Generating Autotetraploid Sporophytes and Their Use in Analyzing Mutations Affecting Gametophyte Development in the Fern Ceratopteris

The haploid gametophytes of the fern Ceratopteris richardii are autotrophic and develop independently of the diploid sporophyte plant. While haploid genetics is useful for screening and characterizing mutations affecting gametophyte development in Ceratopteris, it is difficult to assess whether a gametophytic mutation is dominant or recessive or to determine allelism by complementation analysis in a haploid organism. This report describes how apospory can be used to produce genetically marked polyploid sporophytes whose gametophyte progeny are heterozygous for mutations affecting sex determination in the gametophyte and a known recessive mutation affecting the phenotype of both the gametophyte and sporophyte. The segregation ratios of wild-type to mutant phenotypes in the gametophyte progeny of polyploid sporophyte plants indicate that all of the mutations examined are recessive. The presence of many multivalents and few univalents in meiotic chromosome preparations of spore mother cells confirm that the sporophyte plants assayed are polyploid. The DNA content of the sperm of their progeny gametophytes was also found to be approximately twice that of sperm from wild-type haploid gametophytes.     

CONTROL OF SEX RATIOS IN HAPLOID POPULATIONS OF THE MOSS,CERATODON PURPUREUS

Female biased sex ratios occur in a number of unrelated mosses. Such ratios refer to the relative numbers of male and female gametophytes in moss populations and are therefore more comparable to the numbers of pollen grains and ovules in populations of seed plants than to the numbers of male (microsporangiate) and female (megasporangiate) sporophytes. A survey of 11 populations of the moss, Ceratodon purpureus, showed that sex ratios are heterogeneous, but that female biases occur in more than half the populations. One hundred and sixty single spore isolates representing 40 sporophytes from one population demonstrated that female gametophytes outnumbered males by a ratio of 3:2 at the time of germination. Female gametophytic clones formed significantly more biomass than male clones, and individual female shoots were more robust. Male clones, however, produced more numerous stems. These sexually dimorphic traits may be related to life history differences between male and female gametophytes since females must provide nutritional support to the “parasitic” sporophyte generation, a burden that males do not share.     

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 Kan^R:Kan^S segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.     

T-DNA mediated disruption of essential gametophytic genes in Arabidopsis is unexpectedly rare and cannot be inferred from segregation distortion alone

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 Kan^R:Kan^S segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte. Received: 27 July 1998 / Accepted: 16 September 1998     

Exportin1 genes are essential for development and function of the gametophytes in Arabidopsis thaliana

Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recruited at the kinetochores during mitosis. Here we show that double mutants in Arabidopsis XPO1A and XPO1B are gametophytic defective. In homozygous–heterozygous plants, 50% of the ovules were arrested at different stages according to the parental genotype. Double-mutant female gametophytes of xpo1a-3/+; xpo1b-1/xpo1b-1 plants failed to undergo all the mitotic divisions or failed to complete embryo sac maturation. Double-mutant female gametophytes of xpo1a-3/xpo1a-3; xpo1b-1/+ plants had normal mitotic divisions and fertilization occurred; in most of these embryo sacs the endosperm started to divide but an embryo failed to develop. Distortions in male transmission correlated with the occurrence of smaller pollen grains, poor pollen germination, and shorter pollen tubes. Our results show that mitotic divisions are possible without XPO1 during the haploid phase, but that XPO1 is crucial for the maternal-to-embryonic transition.     

Gametophytic expression of genes controlling endosperm development in maize

Summary An indirect approach was adopted to select viable mutants affecting the male gametophytic generation in maize. This approach consists of a selection of endosperm defective mutants followed by a test for gametophytic gene expression, based on the distortion from mendelian segregation and on the measurement of pollen size and pollen sterility. The material used consisted of 34 endosperm defective viable mutants introgressed in B37 genetic background. Complementation tests indicated that the mutation in the collection of mutants affected different genes controlling endosperm development. The study of the segregation in F2 revealed four classes of de (defective endosperm) mutants: (1) mutants in which the mutation does not affect either gametophytic development or function; (2) mutants in which the effect on the gametophyte affects pollen development processes; (3) mutants showing effects on both pollen development and function, and (4) mutants where only pollen tube growth rate is affected. Positive and negative interactions between pollen and style were detected by means of mixed pollination (pollen produced by de/de plants and pollen from an inbred line used as a standard and carrying genes for colored aleurone), on de/de and de/ + plants. Positive interactions were interpreted as methabolic complementation between defective pollen and normal styles.     

