uniparental organelle inheritance, various mechanisms can eliminate organelles and their DNA during male gametogenesis or after fertilization. Concerning plastid inheritance, two major mechanisms are widespread in green algae: (1) digestion of plastid DNA during male gametogenesis, during fertilization, or after fertilization; and (2) disintegration or fusion of the plastid in the zygote. The first mechanism also eliminates the mitochondrial DNA in anisogamous and oogamous species. These mechanisms would ensure the predominantly uniparental inheritance of organelle genomes in green algae. To trace the evolutionary history of cytoplasmic inheritance in green algae, the relations between uniparental inheritance and sex type were considered in isogamous, anisogamous, and oogamous species using sex-specific features that might be nearly universal among Chlorophyta.     

Occurrence of plastids in the sperm cells of Caprifoliaceae: biparental plastid inheritance in angiosperms is unilaterally derived from maternal inheritance

It is widely held that organelles inherit from the maternal lineage. However, the plastid genome in quite a few angiosperms appears to be biparentally transmitted. It is unclear how and why biparental inheritance of the genome became activated. Here, we detected widespread occurrence of plastids in the sperm cells (a cellular prerequisite for biparental inheritance) of traditional Caprifoliaceae. Of the 12 genera sampled, the sperm cells of Abelia, Dipelta, Heptacodium, Kolkwitzia, Leycesteria, Linnaea, Lonicera, Symphoricarpos, Triosteum and Weigela possessed inheritable plastids. The other genera, Sambucus and Viburnum, lacked plastids in sperm cells. Interestingly, such exclusion of plastids in the sperm cells of some Caprifoliaceae appeared to be associated with the divergence of Dipsacales phylogeny. Closer examination of Weigela florida revealed that both plastids and plastid DNA were highly duplicated in the generative cells. This implies that the appearance of plastids in sperm cells involved cellular mechanisms. Because such mechanisms must enhance the strength of plastid transmission through the paternal lineage and appear ubiquitous in species exhibiting biparental or potential biparental plastid inheritance, we presume that biparental plastid genetics may be a derived trait in angiosperms. This is consistent with our extended phylogenetic analysis using species with recently discovered modes of potential plastid inheritance. The results show that basal and early angiosperms have maternal plastid transmission, whereas all potential biparental transmission occurs at terminal branches of the tree. Thus, unlike previous studies, we suggest that biparental plastid inheritance in angiosperms was unilaterally converted from the maternal transmission mode during late angiosperm evolution.     

Paternal inheritance of plastids in Medicago sativa

Summary Plastids are plant cellular organelles that are generally inherited from the maternal parent in the angiosperms. Many species exhibit biparental inheritance of plastids, but usually with a predominantly maternal influence. In contrast to this, we report strong paternal inheritance of plastids in reciprocal crosses of alfalfa, Medicago sativa, by following restriction fragment length polymorphisms for plastid DNA in two normal green plastids. Mitochondrial inheritance remained exclusively maternal.     

Mechanisms of maternal inheritance of plastids and mitochondria: Developmental and ultrastructural evidence

Summary A number of maternally inherited characters are now known to be associated with mitochondria or chloroplasts, which contain small genomes segregating separately from that of the nucleus. The reason often given for maternal inheritance of plastid-associated characters in plants is the absence of plastids in the generative cell of pollen following an unequal mitosis (Vaughn, 1980). However, fine ultrastructural studies have not established “exclusion” as the sole mechanism for maternal inheritance; in many cases, other mechanisms may be operating. Three lines of evidence concerning the mechanism of maternal inheritance will be discussed: First, while it is true that thorough fine ultrastructural studies have failed to find plastids in generative cells of many seed plants (Cass and Karas, 1975), similar studies in some seed plants have found plastids or structures taken to be plastids in generative cells, and a few studies using serial section electron microscopy to re-examine some plants in the first group have found plastids in generative cells and even in the sperm. Also, the exclusion model fails to account at all for maternal inheritance of mitochondria, which are found nearly universally in the generative cells and sperm which have been studied ultrastructurally. Second, maternal inheritance of plastid characters is seen in many lower plants and algae, despite the presence of plastids and mitochondria in the male gametes and their reported deposition in the zygote. Third, there is evidence for an alternative or additional mechanism which may occur in many plants: mitochondria and plastids in male gametes may be altered during development or syngamy so that, although not excluded, they are genetically and perhaps functionally debilitated, which would result in maternal inheritance. This evidence derives both from ultrastructural studies of pollen and fertilization, and from genetic and developmental analysis of algal zygotes and of embryos derived from pollen tissue culture. This mechanism is logically attractive in that it allows for the observed continuum of variation from strict uniparental inheritance in a number of plants, which cannot be explained by the “all-or-nothing” exclusion hypothesis. Indeed, it may be appropriate to think of both mechanisms as part of a continuum ranging from destruction within the zygote, to exclusion during syngamy, to pre-fertilization debilitation, to absence from male gametes and generative cells (Russell and Cass, 1981).     

