In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

Advantages of sexual reproduction

Despite the obvious efficiencies of many forms of asexual reproduction, sexual reproduction abounds. Asexual species, for the most part, are relatively short-lived offshoots of sexual ancestors. From the nineteenth century, it has been recognized that, since there is no obvious advantage to the individuals involved, the advantages of sexual reproduction must be evolutionary. Furthermore, the advantage must be substantial; for example, producing males entails a two-fold cost, compared to dispensing with them and reproducing by parthenogenetic females. There are a large number of plausible hypotheses. To me the most convincing of these are two. The first hypothesis, and the oldest, is that sexual reproduction offers the opportunity to produce recombinant types that can make the population better able to keep up with changes in the environment. Although the subject of a great deal of work, and despite its great plausibility, the hypothesis has been very difficult to test by critical observations or experiments. Second, species with recombination can bunch harmful mutations together and eliminate several in a single “genetic death.” Asexual species, can eliminate them only in the same genotype in which they occurred. If the rate of occurrence of deleterious mutations is one or more per zygote, some mechanism for eliminating them efficiently must exist. A test of this mutation load hypothesis for sexual reproduction, then, is to find whether deleterious mutation rates in general are this high-as Drosophila data argue. Unfortunately, although molecular and evolutionary studies can give information on the total mutation rate, they cannot determine what fraction are deleterious. In addition, there are short discussions of the advantages of diploidy, anisogamy, and separate sexes. © 1994 Wiley-Liss, Inc.     

Using proteomics to mine genome sequences

We present a method for mining unannotated or annotated genome sequences with proteomic data to identify open reading frames. The region of a genome coding for a protein sequence is identified by using information from the analysis of proteins and peptides with MALDI-TOF mass spectrometry. The raw genome sequence or any unassembled contigs of an organism are theoretically cleaved into a number of equal sized but overlapping fragments, and these are then translated in all six frames into a series of virtual proteins. Each virtual protein is then subjected to a theoretical enzymatic digestion. Standard proteomic sample preparation methods are used to separate, array, and digest the proteins of interest to peptides. The masses of the resulting peptides are measured using mass spectrometry and compared to the theoretical peptide masses of the virtual proteins. The region of the genome responsible for coding for a particular protein can then be identified when there are a large number of hits between peptides from the protein and peptides from the virtual protein. The method makes no assumptions about the location of a protein in a particular gene sequence or the positions or types of start and stop codons. To illustrate this approach, all 773 proteins of Pseudomonas aeruginosa contained in SWISS-PROT were used to theoretically test the method and optimize parameters. Increasing the size of the virtual proteins results in an overall improvement in the ability to detect the coding region, at the cost of decreasing the sensitivity of the method for smaller proteins. Increasing the minimum number of matching peptides, lowering the mass error tolerance, or increasing the signal-to-noise ratio of the simulated mass spectrum, improves the ability to detect coding regions. The method is further demonstrated on experimental data from Mycobacterium tuberculosis and is also shown to work with eukaryotic organisms (e.g., Homo sapiens).     

Using individual-based modeling to investigate whether fluctuating resources help to explain the prevalence of sexual reproduction in animal species