Maternal control of seed development

Maternal control of higher plant seed development is likely to involve female sporophytic as well as female gametophytic genes. While numerous female sporophytic mutants control the production of the ovule and the embryo sac true maternal effect mutations affecting embryo and endosperm development are rare in plants. A new class of female gametophytic mutants has been isolated that controls autonomous development of endosperm. Molecular analyses of these genes, known as FIS class genes, suggest that they repress downstream seed development genes by chromatin remodelling. Expression of the FIS genes in turn is modulated by parent specific expression or genomic imprinting which in turn is controlled by DNA methylation. Thus maternal control of seed development is a complex developmental event influenced by both genetic and epigenetic processes.     

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.     

Cross talk between the sporophyte and the megagametophyte during ovule development

In seed plant ovules, the diploid maternal sporophytic generation embeds and sustains the haploid generation (the female gametophyte); thus, two independent generations coexist in a single organ. Many independent studies on Arabidopsis ovule mutants suggest that embryo sac development requires highly synchronized morphogenesis of the maternal sporophyte surrounding the gametophyte, since megagametogenesis is severely perturbed in most of the known sporophytic ovule development mutants. Which are the messenger molecules involved in the haploid–diploid dialogue? And furthermore, is this one way communication or is a feedback cross talk? In this review, we discuss genetic and molecular evidences supporting the presence of a cross talk between the two generations, starting from the first studies regarding ovule development and ending to the recently sporophytic identified genes whose expression is strictly controlled by the haploid gametophytic generation. We will mainly focus on Arabidopsis studies since it is the species more widely studied for this aspect. Furthermore, possible candidate molecules involved in the diploid–haploid generations dialogue will be presented and discussed.     

The ABNORMAL GAMETOPHYTES (AGM) gene product of Arabidopsis demonstrates a role in mitosis during gamete development

Screening a T-DNA mutagenized population of Arabidopsis thaliana for reduced seed set and segregation distortion led to the isolation of the ABNORMAL GAMETOPHYTES (AGM) mutant. Homozygous plants were never recovered, but heterozygous plants showed mitotic defects during gametogenesis resulting in approximately 50% abortion of both the male and female gametes. Isolation of the genomic sequence flanking the co-segregating T-DNA element led to the identification of a gene located on chromosome 5, predicted to encode a transmembrane protein. BLAST homology searches identified two homologous proteins that are not redundant, as is clear from the existence of the agm mutant. Unexpectedly, expression studies using the beta-glucuronidase reporter gene suggest that AGM and its closest Arabidopsis homolog are mostly expressed in cells undergoing mitosis. Thus, AGM is not a gametophytic gene as originally speculated on the basis of segregation distortion, but rather classified as an essential gene crucial to the process of mitosis in plants.     

Requirement of aminoacyl-tRNA synthetases for gametogenesis and embryo development in Arabidopsis

Aminoacyl-tRNA synthetases (AARSs) are required for translation in three different compartments of the plant cell: chloroplasts, mitochondria and the cytosol. Elimination of this basal function should result in lethality early in development. Phenotypes of individual mutants may vary considerably, depending on patterns of gene expression, functional redundancy, allele strength and protein localization. We describe here a reverse genetic screen of 50 insertion mutants disrupted in 21 of the 45 predicted AARSs in Arabidopsis. Our initial goal was to find additional EMB genes with a loss-of-function phenotype in the seed. Several different classes of knockouts were discovered, with defects in both gametogenesis and seed development. Three major trends were observed. Disruption of translation in chloroplasts often results in seed abortion at the transition stage of embryogenesis with minimal effects on gametophytes. Disruption of translation in mitochondria often results in ovule abortion before and immediately after fertilization. This early phenotype was frequently missed in prior screens for embryo-defective mutants. Knockout alleles of non-redundant cytosolic AARSs were in general not identified, consistent with the absolute requirement of cytosolic translation for development of male and female gametophytes. These results provide a framework for evaluating redundant functions of AARSs in Arabidopsis, a valuable data set of phenotypes resulting from multiple disruptions of a single basal process, and insights into which genes are required for both gametogenesis and embryo development and might therefore escape detection in screens for embryo-defective mutants.     

