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).     

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.     

ULTRASTRUCTURE OF PLASTID INHERITANCE: GREEN ALGAE TO ANGIOSPERMS

1. Plastid inheritance in most green algae and land plants is uniparental. In oogamous species, plastids are usually derived from the maternal parent; even when inheritance is biparental, maternal plastids usually predominate. Only a few species of conifer are known to have essentially paternal plastid inheritance. In spite of the overall strong maternal bias, there exists a spectrum of species in which plastid inheritance ranges from purely maternal to predominantly paternal. 2. Factors that influence the pattern of plastid inheritance operate both before (often long before) and after fertilization. For example, several different mechanisms for exclusion of plastids from particular cells, none of which is completely effective on its own, may operate sequentially during both gametogenesis and embryo-genesis. There appears to exist a general trend such that the more highly evolved the organism, the more numerous the mechanisms employed and the earlier they first come into operation. The pattern of plastid inheritance shown by a species represents the efficiency or lack of efficiency of these combined mechanisms. 3. In the newly-formed zygote of many unicellular algae, the plastids from both gametes are present and there is direct competition between them. Often the plastid from one mating type (usually the ‘invading’ male gamete, where this can be identified) quickly degenerates. Species such as Chlamydomonas are unusual in that the plastids from the two gametes fuse. In spite of this, inheritance of plastid DNA is normally uniparental. How this is accomplished remains unclear. In oogamous algae, the paternal plastids which enter the egg cell are frequently fewer in number and smaller in size than those contributed by the female gamete. The reduced contribution of paternal plastids can result from asymmetrical cell division or from differential timing of cell and plastid division during spermatogenesis. 4. In species ranging from unicellular algae to angiosperms, plastids may be partially or completely debarred from particular cells at critical stages during the reproductive cycle. An important factor in this form of plastid elimination is their postioning with respect to the nucleus prior to a cell division. When plastids closely encircle the nucleus, they are usually incorporated equally into the two daughter cells; when the plastids are concentrated at some distance from the nucleus, they are frequently excluded from one daughter cell. 5. Elimination of plastids from a gamete prior to plasmogamy prevents direct competition between the two types of plastid in the zygote or embryo. Perhaps the most effective method of excluding paternal plastids from the egg cell has been achieved by some lower land plants; the plastids migrate to the posterior part of the spermatozoid, and are discarded from there in a discrete vesicle before the egg is reached. 6. Plastid inheritance in conifers appears to be unique. In those species in which the derivation of plastids in the pro-embryo can be determined, it has been found that they come only from the male gamete. Maternal plastids are positively excluded from the pro-embryo and later degenerate. 7. In most angiosperm species plastid inheritance is maternal; in only a few species is it regularly biparental. The first step towards exclusion of paternal plastids often takes place in the uninucleate pollen grain where the plastids may be concentrated at the pole of the cell farthest from the site of the future generative cell. Any plastids that succeed in entering the generative cell may degenerate before the gametes are released from the pollen tube. Even if paternal plastids reach the egg, they are at a disadvantage because they are (a) entering an environment that is essentially alien, and (b) normally present in much smaller numbers than maternal plastids. Later, when the zygote divides, the few paternal plastids may fail to become incorporated in the small terminal cell which gives rise to the embryo proper. 8. There appears to be no consistent evolutionary progression in the use of more efficient mechanisms to influence plastid inheritance; most of the mechanisms associated with exclusion of paternal plastids in angiosperms, for example, can also be found in one or other species of green alga. The primary factors that influence plastid inheritance appear to be (I) direct competition in the zygote between plastids of the two parental types – the principal mechanism operating in isogamous algae, but also operating in some angiosperms; and (2) the divergent evolution of the two types of gamete - on the one hand a small male gamete with a minimum of cytoplasm which is capable of moving (spermatozoid) or being moved (pollen) efficiently, and, on the other hand, a large egg cell with numerous organelles, which is well able to act as ‘host’ for the future zygote. Many of the additional mechanisms that influence the pattern of plastid inheritance seem to be the more or less ‘accidental’ result of other evolutionary events.     

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。     

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.     

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.     

菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

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.     