The prevalence of sexual reproduction of animal species is a paradox for evolutionary theory since it remains unclear whether the evolutionary benefits of sexual reproduction outweigh the costs. One attempt at explaining the maintenance of sex is the Tangled Bank hypothesis: Sexual reproduction shuffles around alleles through crossing over and recombination, resulting in a wide range of individuals, some of whom will be able to survive in the harshest of environments with low and dwindling food resources. Whereas, with respect to clonally reproduced individuals there is arguably less genetic variation so that if food resources start to fluctuate, these individuals may not be able to survive under the new conditions. In our study, we conducted individual based modeling computer simulations using the program EcoSim to investigate two hypotheses related to fluctuating resources: First, in the context of fluctuating resources, populations of sexual species will outpace the populations of asexual species who are unable to adapt to changing conditions. The second hypothesis that we investigated is that with respect to facultative species there will be an increase in sexual reproduction and a decrease in asexual reproduction as a response to fluctuating resources. The control runs involved relatively stable food resources for obligate sexual, obligate asexual and facultatively reproducing prey species, whereas the experimental runs involved unstable fluctuating resources. Although we found that population levels were higher for obligate sexual prey vs. obligate asexual prey, this was not due to the manipulation of the independent variable, food resources, since these results were consistent across experimental, and control runs. However, in terms of the runs for facultative species, we found that in experimental runs, there was a discernably lower level of asexual reproduction and a slight increase in sexual reproduction in the later stages of the runs, which is likely a response to fluctuating resources. These results tend to confirm the hypothesis that in terms of facultative species, there will be a decrease in asexual reproduction and an increase in sexual reproduction in response to fluctuating resources. Moreover, we found that these features may be evolutionary in nature rather than simply a matter of phenotypic plasticity, which to the best of our knowledge is not a result in any other simulation or empirical study on Tangled Bank with respect to facultative species. Our study therefore contributes to the ongoing debate of whether the switch to sex in facultative species is the result of phenotypic plasticity or evolutionary in character.     

Patterns of sexual reproduction in diatoms

Sexual reproduction takes many forms within the diatoms. The variation has been classified by several authors, but in most cases the distinctions between their main categories have depended on the number of gametes produced per gametangium (and thus on how many zygotes per pair of copulating cells), and upon whether fusion is oogamous, anisogamous or isogamous. These classifications are not themselves an adequate basis for taxonomic comparison, which should be based on individual characteristics of the sexual process. Diatoms seem to be primitively oogamous. In araphid pennate diatoms and some raphid diatoms the gametes and gametangia are morphologically alike but physiologically distinct; one gametangium produces active gametes and the other passive ones. This may be the primitive condition in pennate diatoms, providing a link to the oogamy of centrics via the morphological anisogamy of Rhabdonema Kütz.     

Arabinogalactan proteins in plant sexual reproduction

Summary Arabinogalactan proteins (AGPs) are a class of plant extracellular-matrix proteins believed to participate in a broad range of processes involving the plant cell surface. They are extremely abundant in female reproductive tissues and in pollen tubes, the haploid male structures that traverse the diploid female reproductive tissues to deliver sperms to the egg cells. The prevalence of AGPs in reproductive tissues has led to speculations that they play significant functional roles ranging from serving as nutrient resources to cell-cell recognition in plant reproduction. Recent research from several laboratories demonstrated functional participation by AGPs in reproductive processes and began to examine the mechanisms underlying these functional roles. An overview of these recent studies will be discussed with a historical perspective as well as with a view towards future studies in establishing the significance of AGPs that, as a class, they have prominent roles in plant sexual reproduction in multiple and diverse ways.     

Evolutionarily stable allocation to vegetative and sexual reproduction in plants

I apply the resource allocation approach to construct an ESS model aimed at predicting the rate of vegetative and sexual reproduction in plants. The model provides an integrated explanation of why the mode of reproduction should vary between populations and species, and what factors should affect the trade-off between ramet and seed production. It follows that seed production, usually considered to be less effective and more costly than ramet production, may be maintained in the population. The production of ramets should be favored when their recruitment rate is high or the costs are low. The intensity of vegetative reproduction is also related to conditions which affect seed production directly. The rate of ramet production should be inversely correlated with factors favoring sexual reproduction, such as lower cost of seeds or high opportunity for colonization of new patches, etc. Under such conditions it is beneficial to allocate more resources available for reproduction into seeds, at the expense of ramet production. The model emphasizes the need to consider both modes of plant reproduction whenever one of them is theoretically or empirically studied.     

The requirements for sexual reproduction

Summary Sexual reproduction requires controls for gonadogenesis, genital differentiation, and sexuality. The initiating event is induction of testicular differentiation by an effect of the H-Y locus or a combination of X- and Y-borne genes. This paper reviews the evidence that testosterone, either directly or via regulated conversion to other steroids, controls sexuality as it does male genital differentiation. The point is also stressed that, despite their difference, sexual reproduction and individual homcostasis are intimately linked.     

