A Genetic Screen for Mutations Affecting Cell Division in the Arabidopsis thaliana Embryo Identifies Seven Loci Required for Cytokinesis

Cytokinesis in plants involves the formation of unique cellular structures such as the phragmoplast and the cell plate, both of which are required to divide the cell after nuclear division. In order to isolate genes that are involved in de novo cell wall formation, we performed a large-scale, microscope-based screen for Arabidopsis mutants that severely impair cytokinesis in the embryo. We recovered 35 mutations that form abnormally enlarged cells with multiple, often polyploid nuclei and incomplete cell walls. These mutants represent seven genes, four of which have previously been implicated in phragmoplast or cell plate function. Mutations in two loci show strongly reduced transmission through the haploid gametophytic generation. Molecular cloning of both corresponding genes reveals that one is represented by hypomorphic alleles of the kinesin-5 gene RADIALLY SWOLLEN 7 (homologous to tobacco kinesin-related protein TKRP125), and that the other gene corresponds to the Arabidopsis FUSED ortholog TWO-IN-ONE (originally identified based on its function in pollen development). No mutations that completely abolish the formation of cross walls in diploid cells were found. Our results support the idea that cytokinesis in the diploid and haploid generations involve similar mechanisms.     

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.     

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.     

Gamma-tubulin is essential for microtubule organization and development in Arabidopsis

The process of microtubule nucleation in plant cells is still a major question in plant cell biology. gamma-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, gamma-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis gamma-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of gamma-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both gamma-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of gamma-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of gamma-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of gamma-tubulin in the formation and organization of microtubule arrays in Arabidopsis.     

Hybrid incompatibility in Arabidopsis is determined by a multiple-locus genetic network

The cross between Arabidopsis thaliana and the closely related species Arabidopsis arenosa results in postzygotic hybrid incompatibility, manifested as seed death. Ecotypes of A. thaliana were tested for their ability to produce live seed when crossed to A. arenosa. The identified genetic variation was used to map quantitative trait loci (QTLs) encoded by the A. thaliana genome that affect the frequency of postzygotic lethality and the phenotypes of surviving seeds. Seven QTLs affecting the A. thaliana component of this hybrid incompatibility were identified by crossing a Columbia × C24 recombinant inbred line population to diploid A. arenosa pollen donors. Additional epistatic loci were identified based on their pairwise interaction with one or several of these QTLs. Epistatic interactions were detected for all seven QTLs. The two largest additive QTLs were subjected to fine-mapping, indicating the action of at least two genes in each. The topology of this network reveals a large set of minor-effect loci from the maternal genome controlling hybrid growth and viability at different developmental stages. Our study establishes a framework that will enable the identification and characterization of genes and pathways in A. thaliana responsible for hybrid lethality in the A. thaliana × A. arenosa interspecific cross.     

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。     

The canonical α-SNAP is essential for gametophytic development in Arabidopsis

The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA⁺ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.     

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。     

Trends for Inbreeding in Polyploid Pteridophytes

Abstract Previous studies on the sequence of the gametangium formation in homosporous fern gametophytes (Masuyama, 1975a, b) suggested that diploid and polyploid taxa may favor gametophytic crossing and gametophytic selfing, respectively. In this view, intraspecific polyploids of three fern species ( Phegopteris decursive-pinnata, Lepisorus thunbergianus and Pteris dispar ) have been investigated for the amount of genetic load or the rate of gametophytic selfing. As expected, a marked contrast of mating systems is found between diploids and tetraploids; the former predominantly undergo gametophytic crossing and the latter predominantly undergo gametophytic selfing. It seems likely that diploid homosporous pteridophytes favor gametophytic crossing with some exceptions favoring gametophytic selfing as a derived condition, whereas polyploid homosporous pteridophytes favor gametophytic selfing predominantly.     

Gene expression during the male gametophytic phase

Abstract

In recent years a number of experimental findings have indicated that in higher plants the gametophytic phase is able to express its own genetic information, a large part of which it shares with the sporophytic generation. Quantitative estimates of haploid and haplodiploid gene expression have been obtained by mRNA and isozyme analysis in several plant species: 60-70% of the genes are expressed in both pollen and plant, about 10% are pollen-specific, and 20% represent the sporophytic domain. Moreover, it has been demonstrated that stage-specific genes are expressed in the gametophytic generation: at least two sets of genes are activated during pollen development, others are expressed only in the postshedding period, during germination and tube growth. Studies have been made to ascertain the role played by gametophyte-expressed genes in pollen development; the in vivo and in vitro pollen tube growth rate has been revealed to be controlled by the gametophyte genome itself. Differential effects of specific chromosomal deficiencies on the development of maize pollen grains have indicated that components of normal microspore development are controlled by genes located in specific parts of the genome. For single gene analysis, gene transfer can be used; on the contrary, for traits with a multifactorial genetic control, direct proof of gene expression both in the gametophytic and the sporophytic generation can be obtained when selection is applied to the pollen population of a hybrid plant, and response to selection is observed in the resulting sporophytic progeny. Response to selection, applied at different stages of the gametophytic phase, has been described in the sporophytic progeny and this with regard to many adaptive traits; thus the phenomenon can have an important bearing on the genetic structure of natural populations and on higher plant evolution, it can also be used as a breeding tool to increase the efficiency of conventional selection methods.     

花粉管生长和极性引导的孢子体和配子体控制研究进展

双受精是被子植物特有的生殖方式,精细胞只有通过花粉管穿过花柱才能到达子房、胚珠受精。花粉管在母本组织中的生长和引导包括孢子体控制(sporophytic control)和配子体控制(gametophytic control)两个连续的过程,现已克隆出不同阶段花粉管生长和引导的基因,通过分析其表达调控揭示出花粉管生长和引导的分子机制。该文就近年来国内外有关花粉管生长和极性引导的调控机制研究进展进行综述,并对禾本科(Poaceae)和十字花科(Brassicaceae)植物花粉管引导的异同点进行了比较分析。     

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

The Arabidopsis gene PROLIFERA is required for proper cytokinesis during seed development

The Arabidopsis thaliana (L.) Heynh. gene PROLIFERA (PRL) is a member of the MCM family of genes that are required for DNA replication during the S phase of the cell cycle. PRL is expressed in dividing cells throughout plant development. During reproductive development, PRL is expressed in both the developing megaspore mother cells and microspore mother cells, but is not expressed in the developing microgametophyte, suggesting that it does not function in the final haploid divisions leading to the production of a mature pollen grain. Disruption of PRL leads to megagametophyte and embryo lethality. prl mutant embryos arrest at a variety of stages, and often show defects in cytokinesis. Multinucleate cells and non-stereotypical cell division planes are commonly observed in developing prl mutant embryos, although mcm mutations in other organisms have not been reported to affect cytokinesis. These observations suggest that PRL may play a role in cytokinesis that is distinct from its role in regulating DNA replication. Additionally, a novel cytokinesis checkpoint that monitors cell cycle progression may exist in Arabidopsis.