Reduction in amounts of mitochondrial DNA in the sperm cells as a mechanism for maternal inheritance in Hordeum vulgare

It is known that extranuclear organelle DNA is inherited maternally in the majority of angiosperms. The mechanisms for maternal inheritance have been well studied in plastids but not in mitochondria. In the present study we examined the mitochondrial DNA in the male reproductive cells of Hordeum vulgare L. by immunoelectron microscopy. Our results show that the number of anti-DNA gold particles on sections of sperm cell mitochondria decreased by 97% during pollen development. The reduction occurred rapidly in the generative cells and subsequently in the sperm cells, concomitant with a remarkable reduction in mitochondrial volume. It seems that the copy numbers of mitochondrial DNA were reduced in the male reproductive cells, which may be a possible mechanism by which paternal transmission is inhibited. Unlike mitochondria, plastids are excluded from the generative cells during the first pollen mitosis. These data suggest a mechanism for maternal inheritance of mitochondria in angiosperms and for independent control of inheritance of mitochondria and plastids in H. vulgare.     

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.     

RAPID SCREENING METHOD TO DETECT POTENTIAL BIPARENTAL INHERITANCE OF PLASTID DNA AND RESULTS FOR OVER 200 ANGIOSPERM SPECIES

We have developed a diagnostic method to screen rapidly for plant species potentially capable of biparental inheritance of plastid DNA using the DNA fluorochrome 4′,6-diamidino-2-phenylindole (DAPI) in conjunction with epifluorescence microscopy. Pollen shed from 235 plant species (including about 50 of agronomic importance) representing 80 families were screened. Putative plastid DNA was detected in the generative and/or sperm cells of pollen from 26 genera (43 species) representing 15 families. Plastid DNA was not detected in the generative or sperm cells of pollen from 192 plant species, thereby strongly suggesting that these species have only maternal inheritance. Our cytological diagnosis corroborated the known genetic evidence in 42 plant species and conflicted with the genetic reports in five species, which are discussed. The data suggest that biparental inheritance of plastids is rare; overall, it may occur in about 14% of flowering plant genera, examples of which are scattered among 19% of the families examined. This methodology also readily reveals whether pollen is bi- or trinucleate.     

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.     

Divergent potentials for cytoplasmic inheritance within the genus Syringa. A new trait associated with speciogenesis

Epifluorescence microscopic detection of organelle DNA in the mature generative cell is a rapid method for determining the potential for the mode of cytoplasmic inheritance. We used this method to examine 19 of the known 22 to 27 species in the genus Syringa. Organelle DNA was undetectable in seven species, all in the subgenus Syringa, but was detected in the 12 species examined of the subgenera Syringa and Ligustrina. Therefore, species within the genus Syringa display differences in the potential cytoplasmic inheritance. Closer examination revealed that the mature generative cells of the species in which organelle DNA was detected contained both mitochondria and plastids, but cells of the species lacking detectable organelle DNA contained only mitochondria, and the epifluorescent organelle DNA signals from the mature generative cells corresponded to plastid DNA. In addition, semiquantitative analysis was used to demonstrate that, during pollen development, the amount of mitochondrial DNA decreased greatly in the generative cells of the species examined, but the amount of plastid DNA increased remarkably in the species containing plastids in the generative cell. The results suggest that all Syringa species exhibit potential maternal mitochondrial inheritance, and a number of the species exhibit potential biparental plastid inheritance. The difference between the modes of potential plastid inheritance among the species suggests different phylogenies for the species; it also supports recent conclusions of molecular, systematic studies of the Syringa. In addition, the results provide new evidence for the mechanisms of maternal mitochondrial inheritance in angiosperms.     

Potential cytoplasmic inheritance in Wisteria sinensis and Robinia pseudoacacia (Leguminosae)

We examined pollen cells of Wisteria sinensis and Robinia pseudoacacia (Leguminosae) to determine a possible mode for cytoplasmic inheritance in these species. Epifluorescence microscopy revealed distinct mature generative cells. Mature generative cells of W. sinensis were associated with large numbers of punctuated fluorescent signals corresponding to cytoplasmic DNA aggregates, but no fluorescent signals were observed in the generative cells of R. pseudoacacia. Closer examination showed that the punctate fluorescent signals corresponded to plastid but not mitochondrial DNA. These results suggest a strong potential for paternal transmission of the plastid genome in W. sinensis. Electron microscopy confirmed the presence of plastids in the generative cells of W. sinensis and the absence of plastids in R. pseudoacacia cells due to an unequal distribution of plastids during the first pollen mitosis. Mitochondria were present and intact in the mature generative cells of both species. The lack of fluoresced mitochondrial DNA suggests a very low level of mitochondrial DNA in the cells. Immunoelectron microscopy demonstrated that the labeling of mitochondrial DNA in these cells was reduced by nearly 90% during pollen development. Such a dramatic reduction suggests an active degradation of paternal mitochondrial DNA, which may contribute greatly to the maternal inheritance of mitochondria. In short, we found that W. sinensis exhibits a strong potential for paternal transmission of plastids and that both W. sinensis and R. pseudoacacia appear to share the same mechanism for maternal mitochondrial inheritance.     