Polycomb group gene function in sexual and asexual seed development in angiosperms

In sexually reproducing angiosperms, double fertilization initiates seed development, giving rise to two fertilization products, the embryo and the endosperm. In the endosperm, a terminal nutritive tissue that supports embryo growth, certain genes are expressed differentially depending on their parental origin, and this genomic imbalance is required for proper seed formation. This parent-of-origin effect on gene expression, called genomic imprinting, is controlled epigenetically through histone modifications and DNA methylation. In the sexual model plant Arabidopsis, the Polycomb group (PcG) genes of the plant Fertilization Independent Seed (FIS)-class control genomic imprinting by specifically silencing maternal or paternal target alleles through histone modifications. Mutations in FIS genes can lead to a bypass in the requirement of fertilization for the initiation of endosperm development and seed abortion. In this review, we discuss the role of the FIS complex in establishing and maintaining genomic imprinting, focusing on recent advances in elucidating the expression and function of FIS-related genes in maize, rice, and Hieracium, and particularly including apomictic Hieracium species that do not require paternal contribution and thus form seeds asexually. Surprisingly, not all FIS-mediated functions described in Arabidopsis are conserved. However, the function of some PcG components are required for viable seed formation in seeds formed via sexual and asexual processes (apomixis) in Hieracium, suggesting a conservation of the seed viability function in some eudicots.     

Genes involved in Dictyostelium discoideum sexual reproduction

Macrocyst formation in the cellular slime moulds is a sexual process induced under dark and humid conditions. Normal development life cycle in these organisms involves proliferation by cell division and, upon starvation, formation of multicellular aggregates and fruiting bodies, consisting of spores and stalk cells. Macrocyst formation, cell division by binary fission and spore formation are thus three alternative modes of reproduction, for which it is of interest to understand how a choice is made. The genetic basis of asexual development and fruiting body formation is well known, by contrast information on the genetic control of sexual reproduction during macrocyst formation is scarce. In Dictyostelium discoideum, the most widely used species, several cell-surface proteins relevant to sexual cell fusion have been identified using cell fusion-blocking antibodies, but isolation of the relevant genes has been unsuccessful. Analysis of sexually deficient mutants, some of which are normal for asexual development, has shown that sexual reproduction is regulated by both specific genes and genes that are also involved in asexual development. Reverse genetic analysis of 24 genes highly enriched in a gamete-specific subtraction library has revealed four genes involved in the regulation of sexual cell interactions. One of them was found to be a novel regulator of the cAMP signalling pathway specific to sexual development. Studies on the molecular genetic control of the sexual cycle will be reviewed and their contribution to our understanding of the organization and function of the D. discoideum genome as a whole discussed.     

Parasites and sexual reproduction in psychid moths

Persistence of sexual reproduction among coexisting asexual competitors has been a major paradox in evolutionary biology. The number of empirical studies is still very limited, as few systems with coexisting sexual and strictly asexual lineages have been found. We studied the ecological mechanisms behind the simultaneous coexistence of a sexually and an asexually reproducing closely related species of psychid moth in Central Finland between 1999 and 2001. The two species compete for the same resources and are often infected by the same hymenopteran parasitoids. They are extremely morphologically and behaviorally similar and can be separated only by their reproductive strategy (sexual vs. asexual) or by genetic markers. We compared the life-history traits of these species in two locations where they coexist to test predictions of the cost-of-sex hypothesis. We did not find any difference in female size, number of larvae, or offspring survival between the sexuals and asexuals, indicating that sexuals are subject to cost of sex. We also used genetic markers to check and exclude the possibility of Wolbachia bacteria infection inducing parthenogenesis. None of the samples was infected by Wolbachia and, thus, it is unlikely that these bacteria could affect our results. We sampled 38 locations to study the prevalence of parasitoids and the moths' reproductive strategy. We found a strong positive correlation between prevalence of sexual reproduction and prevalence of parasitoids. In locations where parasitoids are rare asexuals exist in high densities, whereas in locations with a high parasitoid load the sexual species was dominant. Spatial distribution alone does not explain the results. We suggest that the parasite hypothesis for sex may offer an explanation for the persistence of sexual moths in this system.     