植物雄配子体发生和发育的遗传调控

植物雄配子体发生和发育是有性生殖的关键步骤之一, 是高等植物通过有性生殖进行世代交替所必需的。近几年来,随着分离和鉴定配子体型突变体技术的发展, 雄配子体遗传机制研究取得了很大进展, 发现了一些调控雄配子体发生和发育过程中细胞分化和互作的重要基因。本文着重概述和讨论植物雄性生殖细胞和雄配子体发生及其与周边细胞互作遗传机制研究的最新进展。     

植物雄配子体发生和发育的遗传调控

植物雄配子体发生和发育是有性生殖的关键步骤之一,是高等植物通过有性生殖进行世代交替所必需的。近几年来,随着分离和鉴定配子体型突变体技术的发展,雄配子体遗传机制研究取得了很大进展,发现了一些调控雄配子体发生和发育过程中细胞分化和互作的重要基因。本文着重概述和讨论植物雄性生殖细胞和雄配子体发生及其与周边细胞互作遗传机制研究的最新进展。     

On the origin of class B floral homeotic genes: functional substitution and dominant inhibition in Arabidopsis by expression of an orthologue from the gymnosperm Gnetum

Class B floral homeotic genes are involved in specifying stamen and petal identity in angiosperms (flowering plants). Here we report that gymnosperms, the closest relatives of the angiosperms, contain at least two different clades representing putative orthologues of class B genes, termed GGM2-like and DAL12-like genes. To obtain information about the functional conservation of the class B genes in seed plants, the representative of one of these clades from Gnetum, termed GGM2, was expressed under the control of the CaMV 35S promoter in Arabidopsis wild-type plants and in different class B mutants. In wild-type plants and in a conditional mutant grown at a permissive temperature, gain-of-function phenotypes were obtained in whorls 1 and 4, where class B genes are usually not expressed. In contrast, loss-of-function phenotypes were observed in whorls 2 and 3, where class B genes are expressed. In different class B gene null mutants of Arabidopsis, and in the conditional B mutant grown at the non-permissive temperature, a partial complementation of the mutant phenotype was obtained. In situ hybridization studies and class B gene promoter test fusion experiments demonstrated that the gain-of-function phenotypes are not due to an upregulation of the endogenous B genes from Arabidopsis, and hence probably involve interactions between GGM2 protein homodimers and class B protein target genes other than the Arabidopsis class B genes itself. To our knowledge, this is the first time that partial complementation of a homeotic mutant by an orthologous gene from a distantly related species has been reported. These data suggest that GGM2 has a function in the gymnosperm Gnetum which is related to that of class B floral organ identity genes of angiosperms. That function may be in the specification of male reproductive organ identity, and in distinguishing male from female reproductive organs.     

Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

Life history variation in gametophyte populations of the moss Ceratodon purpureus (Ditrichaceae)

The life cycles of mosses and other bryophytes are unique among land plants in that the haploid gametophyte stage is free-living and the diploid sporophyte stage is ephemeral and completes its development attached to the maternal gametophyte. Despite predictions that populations of haploids might contain low levels of genetic variation, moss populations are characterized by substantial variation at isozyme loci. The extent to which this is indicative of ecologically important life history variation is, however, largely unknown. Gametophyte plants from two populations of the moss Ceratodon purpureus were grown from single-spore isolates in order to assess variation in growth rates, biomass accumulation, and reproductive output. The data were analyzed using a nested analysis of variance, with haploid sib families (gametophytes derived from the same sporophyte) nested within populations. High levels of life history variation were observed within both populations, and the populations differed significantly in both growth and reproductive characteristics. Overall gametophytic sex ratios did not depart significantly from 1:1 within either population, but there was significant variation among families in both populations for progeny sex ratio. Some families produced predominantly male gametophytes, while others yielded predominantly females. Because C. purpureus has a chromosomal mechanism of sex determination, these observations suggest differential (but unpredictable) germination of male and female spores. Life history observations showed that male and female gametophytes are dimorphic in size, maturation rates, and reproductive output.     