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.     

Plastid DNA inheritance and plastome-genome incompatibility in interspecific hybrids of Zantedeschia (Araceae)

Plastid DNA (ptDNA) probes were used in RFLP analysis to determine ptDNA inheritance in interspecific hybrids in Zantedeschia. Biparental and maternal ptDNA inheritance was found in albino hybrids between the evergreen species Z. aethiopica and several winter-dormant species. From two albino hybrids, different types of ptDNA were detected in shoots derived from different parts of an embryo. This result indicates that plastids were sorted out during embryo development. Only maternal ptDNA was detected in the hybrids of Z. aethiopica × Z. odorata (a summer-dormant species) but paternal, biparental, and maternal ptDNA were found in the hybrids of the reciprocal cross. Z. odorata × Z. aethiopica. By correlating these ptDNA inheritance patterns with the leaf colour (albino, pale-green, and green) of the hybrids, it is suggested that the Z. odorata plastome is incompatible with the Z. aethiopica genome. The Z. aethiopica plastome is partially compatible with the Z. odorata genome but the development of Z. aethiopica plastids appears to be blocked by the presence of the Z. odorata plastids.     

Plastid inheritance in the planktonic raphid pennate diatom Pseudo-nitzschia delicatissima (Bacillariophyceae)

Plastid inheritance was followed during sexual reproduction in the raphid pennate diatom Pseudo-nitzschia delicatissima, using rbcL haplotypes as plastid identification tools. Pseudo-nitzschia species are dioecious and show functional anisogamy with 'male' mating type+(PNd(+)) cells and 'female' PNd(-) cells. Vegetative cells possess two plastids. In P. delicatissima, meiosis results in two gametes that both contribute two plastids to the zygote. The latter initially contains four plastids, but during auxospore development two of these four seem to disappear, and the initial cell emerging from the auxospore appears to contain only two. Here we assessed if the plastids are inherited strictly unipaternally, strictly biparentally, or randomly. We traced the source of the plastids in the F(1) generation by using PNd(+) and PNd(-) parental strains with different rbcL genotypes, here denoted AA (homoplastidial, with two plastids of rbcL haplotype A) and BB (homoplastidial; two plastids of haplotype B). Results showed that 16 out of 96 strains raised each from single F(1) cells had retained two paternal (PNd(+)) plastids, 20 had two maternal (PNd(-)) plastids and the remaining 60 had one maternal and one paternal plastid. This pattern is in accordance with the hypothesis that either two of the four plastids are eliminated during auxospore formation, or that all plastids are retained in the auxospore and segregate in pairs joining at random during the first mitotic division of the initial cell. Heteroplastidic F(1)-strains retained the AB genotype throughout the vegetative phase of their life cycle. The finding that 60 out of 96 F(1) strains were heteroplastidial contrasts with an absence of such genotypes in our strains raised from single cells sampled in the Gulf of Naples.     

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.     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

INVITED SPECIAL PAPER: The hows and whys of cytoplasmic inheritance in seed plants

Cytoplasmic organelles are inherited in a nonMendelian fashion in all eukaryotic organisms investigated. Among the seed plants, plastids can be inherited strictly from the female parent, strictly from the male parent, or biparentally. Most flowering plants studied to date exhibit maternal plastid inheritance, but approximately one-third of the genera investigated display biparental plastid inheritance to some degree. Among the gymnosperms, paternal plastid inheritance is the rule in the conifers, whereas the other groups appear to have maternal plastid inheritance, although they have been less well studied. Mitochondrial inheritance is predominantly maternal in the seed plants, except for a few coniferous families where it is predominantly paternal. The advent of recombinant DNA technology has allowed restriction fragment length polymorphisms to be used as molecular markers, and has stimulated much research in organelle inheritance and its application to studies of population genetics and phylogenetic biology. This report emphasizes the various mechanisms by which organelles are, or are not, transmitted among the seed plants in order that researchers directly or indirectly involved with organelle inheritance may better understand the potential and the limitations of their investigations. A summary and discussion of the possible evolutionary significance of the various patterns of cytoplasmic inheritance among the seed plants are also included.