Size-dependent allocation to sexual and vegetative reproduction in four clonal composites

Summary Populations of Silphium speciosum, Vernonia baldwinii, Solidago canadensis and Pityopsis graminifolia were studied to determine whether biomass allocation to sexual and vegetative reproduction and the balance between them were size-dependent and whether interpopulation differences in allocation patterns could be predicted from differences in population size distributions. All four species showed strong linear relationships between inflorescence mass and vegetative mass with negative y-intercepts. As a result, sexual reproductive effort (SRE) was a monotonically increasing function of ramet size. Genet size was a poor predictor of SRE. In each species, the regression parameters of these relationships differed significantly between burned and unburned habitats indicating size-independent interpopulation differences in patterns of reproductive effort as well as sizedependent effects. Interpopulation variation in vegetative reproductive effort (VRE) was greater than variation in SRE, but neither VRE nor the pattern of partitioning of VRE among daughter rhizomes showed significant relationships to plant size.     

Mobile genetic elements and sexual reproduction

Transposable elements (TE) are prominent components of most eukaryotic genomes. In addition to their possible participation in the origin of sexual reproduction in eukaryotes, they may be also involved in its maintenance as important contributors to the deleterious mutation load. Comparative analyses of transposon content in the genomes of sexually reproducing and anciently asexual species may help to understand the contribution of different TE classes to the deleterious load. The apparent absence of deleterious retrotransposons from the genomes of ancient asexuals is in agreement with the hypothesis that they may play a special role in the maintenance of sexual reproduction and in early extinction for which most species are destined upon the abandonment of sex.     

A comparative study of genomes in angiosperms

Angiosperms investigated by DNA/DNA reassociation studies were classified and tested for a taxonomic class- and subclass-specifity in a biometrical fashion. Monocotyledons and Dicotyledons differ significantly from each other with respect to a genomic parameter (U/R-ratio;U single copy DNA fraction;R = 1-U fraction of repetitive DNA). This difference is discussed from an evolutionary and molecular point of view.—Intercorrelations between the fraction of fast repeats, slow repeats, and single copy DNA can be detected. The amount of DNA organized in a short period pattern of interspersion is found to depend on the fraction of repetitive and single copy DNA. The number of DNA segments tandemly arranged in a short period pattern is linearly correlated withR/U-values. This correlation allows for a formula suitable for the estimation of the number of active genes in angiosperms. The analytical complexities of repetitive and single copy DNA are linearly correlated with the genome size of higher plants. The ratioU/R depends on the genome size of angiosperms in a hyperbolic fashion.     

The cost of switching from asexual to sexual reproduction in an aphid

The fecundity of asexual and sexual morphs of Sitobion avenae (F.) is compared. The cost of sex is quantified in terms of the reduction in numbers of offspring produced. The cost is much greater than the halving of fecundity that is usually assumed to be associated with the production of males.

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Résumé Les fécondités des virginipares ailés, des gynopares ailés et des ovipares d'un même clone de Sitobion avenae ont été déterminées à 12°C. Bien que les types qui ayant donné naissance à des sexués aient eu une fécondité initiale plus élevée que celle de la lignée asexuée, celle-ci a été significativement plus féconde après 15 jours de reproduction.Les résultats du laboratoire ont été utilisés pour évaluer la fécondité de lignées sexuées et asexuées pendant quatre générations à 12°C. La reproduction parthénogénétique a présenté un avantage numérique sur la reproductions sexuée dans le rapport 320/1. La reproduction sexuée a été accompagnée d'une diminution significative de la fécondité potentielle. Bien que la reproduction sexuée de S. avenae soit avantageuse dans les régions où les hivers sont durs, parce que les oeufs sont résistants au froid, les lignées asexuées présentent un avantage considérable en ce qui concerne la fécondité potentielle pour l'augmentation de la population en automne et elles conservent de plus la capacité d'un accroissement numérique ultérieur si l'hiver est doux.