Western white pine (Pinus monticola Dougl.) reproduction: II. Fertilisation and cytoplasmic inheritance

Fertilisation and proembryo development are described from transmission electron micrographs emphasising the origin and fate of the maternal and paternal mitochondria and plastids. During central cell and egg development mitochondria migrate toward the nuclei, forming a perinuclear zone consisting predominantly of maternal mitochondria and polysomes. At the same time, maternal plastids transformed and at fertilisation are excluded from the neocytoplasm. The pollen tube releases two sperm nuclei into the egg with cytoplasm from the generative cell and the tube cell. The leading sperm nucleus fuses with the egg nucleus and a small number of paternal mitochondria and plastids are taken into the perinuclear zone. The second sperm nucleus degenerates. As the zygote nucleus undergoes mitosis followed by free nuclear division and nuclear migration to the chalazal end of the archegonium, maternal and paternal organelles intermingle within the neocytoplasm. The result is paternal inheritance of plastids and biparental, but predominantly maternal, inheritance of mitochondria. This pattern is consistent within the Pinaceae but differs from some other conifer families. Received: 9 December 1999 / Revision accepted: 30 April 2000     

油松细胞质遗传的细胞学机理：Ⅰ.雄配子的超微结构和细胞器类核

证明了油松（Ｐｉｎｕｓ ｔａｂｕｌａｅｆｏｒｍｉｓ Ｃａｒｒ．）雄配子存在质体物线粒体及细胞器ＤＮＡ,提供了油松具父系质体和线粒体遗传基础的确切的细胞学证据,结果与松科植物在遗传学上已确定的父系质体遗传的一般规律是一致的。但精细胞中的线粒体是否能传递至胚,还需要追踪其后的发育过程。另一重要的结果是揭示了油松的雄配子是细胞,这与以前将松科植物雄配子归入雄核（精核）的类型不同。精细胞无壁,仅被质膜包围     

Mechanisms for independent cytoplasmic inheritance of mitochondria and plastids in angiosperms

The inheritance of mitochondria and plastids in angiosperms has been categorized into three modes: maternal, biparental and paternal. Many mechanisms have been proposed for maternal inheritance, including: (1) physical exclusion of the organelle itself during pollen mitosis I (PMI); (2) elimination of the organelle by formation of enucleated cytoplasmic bodies (ECB); (3) autophagic degradation of organelles during male gametophyte development; (4) digestion of the organelle after fertilization; and (5)—the most likely possibility—digestion of organellar DNA in generative cells just after PMI. In detailed cytological observations, the presence or absence of mitochondrial and plastid DNA in generative cells corresponds to biparental/paternal inheritance or maternal inheritance of the respective organelle examined genetically. These improved cytological observations demonstrate that the replication or digestion of organellar DNA in young generative cells just after PMI is a critical point determining the mode of cytoplasmic inheritance. This review describes the independent control mechanisms in mitochondria and plastids that lead to differences in cytoplasmic inheritance in angiosperms.     

Heterogeneous pollen in Chlorophytum comosum, a species with a unique mode of plastid inheritance intermediate between the maternal and biparental modes

The majority of angiosperms display maternal plastid inheritance. The cytological mechanisms of this mode of inheritance have been well studied, but little is known about its genetic relationship to biparental inheritance. The angiosperm Chlorophytum comosum is unusual in that different pollen grains show traits of different modes of plastid inheritance. About 50% of these pollen grains exhibit the potential for biparental plastid inheritance, whereas the rest exhibit maternal plastid inheritance. There is no morphological difference between these two types of pollen. Pollen grains from different individuals of C. comosum all exhibited this variability. Closer examination revealed that plastid polarization occurs, with plastids being excluded from the generative cell during the first pollen mitosis. However, the exclusion is incomplete in 50% of the pollen grains, and the few plastids distributed to the generative cells divide actively after mitosis. Immunoelectron microscopy using an anti-DNA antibody demonstrated that the plastids contain a large amount of DNA. As there is a considerable discrepancy between the exclusion and duplication of plastids, resulting in plastids with opposite fates occurring simultaneously in C. comosum, we propose that the species is a transitional type with a mode of plastid inheritance that is genetically intermediate between the maternal and biparental modes.     