Embryo and endosperm development is disrupted in the female gametophytic capulet mutants of Arabidopsis

The female gametophyte of higher plants gives rise, by double fertilization, to the diploid embryo and triploid endosperm, which develop in concert to produce the mature seed. What roles gametophytic maternal factors play in this process is not clear. The female-gametophytic effects on embryo and endosperm development in the Arabidopsis mea, fis, and fie mutants appear to be due to gametic imprinting that can be suppressed by METHYL TRANSFERASE1 antisense (MET1 a/s) transgene expression or by mutation of the DECREASE IN DNA METHYLATION1 (DDM1) gene. Here we describe two novel gametophytic maternal-effect mutants, capulet1 (cap1) and capulet2 (cap2). In the cap1 mutant, both embryo and endosperm development are arrested at early stages. In the cap2 mutant, endosperm development is blocked at very early stages, whereas embryos can develop to the early heart stage. The cap mutant phenotypes were not rescued by wild-type pollen nor by pollen from tetraploid plants. Furthermore, removal of silencing barriers from the paternal genome by MET1 a/s transgene expression or by the ddm1 mutation also failed to restore seed development in the cap mutants. Neither cap1 nor cap2 displayed autonomous seed development, in contrast to mea, fis, and fie mutants. In addition, cap2 was epistatic to fis1 in both autonomous endosperm and sexual development. Finally, both cap1 and cap2 mutant endosperms, like wild-type endosperms, expressed the paternally inactive endosperm-specific FIS2 promoter GUS fusion transgene only when the transgene was introduced via the embryo sac, indicating that imprinting was not affected. Our results suggest that the CAP genes represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development.     

Perspective: the origin of flowering plants and their reproductive biology--a tale of two phylogenies

Recently, two areas of plant phylogeny have developed in ways that could not have been anticipated, even a few years ago. Among extant seed plants, new phylogenetic hypotheses suggest that Gnetales, a group of nonflowering seed plants widely hypothesized to be the closest extant relatives of angiosperms, may be less closely related to angiosperms than was believed. In addition, recent phylogenetic analyses of angiosperms have, for the first time, clearly identified the earliest lineages of flowering plants: Amborella, Nymphaeales, and a clade that includes Illiciales/ Trimeniaceae/Austrobaileyaceae. Together, the new seed plant and angiosperm phylogenetic hypotheses have major implications for interpretation of homology and character evolution associated with the origin and early history of flowering plants. As an example of the complex and often unpredictable interplay of phylogenetic and comparative biology, we analyze the evolution of double fertilization, a process that forms a diploid embryo and a triploid endosperm, the embryo-nourishing tissue unique to flowering plants. We demonstrate how the new phylogenetic hypotheses for seed plants and angiosperms can significantly alter previous interpretations of evolutionary homology and firmly entrenched assumptions about what is synapomorphic of flowering plants. In the case of endosperm, a solution to the century-old question of its potential homology with an embryo or a female gametophyte (the haploid egg-producing generation within the life cycle of a seed plant) remains complex and elusive. Too little is known of the comparative reproductive biology of extant nonflowering seed plants (Gnetales, conifers, cycads, and Ginkgo) to analyze definitively the potential homology of endosperm with antecedent structures. Remarkably, the new angiosperm phylogenies reveal that a second fertilization event to yield a biparental endosperm, long assumed to be an important synapomorphy of flowering plants, cannot be conclusively resolved as ancestral for flowering plants. Although substantive progress has been made in the analysis of phylogenetic relationships of seed plants and angiosperms, these efforts have not been matched by comparable levels of activity in comparative biology. The consequence of inadequate comparative biological information in an age of phylogenetic biology is a severe limitation on the potential to reconstruct key evolutionary historical events.