Inheritance of mitochondrial and chloroplast genomes in the isogamous brown alga Scytosiphon lomentaria (Phaeophyceae)

Patterns of inheritance of chloroplasts and mitochondria were examined by fluorescence microscopy and haplotype genome markers in the isogamous brown alga Scytosiphon lomentaria (Lyngbye) Link. Germination of the zygote in this species was unilateral, the growing thallus developed entirely from the germ tube, and the original zygote cell did not develop except for the formation of a hair. Inheritance of chloroplasts was biparental, and partitioning of the two parental chloroplasts into the first sporophytic cells was accidental: either the maternal or the paternal chloroplast was migrated from the zygote into the germ tube cell, whereas the other chloroplast remained in the original cell. In contrast, the mitochondrial genome in all cells of the sporophyte came only from the female gamete (maternal inheritance). These inheritance patterns are similar to those of the isogamous brown alga Ectocarpus siliculosus (Dillwyn) Lyngbye. Maternal inheritance of mitochondria might be universal in brown algae.     

Why does biparental plastid inheritance revive in angiosperms?

It is widely believed that plastid and mitochondrial genomes are inherited through the maternal parent. In plants, however, paternal transmission of these genomes is frequently observed, especially for the plastid genome. A male gametic trait, called potential biparental plastid inheritance (PBPI), occurs in up to 20% of angiosperm genera, implying a strong tendency for plastid transmission from the male lineage. Why do plants receive organelles from the male parents? Are there clues in plastids that will help to elucidate the evolution of plants? Reconstruction of the ancestral state of plastid inheritance patterns in a phylogenetic context provides insights into these questions. In particular, a recent report demonstrated the unilateral occurrence of PBPI in angiosperms. This result implies that nuclear cytoplasmic conflicts, a basic driving force for altering the mode of organelle inheritance, might have arisen specifically in angiosperms. Based on existing evidence, it is likely that biparental inheritance may have occurred to rescue angiosperm species with defective plastids.     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism     

Paternal Inheritance of Plastid DNA in Genus Pharbitis

The inheritance of plastid DNA in Pharbitis was studied by the method of restriction fragment length polymorphisms (RFLP). Experimental results showed that plastid DNA from Pharbitis was paternally inherited in reciprocal crosses, P. nil × P. limbata and P. limbata × P. nil hybrids. But, in the cross of P. limbata × P. nil, the possibility of biparental inheritance of plastid DNA could not be roled out in our preliminary experiment. Thus Pharbitis became the third genus among angiosperms characterized with male plastid transmission. The mechanisms of paternal plastids DNA inheritance in Pharbitis is unclear. The authors proposed that dilution, exclusion and/or degeneration of maternal plastid, including their DNA, after fertilization should be considered.     

Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.     

Exceptional paternal inheritance of plastids in Arabidopsis suggests that low-frequency leakage of plastids via pollen may be universal in plants

Plastid DNA is absent in pollen or sperm cells of Arabidopsis thaliana. Accordingly, plastids and mitochondria, in a standard genetic cross, are transmitted to the seed progeny by the maternal parent only. Our objective was to test whether paternal plastids are transmitted by pollen as an exception. The maternal parent in our cross was a nuclear male sterile (ms1-1/ms1-1), spectinomycin-sensitive Ler plant. It was fertilized with pollen of a male fertile RLD-Spc1 plant carrying a plastid-encoded spectinomycin resistance mutation. Seedlings with paternal plastids were selected by spectinomycin resistance encoded in the paternal plastid DNA. Our data, in general, support maternal inheritance of plastids in A. thaliana. However, we report that paternal plastids are transmitted to the seed progeny in Arabidopsis at a low (3.9 x 10(-5)) frequency. This observation extends previous reports in Antirrhinum majus, Epilobium hirsutum, Nicotiana tabacum, Petunia hybrida, and the cereal crop Setaria italica to a cruciferous species suggesting that low-frequency paternal leakage of plastids via pollen may be universal in plants previously thought to exhibit strict maternal plastid inheritance. The genetic tools employed here will facilitate testing the effect of Arabidopsis nuclear mutations on plastid inheritance and allow for the design of mutant screens to identify nuclear genes controlling plastid inheritance.     

离体受精作为技术平台在被子植物有性生殖研究中的应用

被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质：研究合子细胞被激活的机理：用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。